Current Topics in Membranes and Transport, Volume 14

Current Topics in Membranes and Transport Volume 14 Carriers and Membrane Transport Proteins

Advisory Board

I . S . Edelman Alvin Essig Franklin M . Harold James D. Jamieson Philip A. Knauf Anthony Martonosi Shmuel Razin Martin Rodbell Aser Rothstein Stanley G. Schultz

Contributors

J . P . Bennett L. I . Boguslavskp K . A . McGill S . H . P . Maddrell D . M . Matthews J . W . Papne Peter G. W . Plngeinann Adil E . Shainoo William F. Tivol G. B . Wurren W . F . Widdas Robert M. Wohlhueter

Current Topics in Membranes and Transport VOLUME 14

Carriers and Membrane Transport Proteins Edited b y Felix Bronner Department of Oral Biology University of Connecticut Health Center Fcirrnington, Connecticut und

Arnost Kleinzeller Depurtrnent of Physiology University of Pennsylvania School of Medicine Philadelphia, Pennsylvanio

1980 Academic Press A Subsidiary of Harcourt Brace Jovunovich, Publishers New York

London Toronto Sydney San Francisco

COPYRIGHT @ 1980, BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. NO PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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ISBN 0-12-153314-X PRINTED IN THE UNITED STATES OF AMERICA 80 81 82 83

9 8 7 6 5 4 3 2 1

Contents List of Contributors, ix Preface, xi Yale Membrane Transport Volumes, xiii Erratum, xv Interface between Two Immiscible Liquic; as a Tool for Studying Membrane Enzyme Systems L. I. BOGUSLAVSKY

I. Introduction, 2 11. Potential Jumps at the Interface of Two Immiscible Liquids, 4 111. Experimental Equipment for Measuring the Volta Potential at the Oil/Water Interface, 1 1

IV. Compensation Potential in the Water/Oil Chain, 13 V. Some Approaches to the Study of Enzymatic Reactions Occurring at the Interface, 16 VI . Possible Mechanism of the Potential Generation at the Interface between Two Immiscible Liquids, 25 VII. Chlorophyll and Other Porphyrins at the Interface, 30 VIII. Study of Membrane Enzymatic Systems of the Respiratory Chain of Mitochondria, 42 IX. Rhodopsin and Bacteriorhodopsin at the Interface, 43 X . The Influence of the Dielectric Constant of the Oil Phase on the Efficiency of Charge Transfer through the Interface, 45 XI. Coupling of Membrane-Enzyme Systems, 46 XII. Conclusions, 48 Symbols and Abbreviations, 49 References. 51

Criteria for the Reconstitution of Ion Transport Systems ADIL E. S H A M 0 0 AND WILLIAM F. TIVOL

I. Introduction, 57 11. Reconstitution Experiments, 60 111. Conclusions and the Future of Reconstitution, 108 References, I I 1 V

vi

CONTENTS

The Role of Lipids in the Functioning of a Membrane Protein: The Sarcoplasmic Reticulum Calcium Pump J. P. BENNETT, K. A. McGILL, AND G. B. WARREN 1. 11. 111. IV. V. VI. VII. VIII. IX. X. XI. XII.

Introduction, 128 Sarcoplasmic Reticulum, 128 Purification of Ca-ATPase, 131 Equilibration of Lipid Pools, 133 Which Lipids Support ATPase Activity'?, 137 Reconstitution of Ca-ATPase into Sealed Vesicles, 144 Only 30 Lipid Molecules Modulate Ca-ATPase Function, 148 The Composition of the Lipid Annulus Is Not the Same as the Whole Bilayer, 150 Lipid Asymmetry, 154 Distribution of Lipids across the SR Membrane, 155 Transbilayer Disposition of the Phospholipid Annulus, 157 Concluding Remarks, 158 References. 159

The Asymmetry of the Hexose Transfer System in the Human Red Cell Membrane W. F. WIDDAS I. 11. 111. IV.

Kinetic Asymmetry, 166 Kinetics of Membrane Transfers with Asymmetric Affinities, 181 Morphological Asymmetry, 202 Implications of Asymmetry, 211 References, 215

Permeation of Nucleosides, Nucleic Acid Bases, and Nucleotides in Animal Cells PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER I. Introduction and Technical Principles, 226 11. Carrier Model for Facilitated Diffusion and Tests for Its Applicability to

111. IV. V. VI. VII. VIII.

Nucleoside and Base Transport, 235 Uptake of Nucleosides and Purine Bases, 253 Properties of Nucleoside and Free Base Transport Systems, 271 Transport Inhibitors and Inactivation, 287 Regulation of Nucleoside and Free Base Transport and Uptake, 295 Permeation of Nucleotides, 303 Summary and Conclusions, 310 References. 313

Transmembrane Transport of Small Peptides D. M. MATTHEWS AND J. W. PAYNE I. General Introduction, 332 11. Peptide Transport in Animal Small Intestine, 333

CONTENTS

111. IV. V. VI.

Peptide Transport in Animal Tissues Other Than the Small Intestine, 365 Peptide Transport in Microorganisms, 367 Peptide Transport in Higher Plants, 397 Possible Physiological Advantages of Transmembrane Transport of Small Peptides, 403 VII. Concluding Remarks, 407 References, 408

Characteristics of Epithelial Transport in Insect Malpighian Tubules S. H . P. MADDRELL

I. The Route of Water Transport, 428 The Passive Epithelial Permeability of Malpighian Tubules, 438 Correlation of Structure with Function, 442 Regulatory Properties of Malpighian Tubules, 445 Malpighian Tubule Action in the Absorption of Water Vapor from the Air, 457 Summarizing Remarks, 459 References, 460

11. 111. IV. V. VI.

Subject Index, 465 Contents of Previous Volumes, 473

vii

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List of Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin. J. P. Bennett, Department of Experimental Pathology, University College Hospital Medical School, London WClE 655, England (127)

L. 1. Boguslavsky, Institute of Electrochemistry, Academy of Sciences of the USSR, Leninsky Pr., 31, Moscow, V-71, USSR ( 1 ) K. A. McGill, Department of Biochemistry, University of Leeds, Leeds, England (127)

S. H. P. Maddrell, Agricultural Research Council Unit of Invertebrate Chemistry and Physiology, Department of Zoology, University of Cambridge, Cambridge CB2 3E5, England (427) D. M. Matthews, Department of Experimental Chemical Pathology, The Vincent Square

Laboratories of Westminster Hospital, London SWlV 2RH, England (331) J. W. Payne, Department of Botany, Science Laboratories, University of Durham, Durham DHI 3LE, England (331) Peter G. W. Plagemann, Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455 (225) Adil E. Shamoo,* Department of Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 (57) William F. Tivol,i Department of Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 (57) G. B. Warren, European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany ( 127) W. F. Widdas, Department of Physiology, Bedford College (University of London), Regent’s Park, London NWI 4NS, England (165) Robert M. Wohlhueter, Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455 (225)

* Present address: Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore, Maryland 21201. t Present address: Department of Pharmacology and Toxicology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642. ix

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Preface Volume 14 continues the examination of problems relating to Carriers and Membrane Transport Proteins initiated in Volume 12. A major task in dissecting transport into its molecular components is to retain transport characteristics in the components. To achieve this, it is necessary to understand how transport proteins act at the interface between hydrophilic and hydrophobic domains. In the first chapter, L. I. Boguslavsky analyzes by electrochemical techniques the properties of enzymes and enzyme systems in interphases. Membrane vesicles have proved useful in elucidating some aspects of transport. A . E. Shamoo and W. F. Tivol, in the second chapter, have generalized from such studies in terms of ion transport, describing at the same time the various experimental approaches available to the investigator. Since the lipid environment of membranes profoundly affects membrane proteins, J. P. Bennett, K. A. McGill, and G. B. Warren have discussed in detail how membrane lipids influence the calcium pump in sarcoplasmic reticulum. Using hexose transport in erythrocytes as a model system, W. F. Widdas focuses attention on the evidence for an intrinsic asymmetry of the red blood cell membrane. The rapidly metabolizing cell takes up nucleic acids, nucleosides, and nucleotides; P. G. W. Plagemann and R. M. Wohlhueter have described the kinetic pitfalls one encounters in trying to characterize the entry step of cellular uptake of these important solutes. D. M. Matthews and J. W. Payne present an extensive treatment of how small peptides are transported by cells, both eukaryotic and prokaryotic. Finally, S. H. P. Maddrell analyzes the properties of electrolyte and water transport in the Malpighian tubules of the insect Rhodnius, demonstrating the problems and emphasizing the usefulness of the preparation for such studies.

xi

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Yale Membrane Transport Processes Volumes These volumes originate from the Yale Department of Physiology under the editorial supervision of Joseph F. Hoffman and Gerhard Giebisch.

Joseph F. Hoffman (ed.). (1978). “Membrane Transport Processes,” Vol. I . Raven, New York. Daniel C. Tosteson, Yu. A. Ovchinnikov, and Ramon Latorre (eds.). (1978). “Membrane Transport Processes,” Vol. 2. Raven, New York. Charles F. Stevens and Richard W . Tsien (eds.). (1979). “Membrane Transport Processes,” Vol. 3: Ion Permeation through Membrane Channels. Raven, New York. Emile L. Boulpaep (ed.). (1980). “Cellular Mechanisms of Renal Tubular Ion Transport”: Volume 13 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York.

xiii

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ERRATUM Current Topics in Membranes and Transport, Volume 13 James B. Wade: Chapter 9, Hormonal Modulation of Epithelial Structure

Page 129 The seventh line in paragraph 3 should read: bladders do exist in cytoplasmic vacuoles of granular cells (13,28,

xv

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CURRENT TOPICS I N M E M B R A N E S A N D TRANSPORT, V O L U M E

14

Interface between Two Immiscible Liquids as a Tool for Studying Membrane Enzyme Systems L . I . BOGUSLAVSK Y Institute of Electrochemistry Academy of Sciences of the USSR Moscow. USSR

I. Introduction . . . . . . . . . . . . . . . . . . . 11. Potential Jumps at the Interface of Two Immiscible Liquids . . . . . A. Distribution Potentials . . . . . . . . . . . . . . . B. Adsorption Potential . . . . . . . . . . . . . . . C. Dipole Component of Adsorption Potential . . . . . . . . . D. The Influence of Solution Composition on Adsorption Potential . . . E. Methods for Measuring the Potential at the Interface . . . . . . 111. Experimental Equipment for Measuring the Volta Potential at the Oil/Water Interface . . . . . . . . . . . . . . . . . I I IV. Compensation Potential in the WateriOil Chain . . . . . . . . . 13 V. Some Approaches to the Study of Enzymatic Reactions Occurring at the Interface . . . . . . . . . . . . . . . . . . 16 A. Adsorption of Enzymes at the Interface . . . . . . . . . . 16 9. Equipment for the Study of the Enzymatic Reaction Rate at the WateriAir Interface . . . . . . . . . . . . . . . . 19 C. Acetylcholinesterase at the Aidwater Interface . . . . . . . 20 D. Galactosyltransferase at the Aidwater Interface . . . . . . . 22 E. More Complex Systems Investigated at the Interface . . . . . . 24 VI. Possible Mechanism of the Potential Generation at the Interface between Two Immiscible Liquids . . . . . . . . . . . . . . . . 25 VII. Chlorophyll and Other Porphyrins at the Interface . . . . . . . . 30 A. Oxidation-Reduction Transformations of Chlorophyll and Porphyrins . 30 B. Electron Transfer by Chlorophyll across the Interface between Two Immiscible Liquids . . . . . . . . . . . . . . . . 33 C. Proton Phototransfer Chlorophyll . . . . . . . . . . . . 34 D. Photooxidation of Water in the Presence of Chlorophyll and Ferro Complex of Tetramethyl Ether of Coproporphyrin Adsorbed at the Octane/Water Interface . . . . . . . . . . . . . . . 35 E. Role of Water and DNP in Proton Transfer across the Interface . . . 40 Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN @IZ-l533l4-X

1

2

L. I. BOGUSLAVSKY

VIII. Study of Membrane Enzymatic Systems of the Respiratory Chain of Mitochondria . . . . . . . . . . . . . . . . A. NADH Dehydrogenase . . . . . . . . . . . . . . . B. Succinate-Cytochrome c Reductase . . . . . . . . . . . C. Cytochrome Oxidase . . . . . . . . . . . . . . . IX. Rhodopsin and Bacteriorhodopsin at the Interface . . . . . . . . X. The Influence of the Dielectric Constant of the Oil Phase on the Efficiency of Charge Transfer through the Interface . . . . . . . . . . . . XI. Coupling of Membrane-Enzyme Systems . . . . . . . . . . . XII. Conclusions . . . . . . . . . . . . . . . . . . . Symbols and Abbreviations . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

1.

42 42 43 43 43 45 46 48 49 51

INTRODUCTION

One of the most important properties of biomembranes is their high catalytic activity which is determined by enzymes, some of which are inserted into membranes, others being adsorbed onto the membrane surface. The interface of phases with different dielectric constants facilitates a separation of reaction products provided the latter have different solubilities in contacting phases. The presence of a powerful electric field at the interface of the double layer and the specific orientation of molecules participating in the reaction endow an enzymatic reaction with a new property, which is described as a vectorial process. This allows a conversion of chemical or light energies into electrical energy. An interface between two immiscible liquids is the simplest model for studying the surface properties of biomembranes. Transport of ions through membranes has three stages. The first and third stages are the charge transfer through the membrane interface, whereas the second stage is diffusion through the membrane (Fig. 1). Electrochemical reactions accompanied by spatial separation of charges may occur at the interface. This can assist selective charge transfer through biomembranes. It is therefore necessary to study the ion and electron transport across the interface in order to clarify the selectivity mechanism of the membrane, the generation of transmembrane potential, and other processes which may be essential for understanding of membrane properties as a whole (Boguslavsky, 1971). The transport process through membranes as well as the interdependence of interfaces are supposed to be nonessential. Both interfaces are thought to be independent. This means that the results obtained for monolayers of a single interface are useful when the determining step of the process occurs at the interface of the membrane with an aqueous electrolyte solution. In this case one may

3

MEMBRANE ENZYME SYSTEMS

Three steps of charge transfer through a membrane

One step of charge transfer at the interphase

of two immiscible liquids

FIG. 1. Scheme of charge transfer through the membrane and across the interface of two immiscible liquids.

observe a similarity between some properties of molecules at the interface and their functions in the membrane. On the other hand, when the limiting stages of processes in the model and native systems do not coincide, the data disagree. This can be illustrated by the incompatibility of results obtained when measuring the rate of penetration of water molecules through the lipid monolayer, and the membrane of a squid giant axon (Shanes, 1963). Despite numerous studies of protein-lipid interaction in membranes, much effort is still required to achieve detailed and comprehensive knowledge of this interaction. A Coulomb interaction between charged groups on lipid and protein molecules has been studied in detail. This is much simpler than specific and hydrophobic interactions. Quite a number of investigations of enzymes at the water/oil interface deal with a special type of enzymatic system, i.e., phospholipase. These are water-soluble proteins whose substrates are lipids, i.e., the substances in a condensed state at the interface. This allows us to use the experimental procedure for monolayers to observe protein-lipid interaction and the products of this interaction (Colacicco, 1971). Henceforth, by interaction we mean not only the enzyme-substrate formation, but every chemical reaction occurring at the interface. In the region of a diffuse double layer at the lipid-water interface, when lipids and proteins are separated by more than 6 A, an electrostatic interaction occurs. The total charge of a protein globule is quite essential in this case. Such a conclusion was drawn, for example, when studying hemoglobin and albumin adsorption on a surface covered with lipid monolayers. Intermolecular interaction is essential at distances less than 6 A (Salem, 1962). The functioning of a phospholipase B was investigated by

4

L. I. BOGUSLAVSKY

measuring surface radioactivity of phospholipids containing 32P.The effective functioning of phospholipase was shown to proceed under specific conditions. It was particularly found that a surface-active substance with a negatively charged group should be present in the monolayer. The function of a special class of enzymes, i.e., the electrogenic enzymes at the interface, results in charge separation in the double electric layer. Together with the Laboratory of Bioorganic Chemistry of Moscow State University, we suggested a new approach to the problem of kinetics of enzymatic reactions accompanied by charge transfer across the interface between two immiscible liquids (Boguslavsky et al., 1974a, 197Sa; Kharkatz et NI., 1975). The change in the potential difference at the octane/water interface during enzymatic reactions accompanied by transfer of ions and electrons from one phase to another was measured by the vibrating electrode method. This approach has been used to study not only the enzymatic processes, but the ordinary catalytic reactions as well (Boguslavsky et ul., 1976~). Chlorophyll and other porphyrins were shown to be capable of transferring electrons between two redox systems in different phases (Boguslavsky et al., 1976~). Before discussing the results of the investigation of membrane enzymatic systems at the interface, we shall briefly consider some physicochemical properties of interfaces, which are necessary for the following discussion.

II. POTENTIAL JUMPS AT THE INTERFACE OF TWO IMMISCIBLE LIQUIDS

In the beginning of this century the problem of potential jumps arising at the interface of two immiscible liquids began to attract the attention of many investigators. The impetus to such investigations was given by the study of thick membranes represented by a layer of a polar organic liquid located between aqueous and nonaqueous phases. It was thought that a study of potentials in thick membranes would assist in understanding the function of more complex biological membranes. A thermodynamic theory for electrolyte solutions in two different solvents was developed by Nernst (1892), Luther (1896), Abel (1906), and van Laar (1907). By analyzing a chain of two different solvents the above authors came to a very important conclusion, namely, that the potential difference at the interface can be set up only if the coefficients of cation and anion distributions are unequal. Experimental measurements of po-

5

MEMBRANE ENZYME SYSTEMS

tential jumps at the interface of two immiscible liquids were performed by Haber (1908) and Beutner (1909). Beutner showed that the emf of those chains does not depend on the nature of the common ion. Frumkin (1919) demonstrated that the above results agree well with the theory of potential distribution. Beutner's ideas disagreed with those of Baur and Kornman (1916), who interpreted the origin of potentials in terms of the adsorption theory. The idea of the adsorption potential was introduced by Freundlich, who used it in order to treat electrocapillary phenomena. In Freundlich's opinion, the adsorption potential is superimposed on the potential, which is calculated from the Nernst formula. Frumkin compared experimental results obtained from investigations of solvent chains (I, 11) with data calculated on the basis of Nernst theory (Nernst, 1892). He showed that Baur's data are sufficiently well described by the Nernst theory. From this coincidence of theoretical and experimental results Frumkin concluded that a formal thermodynamic theory of ion distribution is quite sufficient for determining the potential difference between two immiscible liquids, and no additional assumptions about an adsorption potential jump are needed, as was proposed by Freundlich. A. Distribution Potentials For ideal solutions the distribution potential for a salt, M X , in a polar oil/water system depends on the coefficient of cation BM and anion B x distribution. If we express the coefficients of cation and anion distribution in terms of the difference of their standard chemical potentials in the aqueous and nonaqueous phases, then RTln B M=

-

Op."M

(1)

and RTln B x = " p i - ' p i

( 21

where "p0 and ' p 0 are the standard chemical potentials of ions in the aqueous and nonaqueous phases, respectively. The electrochemical potentials of individual ions can be expressed in terms of their standard chemical potentials. Under equilibrium conditions p M=

n-

OFM,

w px -

= "iix

(3)

When in each of the phases the electroneutrality is maintained, then "

c,, = c\,

oc2,

=

OC\

(4)

6

L. I. BOGUSLAVSKY

In order to determine the distribution potential, cpD, through the distribution coefficient for the salt, MX, S,,

OCMX

=-

CMX

it should be borne in mind that

S\n

=

(B\i.B\)''*

( 6)

For two systems containing the salts with a common anion, X

or

The process of ion distribution between phases is closely linked with resolvation, and is accompanied by interpenetration of the solvent molecules into both phases. The derived ratio shows the physical sense of the distribution coefficient as the power factor corresponding to the ion transfer from water into the saturated nonaqueous phase with a different polarity. Thus, cpD is the physical quantity resulting from differences of hydration and solvation energies of potential-determining ions. The concepts described here are particularly important for the interpretation of phenomena occurring at the octane/water interface in the case of membrane enzymatic systems. B. Adsorption Potential The contact of two immiscible liquids results in the interphase distribution of potential schematically shown in Fig. 2. It is assumed that the potential is measured near the aqueous phase, and the point of a test charge is separated from the interface by less than the Debye length. In this case not only the excess charges adsorbed at the interface, but the dipoles as well, will contribute to the measured value. At a n y point near the interface, where the electroneutrality conditions are not observed, the cation and anion concentration is determined by the Boltzmann equation:

(9)

7

MEMBRANE ENZYME SYSTEMS

'. I

/

x-m

t X

FIG.2. Potential distribution at the oil/water interface depending on the distance to the boundary.

Since the electroneutrality equation is not valid at the point near the interface inside the double electric layer, Eq. (7) can be expressed as

By substituting Eqs. (9) and (lo), instead of " C Mand "Cx in Eq. ( 1 I ) , at the condition that cplx+o ='Pads we have 'P =

'PO

-

('PO

-

'Pads)

= 'Pads

(12)

This means that at the interface of water with air or solvent of low polarity the Galvani potential is not determined by the coefficients of B , and B , ion distribution. The change of the observed potential can be attributed to the presence of particles adsorbed at the interface (Davies and Rideal, 1963). C. Dipole Component of Adsorption Potential

For a film composed of uncharged dipole molecules, the change of the potential jump at the interface can be assigned to a vector sum of indi-

8

L. I. BOGUSLAVSKY

vidual dipole moments (Davies and Rideal, 1963): Aqads = 4rrns1pd+ 4rrn,2pd

+ 4rrn,3pd

(13)

Here the effective dipole moment of the water molecule, ' p d , which is assumed to be specifically oriented at the water/air or water/hydrocarbon interface, is taken into account. Moreover, one should allow for the dipole moment of the bond 2 p d ,which connects the polar head with the hydrocarbon chain, or any other liposoluble part of the molecule. The bonds of H,C in the long-chain amines can serve as an example. Finally, in the case of a hydrocarbon chain, there is another component, 3pd, determined by the dipole component of the terminal bond C-H. In order to find the contribution of individual components to overall potential drop, Aqads, it is necessary to examine the adsorption potentials of molecules differing in any one functional group. By changing the terminal , hence Aqads. If the atom in the hydrocarbon chain, one can alter 3 p d and values of Ipd and 2pdare assumed to be constant when replacing the bond C-H by C-Br then the observed alteration of A q a d s may be attributed to the change of IAcPadsI

=

4.rrns*A3Pd

(14)

Gerovich and Frumkin (1936) were the first to show how considerable this alteration can be, taking as an example bromohexadecanoic acid. Insoluble monolayers of halogen-containing aliphatic compounds at the water/air interface are characterized by the negative jumps of the potential, which is explained by the orientation of the negative end of the dipole formed by the halogen atom toward air when an insoluble monolayer is formed by Br-(CH,),,-COOH molecules. A very high negative potential may be ascribed to C-Br bond orientation. The palmitic acid corresponding to this compound is characterized by a potential which is positively shifted by 0.39 V at the water/air interface. The replacement of the C-C bond by C-Br produces a general effect of 1.26 V . Since the dipole moment of C-Br bond p = 1.9 x (CGSE) with a 20 Az area per molecule, the effect would be -2.9 V , if the angle between C-Br dipoles and the water surface is taken into account. The difference of the observed potential jump from the calculated one can be due to the relative polarizability of the C-Br bond. When the surface pressure varies, i.e., the area per molecule changes, the vertical component 3pdalso undergoes certain alterations due to the change of the slope of the hydrophobic chains to the interface. Let a monolayer be adsorbed at the interface. If the polar heads of molecules adsorbed at the interface have an electric charge, then the monolayer is referred to as ionized.

9

MEMBRANE ENZYME SYSTEMS

D. The Influence of Solution Composition on Adsorption Potential

Ions whose charges are opposite to the charge of a monolayer are always active relative to those monolayers. The concepts of the effect of counterions on the properties of charged monolayers are based on the assumption of the existence of particle redistribution in the film-boundary layer due to their potential energy in this layer being different from that in the solution. (This assumption is valid both for charged and uncharged particles.) Therefore the concentration of uncharged particles at the surface may be expressed as

cs =

C.eUlkT

(15)

where U is the decrease of the potential energy of the system upon transition of the particles from the bulk to the surface layer. C', in accordance with the simplifying postulate of the theory, is the function of the surface excess, r:

cs = f(r)

(16)

If the ions on the surface have a charge n , then the potential energy should be altered by the value of the work of a single charge in the electric field:

Cs= Co.exp [ U

-

neAcpo]/kT

(17)

By assuming that a homogeneously charged surface is impenetrable to counterions, i.e., point charges, Gouy has found a solution for the Boltzmann equation of the type (17) for the potential distribution cpG at the interface :

I n principle, the absolute value A q G need not coincide with A v o . The concentration dependences of ApG and A p 0 are experimentally identical. The potential Acpo should not be identified with <-potential, since the plate where the polar heads of surface-active ions are situated does not coincide with the slip plane of the liquid layer. If the relation (19)

APG

is fulfilled, then the change of Volta potential in the aidwater system containing adsorbed ionized molecules should obey the relation: Acpads

= 437nsP5

+ Acpc

(20)

where Ac~Gdenotes the potential drop between the surface of the solution

10

L. I. BOGUSLAVSKY

and its bulk, and pSI is the vertical component of the dipole moment. The equation can be verified in different ways. First, it would be expected that dAcpad,ldcpOshould vary in accordance with the Gouy equation, the area per molecule and the value of p: being kept constant. In this case the slope is:

For positively charged films, "minus" should be taken; for negatively charged films, "plus." Equation (2 1) was previously confirmed (Davies, 1951) for a-bromopalmitate monolayers (with negatively charged polar heads) and trimethyloctadecylammonium monolayers (with a positive charge of hydrophilic heads).

E. Methods for Measuring the Potential at the Interface

The Galvani potential between two points of different phases cannot be experimentally determined. The same can be said about the surface potential jump, which differs from the experimentally observed work function by the value of the chemical potential of the particle, i, in some phases, a . The difference of external potential (Volta potentials) can be experimentally determined. A method for experimental determination of this quantity will be given. Figure 3 shows a scheme for measuring the Volta potential between phases a and p. For the sake of simplicity, let all the leads be made of phase a material. The difference of external potentials at points A and B can be compensated by an external source V, so that the current between a and p equals zero. The current between phases a and p can be obtained by ionizing the space between phases by means of the ionizing source, or due to the displacement current resulting from the relative displacement of phases. Under equilibrium conditions,

bg

-

fig

=

eV

(22)

/Lg - b:' = 0

(23)

0

(24)

$0

-

$0

=

Using, instead of the electrochemical potential of electrons in phase a , the work function in phase alvacuum system c#P, we have /Z = e p . Here $a is the surface potential drop at the interface between phase (Y and the vacuum resulting from the presence at the interface of the free

11

MEMBRANE ENZYME SYSTEMS

FIG. 3 . Scheme of measuring the Volta potential between two metal phases (a), and between metal and solution (b).

charges only. Equation ( 2 2 ) can be transformed into

9"

-

4" + e($O

-

$la)

= -

eV

(25)

By using Eq. (24) one can obtain the relation between the difference of work functions from phases a and p, and the source potential: +a - +B

=

-

eV

(26)

If phases CY and p are equal, but contain different adsorbed molecules, the difference of work function is characterized by an adsorption potential jump in the phase: (p."

111.

+ e$C) - (pU+ e$S) = e($P - $$) = - eV

(27)

EXPERIMENTAL EQUIPMENT FOR MEASURING THE VOLTA POTENTIAL AT THE OILIWATER INTERFACE

A vibrating plate method is one of the ways to determine the potential of a liquid interface. An air reference electrode (gold plate) is placed over the liquid surface. Between these surfaces a plane capacitor is formed, whose capacity changes during the air electrode vibrations. Due to the difference of work function of the electrodes, a constant potential difference in the air gap between the capacitor plates exists. When the capac-

12

L. I. BOGUSLAVSKY

itor is included in the closed circuit, an ac current flows in the circuit due to the periodically varying capacity. If any source supplies such a capacitor with a potential difference, which is equal in value, but opposite in sign to the Volta potential of the chain Au I air I phase I

I phase 2 I reference electrode

(28)

then no current is observed in the external circuit. At any other potential, the current flows in the system. The method of measuring the Volta potential by means of a vibrating electrode for two immiscible liquids has been described by Davies and Rideal (1963) and Gugeshashvili of nl. (1974). The change in the Volta potential between the gold reference electrode and the aqueous phase in the chain ( 2 8 ) is produced by a change in the structure of the electric double layer 1 Au

1 1 2 air

PI2

P23

3 oil. charge acceptor

1

4 water. enzyme substrate

1

P4S

934

sat. KCI

1

6 reference electrode

(29)

PSh

at the oil/water interface. The set-up consists of a Pyrex cell with a bowl in which the oil lies over the aqueous phase (Fig. 4). The vibrating gold reference electrode is located above the oil. A calomel electrode is used as a reference electrode in the aqueous phase. The electrode is connected to the cell via a salt bridge with saturated KCI solution. All aqueous solutions should

3

4

5

FIG.4. Block diagram of experimental set-up for Volta potential measurement by the vibrating electrode method.

13

MEMBRANE ENZYME SYSTEMS

be prepared from twice-distilled water and twice-recrystallized salts. Octane was of standard purity grade. I t was placed in the same vessel as the aqueous phase, and kept there for 36 hours or less, with the system continuously shaken. If q I 2 qZ3. . (P~~ and . q 5 6 remain constant while the potential difference at the interface between phases 3 and 4 changes, then the observed effect can be attributed to the change of the potential difference at the oil/water interface. The charge acceptors were always added in advance to the octane/water system in order to achieve equilibrium distribution of these components between phases. The reaction substrates and enzyme systems were added to the measuring cell directly before measurements. The change of Volta potential was measured 2 minutes after addition of the last component. All the data contained in the following figures show the change of Volta potential in the chain (29), as measured by the vibrating electrode method, plotted against the "supporting electrolyte." The supporting electrolyte was the solution that did not contain one of the components necessary for an enzymatic reaction. As shown by experiments, addition of any of the components, substrate or enzyme, to the oil/water system already containing a charge acceptor leads to a change in the potential difference at the interface. The magnitude of this change is independent of the initiation of the enzymatic reactions, i.e., by addition of either the substrate or the enzyme. The Volta potential is not changed when one of the reaction components is excluded. The observed changes in the Volta potential thus prove to be the result of an enzymatic reaction occurring at the interface. It should be emphasized that the presence of a charge acceptor and all other components in the system is a necessary condition for the change in the Volta potential at the octane/water interface.

IV. COMPENSATION POTENTIAL IN THE WATER/OIL CHAIN

The problem of additivity of potentials in complete concentration chain (30) KCI

KCI

and the potentials of its half-elements

,I; ;

water

half-cell I

half-cell I1

Hg, Hg2C12

1 I 1 1 water oil Mc X, MX

air Au

( 3 1) (32)

14

L. I. BOGUSLAVSKY

always arises when comparing the results of measuring the emf in the chain (30). and the potentials of chains (3 1) and (32), which are the halfcells of chain (30). Figure 5 shows the results of measuring the potentials in the chains that are analogous to chains (30)-(32). The salt, M X , is represented by (But),NCI, and nitrobenzene has been chosen for the oil phase. The emf of the water/nitrobenzene/water concentration chain in the presence of tetrabutylammonium chloride is significantly less then the sum of half-cell potentials measured in the chain by the vibrating electrode and radioactive probe technique. This discrepancy seems to be due to the diffuse potential appearing in the nitrobenzene phase, which can be avoided by introducing a special salt bridge. The salt bridge is chosen in such a way as to fulfill the following conditions. The mobilities of an anion and a cation should be equal, and their concentrations should exceed the concentration of the salt in the phase which the bridge is contacting. The electrolyte of the bridge should be mixed up with the nonaqueous phase. To meet these requirements, a picrate electrode has been chosen, according to the procedure proposed by Karpfen and Randles (1953). A 0. I M solution of tetraethylammonium picrate (Et),NPi in methyl butyl ketone (MBK) has been used as a bridge from the side of nitrobenzene. In this case chain (30)

1 1

y$

KCI

(But),NCI

Hg, Hg2C12 wa;er

1 1 1 I NB

MBK (Et ),NPi

water (Et),NPi

HgPi, Hg

(33)

and chain (33) produced similar potentials (Gugeshashvili et c i l . , 1974). By dividing the nitrobenzene phase with an (Et),NPi bridge the diffusion potential can also be excluded. The compensation potential can arise in the K-ion concentration chain of the heptane phase containing valinomycin. In this case the emf is generated in the electrochemical con-

FIG.5 . Measurement of the Volta potential ( I ) and transmembrane potential (2) versus the concentration of (But)JVCI in the nitrobenzene/water system.

15

MEMBRANE ENZYME SYSTEMS

centration chain (30) exhibiting a linear dependence on the logarithm of the K-ion activity. Investigations of chains (30) and (3 I ) , using a heptane solution of valinomycin, as a nonaqueous phase, showed that the straight line has a slope of 50 mV. In this case one can also observe the nonadditivity of the emf of chains and half-cells (Boguslavsky et ul., 1974b). Evidently, this absence of additivity can be ascribed to the potential drop in the oil phase in the complete chain. Let us examine the set-up of potentials in the system: 3 PI2

P23

In the simplest case the same salt, M X , is dissolved in aqueous phases I and 3. In the case when equilibrium for cation and anion is established at interfaces 1-2 and 2-3, the electrochemical potentials of individually dissolved ions in the aqueous (wb)and nonaqueous ("b) phases may become equal. If, in addition, we assume each phase to be electrically neutral, and consider only one interface between semiinfinite phases oil/ water, we obtain the usual formula relating the distribution coefficients of the cation B,,, and anion B x between oil and water to the potential difference at the interface. If no assumption is made of the electrical neutrality of phases, i.e., the equality of cation and anion concentrations in the nonaqueous phase, but we assume that nonequilibrium (adsorption) potentials are set up at interfaces 1-2 and 2-3, then the equilibrium distribution is established only for the penetrating ion, i.e., the ion which has a larger distribution coefficient, and in a certain layer adjacent to the interface. Let this ion be the cation. Then the cation concentration at a certain point of nonaqueous phase inside this layer can be expressed in terms of the aqueous phase concentration at interfaces 1-2 and 2-3: 'I

C ,,, =

C,,,B ,,,e -nFriRT

(35)

where n is the cation charge and cp is the potential difference at the oil/ water interface (cpI2 or cpZ3). When the chain is closed, a compensation potential difference is set up in the oil phase between points near interfaces 1-2 and 2-3. This is equivalent to an equilibrium inside the oil phase for the penetrating ion. It is significant that a similar assumption is not made for the anion. The equilibrium conditions in the oil phase can then be written as

RT cpk = -In

nF

"Ch ()C&

-

16

L. I. BOGUSLAVSKY

where 1 and 2 refer to interfaces 2-3 and 1-2, respectively. Using Eq. ( 3 9 , we can express the quantity (Pk in terms of the cation concentration in the aqueous phase

The nature of the compensation potential depends on the oil phase. It can arise due to polarization of the dielectric liquid containing colloidal aggregates including water molecules. The total value of the potential difference E between aqueous phases 1 and 3 at the ends of the concentration chain should contain three terms ES

=

(Pl2

+

(P23

+

(Pk

Using Eq. ( 3 7 ) , we have

RT E , = -In nF

"Ck "CL

-

(39)

It is significant that the compensation current responsible for the appearance of (Pk is proportional to the ion concentration in the oil phase, and since the ion concentration is low, and the capacity of the system small, this current may be negligible. An identical :xpression for (Pk can be obtained on the assumption that the distribution potentials are set up at 1-2 and 2-3 interfaces, but the transference number for cation in the oil phase is unity. The simple example previously considered shows hat while the Nernst law is ideally valid in the complete chain, the pots. iti7.s of the half-cells may vary most dramatically. Moreover, it is I 't: 'ifference of these potentials at the interface which correlates with t k dectivity of membranes with respect to individual ions. By the same reasoning, we can arrive at the colLv . A n that it is the consideration of the half-cells with acting enzymes, rather than that of the complete water/oil/water chain, which gives the most valuable information on the characteristics of active transport of charges through biological membranes. L ,

V. SOME APPROACHES TO THE STUDY OF ENZYMATIC REACTIONS OCCURRING AT THE INTERFACE A. Adsorption of Enzymes at the Interface

Most enzymes in nature, even those which are assumed to be watersoluble, are not present in the solution in a free state, but interact with

MEMBRANE ENZYME SYSTEMS

17

a membrane, or the inner part of the cell. Kempner and Miller (1968) showed that intracellular enzymes form part of the cytoplasm. Despite numerous methods for the immobilization of enzymes, two methods are particularly noteworthy when the enzyme is immobilized at any interface. These are covalent binding and molecule adsorption. The enzymes were also deposited on a metal plate, filter paper, and special Millipore filters, or silk. Moreover, it is possible to fix various enzymes at the surface by means of adsorption forces. Various approaches to the problem are considered in a monograph by Zaborsky (1973). Poltorak and Vorobjeva (1966) proposed a model design of the membrane, by making use of lipid adsorption on solid adsorbent. This approach is distinguished by large areas of the actual surface, and a possibility to control the preferential mode of binding. By changing the types of carriers (silica gel, activated carbon), one can produce surfaces in which cephalin or lauric acid are oriented to the surface with hydrophobic tails or polar groups. A study of the activity of alkaline phosphatase relative to the substrate, disodium salt of p-nitrophenyl phosphate, resulted in a stable increase of the reaction rate at the hydrophobic surface of a lipid monolayer. The same approach was developed for hydrogen peroxide decomposition in the presence of catalase. Specific activity of catalase increases with the surface coverage by lauric acid up to 8 = 1, if its hydrocarbon taiis at the interface are solution oriented (Goldfeld et a / . , 1966). In contrast to this, the hydrophobic surface covered with trin-dodecylamine resi ts in a 5-fold inactivation of the enzyme. So, not only a hydrophsl ‘c t>tnding,but the actual structure of the surface is essential for high enzymatic activity. To simulate the enzymatic membrane system, one should allow for the layer structure of the membrane. The use of mcirwla.jers in this case should be rather effective, more so because the technique of monolayers allows for the superposition of different components required for the reaction. Skou (1958) showed that anesthetics derived from amines and alcohols which block the nerve impulse show an ability to penetrate into the lipid monolayer of nervous tissue, and thus increase the surface pressure in the monolayer. Adsorption of enzymes was studied at the water, air, oil/water, and waterholid phase interfaces. These investigations aimed not only at determining the enzymatic activity, but primarily at defining the nature of the adsorption process in order to develop the most appropriate technique for clearing the sample from admixtures. The adsorption properties of an enzyme depend on a number of factors, among which one should note the content of solvent and substrate, the concentration of hydrogen ions,

18

L. I. BOGUSLAVSKY

and temperature. Because of a great number of widely different functional groups, the mechanism of adsorption cannot be based on a single type of adsorbent-adsorbate interaction, but includes a variety of such interactions from van der Waals forces to chemical bond formation. The pH dependence of the enzymatic activity investigated at the interface requires a correction for the interface charge. As a result, a displacement of hydrogen ion concentration toward greater or smaller pH values occurs. In studying enzymes, it is important to know if any changes occur in the protein activity at the interface of a lipid monolayer with air or with some other nonaqueous phase. As a rule, the protein activity changes rather drastically; however, when the active conformation of the proteins is not altered, it is possible to study them at the interface. The Michaelis constant for the enzymes adsorbed at the interface coincides with that in solutions, but both greater and lesser variations are possible. For example, malate dehydrogenase adsorbed onto silica gel exhibits higher values of K , . If this enzyme is adsorbed onto Sephadex, then the K , values are lower then those determined for the soluble enzyme (Tveritinova et a l . , 1971). The enzyme adsorption can be accompanied by a change in the specificity of its action, stability, and heat lability. By investigating enzymatic activity of trypsin, Inbar and Miller (1974) showed that only 2-3% of the original activity in the solution is retained at the water/air interface. As a rule, the interface adsorption changes the enzymatic conformation to such an extent that the process becomes irreversible. The enzymes present at the lipid/water interface under native conditions should inevitably spread out, at least partially, over the water/air interface. The degree of spread depends on temperature, pH, ion composition of the liquid, surface pressure, enzyme structure, interaction with other molecules at the interface, and, finally, on the time during which the protein is present on the surface. Normally, the protein spreads out in a period of several minutes. The orientation of protein groups depends on the surface pressure. At zero pressure the protein groups are in the interface plane. With an increase in the number of particles, the polar groups of a monolayer are immersed in water, and nonpolar groups are ejected from water into air. In addition to common parameters, which control the rate of the enzymatic reaction at the interface, there is another factor characterizing the state of reaction components at the interface. This assertion can be verified by taking as an example the hydrolysis of lecithin molecules which form a monolayer at the water/air interface. Phospholipase A, which can be isolated from different sources, catalyzes the hydrolysis of

MEMBRANE ENZYME SYSTEMS

19

a synthetic phosphatidylcholine with different length of the liposoluble group. Lipids with shorter liposoluble groups produce homogeneous solutions and are not hydrolyzed under these conditions (Haas et ul., 1971). When investigating the reaction kinetics at the interface, one should be sure that the reaction products (fatty acids C8-CI2, in this case) do not produce a monolayer, and are quickly desorbed from the surface. The desorption rate of fatty acids, produced during lipid hydrolysis of CS-Cl2, has been investigated by the technique proposed by Ter-Minassian-Saraga (1973). The fully ionized acids were not found to produce insoluble surface layers, and therefore the desorption rate constant was so large that it could not be measured. The experiments showed that in no case does the rate of product desorption restrict the process as long as lipids with a chain of 12 carbon atoms are not used. Numerous enzymatic reaction investigations have been performed with the use of insoluble monolayers of long-chain glycerides or phospholipids. In order to carry out such experiments, a special setup is needed, where the surface pressure of monolayers can be measured under specific conditions allowing the change of an underlying solution without disturbing the adsorption layer.

B. Equipment for the Study of the Enzymatic Reaction Rate at the Water/Air Interface

Figure 6 illustrates two modifications of the method of investigating the surface pressure at the water/air interface ,(Verger and Pattus, 1976). If the film of the studied substance is spread out at constant surface pressure, the water suspension of liposomes or vesicles will be dropping at the rate of several microliters per minute on a wet glass rod immersed in the first section of the measuring cell. Liposomes or membrane fragments spread out over the interface, and if the surface area of the cell is sufficiently large, the surface pressure does not increase as the membrane suspension is added. Then by means of a movable barrier, the film can be compressed and transferred through section 11, where it is freed from impurities present in the aqueous phase, to section 111. Here the substrate is added to the system. Then the layer is removed to section IV where the surface pressure is measured. Another method of studying a reaction in a monolayer is based on the maintenance of constant surface pressure during experiments. This method is represented in the right-hand side of Fig. 6. The monolayer here is formed in the same way, as in section 111, and the barrier A is moved close to the glass rod, and helps one fill the monolayer. The

20

L. I. BOGUSLAVSKY

4n

1

I

h

4

a

c

1

b

FIG. 6. T w o different methods for the study of enzymatic activity at the wateriair interface using the method of the surface pressure measurement in the monolayer at zero (a) and constant (b) surface pressures. A four-chamber Teflon cell for measuring the surface pressure contains mixers (in three chambers), where the surface pressure is to be measured ( I ) , a movable barrier (2). a glass rod (3), by means of which, using the microsyringe (4). the membrane material ( 5 ) is introduced. The substrate (6) is added in the next to last section (see text). The surface pressure is measured by the Langmuir method in the fourth section. (From Verger and Pattus, 1976.)

increase in the monolayer pressure is immediately compensated by the movement of the barrier. When the whole material is spread over the surface, barrier A , as well as a second barrier B, is used to remove the film to section I. C. Acetylcholinesterase at the AirlWater Interface

Skou (1959) investigated the activity of acetylcholinesterase adsorbed at the water/air interface at different surface pressures. In order to exclude the cholinesterase activity of the enzyme in aqueous solution, the specifically compressed monolayer is transferred to the underlying solution containing no enzyme. The appearance of enzyme at the interface seems to be accompanied by the breaking of hydrogen bonds with a subsequent uncoiling of molecules at the water/air interface. This process occurs concurrently with the enzyme orientation on the surface. Decay of activity after the maximum corresponds to further protein denaturation. As a result, the enzymatic activity at the surface does not exceed

MEMBRANE ENZYME SYSTEMS

21

one-third of its activity in the solution. As shown in Fig. 7, the enzymatic activity has its maximum at a definite surface pressure, amounting to 10 dyne/cm for acetylcholinesterase. The data reported here indicate that the side chains play an important role in ensuring the maximal enzymatic activity at the aidwater interface. This result is typical not only for acetylcholinesterase, but also for other enzymes including phospholipases. For instance, maximal activity of phospholipase A at the water/ air interface is also achieved at a definite surface pressure (Zografi et al., 1971). In accordance with the above viewpoint, the maximum constant of the reaction rate results from two opposite tendencies. On the one hand, the compression of a monolayer leads to an increase in the concentration of substrate molecules on the surface which accelerates the process. On the other hand, the packing of molecules becomes more dense, which prevents an easy delivery of enzymes to necessary groups. An alternative explanation is in the formation of the substrate-enzymatic

P

22

L. I. BOGUSLAVSKY

complex requiring a specific orientation of the substrate. The maximum enzymatic activity is therefore explained by the optimal orientation of substrate molecules at the given surface pressure. The conformational state of the enzyme seems to be also essential in this case, the enzyme being sensitive to the physical state of the substrate. The waterhir interface can be used to reconstruct the complexes containing more than two components. Thus a system has been reconstructed, where the fraction of galactosyltransferase enzyme could be observed (Romeo et ul., 1970a,b).

D. Galactosyltransferase at the AirlWater Interface

The galactosyltransferase enzyme (IDP-galactose-lipopolysaccharide3-galactosyltransferase) belongs to membrane enzymatic systems, and is a catalyst for the transfer of galactose from IDP-galactose to the galactose-deficient lipopolysaccharide. Lipopolysaccharide itself is localized on the outer side of the cell membrane, and is strongly bound with membrane lipids. The reconstruction of galactosyltransferase requires a special mixed monolayer consisting of phosphatidylethanolamine (PE) and lipopolysaccharide (LPS) molecules: LPS

+ PE + LPS.PE

(40)

To this mixed monolayer the enzyme (E) was added, which was found to be strongly bound with the substrate: LPS.PE + E

+ LPS.PE.E

(41)

To obtain the working surface upon which the enzyme can be deposited, a phosphatidylethanolamine monolayer is formed, and then lipopolysaccharide is introduced into the aqueous solution beneath the monolayer. The interaction of both components can be detected by an increase in the surface pressure of the monolayer. The duration is 10-40 minutes, and is accelerated by increasing the temperature and the concentration of lipopolysaccharide. A n increase in the surface pressure is due to the incorporation of polysaccharide molecules in the monolayer, and the efficiency of this process depends on the density of the original packing of phospholipid molecules. The surface potential remains unchanged within 10 mV. The incorporation of lipolysaccharide has been observed directly by using a labeled substrate. The lipopolysaccharide contained 3H and the process strongly depended not only on the magnitude but also on the nature of phospholipid molecules. Both polar and nonpolar parts of molecules participate in lipopolysaccharide-phospholipid interaction.

*

23

MEMBRANE ENZYME SYSTEMS

The participation of polar groups during interaction of both components can be observed when comparing phosphatidylethanolamine and phosphatidylcholine in mixed monolayers with lipopolysaccharide. The decreased activity of phosphatidylcholine compared with phosphatidylethanolamine is not due to the difference in surface charges, since at experimental values of pH 8.5 both phospholipids had zero surface charge. The presence of unsaturated bonds or cyclopropane groups in the nonpolar region of the monolayer promotes the interaction of lipopolysaccharide with lipid. The incorporated lipopolysaccharide is connected with phospholipid molecules in such a way that fatty acid groups protrude in air, while the polar groups are turned in toward the aqueous phase (Romeo et a / . , 1970a). The mixed monolayer can serve as an acceptor for the substrate of IDP-galactose, which then participates in the reaction: LPS.PE.E

+ IDP-galactose

galactosyl.LPS.PE

+ IDP(+E)

(42)

The change with time in the surface pressure of the monolayer (Fig. 8)

I

0

f

f

10

2

I 20

I

I

I

30

40

50

t,m,n

FIG.8. The phosphatidylethanolamine monolayer is formed in a multichamber cell, and compressed down to a pressure of 6 dyneicm. Then lipopolysaccharide is introduced into the base underlying solution, which leads to an increase of the surface pressure of 5.5 dyne/ cm. Then the film is transferred to the next section (the transfer takes 3-4 minutes, and is shown by a dashed line). At point 2 under the two-component system MgCI, is added up to 6 mM, as well as the enzyme in 5 m M phosphate buffer at pH 6.8 containing 20% of glycerin. (From Romeo el al. 1970b.)

24

L. 1. BOGUSLAVSKY

shows how the enzyme interacts with a previously formed mixed monolayer of phosphatidylethanolamine with galactose-deficient lipopolysaccharide. Test experiments with radioactive samples showed that the reaction proceeds for 20-30 minutes, and 1 mole of lipopolysaccharide is used for 0.164 mM of [3H]galactose. Control experiments conducted in the absence of one of the reaction components showed no appreciable incorporation of galactose into the monolayer. The rate of the reaction depends on divalent cations which accelerate the process. Magnesium was found to be the most effective cation. Ca2+and Mn2+ were significantly less effective. Therefore, a stronger interaction of enzyme with a mixed monolayer is dependent upon the participation of divalent ions. The manner in which the enzyme is attached to the monolayer is unknown. One can only note that when adsorbed onto the surface, the enzyme cannot be spontaneously removed from it. In the experiments performed by Romeo et ul. (1970a) a ratio of 10 molecules of polysaccharide per enzyme molecule was used. This ratio was calculated based on the assumption that the enzyme is adsorbed as a single molecule without forming aggregates. Under these conditions, each molecule of enzyme transferred only two galactosyl groups. The simplest explanation for this is that the enzyme catalyzes the galactose transfer only near the adsorption site of the enzyme. The lateral motion of enzyme along the monolayer seems to be excluded.

E. More Complex Systems Investigated at the Interface Data of extreme interest, but difficult to interpret, have been obtained from studying suspensions of different cells placed on the filter surface to form the following chain:

1 1

I

Vibrating air layer of physiological cells solution

electrode

4.2 M KCI

Hg2CI2,Hg

(43)

Measurements were made by the vibrating electrode method (Minc and Dolowy, 1973). The measurements showed that Volta potentials of living and dead spleen cells are different (Fig. 9). Figure 9 shows a change in Volta potential as a function of percentage of dead cells in the filter. The comparison of cells of the mouse Ehrlich carcinoma ascites with other types of cells showed that they produce different values of Volta potential in the chain. As shown by the above data, the functioning of membrane enzymatic systems is controlled by the accumulation of reaction products, variation of the substrate content, if the latter is adsorbed at the interface, by the change in the surface pressure of the monolayer, or the change of the

25

MEMBRANE ENZYME SYSTEMS

20

40

60

80

100

% DEAD CELLS

FIG. 9. Measurement of Volta potential in the chain (43) containing living and dead mouse spleen cells on the filter depending on the percentage of dead cells. (From Minc and Dolowy, 1973.)

surface potential jump at the interface due to the formation of an enzymesubstrate complex. VI.

POSSIBLE MECHANISM OF THE POTENTIAL GENERATION AT THE INTERFACE BETWEEN TWO IMMISCIBLE LIQUIDS

The phenomenological theory of the generation of potential differences at the interface was developed by Kharkhats, 1975. Let us assume that at an oil/water interface an electrochemical reaction occurs accompanied by a transfer of charged particles, e.g., M + , across the interface. Before the reaction starts, positive particles of type M+ are distributed between water and oil, so that a certain potential difference is created (Fig. 10, curve 1). In this case the potential in the aqueous phase is taken as the reference point; when Mf is transferred from water to oil, the nonaqueous phase becomes more positive due to an electrochemical reaction occurring at the interface, and the potential distribution corresponds to curve 2 (Fig. 10). In the steady state the currents Zwo and Zoware equal, and therefore the total charge transfer across the interface is zero. When the reaction affecting the charged particles concentration in the nonaqueous

26

L. I. BOGUSLAVSKY

*P L

Water

J

0

I

FIG. 10. Dependence of the potential jump at the oillwater interface on the distance from the interface. (1) Equilibrium; (2) steady state during the enzymatic action.

phase takes place, the potential difference changes by the quantity Acp = q , which in electrochemical kinetics is defined as overvoltage (Frumkin, 1963): = r ) = cpwo

- eq cpwo

(441

where index 0 stands for equilibrium, and o and w refer to the phase of oil and water, respectively. The rate of an electrochemical reaction accompanied by charge transfer is characterized by the reaction current I . Therefore we can use the kinetic laws which hold for usual chemical reactions, by substituting the quantity Z/nF for the rate of a catalytic reaction with charge transfer. The simplest mechanism of a catalytic reaction accompanied by charge transfer across the interface in the presence of a catalyst K can be written as kl

S + K S S K k-I

where S is the substrate and Py and Pj” are the reaction products. This scheme allows for two possible mechanisms of charge transfer from oil to water, i.e., a direct transfer of products or transfer by means of carriers, i.e., acceptor molecules. For a given reaction, the form of the kinetic equation depends on the nature of the limiting step. The product accumulation rate is given by the

27

MEMBRANE ENZYME SYSTEMS

equation

By using scheme (45) for the accumulation and decomposition of S K , we obtain

d [ K S 1 - k , [ K ] [ S ]- k - , [ K l [ S ] - k , [ K S I dt

~-

(47)

where the reagent concentrations are related by the material balance equations:

For a quasi-steady-state, established after the onset of reactions, when the decomposition of the intermediate product S K is a slow step (in other words, the charge transfer is slow), we have

Since

from Eqs. (47) and (49) we obtain

In this case the initial reaction rate is

where

K,

=

k-1

+ kz kl

The scheme in Fig. 1 I shows the action of a catalyst adsorbed at the interface and transferring charged particles across this interface. The mechanism of such catalysts was studied experimentally for soluble mitochondrial ATPase (factor F , ) (Boguslavsky er a / . , 1975a). The Volta potential was shown to shift in the positive direction by 1.0 V, due to

28

L. I . BOGUSLAVSKY

S

p:

FIG. 11. Scheme of the action of the catalyst at the interface of two immiscible liquids.

transfer of proton from water to octane by the action of ATPase.' This effect can be explained by the fact that the proton transfer causes the change in the charge of the dense part of the electric double layer in octane formed by proton acceptors, e.g., 2,4-dinitrophenol (DNP). According to the plane capacitor formula

where C is the integral capacity of the electric double layer. From Eqs. (46) and (49) we have

From Eqs. (53) and (54) it follows that

Consequently, in the steady-state process, 7 is proportional to the enzymatic reaction rate V , and to the rate of charge transfer across the interface. It was shown that 7) is a constant (Boguslavsky et al., 1974a). The operation of such catalysts has been studied experimentally using as an example the soluble mitochondria1 ATPase (factor F , ) (Boguslavsky et af., 1974a). According to Mitchell's scheme, ATPase transfers protons through membranes, and participates in ATP synthesis on the membrane The high positive charge of the octane observed by Boguslavsky et ul. (l975a) apparently requires a special assumption concerning the nature of the acceptor-proton complex, which should be a positively charged particle.

29

MEMBRANE ENZYME SYSTEMS

(Mitchell, 1966). The catalytic complex, which realizes both the ATP synthesis and the reverse process, can be isolated from the membrane, and, after separation from the hydrophobic part of the membrane, becomes water soluble. The enzyme (factor F , ) prepared in such a way can hydrolyze ATP in water. Investigations of the purified water-soluble ATPase in an aqueous solution did not indicate that the protein adsorbed at the interface would be able to generate the potential difference without the hydrophobic part (Skulachev and Kozlov, 1977). An attempt to investigate spatial separation of charges has been made when studying the functioning of the soluble ATPase in the system of two immiscible liquids by means of the vibrating electrode method:

1 1

Au air

I

1 1

octane water water Hg,CI,, Hg acceptor ATPase sat. KC1 of protons Mgz+,ATP

(56)

The soluble mitochondria1 ATPase ( F J has been obtained by the Horstman and Racker method (1970) from ox heart mitochondria. In most experiments 2,4-DNP has been used as a proton acceptor. However, some other compounds capable of protonation, namely, trifluoromethoxycarbonyl cyanide, phenylhydrazine (FCCP), methyl ester of phenylalanine, 2,3-dinitrophenol, and anisidine, proved to be effective as well. The effect depends on the nature of the acceptor (Boguslavsky et a/., 1975a; Yaguzhinsky er ul., 1976; Skulachev and Kozlov, 1977). Unfortunately, the reproduction of the change of Volta potential due to the action of ATPase is rather poor, though it undoubtedly exists. The difficulties in protein purification, e.g., from detergents used in separation, may be one of the reasons for such a failure. In particular, this follows from the results of experiments in which M CTAB (cetyltrimethylammonium bromide) added to the system before ATPase, leads to an almost complete disappearance of the potential caused by the protein action at the interface. On the other hand, in the case of the reverse procedure of reagent addition, i.e., when the protein is delivered to the interface before addition of the surface-active substance, the value of the potential is not essentially affected. Many enzymatic complexes are suspensions. The adhesion of suspension particles to the interface is a more complex process than ordinary adsorption, i.e., it strongly depends on the dimensions and state of the particle surface. This can be one of the reasons why different samples of adhering protein complexes display different values of the initial potential jump. As opposed to enzymatic complexes forming true solutions, the adhesion of suspensions to the interface cannot be regarded as adsorption, and it is therefore impossible to apply all the terms which have been discussed

30

L. I. BOGUSLAVSKY

in the first part of the article. Therefore in the case of particle adhesion, the potential shift does not have exactly the same physical sense as that discussed in the theoretical model. It should also be noted that since all the membrane enzymatic systems contain lipids, lipid extraction from the adhering complex to the octane phase is inevitable. Therefore, in practice, lipids are present at the interface. This is important because lipids and the products of their interaction with water and oxygen can be both acceptors and donors of protons. VII.

CHLOROPHYLL AND OTHER PORPHYRINS AT THE INTERFACE

A. Oxidation-Reduction and Porphyrins

Transformations of Chlorophyll

Porphyrins are essential functional groups of many enzymes and photosynthetic pigments. The use of porphyrins as catalysts for charge transfer across an interface instead of protein complexes is a convenient means for clarification of the mechanism of this process. The participation of porphyrins in redox reactions in solutions has been studied rather thoroughly. At the same time the information on the participation of porphyrins in redox reactions at the interface is rather scanty. In a thylacoid membrane the pigments are present at the lipid-protein boundary in the form of a monomolecular film. The study of monolayers of the corresponding pigments can therefore be a source of useful information on the processes occurring upon illumination of the interface. If no liposoluble acceptor is present at the interface in the double electric layer then the observed changes in the surface pressure and boundary potential are treated in the conventional way (alteration of orientation, or conformational changes, or the interaction in mixed monolayers accompanied by the corresponding effects). Such an experimental approach proved to be rather useful for determining the surface properties of chlorophyll, plastocyanin, cytochrome f and ferredoxin. All these components which participate in electron transfer during photosynthesis interact with each other within the living system. Therefore, the surface properties of individual components (interphase tension, surface potential) have been compared with the same parameters in mixed monolayers both in darkness and under illumination. In mixed monolayers, in the absence of interaction between molecules for the mean area, S , per molecule, the relation is observed:

s = S , n , + S,n,

(57)

31

MEMBRANE ENZYME SYSTEMS

where Sl,2is the area occupied by molecules 1 and 2 of the components, and n , and n 2 are their mole fractions in the mixed film. The comparison of areas for a chlorophyll molecule at nitrogedwater and heptane/water interfaces shows that at the interface with hydrocarbon, the molecules lie more flatly (100 and 139 A, respectively). When illuminating the interface, one can observe the change in the area occupied by a chlorophyll molecule. This change has different signs at different surface pressures, so that the slope of 7~ = s isotherm changes somewhat, perhaps due to pheophytinization. As shown by Bellamy et a / . (1963), the curve 7 ~ - sfor pheophytin has a steeper slope than the corresponding dependence for chlorophyll. The same 7 ~ - sdependence has been obtained for ferredoxin (Fig. 12). In the case of ascorbate, S,, = 730 A ', and the jump of Volta potential amounts to 170 mV. One can observe an excellent agreement of experimental values for the mean area of a molecule with the data calculated by means of Eq. (57) in the heptane/water system, whereas in the waterhitrogen system a noticeable interaction between molecules has been observed.

>

B a 200

100

I

500

1000

I

1500

1

-

s. A2

5

lo

15

'1s lo4. %-

FIG. 12. Surface pressure n and Volta potential depending on the area per molecule for ferredoxin and mixed films of ferredoxin and mixed films of ferredoxin and chlorophyll (2: I ) . Monolayers are deposited at the heptane/water interface, and illuminated for 15 minutes with white light (2.105 erg/cm2 second). The aqueous solution contained M ascorbate and phosphate buffer, pH 6.7, at an ionic strength of 0.6. Temperature is 15°C. (From Brody and Owens, 1976.)

32

L. 1. BOGUSLAVSKY

+

When illuminating the chlorophyll ferredoxin mixed monolayer in the presence of ascorbate, Brody and Owens (1976) observed a negligible change in the shape of the 7 ~ - sisotherm, which they believe is due to a reaction occurring between the above components. Illuminated mixed films of chlorophyll and oxidized cytochrome reveal 18% reduction of the mean area occupied by a molecule. On the other hand, the illumination of chlorophyll films with cytochrome increases the area per molecule by 10%. I t is difficult to interpret very small effects, because in darkness reduced cytochrome increases. At least for the case of reduced cytochrome, Brody and Owens supposed that they had observed the light-stimulated reaction with electron transfer: CHL + (Cyt c),,,

hi

(CHL)ox + (CYt c)ie<~

(58)

This supposed reaction differs from the reaction occurring during photosynthesis, and is dissimilar to the interaction of cytochrome with chlorophyll at the nitrogedair interface. A study of mixed monolayers of chlorophyll with plastocyanin also revealed insignificant variations in 7 ~ - sand A p I/s dependences on illumination. When illuminating the chlorophyll plus plastocyanin monolayer insignificant reversible variations of Volta potential can be observed. The effect depends on the duration of illumination and indicates the occurrence of an electron-exchange reaction between chlorophyll and plastocyanin CHL + (Pc)rpd

hv

( C H L h + (Pc),,,

(59)

It is believed that cytochrome participates in the coupling of the electron transfer chain with photosystem I. Chin and Brody (1976) therefore investigated the properties of mixed cytochrome f-chlorophyll monolayers at different ratios of both components. Although a strong interaction in a mixture of reduced cytochrome and chlorophyll at a certain ratio of components can be observed, oxidized cytochrome f and chlorophyll show a minimal reaction. This result may favor the assumption that the reaction center of photosystem I consists of reduced cytochrome f and chlorophyll, which is oxidized by an electron. For the chlorophyll-cytochrome f monolayer, the traces of oxygen in the atmosphere of inert gas played the role of an acceptor which oxidized the chlorophyll. Meanwhile, the chlorophyll may be partially oxidized, and then along with cytochrome oxidation, photoreduction of chlorophyll occurs. The assumption that illumination causes cytochrome f photooxidation is supported by the isotherm of surface pressure for the mixed monolayer, taken after a 15-minute illumination of the system (Fig. 13). A decrease in the area occupied by the molecule at constant surface pressure shows that at the interface oxidized cytochrome f occupies a smaller area.

33

MEMBRANE ENZYME SYSTEMS

B. Electron Transfer by Chlorophyll across the Interface between Two Immiscible Liquids Chlorophyll was used as a catalyst for electron transfer. To the nonaqueous phase were added an electron acceptor and chlorophyll, which was adsorbed at the interface, and to the aqueous phase was added a substrate capable of donating electrons. The negative potential difference at the octane/water interface is caused by transfer of electrons from water to octane. During the electron transfer from water to octane, as a result of electrochemical reaction at the interface: (NADH)"+ 2(A)">(NAD+)'"

+ (H')'"' + 2 ( A - ) "

(60)

the nonaqueous phase becomes negative relative to the aqueous solution

lrc C

7

5

3

1

300

I

I

I

400

500

600

S,cm2

FIG. 13. Isotherm of the surface pressure of a mixed chlorophyll-cytochrome f monolayer on the surface of water in nitrogen in darkness (1) and after illumination for IS minutes (2). (From Chin and Brody, 1976.)

34

L. I. BOGUSLAVSKY

in the chain:

Au

I I

oil,

(61)

air

The experimental results suggest that chlorophyll is needed for catalytic participation in the electron transfer across the interface between two immiscible liquids (Boguslavsky et af., 1976~). C. Proton Phototransfer Chlorophyll

Different reactions of porphyrin hydrogenation are known in nonaqueous solvents under anaerobic conditions (Krasnovsky, 1948). The best known reaction is the photochemical reduction of chlorophyll, which was first carried out by A. Krasnovsky in 1948. The photoreaction gives rise to reduced forms of pigments differing in their spectral characteristics and reactivity, depending on the number of accepted electrons and protons, and also on the conditions of acid-base equilibrium in the medium. The mechanism of the observed phototransfer of protons at the interface has not been studied. Removal of the incident light allows the reaction to proceed in the opposite direction. Though both the chlorophyll reduction and its oxidation are known to be accompanied by a release or adsorption of protons, the available experimental data point to the possibility of a sensibilization mechanism. This mechanism is associated with the initial photoreduction and photooxidation of pigments, and a probable intermediate formation of a complex of an excited pigment molecule with an electron donor or acceptor. Neither mechanism excludes the other, and both can be realized with differing efficiencies. The reaction of reversible photoreduction and photooxidation of chlorophyll was usually studied in a homogeneous phase (Krasnovsky, 1974). According to Katz and Norris (1973), the molecular antenna of chlorophyll, which captures the light energy, has hydrophobic surroundings, whereas the photoactive form at the interface is probably a complex of chlorophyll and water. All of these conditions (solvation of chlorophyll molecule both by water molecules and hydrophobic surroundings) are fulfilled at the liquid hydrocarbon/water interface containing adsorbed chlorophyll. On illuminating the interface in the presence of all the necessary components, Ap changes in reaction (61). This change depends on the concentration of all the reaction components, pH of the aqueous solution, and intensity and wavelength of the incident light. Figure 14 shows the potential difference at the interface as a function of the substrate concentration in the aqueous phase. Saturation of the curve is reached at

35

MEMBRANE ENZYME SYSTEMS

-0.31

FIG. 14. Dependence of the change in Volta potential measured in the chain (61) on the concentration of NADH (curves 1 and 4) and ascorbate (curves 2 and 3). The reaction l 2.10-* M Tris-HC1, pH 7.4,for 1 and 2 mM medium is composed of 10 ~ g / m chlorophyll, DNP and I m M K,Fe(CN), for 3 and 4--10-5 M 2N-methylamino-l,4-naphthoquinone.

the NADH concentration of 2 x NADH

+ Fe(CN)W- + RO-T

M. hi,

NAD

+ Fe(CN)$- + ROH

(62)

The proton phototransfer reaction sensitized by chlorophyll or other porphyrins is reversible. On switching on/off the light, the rise/decay time of potential does not exceed 2 seconds. D. Photooxidation of Water in the Presence of Chlorophyll and Ferro Complex of Tetramethyl Ether of Coproporphyrin Adsorbed at the OctanelWater Interface

It is known that chlorophyll extracted from green leaves of plants is capable of water photooxidation, which is accompanied by a reduction of electron acceptors introduced in the incubation medium (Hill reaction). This reaction has been studied in different model systems. In particular, an insulator-electrolyte system has been used, in which the chlorophyll was replaced by ZnO, Ti02, or WOs (Krasnovsky and Brin, 1962). In order to observe oxygen extraction in such a system, it is necessary to use a short-wave source of illumination, while under natural conditions the Hill reaction proceeds in the visual spectral region. If the octane/

36

L. I. BOGUSLAVSKY

water system containing chlorophyll adsorbed at the interface is used, then oxygen is extracted from water when the system is illuminated in the visual spectral region. On studying proton phototransfer, the experimental conditions can be so chosen that the donor of protons or electrons should be water, while NADP, NAD, or potassium ferricyanide should be used as an electron acceptor. If the interface containing adsorbed porphyrin, DNP and electron acceptor is illuminated, a change of the potential takes place in reaction (61) associated with the positive charge transfer in the octane phase. The potential difference that arises depends on the wavelength of incident light and on the intensity of illumination. Saturation is reached when the intensity amounts to 50-100 MW/cm2. The action spectrum obtained in the presence of chlorophyll has its maximum at 520 nm (Fig. 15). Water molecules are the sole source of hydrogen in the system containing HzO, DNP, porphyrin, and buffer. Therefore, the observed potential difference seems to be caused by water decomposition resulting from illumination at the interface. This decomposition is accompanied by hydrogen transfer first to porphyrin and then to DNP anion according to the equation: H,O

hi'

+ (acceptor)" + (RO-)"-fO,

+ (acceptor)" + (ROH)"

(63)

D 15

1.25

1.00

0 75

0.50

0.25

FIG. 15. The action spectrum obtained in the chain (61) for the reaction of water oxidation. Composition of medium: 10 pg/ml chlorophyll, M N A D , and I mM DNP.

MEMBRANE ENZYME SYSTEMS

37

If reaction (63) really takes place, then oxygen will be liberated, and the reduced form of the acceptor is formed which can be detected by means of polarography. However, it was necessary to show that the oxygen released in reaction (63) is actually produced from water molecules. With this purpose we carried out experiments in which the aqueous solution contained H,180. The reaction of water decomposition took place in different cells in the same set-up. The cell for water photodecomposition was a transparent quartz vessel with a cooler which maintained a constant temperature of 20°C. The cell was filled with aqueous solution containing 26% of H2180, octane, and M chlorophyll. The system also included NAD as acceptor of electrons and DNP or PCP as acceptor of protons in lop3 M concentration. The second cell was a n electrolyzer for obtaining oxygen. All the solutions were prepared with the following reagents: pure grade octane, DNP, PCD, PCD, and NAD produced by Calbiochem, California. Before starting the experiments, both cells were evacuated. The pressure of residual gases was lop6Torr. Mass spectrometric measurements were taken with Varian spectrometer MAT-311 A. The cells were attached to the chamber of the device through a Kovar tube. In order to show that the intensity of the line m / e 34 changes due to oxygen evolution from water, the intensity of this line has been measured after electrolytic decomposition of water resulting in the appearance of molecular oxygen 3202-3602 in the system. The results of the mass spectrometer investigation of water electrolysis products make it possible to estimate the isotope composition of water, and calibrate the mass spectrometer against the separate lines m / e 32 and 34. The measurement results showed that the tabular value of the mass m / e 34, corresponding to m / e l60+ l80,is 33.994075, while the experimental value was found to be 33.993673. The deviation of 0.4 millimass confirms the fact that the measurements were actually taken on the line m / e 34. The studied systems were illuminated at 20°C for an hour by an incandescent lamp of 400 W at a distance of 10 cm from the cell. In order to show that the reaction of water photodecomposition by chlorophyll proceeds at the octane/water interface only in the presence of all the ingredients, test experiments have been performed (see Table I ) which show that reaction (63) will not proceed (a) without octane (it occurs at octane/water interface) or (b) in the absence of even one of the reaction components. By analyzing Table I , it can be seen that illumination of the whole system results in the increase of line intensity over that of the background by at least two orders of magnitude. The ratios of the intensities 5 3 2 / 5 3 4 obtained in the complete system, which is necessary for water photooxidation, and in the system subjected to electrolysis, almost fully coincide.

TABLE I MASS SPECTROMETRIC STUDYOF OXYGENPHOTOGENERATION I N THE HILLREACTION MODEL Intensities of lines, J (rel. units) darkness for 30 minutes

.Iszafter keeping in darkness for 30 minutes

0.3

-

J34after keeping in Composition of reaction mixture Complete system" (acceptor DNP) Complete system (acceptor PCP) Complete system without NAD Complete system without chlorophyll Complete system without octane a

0.3

30

after exposure in light for 60 minutes

J34

2 20

Average ratios of intensities J 3 4 / J 3 2after J3* after exposure in exposure in light for 60 light for 60 minutes minutes

660

0.303

0.3

-

0.3

-

-

0.3

43.5

0.3

43.5

0.006

0.3

31

0.3

31

0.009

Complete system contained octane, water, chlorophyll, NAD. DNP, or PCP.

39

MEMBRANE ENZYME SYSTEMS

Knowing the quantity of oxygen released during electrolysis, one can estimate the total quantity of oxygen released during photodecomposition. This value was found to be 2.7 x M . Thus, the experiments suggest that oxygen is released from water upon illumination in the system containing chlorophyll and all the ingredients needed for reaction (63). The oxygen obtained from reaction (63) has been determined by the polarographic method. Figure 16 shows the polarization curves obtained on a dropping an In-Hg electrode in the system containing water, octane, porphyrin, Tris-HCI, DNP, and various electron acceptors. When the system did not contain one of the reaction components, porphyrin, the acceptor of electrons in water, or dinitrophenol, no oxygen was detected. In the absence of octane, neither potential generation in the chain (61) nor oxygen evolution was observed. In addition to oxygen, generation of the reduced acceptor was observed. In this case K,Fe(CN)6 was formed during reaction (64). Under

-40 1

7

FIG.16. Polarograms of Fe(CN)Q-oxidation on Hg-electrode in the system 20 mMTrisM P. Exposure time: ( I ) 0; (2) 0.5 HCI (pH 7.9), 1 m M DNP, I m M K,Fe(CN),, and hours. Polarograms of O2 reduction on Hg-electrode in the system M P, I m M DNP, 20 mMTris-HCl, pH 7.7, and 10 m M K,Fe(CN),. Exposure time: (3) 0; (4) 0.5 hours; InM CHL, I m M NADP, 20 m M Tris-HCI, pH Hg dropping electrode in the system 7.7. Exposure time: ( 5 ) 0; (6) I hour. The same system, but 10 m M K,Fe(CN), is replaced by NADP. Exposure time: (5) 0; (7) 1 hour. The same system, but NADP replaced by NAD. Exposure time: (8) 0; (9) I hour. (From Boguslavsky et a / . , 1976a.)

40

L. I . BOGUSLAVSKY

these conditions the anodic half-wave of ferricyanide oxidation appeared after illumination. The value of this half-wave potential was +0.2 V against a saturated calomel electrode. The polarographic data indicate that during photolysis of water, 4 % 0.5 ferricyanide anions are formed per oxygen molecule: 2 H,O

hi,

+ 4 F e ( C N ) g - T 0, + 4 H+ + 4 Fe(CN)4,-

(64)

So far the mechanism of reaction (64) has not been studied. We may assume, however, that when porphyrin molecules adsorbed at the interface are excited by light, protons and electrons undergo redistribution in the reaction complex (water, acceptor, RO-), which leads to water decomposition. This process is accompanied by a proton transfer to the side of the electric double layer adjacent to the octane phase, and by an electron transfer to the electron acceptor in the aqueous phase. E. Role of Water and DNP in Proton Transfer across the Interface

To study the role of water and proton acceptor (DNP) in the transfer across the interface, four versions of the experiment were run with the octane/water system, in which reaction (64) was to proceed. The cell was filled with aqueous solutions of DNP ( M), dry octane was added to form the interface, and then K3Fe(CN)6( 2 X lo-' M) was added to the aqueous phase and chlorophyll (10 Fg/ml) to octane. Chlorophyll was adsorbed at the interface. Figure 17 shows plots for chlorophyll excitation by light. If chlorophyll is added to the octane/water system immediately, a potential difference is not generated in the system during the entire run of the experiment (90 minutes). If chlorophyll is added to the octane/water system 5 minutes after the formation of the interface, then, as seen from Fig. 17, curve 2, a potential is set up after a long induction period of 20 minutes. The value of the potential reaches 40 mV. Finally, in experiment 3 (Fig. 17) the time dependence of potential generation is shown for the case in which chlorophyll was added to the system 10 minutes after the interface formation. In this case the potential is generated more quickly, and reached a value of 100 mV. In comparison, Fig. 17, curve 4, also shows (straight line parallel to the abscissa) the potential generated instantly when chlorophyll was added to the octane/water system previously equilibrated during 24 hours. If now, in place of dry octane, wet octane' is added to the aqueous solution containing DNP and ferricyanide, the Wet octane is octane which was in contact with water for 24 hours.

MEMBRANE ENZYME SYSTEMS

41

0I

0.05

FIG. 17. Dependence of Volta potential variation on time starting from the moment of M DNP, 2.10-' M octane contact with water. Composition of the reaction medium: K,Fe(CN),, and 2. Mchlorophyll. Curves 1-4: the boundary is formed by "dry" octane with water containing the rest of the components. Chlorophyll is added: (1) at the moment of phase contact: (2) after 5 minutes; (3) after 10 minutes; (4)after 24 hours: ( 5 ) "dry" octane with dissolved D N P and chlorophyll is added to the aqueous medium not containing DNP; (6) "wet" octane with dissolved chlorophyll and D N P is added to the aqueous phase not containing DNP.

potential difference generated upon illumination of the system progresses to a final value of 100 mV in 5-7 minutes, even through chlorophyll was immediately added to the system. The results of the experiments with the aqueous phase free of D N P are shown in Fig. 17, curves 5 and 6. DNP was contained in the octane phase before its contact with water. Curve 5 was plotted for DNP dissolved in wet octane, and curve 6 for DNP dissolved in dry octane. As can readily be seen, the potential is generated only when aqueous ferricyanide solution contacts wet octane containing DNP. All these data suggest the following. Curves 1-3 indicate that the adsorbed chlorophyll prevents the penetration into octane of the ingredients needed for potential generations. These ingredients can be DNP or water. A 15-minute contact of octane with water containing DNP is sufficient to generate the potential upon illumination. The maximum potential difference is developed gradually, unlike the case of the equilibrated octane/water system (curve 4), in which it is developed at once. This may point to the gradual formation of an intermediate layer between octane and water, in which

42

L. I. BOGUSLAVSKY

the redistribution of charges occurs. When wet octane contacts water containing DNP, there is no need for water to be in contact with octane not containing chlorophyll for a definite period of time. The potential, however, is developed only after few minutes when DNP penetrates into the wet octane. Under the same conditions, but with dry octane, no potential is generated. Thus the rate of water penetration into octane limits the rate of potential generation. Chlorophyll at the interface prevents the penetration of water molecules. If the water content in the intermediate layer is insufficient (curve 2 , Fig. 17), the potential does not reach its maximum value. When DNP is contained in dry octane, and water is free from DNP, no potential difference is set up. On the other hand, when wet octane contains DNP, the potential difference arises on contact with water. Chlorophyll at the interface does not prevent removal of DNP to the aqueous phase. This causes a decrease in the DNP concentration in octane, which can be identified by the potential decrease in time (curve 5). These experiments undoubtedly demonstrate that the transition layer should be saturated with water and proton acceptor (DNP) should be present in it.

VIII.

STUDY OF MEMBRANE ENZYMATIC SYSTEMS OF THE RESPIRATORY CHAIN OF MITOCHONDRIA

A. NADH Dehydrogenase Experiments with NADH dehydrogenase (complex I) in the octane/ water system containing liposoluble H+-ion acceptor (for example, DNP) have shown that the positive potential difference is generated in the octane/water system due to transfer of electrons from NADH to ferricyanide. This potential difference evidences that the octane phase is charged positively relative to the aqueous phase. The potential difference caused by the NADH dehydrogenase reaction is stable. Substitution of a liposoluble acceptor (DNP) by a liposoluble electron acceptor (MANQ) leads to a change of the sign of the potential generated by NADH dehydrogenase. It should be noted that the negative charge of octane in the presence of MANQ was sensitive to rotenone. This is an essential difference between electron transfer and proton transfer in a DNP-containing system. The mersalyl-stable part of the activity was responsible for the negative charge of octane. In this case antimycin A did not give rise to any change either (Boguslavsky et al., 1975b).

MEMBRANE ENZYME SYSTEMS

43

6. Succinate-Cytochrome c Reductase

In the presence of succinate-cytochrome c reductase the octane phase is charged positively if the system contains DNP as proton acceptor. This effect is accompanied by the oxidation of succinate at the interface by ferricyanide via the enzyme system: succinate-cytochrome c reductase (complexes I1 and 111). It was found to be inhibited by antimycin, but stable to rotenone and cyanide. In a system free of DNP or Fe(CN)i-, the succinate-cytochrome c reductase transfers electrons to the 2N-methylamino-l,4-naphthoquinone (MANQ) acceptor in the octane phase (Boguslavsky et al., 1975b). C. Cytochrome Oxidase

Experiments in which cytochrome oxidase was introduced into the octane/water interface have shown that in the presence of the watersoluble electron donor (ascorbate) and a liposoluble electron acceptor (MANQ) in the system, this enzyme causes a shift of the Volta potential in the chain toward the negative side.

IX.

RHODOPSIN AND BACTERIORHODOPSIN AT THE INTERFACE

In the bacterial membrane of Halobacterium halobium, the bacteriorhodopsin is asymmetrically oriented in such a way that upon illumination the protons are ejected outside (Oesterhelt and Stoeckenius, 1973). Phospholipid micelles, which had been previously used for incorporation of cytochrome oxidase particles into the membrane (Racker, 1972), were used as a supporting construction for the incorporation of bacteriorhodopsin sheets (Racker and Stoeckenius, 1974). Racker and Stoeckenius (1974) used the membrane of a phospholipid micelle to couple the functioning of two membrane enzymatic systems. Bacteriorhodopsin membranes of Halobacterium halobium were incorporated into phospholipid membranes of micelles, and functioned under these conditions like pumps, delivering H+ inside the particles. This was detected by an increase in pH of the external medium. The ability of bacteriorhodopsin to transfer protons through the membrane has been proved by test experiments with the use of valinomycin (whose addition increased the flow of protons inside the micelle in exchange for the ejection K + ions) and an uncoupler of the oxidative phosphorylation,

44

L. I. BOGUSLAVSKY

whose addition eliminated the shift of pH due to an increase in the leakage of protons in the membrane. By using a 12.5-pm-thick Teflon insulating membrane (Trissl et ( I [ . , 1977), it became possible to observe the displacement current caused by illumination of rhodopsin deposited on one side of the film separating two aqueous solutions. The cell for measuring the photocurrent with the use of an insulating film, one of the faces of which was coated with animal rhodopsin, was a two-chamber vessel divided by the insulating film. The displacement current was connected to the electrometer by means of AgCl reference electrodes placed in the cell sections filled with aqueous NaCl solution. The rhodopsin film was deposited in the following way. At first, one of the cell sections was incompletely filled with aqueous solution in such a way as to avoid contact with the insulating film. Then approximately two drops of proteolipid with animal rhodopsin dissolved in ether were deposited on the surface of the aqueous phase. After ether evaporation, a 60-pm layer of hexane was carefully deposited onto the surface. The level of the aqueous phase was then raised so that the hydrocarbon layer came into contact with the insulating film containing proteolipid. Thus the protein layer contained in the hydrocarbon became attached to the insulating film. As a result of light-pulse illumination, one observed photoresponses with durations of 150-300 psec, depending on the form of protein deposited on the insulating membrane (either in the form of proteolipids or suspensions of separate discs). The intensity of photoresponses coincides with the action spectrum of rhodopsin. Since the displacement currents were not dependent on the ionic strength of solution around the membrane these currents are supposed to be caused only by conformational changes in rhodopsin molecules; in no way can the currents be associated with the ion transfer in the electric double layer at the polar/nonpolar interface. Perhaps, the transfer of protons is the next and slower stage in the chain of events occurring as a result of the illumination of animal rhodopsin. Antanvase et ul. (1977) observed an increase in the conductivity of bilayer membranes with incorporated animal rhodopsin by using the potentiostatic method. The increase in conductivity was observed every 5 seconds, and then a slow decay occurred during several seconds. Conductivity depended on the light intensity, and proton concentration in the aqueous phase. Current-voltage dependences of membrane illumination were linear. No current through the membrane was the same. These results indicate that upon illumination rhodopsin incorporated in the membrane promotes the proton transfer through lipid bilayers. The behavior of bacteriorhodopsin at the octane/water interface was studied by Boguslavsky er ul. (1975a). It was found that the change in

MEMBRANE ENZYME SYSTEMS

45

the potential jump at the octane/water interface occurs in the presence of proton acceptor dinitrophenol. However, further studies showed that quite a number of bacteriorhodopsin samples prove to be inactive or slightly active at the interface, whereas when incorporated into a phospholipid membrane they act normally. Possibly, the instability of the results should be attributed to uncontrolled lipid composition of the material being investigated (lipid can be extracted by octane, so that the active lipid-protein complex will undergo decomposition). Addition of lipid to the octane phase always stabilized the photoresponses as to their magnitude. Such a system (containing in place of octane, decane, and lipids dissolved in it) was used by associates of Stoeckenius (San-BaoHwang et al., 1977b), who also concluded that the arising potential can be due to a change in the ion distribution in the electric double layer at the decane/water interface. Liposoluble anions introduced into the system eliminate the photoresponses. Mention should be made of the structural and spectroscopic studies of bacteriorhodopsin film carried out at the same laboratory (San-Bao-Hwang et al., 1977a), in which the orientation of membrane fragments adhering to the interface was established. The area they occupy on the interface in the presence of lipids is 35%, so that the membranes are not in contact but seem to be frozen into the lipid monolayer.

X.

THE INFLUENCE OF THE DIELECTRIC CONSTANT OF THE OIL PHASE ON THE EFFICIENCY OF CHARGE TRANSFER THROUGH THE INTERFACE

It was of interest to analyze the dependence of interface potential jump Acp on the dielectric constant D of the nonaqueous phase into which the charge was transferred. For this purpose, the effect D in the nonaqueous phase on the value of the Volta potential was studied during the photooxidation of water in the presence of chlorophyll. As the nonaqueous phase decane, benzene, methyl butyl ketone, or propylene carbonate were used with DNP as the proton acceptor. The curve in Fig. 18 illustrates a decrease in the potential jump with the increase of the dielectric constant of the solvent. This decrease in the potential jump may be due to the decrease of the reaction rate, and to the increase in the capacity of the electric double layer, which results in the decrease of potential jump at the same reaction rate. For the case of water photolysis it was shown that the decrease of the potential jump is mainly determined by a decrease in the reaction rate at the interface.

46

L. I. BOGUSLAVSKY

-0.25 -0.2

-0.1

1 '

'

FIG. 18. Dependence of the Volta potential variation on the dielectric constant of the nonaqueous phase in the presence of chlorophyll.

XI.

COUPLING OF MEMBRANE-ENZYME SYSTEMS

The problem of coupling two enzymatic systems, each of which is a catalyst for the conversion of one kind of energy into another, is the key point in any theory of oxidation and photosynthetic phosphorylation. Energy conversion in vivo in both plants and animals takes place in the membranes of chloroplasts and mitochondria as a result of some conjugate redox reactions. In studying the structure of these organelles, one can see that different enzymatic systems are spatially distributed in the membrane. But the mechanism of reaction coupling in the electron transport chain resulting in ATP formation is still not clear. At present there are three well-known hypotheses concerning the mechanism of coupling in oxidation and phosphorylation reactions. In the first the existence of hypothetical intermediate products was put forward by Slater (1971). According to the second, the chemiosmotic hypothesis of Mitchell, the directed motion of protons through the membrane is determined by a specific arrangement of separate elements in the electron transport chain, and results in the proton motion along the electrochemical potential gradient. According to this hypothesis the energy stored as a gradient of the electrochemical potential of hydrogen ions may be used for the ATP synthesis. The reverse process of ATP hydrolysis gives rise to the ap-

47

MEMBRANE ENZYME SYSTEMS

pearance of the gradient of the hydrogen ion electrochemical potential. Various aspects of the chemiosmotic hypothesis have been discussed in review papers by Williams (1961), Skulachev (1972, 1975), and Witt (1971). According to the third, the mechanochemical hypothesis, the proton and electron transfer caused by a redox reaction results in such a change of the charge arrangement that the stored energy can be used for ATP synthesis (Boyer, 1965; Boyer et a / . , 1973). According to the chemiosmotic hypothesis, a membrane containing bacteriorhodopsin, and translocating protons through the hydrophobic barrier represents a "device" in which the light energy is converted into other types of energy. To test this mitochondria1 oligomycin-sensitive ATPase was added to micelles with bacteriorhodopsin together with the corresponding ATP and 4" substrates, in order to couple two membrane enzymatic systems. In the fully reconstructed system, Racker and Stoeckenius observed ATP formation under the action of light. ATP was not observed without illumination; moreover, in the absence of the hydrophobic oligomycin-sensitive protein fraction, i.e., the coupling factor, no noticeable yield of ATP was observed. The uncoupler of oxidative phosphorylation also inhibited the process of ATP formation. In principle, one can couple several membrane pumps at the octane/ water interface in which the product of one of the pumps is the substrate for another. The coupling of enzymatic systems accompanied by ATP synthesis at the octane/water interface in the presence of one of the aforementioned proton pumps can be schematically represented (Fig. 19). It is seen that under appropriate conditions at the interface proton pumps are the source of the partially dehydrated (macroergic) protons, which may be used by the adsorbed ATPase for ATP synthesis. The described approach seems to be supported by direct experiments conducted by Yaguzhinsky ef a / . (1976), who defined the quantity of ATP synthesized at the expense of

Oil

-

ATP

FIG.19. Scheme for the coupling of two membrane enzymatic systems

48

L. I. BOGUSLAVSKY

the excess neutral form of the acid in the octane phase. Further experiments will be required to solve this problem.

XII.

CONCLUSIONS

Intensive studies of the phenomenon of charge transfer across biological and model membranes have greatly contributed to our knowledge of ion penetration and electron injection into the membrane phase and of the migration of these ions across a lipid membrane. All these phenomena constitute the subject matter of a new area of study, i.e., bioelectrochemistry. To date most problems of electrochemistry are related in some way to investigations of membranes and their models. Studies of biological processes, such as oxidative phosphorylation, photosynthesis, or excitation, involves investigations of the properties of the membrane/electrolyte interface. The appearance of a wonderful model such as the bilayer lipid membrane created a new epoch in studies on biological membranes. In many parameters the properties of native membranes and bilayers are quite similar. But in some cases it is the interface between immiscible liquids which proves to be a simplest physicochemical system allowing elucidation of many important problems. For instance, it is rather frequently asserted that the membrane is the primary factor which spatially orients the processes occurring in the system, i.e., endows them with the vector property. But this property already arises in a simpler system-at an interface. This fact is accounted for by the anisotropy of the interfacial region, arising due to orientation of adsorbed molecules at the interface and formation of a strong electric field of the electric double layer, which possibly is the primary orienting factor and may cause further anisotropy of the membrane system. On one hand, the adsorption at the interface of protein macromolecules should lead to a change in the state of other molecules present at the interface. This is actually the case as can be seen from the measurements of the surface pressure of adsorbed particles. On the other hand, when substrate is added, enzyme molecules undergo conformation changes, which can be detected by examining the surface potentials. Finally, enzymatic reactions at the interface can be accompanied by injection of one of the reaction products into another phase. In this case there is a certain analogy with the charge injection into insulators during redox reactions. To use electrochemical terminology, the generation and separation of charges upon illumination of chloroplasts and the adsorption of these charges by the subsequent reaction on the surface of a lamellar lipid-protein complex is an electrochemical

MEMBRANE ENZYME SYSTEMS

49

photoinjection process at the interface between the aqueous phase and the dielectric medium. The change in the dipole moment with changing enzyme conformation as well as the charge separation caused by injection are the simplest manifestations of the vector properties of the process and can be detected by the Volta potential measurements. It is evident from the foregoing that investigation of the monolayers of biologically important objects at their interface with a nonaqueous phase or air can in a sense serve as a model of the biological membrane surface. The examples given in this article represent conclusive evidence for the validity of this assertion. However, for any model of a very complex object such as a biological membrane, the information obtained is rather limited and can even be distorted by the properties of the model. For instance, many proteins undergo denaturation at the water/air interface. Therefore, it should be emphasized that there exists not only a similarity but also a difference between the phenomena to be observed in simple physicochemical models and in those actually occurring in membranes in vivo. The nearer we come to elimination of this difference, the better will be our understanding of the principles of action of natural membranes.

SYMBOLS AND ABBREVIATIONS 10+ cm Distribution coefficient of cation between oil and water Distribution coefficient of anion between oil and water Concentration of cation in water phase Concentration of anion in water phase Concentration of cation in oil phase Concentration of anion in oil phase Total ionic concentration of (uni-univalent) electrolyte in water phase Total ionic concentration of (uni-univalent) electrolyte in oil phase Concentration at surface Total value of potential difference in electrochemical concentration chain Electronic charge Faraday unit Current Reaction velocity constants Michaelis constant Number of molecules or (ions) adsorbed on 1 cmz of interface Ionic charge Gas constant Distribution coefficient of salt (concentration in oil phase/concentration in water phase)

50 S

T

U

Cyt c CHL PC PE LPS IDP (Et)$l (But),NCl MBK DN P

L. I. BOGUSLAVSKY

Area (in A*) occupied by one molecule on interface Temperature (OK) Change of the potential energy of system upon transition of the particles from the bulk to the surface layer Applied (compensation) potential Product accumulation rate Phase Phase Surface excess Electrokinetic potential of interface Overvoltage Standard chemical potential in water phase Standard chemical potential in oil phase Electrochemical potential in water phase Electrochemical potential in oil phase Electrochemical potential of electron in phase a Electrochemical potential of electron in phase P Electrochemical potential of electron in phase a' Effective dipole moment of water molecule Dipole moment of the terminal bond C-H Dipole moment of the polar head of the hydrocarbon chain Surface pressure Electrostatic potential between headgroups charged adsorbed ions and solution Potential 'po calculated by Gouy equation (18) Distribution potential Adsorption potential Potential drop between the surface and its bulk calculated by Gouy equation (18) Compensation potential Work function from phase CY Surface potential drop at the interface between the phase a! and vacuum Surface potential drop at the interface between the phase P and vacuum Acceptor in oil phase Enzyme Catalyst Substrate Reaction products of enzymatic reaction in the oil and water phase, respectively Cytochrome c Chlorophyll Plastoc yanin Phosphatidylethanolamine Lipopoly saccharide Inositoldiphosphate Tetraethylammonium picrate Tetrabutylammonium Methyl butyl ketone 2,4-Dinitrophenol

51

MEMBRANE ENZYME SYSTEMS

PCP MANQ

Pentachlorophenol 2 N-Methylamino- 1,4-naphthoquinone

REFERENCES Abel, E. (1906). Zur Theorie der Elektromotorischen Krafte in phasigen und nicht-wasserigen einphasigen Systemen. Z . Phys. Chem., Stoechiom. Verwandschaftsl. 56, 612623. Aliev, M . K., Boguslavsky, L. I., Volkov, A. G., Kozlov, I. A., Levitsky, D. 0.. and Metelsky, S. T. (1976). Study of the electrogenic function of Caz+, ATPase at the octaneiwater interface. Bioorg. Khirn. 2, 1132-1 137. Antanavase, J., Chien, P., Ching, P., Dunlap, C., and Mueller, P. (1977). Rhodopsin mediated proton fluxes in lipid bilayers. Abstr. Annu. Meet. Biophys. Sect., 2 / s t , New Orleans, TH-PM-C2; Biophys. J . 7 , 182a. Arnon, D. I., Whatley, F. R., and Allen, M. B. (1954). Photosynthesis by isolated chloroplasts. I-Photosynthetic phosphorylation, the conversion of light into phosphate energy. J . A m . Chem. Soc. 76, 6324-6329. Bakeeva, L. E., Grinius, L. L., Jasaitis, A. A,, Kuliene, V. V . , Levitsky, D. O., Liberman, E. A., Severina, I. I., and Skulachev, V. P. (1970). Conversion of biomembraneproduced energy into electric form in mitochondria. 11. Biochim. Biophys. Acra 216, 13-21. Baur, E. (1913). Ein Modell des elektrischen Organs der Fisch. 2. Elektrochem. 19, 590592. Baur, E., and Kornman, S. (1916). Uber die Ionenadsorptionspotentiale. Z . Phys. Chem., Stoechiom. Verwandschaftsl. 92, 81 -97. Bellamy, W . D., Gaines, G. I., and Tweet, A. G. (1963). Preparation and properties of monomolecular films of chlorophyll a and pheophytin a. J . Chem. Phys. 39, 25282538. Beutner, R. (1909). Uber Neue Galvanische elemente. Z . Elektrochem. 13,433-472. Blaurock, A. E . , and Stoeckenius, W . (1971). Rhodopsin-like protein from the purpure membrane of H. Halobium. Nature (London),New B i d . 233, 149-152. Boguslavsky, L . I. (1971). Some problems of bioelectrochemistry. Zh. Vses. Khirn. Ova. No. 6, 711-717. Boguslavsky, L. I., and Yaguzhinsky, L. S. (1975). Bioelectrochemical phenomena and the interface. In “Elektrosintes i Bioelektrokhimiya,” (A. N . Frumkin, ed.). pp. 305-340, Nauka, Moscow. Boguslavsky, L. I., Volkov, A. G., Kozlov, N. A., Metelsky, S. T., and Skulachev, V. P. (1974a). Positive charge injection from water to octane, coupled with ATP hydrolysis in presence of soluble mitochondrial ATPase. Dokl. Akad. Nauk SSSR 218, 963-966. Boguslavsky, L. I., Frumkin, A. N., and Gugeshashvili, M. I. (1974b). Contact phenomena at the heptaneiwater interface in the presence of valinomycins. Bioelectrochem. Bioenerg. 1, 506-514. Boguslavsky, L. I . , Kondrashin, A. A,, Kozlov, I . A,, Metelsky, S. T., Skulachev, V. P., and Volkov, A. G. (1975a). Charge transfer between water and octane phases by soluble mitochondria1 ATPase( F J , bacteriorhodopsin and respiratory chain enzyme. FEES Lett. 60, 223-226.

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Boguslavsky, L. I . , Volkov, A. G., Kondrashin, A. A,, and Skulachev, V. P. (1975b). Charge transfer through octaneiwater interface by enzyme complexes of the respiratory chain. Bioorg. Khim. 1, 1781-1783. Boguslavsky L. I . , Volkov A. G., Kandelaki M. D., and Niznikovsky, E. A. (1976a). Photooxidation of water and proton transport across the interface of two immiscible liquids in the presence of chlorophyll. Dokl. Akad. Nauk S S S R 227, 727-730. Boguslavsky, L. I . , Frumkin, A. N., and Gugeshashvili, M. I. (1976b). Investigation of tetraalkylammonium salts at the nitrobenzeneiwater interface. Elektrokhimiya 12, 856860. Boguslavsky, L. I . , Volkov, A. G., and Kandelaki, M. D. (1976~).Transfer of electrons and protons at the decaneiwater interface in the presence of chlorophyll. FEES Lett. 65, 155-158. Boguslavsky, L. I., Zhyravlev, L. T., Kandelaki, M. D., and Shengelia, K. J . (1978). Investigation of photooxidation of water on the octaneiwater interface in the presence of chlorophyll. Dokl. Akad. Nuuk S S S R 240, 1453-1456. Boyer, P. D. (1965). Carboxyl activation as a possible common reaction in substrate-level and oxidative phosphorylation and in muscle contraction. I n “Oxidases and Related Redox System” (T. E. King, H. S. Mason, and M. Morrison, eds.), Vol. 2, pp. 9941008. Wiley, New York. Boyer, P. D., Gross, L., and Momsen, W. (1973). A new concept for energy coupling in oxidative phosphorylation based on a molecular explanation of the oxygen exchange reaction. Proc. Nurl. Acad. Sci. U . S . A . 70, 2837-2839. Brody, S. S., and Owens, N. F . (1976). Photosynthetic electron carriers of heptane-water surface. Z . Naturforsch., Teil C 31, 569-574. Butler, W. L. (1961). A far-red absorbing from chlorophyll in vivo. Arch. Biochem. Biophys. 93, 413-422. Chin, P., and Brody, S. S. (1976). Mixed monomolecular films of chlorophyll and cytochromes. Z . Naturforsch. Teil C 31, 44-47. Colacicco, G. ( I97 I ) . Significance of surface potential measurements of lipid monolayers during action of phospholipase A on ascitines. Nature (London) 233, 202-204. Davies, J . T . (1950). The distribution of ions under a charged monolayer and a surface equation of state for charged films. Proc. R. Soc. London, Ser. A 208, 224-247. Davies, J . T . (1951). Stabile Kontaktpotentiale an der Grenzflache 01-Wasser. Z . Elektrochem. 55, 559-560. Davies, J . T. (1953). Interfacial potentials. Part 1. Dependence of the character of the nonaqueous phase. Trans. Faraday Soc. 49, 683-686. Davies, J . T., and Rideal, E. K. (1955). Interface potentials. Can. J . Chern. 33, 947-960. Davies, J . T., and Rideal, E. K. (1963). “Interfacial Phenomena,” p. 66. Academic Press, New York. Evstigneev, V. B. (1966). Investigation of the photosensibilization of the redox-reactions of chlorophyll and its analogues by electrochemical methods. In “Elementarnye Fotoprotesy v Molekulakh,” pp. 243-266, Nauka, Moscow-Leningrad. Fomin, G. V . , Brin, G . P., Genkin, M . V., Liubimova, A. K., Blumenfeld, L. A,, and Krasnovsky, A. A. (1973). Mass-spectrometric investigation of photo-decomposition of water in the system inorganic sensibilization-electron acceptor. Dokl. Aknd. Nauk S S S R 212, 424-427. Frumkin, A. N. (1919). “Elektrokapillaynye Issledovaniya i Elektrodnye Potentsialy.” Sapoznikov, Odessa. Frumkin, A. N. (1963). Hydrogen overvoltage and adsorption phenomena. Adv. Electrochem. Electrochem. Eng. 3, 287-491.

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Gerovich, M. A., and Frumkin, A. N. (1936). Electrical properties of films wbromehexadecanoic acid. J. Chem. Phys. 4, 624-629. Goldfeld, M. G., Vorobjeva, E . S., and Poltorak, 0. M. (1966). Investigation of catalase activity of different types adsorbed monolayer. Zh. Fiz. Khim. 40, 2594-2596. Goodal, M. C., and Sacks, G. (1977). A new method of membrane reconstruction. Biophys. J. 17, 182a, TH-PM-CI. Gugeshashvili, M. I., Lozhkin, B. T., and Boguslavsky, L. I. (1974). Elimination of the diffusion potentials in the system water/nitrobenzene/water. Elektrokhimiya 10, 11201124. Haas, De G. H., Bonsen, P. P. M., Peterson, W. A., and van Deenen, L. L. M. (1971). Studies on phospholipase A and its zymogen from porcine pancreas. 111. Action of the enzyme on short-chain lecitins. Biochim. Biophys Acta 239, 252-266. Haber, F . (1908). Theorie Galvanische Elemente. Ann. Phys. (Leipzig) 26, 927-947. Hartree, E. F. (1972). Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal. Biochem. 48, 422-427. Haugen, G. R., and Friedman, H. L. (1956). The partition of electrolytes between aqueous and organic phases. 11. Free energy of transfer from pure water to water saturated with nitromethane. Phys. Chem. 60, 1363-1367. Hendrikx, Y., and Ter-Minassian-Saraga, L. (1970). Adsorption d’un sel d’ammonium quaternaire substitue par des films etales de lecithine. J . Chim. Phys. 67, 1620-1628. Horstman, L. L., and Racker, E . (1970). Partial resolution of the enzyme catalyzing oxidative phosphorylation. J. Biol. Chem. 215, 1334- 1336. Inbar, L., and Miller, I. R. (1974). Enzymatic activity of trypsin at the air-water interface. Biochim. Biophys. Acta 364, 146-158. Karpfen, F. M., and Randles, J . E. B. (1953). Ionic equilibriaand phase boundary potentials in oil-water systems. Trans. Faraday Soc. 49, 823-831. Katz, J . J . , and Norris, J. R. (1973). Chlorophyll and light energy transduction in photosynthesis. Curr. Top. Bioenerg. 5 , 41-75. Kembell, C. (1950). The adsorption of vapours on mercury. I. Surface potentials in chemisorption. Proc. R . Soc. London, Ser. A 201, 377-391. Kempner, E. S . , and Miller, J . H. (1968). The molecular biology of Euglena Gracilis. Enzyme localization. Exp. Cell Res. 51, 150-156. Kenric, C. B., and Frank, B. (1896). Die Potentialspiinge zwischen Gasen und Fussigkeiten. Z. Phys. Chem., Stoechiom. Verwandschaftsl. 19, 625-626. Kharkatz, Y. I., Volkov, A. G., and Boguslavsky, L. I. (1975). On the possibility of studying the kinetics of the catalytic reactions with charge transfer through the water/ oil interface by the vibrating capacitor method. Dokl. Akad. Nauk SSSR 220, 14411444. Korenbrot, J . I., and Pramik, M. J . (1977). Spectrophotometry of formation, structure and spectrophotometry of air-water interface films containing rhodopsin. J . Membr. Biol. 37, 235-262. Krasnovsky, A. A. (1948). Reversible photochemical reduction of chlorophyll in ascorbic acid. Dok. Akad. Nauk SSSR 60, 421-424. Krasnovsky, A. A. (1966). Photochemistry of chlorophyll and its analogues. In “Elementarnye Fotoprotsesy v Molekulakh,” pp. 213-242. Nauka, Moscow-Leningrad. Krasnovsky, A. A. (1974). “Preobrazovanie Energii pri Fotosinteze.” Nauka, Moscow. Krasnovsky, A. A., and Brin, G. P. (1962). Unorganic models of Hill reactions. Dokl. Akad. Nauk SSSR 142, 656-659. Luther, R. (1896). Elektromotorische Kraft und Verteilungsgleckgewicht. Z . Phys. Chern., Stoechiom. Verwandschaftsl. 19, 529-571.

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Criteria for the Reconstitution of Ion Transport Systems1 ADIL E . S H A M O 0 2 A N D WILLIAM F. T1VOL3 Depurtment of Radicition Biology rind Biophysics University of Rochester School of’Medicine rind Dentistry Rochester, N e w Yorh

Introduction . . . . . . . . . . . Reconstitution Experiments . . . . . A. Preparation of Vesicles and Bilayers . B. Measurement and Control of Parameters C. Reconstitution of Ion Transport Systems 111. Conclusions and the Future of Reconstitution . . . . . . . . . . . References

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Biochemical studies in general consist of observations of a system of interest in sitrr, isolation and characterization of the components of the system, and reassembly of these components to re-create a working system in v i m . This article is concerned with the reassembly of ion transport systems. The term “reconstitution” refers to such reassembly and has been used broadly in the literature. In the most limited sense reconstitution implies the rebuilding of a system from isolated, molecularly welldefined, components such that the biological activities of the reconstiThis contribution is based on work performed under contract with the U.S. Department of Energy at the University of Rochester Department of Radiation Biology and Biophysics and has been assigned Report No. UR-3490-1596. This work was also supported in part by NIH Grant 1 R 0 1 18892, Program Project ES-10248 from NIEHS, and the Muscular Dystrophy Association (USA). An Established Investigator of the American Heart Association. Present address: Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore, Maryland. Present address: Department of Pharmacology and Toxicology, University of Rochester School of Medicine and Dentistry, Rochester, New York. Copyright @ 1980 by Academic Press. Inc. All rights of reproduction in any form reserved.

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ADlL E. S H A M 0 0 AND WILLIAM F. TIVOL

tuted system are identical to those of the same system in \,iiv. For example, if one combines Ca2+ + Mg2+-ATPase, phosphatidylethanolamine, and phosphatidylcholine, and if one sonicates these ingredients in an appropriate aqueous buffered salt solution, vesicles will form which will efficiently translocate calcium ions when magnesium ions and ATP are present, if the p H and ionic strength of the suspension are maintained at physiological values (Racker and Eytan, 1973). Less successful reassembly which recreates only some of the biological activities is also referred to as reconstitution. For example, Bradley et (11. (1976) report a reconstitution of rabbit skeletal muscle acetylcholine receptor ( A c ~ R )in~ a planar bilayer which gives quanta1 conductance increases when stimulated by carbamylcholine and is antagonized by curare, a-bungarotoxin, DTT, and Con A ; however, quantitative properties such as agonist independent conductance, channel magnitude, and lifetime are different from the values found in the intact receptor. Reconstitution using only some of the components of a system or using fragments of a system can also recreate only a part of the biological activities. In this case, of course, one expects to recover only a specific fraction of the activity, and such partial reconstitutions are useful in assigning particular biological activities to particular components of a system or in studying the interactions between components of a system. Razin (1972) suggests that the term reconstitution be used to refer to cases in which both membrane structure and normal enzymic activities have been restored, that reformation be used when membrane structure, but not necessarily function, is restored, and that reaggregation be used when no firm conclusions can be drawn regarding the structure or function of the end product. He also suggests reserving the term recombiAbbreviations: Ach, acetylcholine: AchR, acetylcholine receptor: ANS, I-anilino-

naphthalene-8-sulfonate: 12-AS. 12(9-anthroyl) stearate; ATP, adenosine triphosphate; ATPase, ATPdiphosphohydrolase; Bch, bacteriochlorophyll: BLM, black lipid membrane or bilayer lipid membrane: BR, bacteriorhodopsin: BTX, a-bungarotoxin; CCCP, trichlorocarbonylcyanide phenylhydrazone: Con A, concanavalin A: DCCD, dicyclohexyl carbodiimide: DSC, differential scanning calorimetry; d-TC, d-tubocurarine; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid): DTT, dithiothreitol: EDTA, (ethylenedinitri1o)tetraacetic acid: EGTA, ethyleneglycol-bis-(P-aminoethylether)-N,N'-tetraacetic acid: EM, electron microscope, electron microscopy, etc.; EPR, electron paramagnetic resonance; FCCP, trifluoromethoxycarbonylcyanide phenylhydrazone; F,.F,, oligomycin-sensitive mitochondria1 ATPase; NADH, nicotinamide adenine dinucleotide (reduced form): NADPH, nicotinamide adenine dinucleotide phosphate (reduced form): NMR, nuclear magnetic resonance; PC, phosphatidylcholine; pCMB, p-chloromercuribenzoic acid: PE, phosphatidylethanolamine: P-G border, Plateau-Gibbs border; PI, phosphatidylinositol: pNPP, p-nitrophenyl phosphate: PS, phosphatidylserine; SDS, sodium dodecyl sulfate: SR, sarcoplasmic reticulum: TLM, thick lipid membrane: Tris, tris-(hydroxymethyl) amino methane: VDAC, voltagedependent anion channel, derived from paramecium mitochondria.

RECONSTITUTION OF ION TRANSPORT SYSTEMS

59

nation for those cases in which the proteins and lipids had been isolated prior to mixing. I n spite of the many terms and their many uses in the literature, it would be valuable to distinguish among degrees of success in recreating membrane structure and function. We endorse Razin‘s suggestions and propose that “partial reconstitution” refer to the case in which some but not all of the biological activities are restored. If phospholipids are suspended in an aqueous phase, the lipid molecules spontaneously aggregate into structures in which all the polar head groups are in contact with the aqueous phase, and the nonpolar fatty acid chains are not. Considerations of optimal areas for both head groups and acyl chains lead to the conclusion that two related structures have the lowest chemical potential: extended bilayer sheets and bilayer surfaces enclosing an aqueous cavity, which are called vesicles or liposomes (Tanford, 1978). By varying the conditions of formation either open bilayer membranes (bounded by suitable hydrophobic supports, usually a polyethylene or Teflon frame) or vesicles can be formed. Under appropriate conditions, the vesicles will be single-walled, as opposed to multilamellar, onion-like structures. Unless otherwise stated, the term “vesicles” will be used to mean closed, reconstituted, lipid bilayer structures. Both open lipid bilayer membranes and closed vesicles have been used in reconstitution and each structure has its own advantages and disadvantages. Open bilayers have the advantage that both sides of the bilayer are accessible to measurement or modification. This allows the measurement of transmembrane differences, for example, voltage or osmotic pressure. Openness has the disadvantage that there is no enclosed volume so that any transported ions are diluted instead of being collected in a small space. This renders the measurement of transmembrane ion currents very difficult even with radioactive isotopes. Other disadvantages of the open bilayer come from the usual method of formation. An unknown amount of organic solvent is usually present which vastly complicates the interpretation of any measurements-especially when the o Little is known about reconstituted system differs from the in ~ ~ i vone. the properties of the lipid torus, called the Plateau-Gibbs border (P-G border), which is found at the edge of the frame used to support the bilayer. This thick lipid structure can affect membrane area measurements and may contribute errors to current measurements. The advantages of vesicles are that there is generally no organic solvent present and that they enclose a volume which is smaller relative to surface area than in any practical open bilayer system. Electrical measurements are notoriously difficult, however, due both to the problems associated with implanting a sufficiently small electrode inside the vesicle and to uncertainties about the region where the lipid joins the electrode wall. Another disadvantage in vesicles is that some parameters are very

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ADlL E. S H A M 0 0 AND WILLIAM F. TIVOL

poorly defined in the inside of a vesicle. Typical methods of preparation give vesicles smaller than 1 p m in diameter. A I-pm-diameter vesicle at pH = 7 contains about 30 Hf ions and since many ion concentration ranges of physiological interest are in the 1- 10 p M range this means that there are at most only a few hundred or perhaps a few thousand ions inside a vesicle when a measurement is of interest. Application of a theoretical equation describing a macroscopic solution to such a situation is either dubious or completely inappropriate. Recent developments have led to the production of much larger vesicles, typically having a diameter of about 100 pm (Antanavage et ~ J I . , 1978; Murphy and Shamoo, 1978). In these “giant” vesicles such quantities as pH are probably well defined. Recent measurements in this laboratory (Murphy and Shamoo, 1978) have shown that whereas membrane capacitance can be readily measured in giant vesicles, membrane resistance measurements are inconsistent with the values reported in the literature and have a large scatter. There is no organic solvent in giant vesicles and the geometric uncertainties are absent (except in electrical resistance measurements, etc.). There are, no doubt, limitations on the use of giant vesicles which will become apparent with sufficient experience in their use. At this time, though, they seem to offer great promise for reconstitution studies.

I I.

RECONSTlTUTlON EXPERIMENTS

A. Preparation of Vesicles and Bilayers

The preparation of bilayer membranes usually goes smoothly due to the fact that the bilayer is a thermodynamically stable system. There are several cautions which should be observed however, Careful purification of lipids and solvents is important, in particular when attempting to reproduce a bilayer system from another laboratory (Tien, 1974). For example, a contaminant in the organic solvent used to dissolve lipids can distribute itself unevenly between the bilayer and the P-G border. In this case, the effect of the contaminant can be magnified greatly. Temperature is another particularly important variable in open bilayer systems; the formation and stability of the bilayer is very sensitive to temperature. Oxidation of lipids by atmospheric oxygen can also be a serious problem. Lipids extracted from biological systems, such as mixed brain lipids or sarcoplasmic reticulum (SR) lipids, seem to be especially susceptible to oxidation. Optimally, such extractions should be performed under an inert atmosphere, and the resulting lipids should be stored at 4°C at a

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61

concentration in the range 0.5-2% in a (26 to c16 hydrocarbon solvent sealed under inert atmosphere in glass ampoules. When stored in such a fashion lipids have a lifetime of greater than 1 year. Bilayers formed from lipids susceptible t o oxidation ideally should be formed under inert atmosphere and the aqueous phase on either side deoxygenated; however, relatively few laboratories take this precaution. In order to preserve the homogeneity of the aqueous phases, these should be stirred vigorouslybut, of course, not so vigorously as to rupture the bilayer. I . VESICLES

The preparation of lipid vesicles consists of dispersing the lipids in an appropriate aqueous phase and evaporating, diluting, or otherwise removing any solubilizing agent used. If the lipids have been stored under nitrogen in a volatile hydrocarbon solvent, this solvent can be removed by evaporation under an inert atmosphere. The lipid is then swelled in a stream of water-saturated nitrogen. Upon addition of an aqueous phase, vesicles will form whose properties depend on the kind of lipid used, the composition of the aqueous phase added, and the amount of agitation to which the sample is subjected. Stirring typically gives vesicles whose diameter is on the order of 10 p m , while sonication yields vesicles whose diameter is on the order of 10 nm (Tien, 1974; Bangham et al., 1974). If salt is rigorously excluded and the sample is incubated at 4°C with no agitation, "giant" vesicles are formed whose diameter is on the order of 100 p m (Antanavage et ul., 1978; Murphy and Shamoo, 1978). For example, Racker (1973) takes phospholipid which is stored under nitrogen in chloroform-methanol, evaporates the solvent under N2, then adds a buffered salt or sucrose solution (pH 7.5) and protein. The mixture is sonicated with a 90-W sonicator. The optimal sonication time is between 3 and 16 minutes depending upon the protein used. Hyono el al. (1975) evaporate solvent from phospholipid on a rotary evaporator and add buffered salt solution to the resulting thin film of lipid. The sample is shaken vigorously under nitrogen for 30 minutes and left at room temperature for 2 hours. Murphy and Shamoo (1978) wash ox-brain phospholipids with distilled water, deposit a thin film on the bottom of a glass vessel, hydrate the film with water-saturated nitrogen, add distilled water or sucrose, and incubate at 5°C overnight. Vesicles form with diameters up to 300 pm. Salt can be added to vesicles formed in sucrose by diluting the vesicle suspension in isosmolar (or slightly hypotonic) salt solution. It is sometimes useful to prepare vesicles from detergent-containing

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ADlL E. S H A M 0 0 AND WILLIAM F. TIVOL

solutions (e.g., the protein to be incorporated may otherwise aggregate and fail to enter the membrane). Detergents have been successfully removed by dilution and centrifugation, dialysis, or column chromatography. Since cholate has an aggregation number of only two to four, it is the detergent of choice when dialysis i s used (Kagawa, 1978). Kalbitzer et 01. (1978) solubilized native vesicles using 0.3 mg deoxycholate per mg protein, then diluted 5- to 10-fold, and pelleted the resulting vesicles by centrifugation. This procedure should also work if purified protein and lipid moieties are used. The most common method of detergent removal is dialysis (Kagawa and Racker, 1971). Jilka et al. (1975) dried an aliquot of phospholipid under nitrogen, dispersed the residue by nine or ten 10-second 60-W bursts from a Branson sonifier at 2-10°C in a solution of buffered KCI with 1.6% sodium cholate to give 60-90 mM phospholipid. Protein (in this case Ca2+pump from SR) was dissolved in buffered salt and sucrose solution and added to a few mg/ml final concentration. The phospholipid, cholate, and salt concentrations of the solution were adjusted, and the solution was dialyzed and chromatographed on a Sepharose 4B column. The void volume fraction was used for further experimentation. MacLennan et al. (1976) removed sodium dodecyl sulfate from sarcoplasmic reticulum Ca2+ pump using ion exchange chromatography as follows: the protein was dissolved in buffered salt and sucrose solution and one-quarter volume of buffer containing 5% SDS and 5% 2-mercaptoethanol was added and the solution was incubated at 24°C for various periods of time. This solution was then diluted into five volumes of phospholipid suspension. The combined protein and phospholipids were incubated at 0°C for 30 minutes and passed through a column of anion exchange resin. The column was eluted with a buffer containing 0.75% sodium cholate and DTT. When the room temperature incubation of protein with SDS was 20 minutes or less, high Ca2+ transport activity was reconstituted. Brunner et al. (1977) reconstituted pure hydrolases from brush border membranes by recombination with egg lecithin in the presence of cholate, and then removed the detergent by passage over Sephadex. Unsonicated egg lecithin was dispersed in buffer at a concentration of I-2% and the suspension was made 2.5% in sodium cholate. Isolated hydrolases were dissolved in the same buffer and again the solutions were made 2.5% in sodium cholate. The addition of cholate disintegrated both the multilamellar lecithin structures and the aggregations of the proteins which form in the absence of detergent. The lipid and protein solutions were mixed in various ratios, and the mixed solutions were passed through a Sephadex (3-50 column and eluted with the same buffer which removed 99.9% of the cholate and led to the formation of a stable lipid-protein complex.

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63

When using sonication to produce vesicles there are several general 1974). Direct contact cautions to be observed (Tien, 1974; Bangham et d., between the metallic sonicator probe and the lipid suspension should be avoided. Not only does cavitation produced by such contact increase the rate of reaction of dissolved oxygen with the sample, but metal atoms could become dissolved in the sample where they will serve as catalysts for lipid oxidation. It follows that the sonicated sample should be thoroughly deoxygenated and sonication performed under an inert atmosphere. Reducing the surface tension of a sample to be sonicated will reduce cavitation and the denaturation caused thereby, but addition of an agent to reduce surface tension may lead to unknown alterations in the composition of the vesicles if the agent is lipid soluble. PC and mixtures of PC and cholesterol are resistant to dispersion by sonication and if a sonication sensitive protein is to be reconstituted into PC or PCcholesterol vesicles other dispersion techniques are preferred (Bangham el ul., 1974). 2. OPEN BILAYERSYSTEMS

There are four techniques currently being used in the preparation of open bilayer systems. The oldest and still one of the most satisfactory is the brush technique. The apparatus used is quite simple, consisting of an outer chamber made of glass or Lucite within which is a 10-ml Teflon cup which has had part of one side machined to about 0.2 mm thickness. A hole (usually 1-2 mm in diameter) is then punched or bored through the thin section of the cup. There should be provisions to observe the hole to determine when the lipid layer has thinned and to make any measurements (e.g., electrical) required during the experiment. Both chambers are filled with aqueous phase, and the lipid solution is painted across the hole in the inner chamber using either a trimmed sable brush or a polyethylene or Teflon stick trimmed flat, or a microsyringe is used to inject small increments (e.g., 2 PI) of lipid solution to form the bilayer. A good reference for formation of bilayers by the brush technique is Finkelstein (1974). Another technique is the dipping technique. A thin ring is lowered through an interface below which is an aqueous phase and above which is a solution of lipid in hydrocarbon. A modification of this technique used a lipid monolayer instead of a lipid solution (Montal, 1974b). The Teflon frame is stationary and initially the aqueous phase is at a height so that its surface is just below the hole in the Teflon. A few drops of lipid solution are introduced onto the aqueous surface and a monolayer is formed on each surface as the lipid solvent evaporates. The height of the interface is raised by careful infusion of aqueous phase from below

64

ADlL E. SHAM00 AND WILLIAM F. TIVOL

and a bilayer forms across the hole. The advantages of using a monolayer are that ( I ) the bilayer is formed immediately with no thinning required, ( 2 ) no organic solvent need be present in the bilayer since it can be allowed to evaporate prior to formation, and (3) each monolayer comprising the bilayer can have a different composition. A third technique uses a much different geometry. A microsyringe with Teflon tubing attached is dipped in lipid solution. The end of the tubing is immersed in aqueous solution and aqueous solution (the same or different) is carefully forced through the tubing using the syringe to "blow a bubble." A second tube can be placed at the lower surface of the bilayer and aqueous phase simultaneously sucked out the top and forced in at the bottom. This will break that part of the membrane which spans the lower tube leaving a cylindrical bilayer (Murphy, 1977). Spherical bilayers have a high surface-to-enclosed volume ratio and are useful for tracer or flux studies. The inner aqueous volume is connected to the syringe so that electrical measurements can be made, but ions will diffuse only slowly up the tubing. Cylindrical bilayers through which a steady flow of aqueous phase has been established can be used to study kinetics or, by changing the composition of the aqueous phase, to study changes in bilayer properties. The fourth bilayer-forming technique is quite recent (Antanavage et a/., 1978; Murphy and Shamoo, 1978). Open bilayers can be formed from giant vesicles by trapping a vesicle in a hole and then breaking one side. Aqueous solution is placed in both chambers of a bilayer-forming apparatus, and allowed to reach equilibrium height. A suspension of giant vesicles is introduced into the inner chamber. Fluid will flow through the hole until a vesicle is caught. Of course, the diameter of the hole must be smaller than the largest vesicles in the suspension. A single pulse of about 5 V will serve to rupture one wall of the trapped vesicle, and the remaining wall will relax until a planar bilayer is formed. This can be followed by monitoring the capacitance across the hole. Care must be taken that no osmotic pressure is developed across the vesicle walls. Otherwise, either the vesicles will break before trapping or shrunken nonspherical vesicles will be produced which may not trap or break properly.

6. Measurement and Control of Parameters

The great advantage of reconstitution is that an isolated system can be studied under a variety of experimental conditions and that these conditions can be controlled by the experimenter. Measurement of the re-

RECONSTITUTION OF ION TRANSPORT SYSTEMS

65

sponse of the system under investigation to these conditions gives information both about the properties of the system and the extent to which the reconstitution has been successful. It is essential to the success of a reconstitution experiment that those parameters which determine the behavior of the system be accurately measured and controlled. In this section the kinds of measurement techniques available, their advantages, and the problems associated with them will be reviewed. Since each of the techniques considered gives only incomplete information, it is wise to design an experiment using as many independent methods of measurement as is feasible. The overlap from redundant measurements will give confidence that the effects measured are real rather than artifacts of the measuring technique used. 1. OPTICAL MEASUREMENTS

a . Direct Measurements. Observation of the reflectance of an open lipid membrane gives a qualitative indication of the progress of the thinning process which culminates in the formation of a bilayer. As the membrane thins, interference effects give rise to the appearance of colors-exactly like those visible in soap bubbles and oil slicks. These colors appear when the membrane is a few wavelengths (-1 pm) thick, and as the membrane thins further it first appears silver (- 100 nm), then grey, then black (-30 nm). Further thinning to bilayer thickness ( < l o nm) cannot be followed by monitoring reflectance, since the membrane appearance does not change (Tien, 1974). It is not sufficient to assume that the bilayer area is equal to the area of the hole across which the bilayer was formed due to the existence of the P-G border. Near the edge of the P-G border, however, the membrane is highly reflective (that part of the border is -100 nm thick), and the area bounded by this reflective band is essentially equal to the bilayer area. Although bilayers often have thick regions called lenses present, these cover a minute fraction of the surface and no significant error is introduced by ignoring them (Fettiplace et al., 1975). The diameter of the silver ring at the P-G border is measured using a reticle and a microscope, or calculated from a photograph taken at known magnification. In order to make these measurements, a strong light source which illuminates a small, well-defined area is required, for example, a high-intensity lamp with a fiber optic light pipe. The source should be placed so that the bilayer is illuminated while stray light is minimized, and the system should allow the location and orientation of the light with respect to the bilayer to be adjusted readily (Tien, 1974). Another simple optical measurement is the measurement of light scat-

66

ADlL E. SHAM00 AND WILLIAM F. TIVOL

tering. The amount of light scattered by a small vesicle (1-20 p m ) is proportional to the square of the wavelength (for visible light) and simply related to the mean cross-sectional area of the vesicle. For a suspension of vesicles, measurement of the light transmitted (and therefore also light scattered) before and after osmotic changes, can be related to the changes in interior volume. These changes in volume are directly related to the permeabilities of the vesicles to water or to permeant solutes in the case of protein-containing vesicles (Reeves and Dowben, 1970). More complicated optical measurements such as circular dichroism (CD) can yield important information. CD is especially promising to monitor conformational changes in reconstitution. However, the usual difficulties in interpreting CD spectra are made more severe due to the turbidity of membrane suspensions. Furthermore, membrane proteins have functions which are often very different from those of soluble proteins and this is reflected in their structures. Thus, although changes in CD due to changes in experimental conditions indicate conformational changes in the system under investigation, these changes do not necessarily reflect changes in the a-helical content. A good review by Urey and Long (1974) is available. b. Optical Probes. Fluorescent probes have found great use in the study of membrane structure and function. As in CD experiments, however, fluorescence studies are easy to perform but difficult to interpret. A thorough review has been written by Radda (1975). Lipophilic fluorescent molecules either with or without a polar group have been used to investigate membrane fluidity, phase transitions, and lipid-protein interactions. Differences in the structures of fluorescent probes lead to different locations of the chromophoric groups within the membrane. For example, 1-anilinonaphthalene-8-sulfonate(ANS) binds to a bilayer with the sulfonate group at the surface and the aromatic moiety within the hydrophobic region, but located near the phospholipid head groups. Pyrene or perylene have no polar group and are sensitive to changes throughout the hydrophobic region. Various 9-anthroyl fatty acids have been synthesized with the chromophore attached at a range of distances from the head group. Comparisons among this class of probe can elucidate very localized changes in the properties of the bilayer. Other suggested uses of the 9-anthroyl fatty acid probes are in the study of diffusion of small molecules through membranes and the location of fluorescent residues (e.g., tryptophan) in a membrane-bound protein by means of fluorescent energy transfer. There are also probes whose chromophore is located at the surface of the bilayer such as dansyl-phosphatidylethanolamine. Photobleaching techniques are useful for measuring lateral diffusion in

RECONSTITUTION OF ION TRANSPORT SYSTEMS

67

membranes. In this technique, a localized intense burst of light is used to inactivate a large fraction of the fluorescent molecules within a small, well-defined area. Fluorescence is observed as molecules which were not inactivated diffuse or flow into the bleached area. By following the time course of fluorescence recovery-which is observed at lower irradiation levels-diffusion can be differentiated from flow (e.g., connective transport, cytoplasmic streaming, etc.) or from a mixture of flow and diffusion (Elson et a / . , 1976), and the diffusion coefficient or flow velocity (or both) can be determined (Axelrod et al., 1976a). Fluorescent recovery after photobleaching has been used to characterize diffusion in solution (Jacobson et ({/., 1976; Koppel et "/., 1976; McGrath et ( I / . , 19761, biomembranes and cells (Schlessinger et d . , 1976: Axelrod et NI., 1976a,b; Elson et u / . , 1976; Jacobson et uI., 1976; Koppel et ([I., 19761, liposomes (Wu et ( I / . , 1977; Smith and McConnell, 1978), and monolayers (Teissie et ( I / . , 1978). Diffusion coefficients can be measured in the range lo-" < D < cm2/secondand flow rates of the order of 1 pm/minute can be measured. Major limitations to the technique are the uncertainty of beam size and shape, chemical recovery of fluorescence, and sample damage from the bleaching burst of light. The last limitation can be avoided by choosing a bleaching wavelength that is not absorbed by the sample, and chemical recovery occurs in few of the systems reported in the literature. One interesting fluorophore in this respect is 12(9-anthroyl) stearate (12-AS) which is dimerized to a nonfluorescent molecule by irradiation at a wavelength of 366 nm. The dimer can be converted to a monomer by irradiation at 254 nm. This would allow a check to be made for other inactivating reactions such as covalent binding of 12-AS to lipid or protein if such conjugates do not dissociate at 254 nm. Both beam size uncertainties and motion artifacts can be much reduced by the use of a periodic pattern of photobleaching (Smith and McConnell, 1978). Photobleaching by dimerization-as occurs with 12-AS-may be subject to difficulties in interpretation. These difficulties arise due to the kinetics of dimerization. At temperatures below the liquid-gel phase transition, 12-AS is excluded to some extent from the gel phase, and is postulated to form microdomains (McGrath et a/., 1976). Within these microdomains the 12-AS monomer concentration and the diffusion coefficients (both translational and rotational) are relatively high. Dimerization is thus rapid, but occurs largely in regions where the probe is clustered. The sizes and properties of these microdomains have yet to be defined (Teissie et a / . , 1978), and information obtained from such a system is subject to the difficulty that fluorescence (or lack thereof) arises from a particular ill-defined compartment of the membrane under inves-

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ADlL E. SHAM00 AND WILLIAM F. TIVOL

tigation. Studies such as fluorescence energy transfer and membrane fluidity might be most seriously affected (Teissie et a l . , 1978). The former relies on the close approach of a fluorescent probe to a fluorophore with a membrane protein, and membrane ordering effects due to the protein could change the effective probe concentration. If the fluorophores reporting on membrane fluidity are clustered in the most fluid compartment of the membrane, determinations of membrane fluidity will be of limited use. The silver lining of this small cloud is that dimerization kinetics can be used to measure probe aggregation and thus lateral phase separation (McGrath et a / . , 1976). This might be especially valuable in mixed lipid systems such as biological membranes. Gaber et al. (1978) report the use of deuterated phospholipids as nonperturbing probes for Raman spectroscopy of membranes. The deuterated lipids mix nearly ideally with those of the bilayer, minimizing or eliminating artifacts due to uneven distribution of probe molecules (see above and Section 11,BS). Since Raman spectra are sensitive to probe environment, melting transitions can be measured. Resonances arising from the head group regions can be differentiated from those due to the deuterated acyl chains, suggesting that protein incorporation and organization or requirements for specific head groups could be investigated by examining changes in Raman spectra. 2. ELECTRICAL MEASUREMENTS

Due to size limitations, electrical measurements on closed bilayer systems are not possible except for some measurements on giant vesicles. Unless otherwise stated the measurements and techniques described in this section will refer to open bilayer systems. Suggestions for equipment useful in electrical measurements are listed in Table I. In addition to building electrical measuring apparatus whose specifications are sufficiently rigorous to give precise results, care must be taken to eliminate other sources of error. The entire apparatus must be well insulated, i.e., at any point the resistance to ground should be more than 10'' R (Tien, 1974). Films of body oils, machining oils, and/or residue from the ambient atmosphere should be removed-cleaning the exposed parts with ethanol is sufficient. Stray capacitance should be minimized as should capacitive coupling (e.g., between wire leads, etc.). This is especially important for ac measurements and becomes more so for higher frequency. To accomplish this, low-noise shielded cable should be used and leads should be immobilized. The entire apparatus should be electrically and mechanically isolated so that stray electromagnetic fields and vibrations will not influence the measurements (Tien, 1974).

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69

OF ION TRANSPORT SYSTEMS TABLE I

Item

Desired characteristics

Examples

Electrode

Low emf induced, nonpolarizable, well-insulated, low capacitance

Calomel, Beckman 41239, Ag-AgC1," Pt-black, Pt

Resistors

Stable, high value

Victoreen"

Capacitors

Low leakage

Many

Amplifier

High-input impedance, low-input bias current, large bandwidth, low noise

Analogue Devices 42, Philbrick 1011, Analogue Devices 48K, Keithley 427"

Faraday cage

Good shielding of stray fields

Wire mesh," metal cage"

Cable

N o ground loops, immobilized leads

Low-noise coaxial cable," BNC connectors

Electrometer

High-input impedance, low-output impedance

Keithley 616"

Voltage source

IEC Function,o Generator F 84

Used in our laboratory.

The bilayer and associated electrical apparatus should be enclosed in a metal or wire mesh cage (Faraday cage) which is very well grounded. This cage should be placed on or anchored to a massive support slab which in turn is supported by foam or inflatable tires and placed on as massive a support as is available. An alternative scheme is to suspend the support slab from girders. In each case the objective is to provide a high-inertia support which is isolated by being attached to the earth through springs with low elastic constant. In this way if the earth (or lab building or lab bench) moves, the springs will change length but the force on the massive support slab will cause little motion. Electrical quantities which can be measured include membrane conductance, capacitance and potential, and currents due to specific ions, and changes in these currents ranging from small statistical fluctuations (noise measurements) to current jumps caused by changes in membrane potential [as much as a lo8fold increase in current can be measured in a circuit used to measure current logarithmically (Fettiplace et a / ., 1975)l. a . Conductance. The conductance of an unmodified lipid bilayer lies in the range 10-4-10-10 R-' cmP2 (Tien, 1974). The smallest value is

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ADlL E. SHAM00 AND WILLIAM F. TIVOL

equivalent to the conductance of bulk hydrocarbon and is the minimum conductance expected. Many factors influence conductance including the lipid composition, the lipid solvent used (polar solvents such as methanol give membranes with higher conductance-not surprisingly), the composition of the aqueous bathing fluid, and details of the membrane such as the distribution of material between the P-G border and the BLM and the micro-roughness of the hole across which the bilayer is formed. Although the conductance of a given membrane is constant once thinning is completed, conductances of different membranes, even if made on the same apparatus using the same lipid mixture, are unreproducible. Changes in conductance, however, are quite reproducible if sufficient care is taken (Tien, 1974). Pretreatment of the hole in the bilayer frame with a dilute lipid solution minimizes effects due to the roughness of the hole. Impurities in the lipid solution can have a dramatic effect on conductance: e.g., inclusion of 2% lysolecithin in a lecithin bilayer changes the specific resistance from lo7 Cl-cm2 to lo5 Cl-crn, (Van Zutphen and Van Deenan, 1967). Since lysolecithin is derived from lecithin by phosphohydrolase, the presence of this enzyme will lead to increases in bilayer conductance which can mask increases due to the incorporation of an ion transport protein and which can give much different selectivity properties than the transport protein incorporated in a bilayer where there is no phosphohydrolase activity. Differences in bathing solution can give conductivity differences of as much as three orders of magnitude, and in the case of I,, I - systems, as much as seven orders (Lauger et u / . , 1967). In the latter case, the conductance is selective for I-. There are two models to account for this effect; in one the current is carried by polyiodide ions (I;, I;, etc. formed from aqueous I- and I, dissolved in the bilayer), and in the other, I- is oxidized at one face and I2 is reduced at the other. Anions which lower bilayer resistance can be listed in order of effectiveness as follows (Tien and Diana, 1967a,b): I- > Br- > SO:- > CI- > F-

Fe2+ and Fe3+ also lower bilayer resistance at concentrations of about M (Miyamoto and Thompson, 1967). In a bilayer formed from a 3 : 2 CHC1,-CH,OH solution of egg PC and n-tetradecane, addition of Fez+or Fe3+ caused a sudden drop in membrane resistance by three orders of magnitude. The effect of FeCl, is both time and voltage dependent and the voltage dependence is exponential. The current is selective for Cl-; however, the selectivity is abolished in the presence of chelating agents. The explanation of this effect is thought to be that FeCI, dissolved in the

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bilayer facilitates the transport of CI- ion as the FeCI; species (MacDonald and Thompson, 1972). Divalent metals increase bilayer resistance when added to the bathing solution on both sides, but this is a small effect. Cd2+,Mn2+, and Cu2+ increased bilayer resistance by about a factor of two; Mg2+, Ca2+, and Sr2+had no such effect (Miyamoto and Thompson, 1967). For PS bilayers, however, inclusion of Ca2+ on one side of the bilayer lowered the resistance and led to the breaking of the membrane, whereas with Ca2+ on both sides the bilayers were stable and had high resistance (Papahadjopoulos and Ohki, 1969). This effect is due to the interaction of CaZ+ with the head groups of the phospholipids. Other salts can affect bilayer stability; brain lipid bilayers with 0.1 M KCI in the bathing fluid were quite unstable having an average lifetime of 21 seconds, while those with distilled water pH 7.0 as a bathing fluid had lifetimes of about 30 minutes. Of course inclusion of lipophilic ions such as picrate or tetraphenyl boron will lower bilayer resistance because the complexes formed with metals will diffuse easily across membranes. h. Cupcrcitance. Membrane capacitance can also be measured, and unlike conductance this quality is very reproducible from bilayer to bilayer (Tien, 1974); however, specific capacitance measurements on the same system in different laboratories can vary by 15-20% (Lauger et a / . , 1967). For unmodified bilayers the capacitance is in the range of 0.3- 1.3 FF/cm2 and is independent of frequency (lo3 < w > lo6) (Tien, 1974; Fettiplace et N / . , 1975).The greatest source of error in capacitance measurements is in the determination of the bilayer area (Fettiplace ef a / . , 1975). DC measurement of capacitance is quite simply performed by establishing a potential difference across the bilayer and allowing the system to come to equilibrium. The voltage is then removed and the bilayer allowed to discharge through a known resistance. Measurement of the time constant of the decay, T , of the voltage gives the membrane capacitance C, = T / R , where IIR, = 1/R, + 1/Ri and R , is the membrane resistance and R i is the resistance inserted between one chamber and the other. Capacitance can be even more accurately measured on the ac bridge circuit. Furthermore, this circuit can be used to follow the thinning of the bilayer (Tien, 1974). The circuit is adjusted to null output before the bilayer is formed. The output is then followed as a function of time after formation (this output is a monotonic but nonlinear function of membrane impedance). As the membrane thins, the output decreases, and becomes stable when the bilayer state is reached. Another method of following film thickness is to voltage clamp the film with a sine wave of high enough frequency ( w = a few hundred hertz) that the

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capacitive current is much larger than the resistive current. Thinning is complete when wZ/V (= C ) has the value expected for the capacitance of a bilayer. c . Specific Ion Currents. Potentials across bilayers are related to differences in the concentrations and permeabilities of an ion or ions on different sides of the bilayer (Shamoo and Goldstein, 1977). The relevant ion or ions must be able to cross the bilayer to establish contact between the two aqueous phases otherwise there would be no potential developed. The equations describing the relationship between potential and ion conductivity are the Nernst equation and its generalizations. The Nernst equation refers to a single ion: RT C l AV=-lnZF C , where A V = V, - V2, the voltage drop across the bilayer, R is the gas constant, T is the absolute temperature, Z is the valence of the ion (negative for anions), F is the faraday (the charge per mole of electrons), C is the concentration of the ion, and the subscripts refer to the two sides of the membrane. The Goldman equation refers to a system with any number of ions; however, all the ions are univalent:

AV

=

RT -In F

n

m

i= 1

i=l

n

m

where U ; is the mobility of the j t h cation, U ; is the mobility of the j t h anion, the C$ are the concentrations of the j t h ion on the ith side of the membrane and the sums are taken over all the relevant ions. The expressions become much more complicated when ions of different valence are present. However, we can write the generalized Goldman equation in the following form: FAV [Cil - Ci2exp (ZiFAV/RT)] Zf=o (3 1 i RT p i [exp(ZiFAV/RT) - I ]

2

where P iis the permeability of the ith ion and the other symbols have the same meaning as in Eqs. ( I ) and ( 2 ) . I n this form Eq. (3) says that the total current due to all the ions is zero. It is appropriate therefore to measure the voltage under conditions where no net current flows in order to determine the permeabilities of the ions which carry current in the system under investigation. Knowledge of the permeabilities and com-

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parisons with the permeabilities without a bilayer present gives direct information on the selectivity of the membrane. Although the Goldman equation was derived using the assumption that the voltage drop across the membrane is linear (implying that the electric field is constant), that assumption is not necessary (Barr, 1965). Other conditions when the Goldman equation is applicable include electroneutral active transport of anions or cations only, membrane permeabilities to cations (or anions) much greater than those to anions (or cations), and a neutral membrane with equal total ionic concentrations at either edge. Thus, the Goldman equation is applicable to a wide range of biophysically interesting membranes. Experimentally, the voltage at zero current is easy to measure and the measurement is very consistent. Different membranes in different laboratories give the same voltage intercept once corrections have been made for non-zero voltages (e.g., electrode induced emfs, etc.). A circuit with a triangle wave generator as a voltage source is suitable for this measurement using an amplifier whose output is connected to an X-Y plotter. If such a circuit is used, I-V curves will be obtained which will all pass through the same voltage intercept, although for nonzero current the curves can be quite different. A typical series of measurements for a suspected ionophore would entail first a confirmation that bilayer conductance increases when the suspected ionophore is incorporated, then an anion-cation selectivity measurement using a single salt at different concentrations on the two sides of the bilayer, for example 5 mM NaCI on side 1, 50 mM NaCl on side 2. If the membrane is found to be cation selective, that is, zero current occurs when side 1 is somewhat positive, experiments will then be performed to compare one cation with another. For a perfectly selective membrane which is completely impermeable to one ion (say CI-) and the sodium permeability, = l), which allows another to pass freely (PNar there will be a potential of 58 m V for a concentration ratio of 10 between the two sides. For a divalent ion under similar conditions, there will be a potential across the membrane of only 29 mV for a concentration ratio of 10 due to the factor Z i which always occurs multiplying A V [see Eq.

(31. In order to determine permeability ratios among cations, different salts are used on the two sides of the bilayer at the same concentration to cancel the contribution of the anion current, (for example, 5 mM NaCl on one side and 5 m M KCI on the other). In this way selectivity sequence for an ionophore can be determined. Typically there will be large permeability ratios for cations compared to anions, and for monovalent ions compared to divalent ions, but much smaller ratios within a class-Na'

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ADlL E. S H A M 0 0 AND WILLIAM F. TIVOL

compared to K + or Mg2+compared to Ca2+.An important class of measurements is conductance changes due to specific inhibition of an ionophore. In order to be sure that conductance increases are caused by incorporation of the ionophore of interest rather than by leakage of the membrane or by an impurity in the sample, any specific inhibitors of transport in vivo should be tested in the bilayer to see that they reverse increases in conductance to the same extent and at the same concentration as in the intact system. Recalling that transmembrane potentials are generated only by concentration gradients of ions which can cross the membrane, if an inhibitor shifts the voltage intercept then there must also be a shift in one or more permeabilities. This shift should be proportional to the fraction of ionophore to which inhibitor is bound rather than to membrane area, inhibitor concentration, or any other parameter. d . Potentials. Oxidation-reduction potentials can also be measured in bilayers. Tien and Verma (1970) report a titration of Fe2+ with K M n 0 4 using transmembrane potential as an endpoint indicator. This is in every way equivalent to the usual potentiometric titration with the bilayer behaving as a redox electrode (see Section II,C,4 for other examples). Photoinduced emfs have been observed in chloroplast bilayers. When Fe3+ is added to one side of the bilayer and ascorbate to the other, open circuit potentials greater than 100 mV can be generated. The Fe3+, an electron acceptor, and the ascorbate, an electron donor, trap electrons and holes generated in the membrane by light and thus prevent recombination. The potential, V, is related to the intensity of the incident light, I , V = A In( 1 + Z/B) where A and B are constants for a given membrane at a fixed temperature (Tien, 1974). Most semiconductor photovoltaic cells give the same response (see Section II,C,4 for other examples). e . Fluctuations. Studies of current fluctuations can be divided into two classes: noise analysis and relaxation analysis. If a bilayer has channels which open and close or carriers which ferry ions across the bilayer, the current through the membrane will fluctuate around its mean value in a predictable way. With appropriate assumptions about the characteristics of the channels or carriers, parameters such as single channel conductance and lifetime can be determined from accurate measurements of the current. A rigorous treatment of fluctuation analysis is presented in a recent review by Neher and Stevens (1977). For the simple case of a channel which can only be opened or closed and for which the probability that it is open is p , an expression for the conductance of a membrane containing m channels and one for the variance of the conductance is written ( G )= Y m P

(4) = Y2MP(l - P )

(41

(5)

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75

where ( G ) is the mean conductance, (cg)is the variance of the conductance, and y is the conductance of a single open channel. From the ratio of the variance to the conductance the single channel conductance can be obtained under conditions where p + I for channels which close at a time T after they open,

Knowledge of the elementary conductance-changing - events is necessary in order to derive rigorous conclusions from noise analysis. The covariance or autocorrelation function C ( T ) is defined by C ( T ) = lim L+rn

_f_

L

10''

y(t)y(t + T ) dt

(7)

C ( T ) contains information regarding the average time course of a fluctuation: note that C(0) = 08 and that C ( T ) decreases becoming zero for large T . Experimentally C ( T ) is calculated for large (but finite) L with y ( t ) = G ( t ) - ( G ) . The Fourier transform of C ( T ) gives the spectral density of the noise. A log-log plot of the spectral density for conducting channels displays two features: the spectrum is roughly constant until a frequency f , = 1 / 2 7 r ~at which point the noise falls off rapidly, and the integrated spectral density is equal to C(0). For processes involving channels with more than one characteristic open time, the theoretical fits to the data are much less good. Neher and Stevens (1977) show one case where apparently equally good fits may be obtained for two sets of parameters, one of which agrees with the theoretically expected values and the other of which differs by up to 70% from these values. Carriers exhibit a different spectral density from channels (Kolb and Lauger, 1978). At both low and high frequency, the current noise is independent of frequency but with different amplitudes. The transition region occurs typically at about 1 kHz. For the cases of both channels and carriers the transition frequency of the spectrum is related to the relaxation time constant. If the transmembrane voltage is jumped from one value to another, the current relaxes to a new steady-state value. The time constant for this relaxation, T r = 1/27rf, where f , is the corner frequency of the Lorenzian noise spectrum for channels or the transition frequency for the noise spectrum for carriers. Nongated channels have a spectrum proportional to l/f with no characteristic frequency. This might be expected since there is no time constant which characterizes the channel. Noise whose spectrum is of the form Ilfis not too well understood, but seems to be related to processes which look the same when scaled up or down. The rms amplitude of the noise is proportional to the average

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ADlL E. S H A M 0 0 AND WILLIAM F. TIVOL

current, that is, the "roughness" of the current trace is the same regardless of the magnitude of the average current. 3. CALORIMETRY Differential scanning calorimetry (DSC) is used to study changes in heat capacity with temperature. Due to sensitivity limitations abrupt changes corresponding to phase transitions are generally all that can be measured in biological membranes. In DSC the temperature of a sample and a reference are changed equally and the difference in heat input is measured. In a well-calibrated calorimeter this heat difference will be constant except at a phase transition. By subtracting the constant baseline and integrating the area under the peak, the heat of transition is obtained. A short review of DSC techniques and instrumentation has been written by Scheidler and Stein (1975), and a recent review covering high sensitivity DSC has been written by Mabrey and Sturdivant (1978). 4. ELECTRON MICROSCOPY

Electron microscopy (EM) is unique in that it provides a direct method to obtain unaveraged information about a membrane system or any other irregular preparation at the molecular level. In order to obtain sufficient contrast for most observations, however, the sample must be prepared and stained so even this technique often looks not at the membrane directly, but at an altered preparation or a replica. Furthermore, an electron micrograph gives only an instantaneous view rather than a motion picture, so additional information must be gathered in order to obtain a view of the dynamic, functioning system. In addition to enhancing contrast in biological specimens by staining or shadowing techniques, EM images can be manipulated by computer-particularly in those cases where there is a periodicity in the membrane structure. 0 . Preparation of Conventional Sumples. One overriding difficulty in EM is assuring that the preparation of the sample has not introduced artifacts which will be mistaken for features of the sample. Knowledge of the mechanisms involved in the procedures used in sample preparation is important in this regard: however, such knowledge is generally not yet available, so caution is required in the interpretation of EM images, and as much independent evidence as possible should be gathered to support any conclusions reached. An excellent review (Zingsheim and Plattner, 1976) discusses many of the mechanisms which have been studied as well as other aspects of EM. Since an initial step in the preparation of biological samples is fixation to prevent motion of the components during subsequent preparation

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steps, and since fixation usually (but not always) destroys biological activity, it is often impossible to check that the conformation of the membrane is unaltered by this step. In those cases in which checks have been made it has been found that the ultrastructure of the membrane has usually been preserved by fixation even when no enzymatic activity remains (Sabatini er al., 1963; Hopwood, 1972). Model compounds and artificial and natural membranes have been fixed with OsO, and examined (Bahr, 1954, 1955; Stoeckenius, 1960, 1962). The electron micrographs show the usual “railroad track” appearance of bilayers with the electron-dense areas corresponding to the polar head group regions. Changes in lipid organization after fixation have been investigated (Jost and Griffith, 1973; Jost et al., 1971, 1973) by spin label and X-ray diffraction. Restriction of lipid mobility by fixation and lipid disordering were found. Hackenbrock and Hochli (1977) examined fixation of mitochondria by several methods. They found that 1 % glutaraldehyde prevents protein movement and cross-links PE preventing its movement as well. Ferritin in the polycationic form fixes much less well, but if the outer surface of the membrane is covered by a mixture of polycationic and polyanionic, metal-free ferritin, fixation is moderately good. Specific antibody to cytochrome oxidase fixes 18 nMparticles in inner mitochondria1 membrane, thus these are associated with cytochrome oxidase. Other particles are mobile and appear to be confined to the inner leaflet of the inner mitochondrial membrane. After fixation, the next steps in membrane preparation are drying, embedding, sectioning, and staining. These steps are performed by stateof-the-art methods and have not been thoroughly analyzed. These processes should be viewed as necessary evils since many artifacts can arise (see Zingsheim and Plattner, 1976, for a particularly good discussion). Thin section EM has been very successful when combined with negative staining, since dimensional information obtained with this technique seems reliable. In this method, a heavy metal salt such as phosphotungstate is added to the sample, and during drying the metal concentrates around protrusions. The dry sample is then embedded in epoxide resin and sectioned. Care must be used to avoid alteration of the sample. For example, silicotungstic acid removes the 9-nm particles from inner mitochondrial membrane (Rikker er ul., 1969), whereas they remain visible under other conditions (Racker et ul., 1965). Another useful staining procedure is tracer staining. For example, lanthanides have been used to stain pores (Revel and Karnovsky, 1967), and it can be inferred that the location of the stain is related to the ion channel in a system that is inhibited by lanthanides. To make this infer-

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ADlL E. S H A M 0 0 AND WILLIAM F. TIVOL

ence it must be known that no movement of the metal atom takes place between the time that it binds to the transport system and the time the micrograph is taken. The fact that such tracer stains are found in a particular location with respect to protein particles-viz. the centers of pits in the membrane-make this likely, but not certain. b. Freeze-Fracture. Thin section techniques give views of random slices of the sample, and from examination of several such slices a threedimensional representation of the sample can be constructed. An alternative procedure, freeze-fracture, gives a very nonrandom view of membrane samples. Fracture planes occur between ridge-like features which may be the polar regions of the bilayer or added layers (occurring on the membrane or generated by sample preparation). Evidence on the location of the fracture surface has been obtained by examining the location of radioactive labels subsequent to fracture (Deamer and Branton, 1967) and by looking for membrane surface markers (Pinto Da Silva and Branton, 1970; Tillack and Marchesi, 1970). Since radioactive labels were found on both fracture faces, and since markers only appeared after etching of the fracture surface, it was concluded that fracture occurs bet ween leaflets, which is reasonable since the cohesive forces between leaflets are small. The amount of shadowing of a freeze-fractured specimen is an important parameter and represents a compromise. The greater the shadowing the greater the contrast (within the useful shadowing range) but the poorer the resolution. Freeze-cleaved samples must be adequately shadowed within 5 to 10 seconds to avoid unacceptable sample damage and to keep contamination due to outgassing to a minimum. Ideally, shadowing should have an equal probability of deposition at any point on the sample. Departure from ideality leads to differences in shadowing called decoration. This seems to arise from nucleation centers in a regular crystal lattice and may contain significant information, but this cannot as yet be interpreted. Dimensional information is important to the interpretation of micrograms, and decoration impairs the fidelity of this information to an unknown extent. A major source of artifacts in freeze-fracture is damage due to the formation of large ice crystals. Slow freezing leads to phase separation and concentration of solutes which are excluded from growing ice crystals. Although some cells survive this process and are viable when thawed, the ultrastructure is generally distorted, and rapid freezing is necessary to preserve structure. Two procedures, high-pressure freezing and spray freezing, do not require cryoprotectants and prevent the formation of large ice crystals. Freezing at high pressure allows supercooling. Under this condition the specific volume of solid water is not larger

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79

than the specific volume of liquid water, as it is at atmospheric pressure, and ice is not nearly as destructive to membrane structure; however, the sample must not be kept long at elevated pressure before freezing or the pressure itself will create structural distortions. In spray freezing an atomizer is used to prepare droplets of the sample which have diameters of 50 p n or less. These droplets are sprayed into liquid propane where the large surface-to-volume ratio and the good thermal contact lead to extremely rapid cooling rates (- lo5 Khecond), and ice crystal formation is negligible under these circumstances. Then the sample can be cleaved, etched if desired by allowing ice to sublime, and shadowed. Cryoprotection of the sample is not required, nor is any other pretreatment necessary. This method has been successful with all samples studied including both intact cells and liposomes. Thus, direct comparisons between in vivo and reconstituted systems can be made. Quick freezing from a temperature above the lipid phase transition leads to membrane replicas whose structure shows all the features of the fluid state, whereas slow cooling to a temperature below the lipid phase transition and then quick freezing yields membrane replicas whose structure shows all the features of the liquid crystal state. Protein motion effects for all forms of freezing are slower than lipid motion effects so that in some cases in which the lipids are well fixed, conventional freezing methods give adequate results, but spray freezing is preferred even in these cases. Cleaving artifacts arise from plastic deformation in the sample (Clark and Branton, 1968; Schmitt et ul., 1970), even at liquid helium temperature, and evidence of the breakage of covalent bonds in macromolecules has been found (Sleyter and Robards, 1977). Such deformations lead to uncertainties in the interpretation of the features seen in the exposed bilayer-especially in regard to the dimensions of these features. Particles found in one leaflet do not in general match pits found in the other. If these features represent real structures in the bilayer, it is difficult, often impossible, to say which leaflet portrays the features more accurately. Difficulties in shadowing due to the uneven, nonplanar shape of the cleavage surface may account for this mismatch, or the “particles“ and “pits” may be pseudogranules formed by plastic deformation. Membranes which exhibit 20-nm-diameter holes when negatively stained can show only shallow 10-nm pits when prepared by freeze etch. High resolution Ta/W shadowing of freeze-cleaved membranes show 5-nm pits whereas negatively shadowed preparations show 7-nm holes. The shadow thickness must be small enough to give good contrast, and this might explain why the holes appear to be shallow depressions. Possibly contamination of the surface by residual vapors can explain some of the

80

ADlL E. S H A M 0 0 AND WILLIAM F. TIVOL

other differences. Materials coating the outer surfaces of the bilayers are not well visualized by freeze etch techniques. After freeze etching 10-nm “warts’‘ can be found which can be mistaken for particles; however, they have been found in liposomes which are free of protein. The source of these artifacts is not known, although they are thought to be related to the orientation of ice crystals in the sample. They occur less frequently in spray-frozen samples which may be due to the higher rates of cooling obtained with that technique. Particles associated with proteins have been found in many membrane preparations. Vanderkooi (1974) found a crystalline array of 8.7 nm by 11.5nm particles by X-ray diffraction and negatively stained electron micrographs in reconstituted cytochrome oxidase membranes; Montal ( 1974a) found 10-nm particles in freeze-fractured membranes; Hong and Hubble (1972) found 20-nm particles in freeze-fractured reconstituted rhodopsin membranes, and 8-nm particles seen by Segrist et al. (1974) and by Grant and McConnell (1974) were attributed to glycophorin aggregates. An experiment was performed by DiPauli and Brdiczka (1974) in which the proteins in erythrocyte membranes were aggregated and membrane vesicles were formed and subjected to density gradient centrifugation. Freeze-fracture revealed membrane particles whose density was correlated with the protein content of the vesicles. This simple experiment is notable because it is a rare instance where such a correlation has been found (see Sections II,C,3 and II,C,4 for other examples). Rash and Ellisman (1974) found rows of 11 by 14-nm granules on the upper half of the postsynaptic folds when they examined the neuromuscular junction, which corresponded to the a-bungarotoxin (BTX) binding regions of these junctions. Albuquerque et ul. (1974)measured the density of AchR in the postsynaptic membrane of muscle endplate, and Fertuck and Salpeter ( 1974a,b) used lZ5I autoradiography of 1251-labeleda-bungarotoxin to locate the toxin binding sites on electron micrographs (see Section 11,B,3). Rang (1975) found that the AchR complex of molecular weight 500,000 binds on the order of 10 BTX molecules and that the density of particles found in this membrane is equal to the number of such complexes and thus about one-tenth the density of binding sites seen in autoradiography. Such aggregation is typical of membrane micrographs, but whether this represents in viw organization or an artifact of lateral phase separation is not known. L‘. Auxiliury Techniques. Several auxiliary techniques have been used with EM. Autoradiography is an important technique since quantitative information can be obtained. Resolution is limited to greater than 100 nm, but quantitation is accurate when technical problems with the emulsion are overcome. Location by electron probe X-ray microanalysis is

RECONSTITUTION OF ION TRANSPORT SYSTEMS

81

another useful technique (Gupta and Hall, 1978), especially when used with tracer staining. There are still problems fixing tracers and some ancertainty and skepticism are appropriate when interpreting these data; however, this is a rapidly advancing area and results found ultimately will be exceedingly useful for the interpretation of electron micrographs, especially in relating ion-binding functions to particular structures. Fortunately, one of the most interesting metals, calcium, is well suited for precipitation with Os0,-oxalate during membrane fixation. Scanning electron microscopy (SEM) is a relatively new technique which is starting to find use with membrane samples. With this technique it is possible to obtain many kinds of information simultaneously. The focusing problems found in conventional electron microscopy are absent in SEM so elastically and inelastically scattered electrons can be examined as well as unscattered electrons, and X-rays produced by the electron beam can be counted as well. From these data, the atomic composition of the sample can be obtained as a function of position. Staining is not necessary but tracer staining is quite useful since the tracers are easy to locate. One disadvantage of S E M is that the intense electron beam used causes rapid degradation of the sample, and another is that resolution is not as good as with conventional E M . For systems in which there is periodicity, computer enhancement of EM images can be used to provide high-resolution maps of the protein molecules in a membrane (Unwin and Henderson, 1975). This has been done, for example, in the cases of BR (Henderson and Unwin, 1975), where a 0.7-nm resolution map of BR is presented showing seven ahelical regions and their orientation, and where the packing of BR in purple membrane is shown (see Unwin and Henderson, 1975, for a better picture), cytochrome oxidase (Henderson et al., 1977), where a 1.2-nm resolution map of the structure and packing is shown, and F1ATPase (Wakabayashi et al., 1977), where a map of similar resolution is presented. Information in this last report was used to support the hypothesis that there are six major subunits (3a, 3p) in F1 ATPase. This method provides a sensitive measure of the extent to which the higher order structure of a reconstituted system matches that of the native system (however, see Section II,C,3; the higher order structure can be difficult to reassemble). In principle EM techniques can give a great deal of valuable information regarding membrane structures. In practice, the contributions of E M to the understanding of membranes have been disappointing due to the artifacts which arise and the uncertainties in the interpretation of the images produced. The future, however, looks more hopeful since E M is a rapidly advancing field and new techniques which minimize artifacts or

82

ADlL E. S H A M 0 0 AND WILLIAM F. TIVOL

extend the range of measurable parameters appear frequently. EM used to investigate reconstitution should be especially promising, since in the comparison of the in vivo with the reconstituted systems the same artifacts should be expected to arise, and measurements of differences can be interpreted with more confidence than measurements of absolute quantities.

5. MAGNETICRESONANCE The two most familiar magnetic resonance techniques, electron paramagnetic resonance (EPR) and nuclear magnetic resonance (N MR), are based on the same physical principles. In both cases differences in spin state can be measured by inducing transitions between the states. A magnetic resonance spectrometer consists of a large magnet which produces a uniform magnetic field into which the sample is placed, and a magnet which produces a smaller, time varying magnetic field perpendicular to the constant field. The frequency of this smaller field can be swept through a range, and when the frequency matches a transition frequency of the sample energy will be absorbed. The transition frequency is a function of the total magnetic field experienced by the resonating electron or nucleus. This total field consists of the applied field and the fields due to the spins of other atoms in the neighborhood. Different chemical structures will lead to different transition energies so that the resonance measured can be identified. The width of a transition is determined by the relaxation mechanisms whereby the energy contained in the orientation of a spin is dissipated. The relaxation times can be related to molecular motions and local order, so that measurements of membrane fluidity or of protein rotation or diffusion can be made. Cadenhead and Katti (1971) examined whether spin labeled probes perturb the structures of lipid monolayers. They find significant differences in the mean area per molecule in lipid films composed of myristic acid and either cholesterol or a spin labeled cholesterol analog. Thus, measurements using this probe may be unreliable since the probe perturbs the system to be measured. They argue that these results should also apply to bilayer membranes and that even the use of low probe concentrations will not improve the situation since the EPR probe only reports on its own immediate environment. Notwithstanding this limitation there have been successful applications of magnetic resonance techniques in the investigation of membrane structures and comparisons between native and reconstituted systems. Birdsall et al. (1971) report data on the 19F relaxation rates of monofluorostearates with the fluorine atom located at various positions along the

RECONSTITUTION OF ION TRANSPORT SYSTEMS

83

hydrocarbon chain. Monofluorostearic acid and lecithin were sonicated under nitrogen until the suspension was translucent and light scattering was minimal, and aliquots of the vesicle suspension were examined. Chemical shifts relative to a perfluorobenzene standard were found to be the same for the vesicles as for a CDCIBsolution of monofluorostearate, which indicates that the 19Fnuclei in each derivative experience a similar lecithin “solvent” environment and that the polar interface plays little part in the relaxation process. Relaxation times were found to increase as a function of temperature between 25 and 50°C and as a function of distance from the polar interface. This shows that the interior of the bilayer is more fluid than the polar region and that the fluidity is maximum at the center of the bilayer. In order to examine possible perturbations of the lecithin bilayer structure by the monofluorostearates, a spin labeled stearate was incorporated in vesicles composed of lecithin or lecithin plus monofluorostearate. No differences were observed in either line shape or resonance energy between samples; this indicates that the monofluorostearates do not affect the lecithin bilayer structure. The results are interpreted as showing that the relaxation is dominated by motion of the chain segment carrying the 19F,and that there is a progressive and approximately linear increase in motional freedom in going toward the center of the bilayer. A palmitoylcholine derivative spin labeled in the 8-position was used as an amphipathic acetylcholine antagonist to measure the lipid environment of AchR by EPR (Brisson et al., 1975). It was found that the immediate lipid environment was immobile indicating strong lipid-protein interactions, and the existence of these interactions is suggested as an explanation of the many failures to reconstitute active AchR (see Section II,C,3). Nuclear magnetic resonance studies of SR membrane (Davis and Inesi, 1971) showed a correlation between Ca2+efflux and a reversible structural transition as the temperature was varied between room temperature and 50°C. Further heating to 80°C denatured the Ca2+ + Mg2+-ATPaseand altered the temperature dependence of the transition. The interpretation of these results is that the lipid mobility observed depends on lipidprotein interactions and that the increase in hydrolytic activity observed between 40 and 50°C corresponds to the reversible uncoupling of the hydrolytic function from the ion transport function. The NMR spectra are consistent with a membrane in which about 80% of the lipid fatty acid chains are organized in a fashion which allows only limited motion while about 20% of the lipid chains are in a fluid region where isotropic motion occurs.

a4

ADlL E. S H A M 0 0 AND WILLIAM F. TIVOL

J . C. Metcalfe et al. (1971) and S . M. Metcalfe et al. (1971) compared the structures of native, treated, and reaggregated membranes from M y oplasma luidlawii and erythrocytes by many different techniques. The reaggregated membranes were indistinguishable from untreated isolated membranes by EM, lipid and protein composition, and density gradient centrifugation. Reaggregated M . laidlawii membranes were prepared by dissolving the isolated membranes in 10 mM sodium dodecyl sulfate then dialyzing out the detergent for three days at 4°C (see Section II,C,l for a discussion of renaturation from SDS solution). Reaggregated erythrocyte membranes were prepared by dissolving erythrocyte membranes in methyl cellosolve at pH 2 and dialyzing against 10 mM Tris buffer pH 7.4 at 4°C. The reaggregated vesicles were smaller than the isolated vesicles by about a factor of two to ten, and the contained some residual SDS, but not more than 0.5% of the membrane dry weight. Though EM showed no structural differences between isolated and reaggregated membranes by both positive and negative staining, the membranes were easily distinguished from either isolated membrane lipids or proteins. Density gradient centrifugation gave a single band for both isolated and reaggregated membranes and all the original protein and lipid appeared in the reaggregated membrane band. X-Ray diffraction at various temperatures showed distinct changes corresponding to a broad phase transition whose high temperature limit was 23 k 2°C and whose width was about 10" in both isolated and reaggregated membranes. However, it was found that the ordered regions are smaller in reaggregated membranes than in isolated membranes. Nuclear magnetic resonance studies were carried out using the phenyl proton resonance of benzyl alcohol as a probe of membrane structure. Line widths were measured as a function of benzyl alcohol concentration for isolated and reaggregated membranes, pure lipids, pure proteins, and membranes pretreated with lytic concentrations (200 mM) of benzyl alcohol. Isolated membranes give line widths which decrease with increasing benzyl alcohol concentration up to 60-80 mM then increase with higher concentrations. This biphasic behavior was shown to correspond to increasing benzyl alcohol mobility (and therefore greater membrane fluidity) at low concentrations, and to the exposure of the new binding sites for benzyl alcohol, located primarily on the membrane proteins, at high concentrations. The weighted mean line width for pure protein plus lipid is considerably larger than the line width of the membrane indicating considerable ordering in the intact membrane. Reaggregated erythrocyte membranes give line widths which are the same as those measured with pretreated erythrocyte membranes and essentially identical to the

d

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weighted mean line widths of separated protein and lipid components. Reaggregated M . laidfawii membranes, however, give line widths which are intermediate between those of isolated membranes and those of pretreated membranes, indicating some ordering in the reaggregated structure. It was found that different preparations gave somewhat different line widths, and this was interpreted as indicating a gradation of success in reaggregation and two possible explanations were offered: varying residual SDS or the requirement for precise (but unknown) conditions necessary for successful reaggregation. Similar results were found with the fluorescent probe ANS (S. M. Metcalfe et a/., 1971). These results indicated that the major difficulty in reaggregation is that the protein component does not reassemble properly, and that the organization of.the lipid is insensitive to the protein conformation. Both NMR and fluorescence depend on how the probe molecule binds, and differences found with the two probes can be related to differences in binding properties. In both cases, however, the binding was determined primarily by the state of membrane organization. The two techniques report on independent parameters of membrane organization: N M R relaxation is determined primarily by rotational correlation times, whereas ANS fluorescence is determined by the hydrophobicity of binding sites. Another feature of the fluorescence study which does not occur in the NMR measurements is the quenching of fluorescence (in this case by carotinoids in the M . Iaidlawii membranes). This lowers the overall observed fluorescence, and, if not taken into account, could lead to misinterpretation of the data. J. C. Metcalfe et a / . (1971) offer the suggestion that if reconstitution of a membrane system is possible, the labeling of a specific component will give more useful information than studying the system with a probe whose location is not precisely known. They also suggest the removal, labeling, and reintroduction of a specific component as a potentially useful technique. Tourtellotte et a / . (1970) studied M . laidlawii membranes by EPR measurements of a biosynthetically incorporated stearic acid analog and found that the EPR spectra of isolated membranes are quite similar to those of liposomes made from extracted M . laidlawii lipids. Rottem et a / . (1970) obtained similar results with spin labels incorporated in v i m . Both these results suggest that EPR spectra of membranes using lipid probes are sensitive primarily to lipid-lipid interactions. Unless the probe can be localized [see the discussion in this section of the work of Brisson et a / . (1975)], EPR appears to be sensitive to the wrong parameters. Therefore it is less useful than N M R in deciding the fidelity of reaggregation.

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6. SURFACE PRESSURE Measurement of the surface pressure of a monolayer with a film balance gives information on lipid-lipid interactions and the thermodynamic state of the monolayer. Such measurements are usually made on films whose area and lipid content are known and are often displayed as curves in the surface pressure-specific area plane. Perturbations of the overall lipid structure can be easily measured and can be used to follow the incorporation of protein into a monolayer. When there is no available probe (fluorescent, EPR, or NMR), this is the only simple, real-time method to measure incorporation. Surface pressure measurements are also useful in determining the effects of surface active agents such as solvents or detergents which may be present in reconstituted systems, and in determining the P-G border contact angle, which gives information about the thermodynamic properties of open bilayer systems. The equipment used to measure surface pressure is quite simple. For monolayers it consists of a trough whose surface area can be measured, and over which a lipid film can be spread. One edge of the film is attached to a movable rod and a known force is applied to the rod to measure the area as a function of surface pressure. Another method is more suitable for use with open bilayer systems. A bilayer is spread across a hole in the usual manner and hydrostatic pressure is developed between the chambers on either side of the bilayer until the bilayer is in the shape of a hemispherical bubble. At equilibrium,

II = P d / 8 where II is the surface pressure, d is the diameter of the bubble, and P is the hydrostatic pressure needed to form the bubble (Tien, 1974). I n closed bilayer systems, osmotic pressure can be used instead of a hydrostatic head. In both cases, however, the applied pressure must be small and controllable since bilayer surface pressures are small. C. Reconstitution of Ion Transport Systems 1. Ca2+

+ Mg2+-ATPAsE

The calcium pump from skeletal muscle SR is in many ways one of the simplest active ion transport systems. The pump consists of a single protein (MacLennan, 1970), which can be readily isolated (MacLennan, 1970; Meisner et a / . , 1973; le Maire et a / . , 1976; Banerjee ef a / . , 1979), and which has been well characterized by its amino acid composition (Thorley-Lawson and Green, 1975), partial sequence (Allen and Green,

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1976, 1978), substrate and co-factor binding properties (Meisner ef a / . , 1973; Yates and Duance, 1976: Dupont, 1977; Kalbitzer et a / . , 1978),and kinetics and stoichiometry of ATP hydrolysis and Ca2+transport (Masuda and DeMeis, 1974; Sumida and Tonomura, 1974; Coffey et u / . , 1975; Ikemoto, 1975, 1976; Inesi e f a / . , 1976; Hidalgo et a / . , 1976; Duggan, 1977; Dupont, 1977: Sorenson and DeMeis, 1977). Reconstitution of the calcium pump protein has been successfully accomplished, and many features of this ion transport system have been elucidated in reconstituted systems. Racker (1973) initially reconstituted the pump with soybean phospholipid by dissolving both protein and lipid with sodium cholate and then dialyzing out the detergent. A similar procedure was successfully used to reconstitute the pump in endogenous microsomal lipid (Meissner ef a / . , 1973). Racker and Eytan (1973) were able to reconstitute efficient Ca2+ active transport without the use of detergent by sonicating the pump protein with various phospholipid mixtures. They determined that the reconstituted system required PE for significant Ca2+uptake. This result was confirmed (Knowles et N / . , 1975: Hidalgo and Tong, 1978) in studies in which modifications of PE lead to loss of Ca2+transport activity. The loss of activity could be prevented by inclusion of an alkylamine in t h e phospholipid mixture (Knowles ef N / . , 1975). Warren et a / . (1974a,b,d, 1975) found that upon isolation ATPase retained 30 molecules of phospholipid in an ordered annulus and that these phospholipids could be exchanged for known phospholipids without irreversible loss of activity. When lipids in the annulus were replaced with dioleoyl PC the ATPase retained activity, whereas when the saturated phospholipids dipalmitoyl PC or dimyristoyl PC were used in addition, activity decreased linearly depending upon the fraction of saturated lipid in the annulus. The activities measured ranged from 150% of the activity of SR with all dioleoyl PC to 50% of the activity of SR with all saturated phospholipid. These changes in activity were found to be reversible. When lipid mixtures including cholesterol were used (Warren et u/., 1975), it was found that the cholesterol was excluded from the annulus unless high detergent concentrations were used. The activities of those preparations in which cholesterol was substituted into the annulus decreased linearly with increasing cholesterol until only 15 molecules of phospholipid remained in the annulus at which point the activity was zero. This pattern was also observed in the presence of cholate alone; however, only in the case in which cholesterol was present could the activity be restored by reassembly of the annulus with phospholipid. Detergent alone irreversibly denatured the ATPase leading to irreversible loss of activity.

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These results are important for two reasons. First, the effects of changing the length and saturation of phospholipid hydrocarbon chains and of substituting cholesterol or detergent for phospholipid indicate the specific lipid requirements for pump activity and demonstrate that strong lipidprotein interactions are fundamental to the organization of the membrane. Second, the technique of lipid substitution should prove useful in other systems, and conditions whereby such substitutions can be made in a given system will allow the reconstitution of that system. In the case of acetylcholine receptor (see Section 11,C,3), where functional reconstitution has not yet been accomplished, studies of changes in receptor conformation upon attempted lipid substitution might be used to great advantage in determining the appropriate conditions for reconstitution. I n Section 11 ,B,5 comparisons between native and reaggregated membranes showed that the protein is the membrane component which does not assemble properly. Lipid substitution should alleviate this problem since the protein is never completely stripped of lipid during the process, and thus a stable, active conformation is preserved. Hidalgo er u / . (1976) investigated phosphoenzyme formation and dephosphorylation in Ca2+ + Mg2+-ATPaseusing lipid substitution. They compared four preparations: SR vesicles, partially delipidated SR vesicles, and vesicles reconstituted predominantly with dioleoyl PC or dipalmitoyl PC. I n all cases, the rate of phosphoenzyme formation was essentially the same, but inorganic phosphate release varied. EPR using spin-labeled stearic acid was measured, and transition temperatures obtained were compared to Arrhenius plots of calcium-stimulated ATPase activity. Transitions observed in both techniques agreed closely. In the cases of both dioleoyl and dipalmitoyl PC the transition temperatures were not the same in pure lipid as in reconstituted lipid-protein membranes. Thus, strong lipid-protein interactions fundamental to membrane organization were demonstrated. The conclusions reached were that the phosphoenzyme was stabilized by the ordered lipid environment and by the lack of mobility of the saturated hydrocarbon chains in the case of dipalmitoyl PC (although the reduced lipid content could also play a part). This agrees with other work showing that membrane fluidity is important to ATPase function (Inesi er ( I / . , 1973; Coan and Inesi, 1977). Racker and Eytan (1975) found that during isolation of the calcium pump different fractions were produced which differed in their ability to translocate calcium although the hydrolytic activities were similar. Those preparations which were efficient transporters contained high amounts of a proteolipid, and this was proposed to form an ion translocating complex with Ca2+ + Mg2+-ATPase.However, Laggner and Graham (1976)found that the proteolipid showed no ionophoric properties but instead reduced

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the water and nonspecific ion permeabilities of artificial planar phospholipid bilayers. They also found refractive index changes upon incorporation of proteolipid which indicated an ordering of the hydrocarbon chains in the bilayer. In view of the effect of chain ordering on the membrane permeability and the dephosphorylation step of the calcium transport, it is reasonable that a specific molecule should have developed which orders the lipid chains so that the leakage of the membrane is minimal but the dephosphorylation is not impeded. This allows a membrane to be synthesized which is fluid enough to let the calcium pump function unimpeded, but tight enough to assure that calcium will not leak back out. Studies of the structure and organization of Ca2++ Mg2+-ATPaseusing reconstitution or partial reconstitution have been very successful. The calcium pump can be subjected to controlled tryptic digestion (Stewart and MacLennan, 1974; Thorley-Lawson and Green, 1973) first yielding two fragments, A and B, of molecular weights 55,000 and 45,000, respectively. Subsequently, the A fragment is cleaved into two fragments, A, and A2, of molecular weights 30,000 and 20,000, respectively. The A , fragment contains the hydrolytic function (Thorley-Lawson and Green, 1973; Stewart et al., 1976) and the A2 fragment contains the ionophore function, and by implication, the high-affinity Ca2+binding sites (Stewart et al., 1976; Shamoo et al., 1976, 1977, 1978). The rate of ATP hydrolysis is not significantly affected by either tryptic cleavage, and calcium transport is affected only by the cleavage of the A fragment into A, and A2 (Shamoo et al., 1977; Scott and Shamoo, unpublished observations). MacLennan et ul. ( I 976) found that calcium transport activity could be restored in Ca2++ Mg2+-ATPasewhich had been tryptically cleaved into A and B fragments and dissolved in 1% SDS as long as the SDS was removed within I hour. Under these conditions, the A and B fragments were dissociated. The renaturation procedure consisted simply of diluting the SDS solution into five volumes of sonicated phospholipid (see Section II,A,I for details), incubating on ice for 30 minutes, and passing the suspension through an anion exchange resin column. Exposure to 0°C (Sumida and Tonomura, 1974) or to trypsin (Scott and Shamoo, unpublished observations) results in the change of one of the two high-affinity Ca2+binding sites to a lower affinity site. We found that the temperature-sensitive site is different from the trypsin-sensitive site. Shamoo and co-workers have investigated the conductance properties of Ca2++ Mg2+-ATPaseand its tryptic fragments in open bilayer systems. They found ( 1 ) the specific ionophore site is in the A2 fragment (Shamoo et al., 1976, 1977, 1978), ( 2 ) the A , fragment has no specific ionophorous properties (Shamoo et al., 1976), (3) the B fragment is a nonspecific

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ionophore having properties similar to an aqueous pore, but selective against monovalent ions (Abramson and Shamoo, 1978), (4) the ion selectivities of the undigested, succinylated protein and the A fragment were essentially identical and very similar to the selectivity of the A2 fragment (Abramson and Shamoo, 1978), and (5) reduction of succinylated ATPase or of the B fragment abolished ionophorous properties whereas reduction of the A fragment had no effect (Abramson and Shamoo, 1978). These partial reconstitution experiments have located the functional sites and shown the interrelations necessary for active transport (Shamoo et al., 1977). The results of Abramson and Shamoo (1978) imply that the A and B fragments were aligned functionally in series in the membrane. Dean and Tanford (1978) have shown that the ATPase is active in the monomer state and experiments are under way to determine the minimal aggregation state necessary for transport. There is evidence, however, showing cooperative interactions between pump molecules and favoring an oligomeric association in SR vesicles (Coffey et al., 1975; Kalbitzer et al., 1978) and reconstituted vesicles (Dean and Tanford, 1978). Perhaps eludication of the quinternary structure necessary for transport can be obtained by reaggregation of the pump using a vast excess of lipid to obtain vesicles with one pump molecule per vesicle then progressively fusing such vesicles until calcium is taken up. Reconstitution has been successfully used in this Ca2+transport system to elucidate many aspects of the system: lipid requirements and effects of membrane fluidity, location of functional sites, and higher organization of protein and membranes. These successes give hope of similar success in other systems of interest and should be noted by those attempting reconstitution experiments.

2. Na+

+ K+-ATPAsE

The sodium-potassium pump is found in cell plasma membranes and in many organs. This pump provides cells with a mechanism for regulating their internal sodium and potassium concentrations, which is necessary for proper function of the cells' metabolic processes. Salt glands of marine organisms, electric organs of electric fish, and kidneys are rich in Na+ + K+-ATPase as are nerve cells. This is expected since these tissues either regulate salt within the organism or use monovalent cation gradients to control membrane electrical properties. The Na- K pumps from these various sources are very similar (Perrone et al., 1975). The amino acid compositions of pumps from different sources are indistinguishable

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indicating a highly conserved sequence; however, the amino terminal residue is different in different species. The pump consists of two different polypeptide chains, but the quaternary structure is unknown as yet. Values ranging from one large peptide to two small peptides (Shamoo, 1974: Hastings and Reynolds, 1979) to two large peptides to one small peptide (Perrone et a / . , 1975; Jorgensen, 1977; Hokin, 1977) have been reported. The pump from various sources has been purified: rectal gland of S. acanthius (Hokin et a / . , 1973),eel electric organ (Perrone et a / . , 1975: Swann et a/., 1975), kidney (Kyte, 1971: Jorgensen, 1974a,b), duck salt gland (Hopkins et a / . , 1976), and brain (Klodos et a / . , 1975; Fukushima and Tonomura, 1973; Nakao et a / . , 1973a,b). The stoichiometry, binding properties, and kinetics of Na+ + K+-ATPase have been widely studied (Shamoo et d.,1970a,b, 1971; Shamoo, 1971; Shamoo and Brodsky, 1971, 1972, Brodsky and Shamoo, 1973; Fukushima and Tonomura, 1975a,b; Taniguchi and Post, 1975; Patzelt-Wenczler et a / . , 1975; Calvieres and Ellory, 1975; Massa et a/., 1975; Bond and Hudgins, 1975; Reddy et a / . , 1976: Hansen, 1976; Gache et a / . , 1976: Hobbs and Dunham, 1976; Henderson and Askari, 1976; Kuriki et a / . , 1976; Karlish ef ul., 1976; Mardh and Post, 1977; Akera, 1977; Blostein and Chu, 1977; Post, 1977). Like the case of Ca2+ + Mg2+-ATPase,controlled typtic digestion of Na' + K+-ATPase has proved useful (Shamoo, 1974; Giotta, 1975; Jorgensen, 1975, 1977). Reconstitution of Na+ + K+-ATPase has proceeded along similar lines as reconstitution of the Ca2+pump, but appears to be a few years behind. Goldin and Tong (1974) attempted to reconstitute Na+ + K+-ATPase from canine renal medulla into phospholipid vesicles by the cholate dialysis technique. They were able to reconstitute Na+ transport, but not K f transport, although K+ was still required for hydrolytic activity. Instead, they found Na+ and C1- appeared to be cotransported. Sweadner and Goldin (1975) reconstituted coupled Na+ and K + transport with approximately a 3 :2 stoichiometry using Na+ + K+-ATPase extracted from canine brain, again using cholate dialysis. Although they obtained low yield of active enzyme (7-13% of the initial activity), the properties of the reconstituted system are not significantly different from those of the native enzyme. Goldin (1977) reconstituted Na+ + K+-ATPase from canine renal medulla using a modification of this earlier technique (Goldin and Tong, 1974) and obtained coupled Na+ and K + transport. The major modifications from the earlier procedure were a shorter dialysis time and the use of a more highly purified lipid preparation with which care was taken to prevent oxidation. Comparisons of cation permeabilities between the vesicles produced by the earlier technique and those produced in this

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work showed that the low level of lipid oxidation significantly reduced passive cation permeabilities, i.e., low levels of lipid oxidation give tight vesicles (see Section II,A,l). Goldin concluded that there is no essential functional difference between the Na+ + K + pump from kidney and that from other sources, and that other factors in the kidney produce net NaCl transport. He showed that the pump inserts into vesicles in random orientation, that most if not all the appropriately oriented (inside out in this case) pump molecules retain transport activity, and that this activity is characterized by a substantially lower turnover number than in ~'ivo. This lower V,,, could explain the lower activity found by Sweadner and Goldin (1975). Goldin was also able to exclude the possibility that any polypeptide other than the large and small subunits already known could be required for the pump activity. Very conservative estimates gives a maximum molecular weight for such a hypothetical component of 12,000. Hokin (1977) reported the purification and reconstitution of the Na' + K + pump from the rectal gland of S. acanthias. The cholate dialysis technique was again used; however, sonication in the absence of detergent was investigated and it was found that the protein failed to incorporate in the liposomes. Hokin recognized the relation between his reconstitution procedure and the lipid exchange procedure of Warren ef crl. (1974a,b,c,d, 1975) and attempted lipid exchange. This was successful and vesicles whose lipids had been twice exchanged with egg lecithin (and which showed only egg lecithin on thin layer chromatography) were actually twice as efficient as native vesicles (Hilden and Hokin, 1976). Cross-linking studies using fluorodinitrobenzene have shown that PS and PE can be cross-linked to the pump and may be boundary lipids (Hokin, 1977); however, no experiments on lipid requirements have yet been reported. Judging from the successes in the case of Ca2++ Mg2'-ATPase, such experiments should prove quite fruitful. For example, the optimal temperature range for transport activity in the pump from S. ucanthias is between 4 and 25°C with highest activity at 25". The optimal temperature for microsomes from turtle bladder is 60-63°C (Shamoo et a / . , 1971). It should be surprising if the same optimal temperature were found for the pump from mammalian sources. Differences in optimal temperature may arise from the protein structure or from the interaction of the protein with its lipid environment, and determination of the source of the differences will give us information about the workings of evolution. Shamoo and co-workers (Shamoo and Albers, 1973; Shamoo and Myers, 1974; Shamoo ef al., 1974; Blumenthal and Shamoo, 1974; Shamoo, 1974; Shamoo and Ryan, 1975) have used partial reconstitution of tryptic fragments of Na' + K+-ATPase to show that the mixture of fragments has ionophoric activity. They characterized the ionophoric

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activity showing that it resides in one or more peptides (Shamoo et a/., 1974) and was abolished by treatment with Pronase, dansyl chloride, and photooxidation catalyzed by methylene blue, but not affected by treatment with N-ethylmaleimide, DTNB, or oxidation by H 2 0 2or 0,. These results indicate that histidine may play an essential role in the ionophore as well as amino group(s), and that no sulfhydryl group is essential to ionophoric activity. Comparisons of the successes of reconstitution in elucidating the structure of Na+ + K+-ATPase with those in the case of Ca2++ Mg2+-ATPase indicate that reconstitution should be very useful in future investigations of Na' + K+-ATPase but that as yet reconstitution has not been used to its fullest extent in this system. 3. ACETYLCHOLINE RECEPTOR

In contrast to the first two systems discussed, the AchR is a very complex structure. Since the complexity of the system seems to be related to the difficulties found in its reconstitution, its structure will be discussed in some detail. There are different classes of AchR which respond to quaternary ammonium compounds in different ways. The two best characterized classes are nicotinic receptor, which responds to nicotine and is blocked by curare and a-bungarotoxin (BTX) (see Rang 1975 for an extensive review and Changeux et a/., 1976 for another excellent review), and muscarinic receptor, which responds to muscarine and is blocked by atropine (for a review, see Massoulie et a / . , 1977). In addition there are receptors derived from housefly head which have neither nicotinic nor muscarinic responses (O'Brien, 1979). Since most of the work (and particularly reconstitution attempts) has been done with nicotinic receptor, this section will be restricted to that class. Most preparations consist of four distinct polypeptide chains for AchR derived from lorpedo and three chains for AchR derived from Electrophorus; however, other numbers have been reported: Carol1 et n / ., (1973) and Potter (1973) find one, Hucho and Changeux (1973). Heilbronn and Mattson (1974), and Michaelson et a / . , (1974) find two, Raftery et a / . (1974) and Karlin et a / . (1976a)find three, and Cohen et a / . (1974), Duguid and Raftery (1973), Karlin et a / . (1976a). Reed et a / . (1975). Raftery et a / . (1976). and Hucho et a / . (1976) find four. Raftery et a / . (1976)implicate endogenous proteases as a cause of varying subunit patterns and find that the inclusion of protease inhibitors or EDTA in the purification eliminates this variability. Other possible causes of variability in quaternary structure are that AchR from different sources may have different subunits (although the differences found between Torpedo and Electro-

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phovus do not necessarily reflect different numbers of subunits since two distinct peptides can coelectrophorese), and that different methods of preparation may copurify different aggregations of subunits. Preparations from different sources have somewhat different molecular weights: however, few rigorous studies have been performed (most of the weight’s have been found by SDS gel electrophoresis which is often inaccurate for membrane proteins), so how much of the apparent difference is related to primary sequence difference and how much is related to difference in preparation or measurement is difficult to estimate. Typical values for the molecular weights of the subunits are 37,000 to 42,000 for the a-subunit, 43,000 to 50,000 for the p-subunit, 50,000 to 68,000 for the y-subunit, and 64,000 to 81,000 for the 6-subunit (Gordon et al., 1974; Karlin et al., 1976b; Rubsamen et al., 1976; Sobel and Changeux, 1977: Reynolds and Karlin, 1978; Hucho et al., 1978). Apparently in situ the AchR exists as a dimer joined by a disulfide bond between the two 6chains (Hucho et al., 1978); thus molecular weights of about 140,000 have been reported for an AchR subunit. However, reduction of the disulfide bridge yields monomers with identical &chains, and AchR preparations giving two protein peaks with receptor activity and molecular weights consistent with a monomer-dimer relationship can be reduced and only the monomer peak remains. There is at least one disulfide bond which is important for the structure of the ion channel (Karlin, 1973; Chang, 1974; Landau and Ben-Heim, 1974). Landau and Ben-Heim (1 974) investigated frog neuromuscular junction acetylcholine noise in both innervated and denervated frog sartorius muscle. In innervated muscle noise was reduced to 60% of the control value when the preparation had been incubated with 1 mM DTT for 45 minutes, and in denervated muscle, whose AchR differs from that in normal muscle in location, kinetics, and binding properties, the noise was reduced to 38% of the control value. Incubation with 1 mM DTNB for 75 minutes restored the noise nearly to its control value (92% for denervated muscle, no value given in that paper for innervated muscle). A further complication noted in this experiment is that the half-time for channel closing fell to 59% of the control when incubation with DTT proceeded for 35 to 65 minutes, but remained at 93% of the control for shorter incubations. The amplitude of the noise however decreased mostly during the first 30 minutes of incubation. Thus more than one process is involved in the DTT effect, and all these processes can be reversed by DTNB. The stoichiometry of AchR is at present uncertain. Molecular weights from 230,000 to 390,000 have been reported (Hucho and Changeux, 1973; Edelstein et al., 1975; Raftery et al., 1976; Gibson et al., 1976; Reynolds

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and Karlin, 1978; Hucho et ul., 1978), but the most reliable of these are at the lower end of the range, i.e., 250,000 to 270,000. Stoichiometries of 1 : 1 : 1 : 1 (O'Brien, personal communication), 2 : 1 : 1 : 1 (Reynolds and Karlin. 1978), 4 : 2 : 1 : 1 (Raftery et a / . , 1976). 5 : I : 5 : 1: I . 5 (Karlin et a l . , 1976b), and 3.1 : 1.4: 1 : 1.1 (Rubsamen el al., 1976) have been suggested, and Albuquerque e f al. (1974), by labeling mouse motor end plates with '251-labeled BTX, finds about six toxin binding sites per membrane particle or one to two per ion channel. The a-chain contains the cholinergic binding site, but the functions of the other subunits and the locations of any other functional sites (including the ion channel) are not known. Finally, there is higher order structure in the membrane (quinternary structure). In innervated muscle end plate the AchR is located at the upper portion of the junctional folds (Albuquerque et al., 1974; Fertuck and Salpeter, 1974a,b). The density of toxin binding sites is consistent with the hypothesis that the receptor forms a closely packed pseudocrystalline array where it is densest. A similar result was obtained by examining receptor-rich preparations from Eleczrophorus and Torpedo by EM (Cartaud et a / . , 1973; Meunier et a / . , 1974; Changeux et a / . , 1976) and X-ray diffraction (Dupont et a/., 1974). Brisson et a / . (1975) using a spin-labeled Ach analog found the lipid in the neighborhood of the binding site to be completely immobile. Dupont et al. (1974)found a much larger spacing between polar head groups of subsynaptic membrane (6.0 nm) than in the usual lipid bilayer (3.5-4.0 nm). Changeux el a / . (1976) interpret this as distortion of the bilayer by the densely packed receptor. In denervated muscle or muscle tissue culture AchR is present which is not organized into arrays. This extrajunctional receptor differs from junctional receptor in the equilibrium constants for cholinergic agonists and antagonists (Chiu e f a/., 1974; Lapa et a / . , 1974; Brockes and Hall, 1975). The mean time for channel closing is also different for the two kinds of receptor, and this fact has been used to show that both kinds of receptor are present in denervated muscle (Neher and Sackmann, 1976). This indicates that whatever process causes the organization of AchR effects some change in AchR which persists after the organization has been abolished by denervation. The nature of the organization process and of the change induced in AchR are not known. The reactions of junctional and extrajunctional AchR with antiserum from myasthenia gravis patients are different (Almon and Appel, 1975) in that antigenic determinants exist on extrajunctional AchR which are not found on junctional AchR (Weinberg and Hall, 1979). so that at least the part of AchR which is accessible to the extracellular fluid is different for the two kinds of receptor. It is not known whether this is due to a difference in primary structure or of positioning of the AchR in the membrane.

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That quinternary structure affects AchR function is shown by the results with denervated preparations and by observations made when detergent is added as it is in the isolation of AchR. O'Brien and Gibson (1977) examined studies of Ach binding and suggested that differences between isolated AchR and membrane preparations could be explained by changes in the cooperativity of Ach binding rather than changes in the equilibrium binding constant. They also argue that certain studies on purified AchR, including reconstitution, are premature at this point, but this may be too pessimistic. Reconstitution attempts can elucidate those very aspects of AchR structure which have led to failures in past attempts. Karlin et al. (1976a) find that AchR from Torpedo or Electrophorus is immunogenic in rabbit when injected with Freund's complete adjuvant. Furthermore, the rabbit inoculated with AchR develops an autoimmune paralysis and death ensues. However, when the AchR was first incubated in SDS, although the preparation was still immunogenic, no paralysis or death resulted. Antisera made with Torpedo AchR, Torpedo AchR denatured with SDS, and Electrophorus AchR cross-react with each of the three antigens: thus it is a subtle property of the AchR which leads to the paralysis. Changeux et (11. (1976) examined the effects of cholate. I n AchR-enriched membranes local anesthetics or Ca2+increase the apparent affinity for Ach. Exposure to cholate abolishes this increase, and furthermore, lowers the affinity of AchR for agonists while leaving the affinity for antagonists unchanged. Dilution of the cholate-containing suspension restores the Ca2+and local anesthetic effects unless the receptor has been exposed to detergent for a long time. This sometimes bewildering collection of observations on AchR structure indicates that the higher order structure affects the function of the receptor. An explanation of the failure to reconstitute functional AchR whose properties match those of the in vivo system can probably be found in distortions of higher order structure during isolation or reaggregation. Isolation is accomplished for nicotinic AchR only in the presence of detergent. Triton, cholate, and organic solvents have been used to isolate receptor. However, organic solvent extractions were less successful and have not been used often. Following detergent solubilization, detergent exchange on DEAE is sometimes used, followed by density gradient centrifugation and affinity chromatography. These procedures yield high-affinity preparations which resemble desensitized AchR (but see O'Brien and Gibson, 1977). Yields are low (20-50%) raising the possibility that a particular fraction of AchR is being selected by the isolation procedure (Briley and Changeux. 1977).

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Reconstitution has been attempted in monolayers and closed and open bilayers. Wiedmer et al. (1978) incorporated AchR into a monolayer made from human erythrocyte lipids. They injected AchR into the subphase of a Wilhelmy trough and measured the surface pressure with and without a lipid monolayer present. They also measured the incorporation using a radioactively labeled AchR preparation. They found that at most 3.5% of the AchR was incorporated, and that a low pH (pH < pZ) and the presence of 2 mM Ca2+ in the subphase gave better incorporation than pH = 7 and/or absence of Ca2+. A small amount of Triton X-100 was necessary to maintain AchR solubility in the subphase, and they caution that this means that AchR may not be interacting with the monolayer as it does in its native environment. The AchR bound in a manner such that the toxin-binding site was accessible to the subphase. The purpose of the study was to determine optimal conditions for incorporation with the end goal of reconstituting AchR in a monolayer, then forming a bilayer from two monolayers. Partial reconstitution of AchR into vesicles has been reported by several groups (Hazelbauer and Changeux, 1974; Michaelson and Raftery, 1974; Michaelson et al., 1978; Schlieper and DeRobertis, 1977). Hazelbauer and Changeux (1974) used the cholate dialysis procedure (dialyzing under argon to prevent oxidation) to reconstitute Torpedo AchR. This was followed by addition of Torpedo lipids and of Mg2+ and Ca2+ and centrifugation to pellet the reaggregated vesicles. These vesicles can be stimulated by carbamylcholine to release "Na; however, the rate of 22Na release in the absence of agonist is high and chemical excitability is not measurable in all preparations. The maximum excitability shown is less than two. The excitability could be blocked by incubation with Naja nigricolis a-toxin. Michaelson and Raftery (1974) used a quite similar procedure to reconstitute AchR from a different species of Torpedo. The dialysis was performed at room temperature rather than 4°C and butylated hydroxytoluene was used as an antioxidant rather than carrying out the procedure in an argon atmosphere. Once again variability was observed in the chemical excitability of the preparations; the maximum excitability observed was ten. The excitability could be blocked by incubation with BTX. Michaelson et al. (1978) use essentially the same procedure but detergent exchange used by Michaelson and Raftery (1974) was not used. Instead, the AchR-Triton complex was added to Torpedo lipids and dialyzed. Variability in excitability was again observed with the best preparation having an excitability of ten. Michaelson et al. (1978) concluded that reconstitution probably depends upon subtle properties of AchR, and that possibly even in the best preparations only a fraction of the AchR is excitable. They note the similarity of isolated AchR to

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desensitized AchR and suggest that the lower ligand affinity form of AchR may be more suitable for reconstitution. Schlieper and DeRobertis (1977) reaggregate AchR by combining a CHC1,-CH,OH extract of electroplax from Torpedo with egg PC, evaporating the solvent, adding saline solution, and sonicating. The vesicles were added to a BLM bathing solution and they subsequently became integrated into the BLM as shown by conductance measurements in the presence or absence of Ach and d-TC. A cautionary note in the interpretation of such experiments can be found in the report (Parisi et a/., 1975) that in membranes containing 1-10% PS chemical excitability by Ach could be demonstrated in the absence of protein; this excitability could be abolished by d-TC. Reconstitution of AchR into bilayers allows electrical measurements to be made, which is especially useful for a protein whose electrical properties have been so well studied. Reports of partial reconstitution of AchR into open bilayers have been published by Parisi rt al. (1971, 1972), Kemp et al. (1973), Jain (1974), Shamoo and Eldefrawi (1973, Bradley et al. (1976), and Schlieper and DeRobertis (1977). The papers of Parisi et a / . (1971, 1972, 1975) report on the reaggregation of a proteoplipid extract from Efectrophorus into a BLM. The proteolipid was added to the BLM-forming solution prior to formation of the bilayer. The resulting bilayer had cationic selectivity and lower resistance compared to controls and responded to cholinergic agonists with a rapid and transient increase in conductance which could be blocked by d-TC. Kemp er al. (1973) added Triton-solubilized AchR from rat diaphragm to one side of a BLM. Addition of 50 p M Ach to the opposite side caused a conductance increase which could be prevented by d-TC or BTX. No effect was observed if Ach was added to the same side of the BLM as the AchR. Jain ( 1974) used an impure eel electroplax acetylcholinesterase preparation as the starting material in his reconstitution attempt. He found concentration-dependent increases in conductance with several agonists with halfmaximal exciting concentrations in reasonable agreement with those for isolated electroplax (except for the bifunctional agonist decamethonium). He notes that it is an impurity in the esterase which is responsible for these results rather than the esterase itself. Shamoo and Eldefrawi (1975) incubated purified AchR from Torpedo with trypsin and incorporated undigested or digested AchR into bilayers. With untreated AchR conductance increases were found with Ca2+or Na+ in the bathing solution, but no sensitivity to agonist was found. Digested AchR in the bilayer and Na+ in the bathing solution give marked conductance increases upon application of carbamylcholine in every preparation tested. These increases were absent when curare was included in the bathing solution. Bradley et a f . (1976) partially reconstituted rabbit skeletal muscle AchR

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in open bilayers formed from soybean phospholipid. After the bilayer had been monitored for at least 60 minutes AchR in 0.1% Triton was added to one side of the bilayer. Carbamylcholine added to both sides caused marked conductance increases when small amounts of AchR were used. They report that their reconstitution attempt was about as successful as that of Michaelson and Raftery , although direct comparisons are difficult due to the different goemetry and measurements. Schlieper and DeRobertis (1977), in addition to the aggregation of Torpedo AchR in vesicles (see above), reaggregated AchR into open bilayers by mixing the CHC1,-CH,OH extract with the bilayer-forming solution. Addition of Ach greatly increased membrane noise and lowered membrane resistance. The effects were reversed when d-TC was added-the noise and resistance returned to their original values. The most likely reason for the lack of success of various reconstitution attempts is that the structure of AchR is not preserved during isolation. In contrast to the case of Ca2+ + Mg2+-ATPase which is isolatable in active form as a monomer (Dean and Tanford, 1978) and which can proceed through a reversible series of delipidation and relipidation steps (Warren et o/., 1974a,b,c,d, 1973, there is evidence that when AchR is isolated its agonist-binding properties change (see, e.g., Changeux et it/., 1976). Electron micrographs of reaggregated AchR (Michaelson et ( I / . , 1978) fail to show the array structure seen in micrographs of subsynaptic membrane fragments (Briley and Changeux, 1977). 4. OTHERSYSTEMS

This section deals with various reconstitution and partial reconstitution experiments. Since the purpose is to evaluate methods of reconstitution, no attempt at completeness is made, rather those publications which provide novel methods, more or less thorough comparisons between native and reconstituted systems, or interesting and fruitful measurements displaying the range of properties which can be measured in reconstitution attempts have been selected. Reconstitutions of TFo.F,,the protein which can either synthesize ATP using an H+-gradient as an energy source or create a proton gradient by ATP hydrolysis, have been used to investigate many properties of the TF,.FI and to give evidence regarding the chemiosmotic theory (Mitchell, 1976). Kagawa (1972) gives a thorough review of isolation of components of the oxidative phosphorylation system and their reconstitution into vesicles capable of energy transduction. Many of the aspects covered have general applicability, e.g., a detailed discussion of detergents and their properties, properties of phospholipids, and reversibility of changes

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in protein properties found during isolation steps (see also Kagawa, 1978). Drachev et a / . (1976a,b,c; Barsky et a/., 1976) reconstituted a number of ion transport systems in vesicles by cholate dialysis and then fused the vesicles to one side of a thick lipid membrane in order to make ) electrical measurements. In the case of Fo.F,(Drachev et al., 1 9 7 6 ~they isolated oligomycin-sensitive ATPase and coupling factors from beef heart mitochondria, then reconstituted using mitochondrial phospholipids, PC, PE, or PS. They measured transmembrane voltage generated when ATP is added to the same side of the lipid membrane as the vesicles either by direct electrode measurements o r by incorporation of a permeant anion, phenyldicarbaundecaborane, in the vesicles. The data obtained by the two methods are consistent and show electric generation upon ATP hydrolysis when PE or mitochondria1 phospholipid was used in the reconstitution, but not when PS or PC was used. EM studies revealed closed proteoliposomes in the reconstituted preparations except when PC was used in which case only small amounts of vesicles were found. Other lipid composition effects are attributed to requirements of the proteins for a specific lipid environment. Sone et al. (1977) reconstituted TF,.F, from a thermophilic, aerobic bacterium, PS3, in lipid derived from the bacterium by cholate dialysis. These vesicles generate a pH and voltage gradient across the membrane upon addition of ATP and synthesize ATP if an electrochemical potential gradient is imposed across the vesicles. This shows that TFo.F, is a reversible energy transducing system which can interconvert scalar energy stored as ATP and vector energy stored as transmembrane differences in H+ chemical potential. The voltage gradient across the vesicles was provided by incorporating valinomycin in the vesicles and adding 0.2 M K+ outside. Inclusion of bacteriorhodopsin and TFo.F, in the same vesicle leads to the generation of ATP when the vesicles are illuminated. The optimal conditions for ATP synthesis were found to be an inside pH of 5.5 (obtained by incubation in malonate buffer), an outside pH of 8.33, an outside [K+] of 0.2 M , and [K+] inside of zero. Under these conditions, the reaction is complete in about 40 seconds and half complete in less than 5 seconds. Typically, 100 nmole ATP was synthesized per mg of ATPase with values ranging between 40 and 150 nmole/mg. The quality of the lipid was found to be important and vesicles reconstituted from soybean phospholipid did not generate ATP. This difference between the work of Sone er al. (1977) and Drachev et al. (1976~)is probably due to the different requirements of the proteins from the two sources. Sone et al. (1977) investigated the contributions of various parameters

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to ATP generation by leaving out low pH incubation, K+, and ADP and by adding inhibitors (DCCD or tri-n-butyltin) or uncouplers (FCCP, nigericin, or Tris, which collapse the pH gradient). In the absence of a pH gradient synthesis was reduced to 10% or less, while in the absence of a voltage gradient ATP synthesis was reduced to 25% of the control. Internal concentrations of potassium of 1 , 10, and 40 mM reduced ATP synthesis to 85%, 60%, and ~ W O respectively. , The contributions of the pH and voltage gradients to the transmembrane difference in H + chemical potential were evaluated using 9-aminoacridine or ANS, respectively, to monitor these gradients, and the rate of ATP synthesis was determined as a function of chemical potential. They found that the rate was zero for ApH+ < 180 mV and rose sharply for 220 mV < ApH+ < 260 mV. The rate was half maximal for V = 250 mV and appeared to level off for V 2 275 mV. The rate appeared to approach the rate of phosphate-ATP exchange as a limit indicating that enzyme turnover is rate limiting in the presence of a very large chemical potential difference (i.e., the process not requiring energy represents the limit of the energy-requiring process when excess energy is avialable). Thermodynamic calculations give a threshold of 204 mV for ATP synthesis which agrees with the experimental measurements and which shows the TF,.F, reconstituted system to be very efficient. Drachev et al. (1976b) reconstituted beef heart cytochrome oxidase by mixing I5 mg with 100 mg soybean phospholipid in buffered sucrose then adding an additional 100 mg soybean phospholipid in decane. A planar membrane was formed by blowing a bubble into an aperture in a Teflon partition. Ascorbate was included in the bathing solution on both sides of the BLM, and when cytochrome c was added to one side a voltage was generated which was positive on the side to which the cytochrome c had been added (cis side). CN- added cis immediately abolished the voltage difference and CN- added trans lowered the voltage more slowly. Drachev ef al. (1976b) reconstituted cytochrome oxidase in vesicles by cholate dialysis and then added the vesicles to a thick lipid membrane (see above). Cytochrome c was added to the outside of the vesicles by adding it to the TLM bathing solution on the same side as the vesicles (cis) and to the inside of the vesicles by inclusion of cytochrome c in the cholate dialysate and washing the vesicles in buffered sucrose to remove externally bound cytochrome c. When ascorbate was added this system generated voltages of up to 100 mV positive on the side cis to cytochrome c when cytochrome c was outside the vesicles and with the reverse sign when cytochrome c was inside the vesicles. When a membrane was interposed between the ascorbate and the cytochrome, voltage was generated only when a penetrating hydrogen carrier such as phenazine meth-

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osulfate was added. Experiments using rabbit antiserum to cytochrome oxidase show that half the cytochrome oxidase is activated by cytochrome c within the vesicles and that the incorporation of cytochrome oxidase is essentially symmetric with respect to orientation in the membrane. Measurements made in the presence of an external voltage reveal that the transmembrane voltage generated by cytochrome oxidase is a linear function of external voltage and that the emf of cytochrome oxidase (that externally applied voltage which abolishes the electrical response) is 210 mV. Vesicles reconstituted with PC were inactive whereas those made with PE, PS, or mitochondria1 phospholipids were active (Drachev et al., 1976b). Gutweniger et al. (1977) used spectral changes in the dye safranine to measure the activity of cytochrome oxidase vesicles. In the presence of cytochrome c and ascorbate the dye was taken up by the vesicles where it aggregated. The absorbance changes found were proportional to the amount of dye taken up, and a stoichiometry of 1 dye molecule per 6-7 H+ extruded was found. Archakov et al. (1974) compared three methods of reconstitution of rat liver microsomal redox chains. The methods are referred to as self-assembly, nonspecific template, and specific template procedures. All three procedures involve cholate dialysis; the first is dialysis of microsomes completely solubilized in 4% cholate then dialyzed against 0.1% cholate (necessary to keep the components in solution), the second is dialysis of a mixture of solubilized microsomes (in 0.1% cholate) and PC liposomes, and the third is dialysis of a mixture of solubilized microsomes and “ghosts” prepared by treating microsomes with 0.15% cholate and collecting the pellet after centrifugation. E M examination showed that “ghosts” are vesicular but poorly staining and depleted of ribosomes and some protein (60% of the microsomal protein is retained in the “ghost” fraction), the liposomes were often multilayered; however, treatment with 0.1% cholate resulted in the formation of smaller, single-walled vesicles. The reconstituted systems consist of a network of fused vesicles in the case of self-assembly, and of small, single-walled vesicles resembling the original microsomes in the cases of specific or nonspecific template assembly. The protein content and various enzymatic activities were compared for the three procedures. The specific template procedure resulted in somewhat higher protein incorporation than the other two procedures. Reactivation of cytochrome P-420 to P-450 did not occur in the specific template procedure and therefore NADPH-dependent hydroxylation was not reconstituted in that procedure. However, self-assembly and nonspecific template procedures reconstituted 60 and 30% of this activity, respectively. Ascorbate dependent lipid peroxidation activity was reconstituted by all three methods to levels higher than in the

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original microsomes. Either template procedure gave higher activity than self-assembly. This is thought to result from the greater lipid/protein ratio in the reconstituted system (the excess lipid is a source of substrate). NADPH-dependent lipid peroxidation is reconstituted effectively by all three procedures. NADPH oxidase activity is highest in the “ghost” fraction and is also reconstituted by all three procedures. The claim that NADPH oxidase activity is a sensitive test of reconstitution does not seem to be borne out by the data presented: approximately the same activity is present in all three reconstituted systems. In short, all three procedures should prove useful for reconstitution, and comparisons among the procedures and with native or modified vesicles can give information about structural requirements for reconstitution. The well-known “purple membrane” of certain halophilic bacteria consists of a single protein, bacteriorhodopsin (BR) which forms a twodimensional hexagonal lattice. The membrane is isolated in the form of rigid sheets roughly 0.5 pm in diameter, each containing about lo5 oriented protein molecules, as well as phospholipids. The size of these sheets, their rigidity, and their extent of orientation make BR unusual from the reconstitution standpoint. This light-driven proton pumping protein is extremely stable, remaining functional over large pH and temperature ranges. BR has been reconstituted into phospholipid vesicles by both the sonication and the solubilization/dialysis techniques described earlier. These preparations usually exhibit uptake of protons into the vesicles. However, Happe et al. (1977) have shown that by using acidic phospholipids and carefully controlling pH, BR can be reconstituted in the opposite orientation, pumping protons out of the vesicles. The extent of orientation in vesicle preparations is not perfect. EM reveals that a significant fraction of BR in vesicle preparations is misoriented (Lozier et al., 1976: Hwang and Stoeckenins, 1977). Reconstitution in planar lipid membranes has been done by three methods. One of these is to mix a wet pellet of purple membrane with the membrane-forming phospholipid solution, and form a planar membrane from this (Drachev et al., 1975, 1976a; Herrmann and Rayfield, 1976). The resulting membrane exhibits a light-induced short-circuit current and open-circuit potential. The direction of the net charge transfer varies from membrane to membrane, apparently due to random orientation of purple membrane sheets. A second method of incorporation is first to incorporate BR into vesicles and add these to the solution bathing the planar membrane (Drachev et al., 1974, 1976a). Vesicle interaction with the planar membrane does not occur without particular cations: usually CaZ+is used, although Drachev et al. (1976a) report that others are

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effective in the order La3+ > Ca2+ > Mg2+ > NHZ. Herrmann and Rayfield (1978) used 5 mM CaCl, to cause vesicle interaction with planar lipid membranes. They concluded that vesicles adsorbed onto the membrane and remained closed. The direction of the light-induced shortcircuit current was consistent with inward proton pumping in the vesicles. A third method for incorporating BR is to add purple membrane sheets to the solution bathing the planar membrane. Shieh and Packer (1976) obtained photopotentials in this manner with an oxidized cholesterol membrane stabilized with polystyrene. Dancshazy and Karvaly ( 1976) used this method with a planar membrane given a positive surface charge by octadecylamine. They report no incorporation of BR if octadecylamine is not present in the membrane-forming solution. Herrmann (1978) reports using this method with a planar membrane containing PS as well as egg lecithin. The presence of Ca2+(5 mM) was necessary for incorporation. He concludes that the sheets adsorb onto the surface of the planar membrane in an oriented fashion, but do not become coplanar with the lipid bilayer. Bamberg et al. (1979) have suggested the same structure. In none of the above methods is BR incorporated in a planar membrane so that it is coplanar with the membrane (Herrmann, 1978). In each case addition of a protonophore such as CCCP dramatically increases the short-circuit current. This would not occur if BR were coplanar with the supporting planar membrane. BR has been studied in systems other than vesicles and planar lipid films. Boguslavsky et al. (1975) and Hwang et al. (1977) have observed photopotentials from BR incorporated at an oil-water interface. Other supporting structures for vesicles and BR fragments have been used as well, such as Millipore filters impregnated with lipids (Blok et al., 1977), collodion films (Drachev et al., 1978), and Teflon membranes (Trissl and Montal, 1977). The mechanical stability and large surface area of these systems provides advantages over planar lipid films. The collodion and Teflon structures, in particular, have proved useful in measuring fast, transient photovoltages. Similar results were found with bacteriochlorophyll (Bch) and photosystem I (Barsky et al., 1976). The action spectrum for the photoresponse of Bch corresponds well with the absorption spectrum; the emf of Bch was found to be 180 mV, and 0-phenanthroline and oxidation by Fe (CN)g- inhibited the photoresponse. An amusing effect was observed in vesicles containing both BR and Bch. When illuminated with red light a voltage was produced negative to the side where the vesicles were added, and when illuminated with green light a voltage of the opposite sign was generated. Goodall and Sachs (1977) used a similar procedure to incorporate a

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H+-K+ pump in a planar lipid membrane; however, in this case a thin membrane was used rather than a thick one, and no determination of the structure of the membrane system was reported. The membrane was monitored visually until it was completely black (1-3 minutes) and electrically by plotting the I-V curve on an X-Y plotter (see Sections II,B,l,a and II,B,2,b). The capacitance was found to increase approximately linearly for 20 minutes. Conductance increases were observed when vesicles enriched in the H+-K+ pump were added to one side of the membrane. The time course of the conductance increase depended on the state of the membrane and not on the time when the vesicles were added. The greatest activity was observed with PS bilayers. The conductance increased exponentially with time; the final conductance was a function of the concentration of protein added, but the rate of increase was not. Thus it was concluded that the rate of incorporation was not limiting. The selectivity sequence of the conductance was determined and was the same as that found for H+ uptake and K+-ATPase activity except that T1+ is the most active ion for the latter process. ATP added cis resulted in a transmembrane voltage. When the bathing solution contained Na+ the voltage persisted, but when K + was added the voltage declined sharply after 15 min. The sign of the voltage was consistent with a pump which transported protons from the cis to the trans side of the bilayer. Addition of pCMB, an inhibitor of the ATPase, reduced the transmembrane voltage but had little effect on the conductance (Goodall and Sachs, 1977). Asymmetry of incorporation of the pump was shown by the addition of ATP trans to the vesicles. A potential was generated of opposite sign to that found with ATP added cis but of smaller magnitude. The short circuit current obtained was calculated to be about 1% of that in native vesicles which indicates that relatively few of the added vesicles fuse in such a way that they incorporate into the BLM. The experimental data can be fit using a model in which there is obligatory H+-K+ exchange. This model proposes that the pump exists as a dimer in the native state in which each monomer is in a different conformation; one conformation transports H + and one transports K+. Evidence for this is a Hill coefficient of about 2 when p-nitophenylphosphate (pNPP) is used as the substrate, the doubling of the effectiveness of ATP to compete with pNPP when native vesicles are ruptured, and the 2-fold faster rates of inactivation of ATPase and H+-transport compared with pNPPase. Evidence for the existence of two different conformations comes from differences in the conductance increases in the presence and absence of ATP. These differences can be explained if there is an activation energy for the K + transport site to become available for binding which increases

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from ll4kTd to ll2kTd when ATP is present and d is the membrane thickness in A. The calcium pump from human erythrocyte membrane has been investigated by Wolf et al. (1976). They solubilized erythrocyte membranes in Triton X-100 then transferred the protein to mixed micelles of PC and Triton. The Michaelis constant and pH profile for the (Ca2+ + Mg2+)ATPase and the affinity for Ca2+are the same in the reconstituted system as in the native environment. ATPase activity is increased in the presence of monovalent cations (although they are not required for transport) in both the native and reconstituted preparations. Roelofsen and Schatzmann ( 1977) treated erythrocytes and erythrocyte ghosts with phospholipase C and found that although the lipids in the outer leaflet could be degraded with no effect on (Ca2++ Mg2+)-ATPase activity, this activity was lost in direct proportion to the loss of PC, PE, and PS from the inner leaflet. They then added back phospholipids to inactivated ghosts and found that each of the phospholipids could restore (Ca2+ + Mg2+)-ATPaseactivity, and that this reconstituted activity had the same Mg2+requirement and affinity for Ca2+as the original system (Schatzmann and Roelofsen, 1977). The anion transport protein (Band 3) from erythrocyte membrane has been reconstituted by several laboratories. Yu and Branton (1976) dissolved erythrocyte ghosts in 0.75% Triton X-100, reduced the Triton concentrations to the critical micelle concentration with SM-2 beads, then added 2% cholate and egg yolk PC and dialyzed. In other experiments, purified Band 3 was reconstituted with egg yolk PC by cholate dialysis. EM examination of the reconstituted systems by both negative staining and freeze-fracture showed 8.5-nm particles whose density agreed with that calculated assuming each particle contained a dimer of Band 3 . The particle density was the same when Band 3 had been crosslinked with Cu2+-phenanthrolinebefore Triton extraction. When a spectrin-actin extract from erythrocytes was included in a reconstitution, this extract bound to vesicles containing Band 3 and caused aggregation of Band 3 when the vesicles were incubated at pH 5.5. The Band 3 particles were seen to be clustered in freeze-fracture EM. Limited quantities of purified Band 3 precluded experiments involving spectrin-actin association with a purified, reconstituted system, so the possibility that spectrin associates with another protein in the erythrocyte membrane cannot be ruled out. Ross and McConnell (1977) reconstituted Band 3 , in egg PC, erythrocyte lipid, glycophorin, and cholesterol with buffer containing 0.1 M dodecyl trimethyl ammonium bromide. This mixture was incubated on ice under argon for 4-5 hours then dialyzed with N z bubbled through the

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dialysate to prevent lipid oxidation. Attempts at reconstitution using egg PC and Band 3 failed, apparently due to lack of formation of closed vesicles [see the previous discussion of the work of Drachev et al. (1976c)l and inclusion of all three of erythrocyte lipid, glycophorin, and cholesterol was more effective than inclusion of any one or two. Rough calculations show a SO:- flux in the reconstituted system which is four times the PO$- flux in erythrocytes. Wolosin et al. (1977) incubated erythrocytes ghost in 0.045% Triton X100 for 20 minutes then centrifuged to remove sialoglycoproteins. Excess detergent was removed by resuspending the pellet in dilute phosphatebuffered saline and centrifuging, then resuspending again, incubating the suspension with SM-2 beads then removing the beads and centrifuging. The material was frozen in liquid nitrogen then stored at -20°C. Prior to flux measurements, the sample was thawed and sonicated under N, for 10 minutes, in a bath sonicator. The activation energy for transport was found to be 26 kcalhole in good agreement with the value of 28-33 kcal/ mole found in the intact cells (Schwoch et al., 1974). The difference in activation energy was interpreted as arising from the loss of carbohydrate from the membrane in the reconstituted system (the loss of negative charge would lower the activation energy). The pH profile for transport was similar to that in intact cells, having the same pH optimum, but the variation with pH was not as pronounced. It is not known if the difference is due to differences in a leakage pathway or net SO:- transport. A more recent report from the same group (Cabantchik et al., 1977) points out that the pH profile for net flux differs from that for exchange flux and resembles that for flux in vesicles. Possibly a different ratio of contributions of net and exchange fluxes makes up the total flux in vesicles than in intact cells. An additional possibility mentioned is that subtle alterations in structure during reconstitution lead to this difference in pH profile. Selectivity between SO:- and PO:- is similar in reconstituted and native systems; phosphate is transported 3.5 times faster in intact cells and 4.7 times in vesicles. DIDS and persantine inhibit the reconstituted system to the same extent as the native. They determined the rates of anion exchange in reconstituted vesicles and erythrocyte ghosts and compared this with the surface-to-volume ratios. They found a 30% difference, but their assumptions include equal protein density in vesicles and ghosts and uniformity of size of vesicles which are admittedly not valid. The agreement is better assuming some loss of protein and either better or worse depending on the distribution of vesicle sizes, and in any event is quite good. Rothstein et al. (1975) and Grinstein et al. (1978) studied the effects of protease digestion on anion transport and inhibition by DIDS of Band 3 .

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Rothstein et ul. (1975) dissolved normal, Pronase-treated, and DIDStreated ghosts in 0.75% Triton X-100. They added an equal volume of egg PC in toluene, removed the aqueous phase, and bubbled it with N, to remove the remaining traces of toluene and concentrated 3- to 5-fold. The protein was mixed with egg or bovine brain PC [Rothstein et al. (1975) report I ml mixed with 200 gm lipid, but this seems impossible], shaken vigorously under Nz,and sonicated for 20 to 30 minutes under Nz in a 100 W sonicator. They found that the reconstituted preparation transported SO:- in the cases of both normal and Pronase-treated ghosts, but that DIDS-treated ghosts displayed no ability to transport SO:-. Cells labeled with [3H]DIDS where found to have the label in the 95,000-dalton region in normal cells and the 65,000-dalton region in Pronase-treated cells, corresponding to Band 3 and the Pronase-resistant fragment of Band 3 , respectively. Grinstein ef ul. (1978) prepared inside-out vesicles from normal and chymotrypsin-treated erythrocyte ghosts and subjected the vesicles to trypsin treatment. Anion transport was unaffected in trypsinized vesicles from normal ghosts and was blocked by DIDS which was found to be associated with a 55,000-dalton fragment. Anion transport was little reduced in trypsinized or untreated vesicles from chymotrypsin-treated ghosts, and was also blocked by DIDS which was found to be associated with a 17,000-dalton fragment. N o reports of reconstitution of these fragments or their selectivity and transport properties have come out, but experiments such as those performed by Shamoo et rrl. (1976, 1977, 1978) on (Ca2+ + Mg2+)-ATPase(see Section II,C,l) could elucidate the properties of the anion binding site. Schein et al. (1976) reported the reconstitution of another anion transport protein, a voltage-dependent, anion-selective channel (VDAC) from paramecium mitochondria. A similar protein was found in small amounts in rat liver mitochondria as well (see also Lynch and Colombini, 1979). Paramecium membranes were prepared and fractionated on a sucrose density gradient. However, this protein is at a primitive stage of purification and thus no conclusion can be drawn regarding what protein is responsible for the ionophoric activity.

111.

CONCLUSIONS AND THE FUTURE OF RECONSTITUTION

The reconstitution of ion transport systems allows detailed investigation of their properties (Kornbrot, 1977; Racker, 1977), but in order for these investigations to be meaningful it must be shown how the system in question is related to the native membrane. Since the original membrane may contain components which interact with the system of interest

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and modify its properties, measurements of membrane function are not sufficient and methods of measurement which yield other information about the structure and function of both the native and reconstituted systems must be used to compare the systems. Although native and reconstituted systems may appear identical by certain criteria they may well differ in detail (see the discussion at the end of Section II,B,5, for example), so as many independent measurements as practical should be performed. The range of properties measurable in reconstituted systems is extremely broad (see Section II,C, 1) encompassing specific lipid requirements, kinetics, stoichiometry, and variations in properties as functions of pH, transmembrane voltage, temperature, o r composition of aqueous phase on each side of the membrane. Reassembly of fragments of an ion transport system can give information regarding the localization of functional sites within the system. In this case reassembly will not yield a system identical to the native system and measurements relating the partially reconstituted system to the native system are particularly important to assure that any conclusions derived are not based on artifacts. Another technique involves direct injection of lipid solution into an aqueous phase. Rapid injection of a 20-40, mM ethanol solution of phospholipid gives single walled vesicles of about 25 nm diameter (Batzri and Korn, 1973). Larger vesicles are formed by injecting a 2 mM solution of lipid in diethyl ether at a rate of 0.25 ml/minute into 4 ml of aqueous phase. The aqueous phase is maintained at 55-65°C so that the ether vaporizes immediately upon injection. The aqueous phase can also be maintained at 30°C under vacuum to protect thermolabile proteins. The vesicles obtained are about 130 nm in diameter (Deamer and Bangham, 1976). The same technique was used by Schieren et al. (1978) with petroleum ether rather than diethyl ether as the lipid solvent. They obtained vesicles about 210 nm in diameter. Schieren et al. (1978) also have a good diagram and description of their apparatus, which will be helpful for those wishing to use this technique. A related technique was described by Szoka and Papahadjopoulos (1978). Three milliliters of diethyl ether was added to phospholipid to make a solution of about 30 mM CHC I or MeOH was added if necessary to solubilize the lipid and 1 ml of aqueous phase was added. The mixture was sonicated in a bath sonicator for 2 to 5 minutes, and the organic solvent was removed in a rotary evaporator at room temperature. This technique gives vesicles whose diameters are 200 to 500 nm and is particularly useful for encapsulating solutes within the liposomes. The criteria for determining when a reconstituted system gives infor-

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mation relevant to the native membrane can be somewhat vague. In many circumstances, the biological activities must be the same in the reconstituted system as in the native system; included in this criterion is the requirement that kinetic parameters be equal in the two systems. However, this is not a reasonable criterion for the partial reconstitution of fragments of ion transport proteins or for investigations of lipid specificities, since it is just those differences between native and reconstituted systems which yield the information sought. Likewise, structural criteria, while appropriate in many cases, are unsuitable when structural changes (e.g., upon lipid substitution) are the sources of the changes in biological activities. In addition, there is no reason why a particular fragment must have the same structure in the partially reconstituted system as in the native, since protein-protein interactions may affect the structure. “Reasonable” agreement between the structures and activities of the native and reconstituted systems can be used as a criterion, however, as long as “reasonable” is carefully and explicitly defined. That is, arguments should be presented to explain the origin of any differences between native and reconstituted systems. Another approach is to consider the reconstitution process. It is reasonable to believe that a thermodynamically reversible reconstitution procedure will lead to minimal distortions of the system. For example, if (Cazf + Mg2+)-ATPase is reconstituted by lipid exchange, measurements are made, and lipid exchange is again used; recovery of full biological activity after the second lipid exchange implies that any differences found in the first lipid exchange are due to the differences in lipid, not to reconstitution artifacts (see Warren et al., 1975). It is easy to see that the case of (Ca2+ + Mg2+)-ATPase meets both sets of criteria. No difference in structure or activities between native and reconstituted systems has been found and the reconstitution can proceed adiabatically through a series of thermodynamic equilibria. The case of AchR illustrates a failure of reconstitution attempts. Although reconstituted preparations show excitability, this is much less of an effect than seen in vivo and is present only in some of the preparations. Moreover, the high leak rate of Na+ in the absence of cholinergic agonists indicates a structural difference between the reconstituted and native membranes. The mechanism which produces these differences is not known and thermodynamic analysis of the reconstitution process has not been reported. The future of reconstitution looks particularly bright. As information comes to light about the processes involved in the isolation and reassembly of membrane components, better procedures introducing fewer distortions are being used to reconstitute (as one example, see Warren et

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al., 1974a,b,c,d, 1975). The reconstitution of a system involving one protein has been successful (see Section ll,C,l) and that of a system involving two proteins is not far behind (see Section ll,C,2). The major improvements in the next decade will probably involve the reconstitution of more complex systems (some impressive work has already been done-see Section II,C,4) and especially systems in which higher order structure of the membrane is essential to function (see Section ll,C,3). REFERENCES Abramson, J. J., and Shamoo, A. E. (1978). Purification and characterization of the 45,000 dalton fragment from tryptic digestion of CaZ++ Mgz+-adenosine triphosphatase of sacroplasmic reticulum. J . Mernhr. B i d . 44, 233-257. Akera, T. (1977). Membrane adenosine triphosphatase: a digitalis receptor? Science 198, 569- 574. Albuquerque, E. X . , Barnard, E. A., Porter, C. W., and Warwick, J. E. (1974). The density of acetylcholine receptors and their senstivity in the postsynaptic membrane of muscle end plates. Proc. N o t / . Acud. Sci. U.S.A. 71, 2818-2822. Allen, G., and Green, N. M. (1976). A 31-residue tryptic peptide from the active site of the Caz+-transporting adenosine triphosphatase of rabbit sarcoplasmic reticulum. FEBS Lett. 63, 188-192. Allen, G., and Green, N. M. (1978). Primary structures of cysteine-containing peptides from the calcium ion-transporting adenosine triphosphatase of rabbit sarcoplasmic reticulum. Biochern. J . 173, 393-402. Almon, R. R., and Appel, S. H. (1975). Interaction of myasthenic serum globulin with the acetylcholine receptor. Biochirn. Biophys. Actu 393, 66-77. Antanavage, J., Chien, T. F., Ching, Y . C., Dunlap, L., Mueller, P . , and Rudy, B. (1978). Formation and properties of cell-size single bilayer vesicles. Biophys. J . 21, 122a. Archakov, A. I., Bachmanova, G. I., Devichensky, V . M., Karuzina, L. I., Zherebkova, N. S., Alimov, G. A., Kuznetsova, G. P., and Karyakin, A. V . (1974). The reconstitution of rnicrosornal redox chains: a comparative analysis of the effectiveness of membrane self-assembly and template binding of electron carriers. Biochern. J . 144, 1-9. Axelrod, D., Koppel, D. E., Schlessinger, J., Elson, E., and Webb, W. W. (1976a). Mobility measurement by analysis of fluorescence photo bleaching recovery kinetics. Biophys. 1. 16, 1055-1069. Axelrod, D., Ravdin, P., Koppel, D. E., Schlessinger, J., Webb, W. W., Elson, E . L., and Podleski, T. R. (197613). Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers. Proc. Nutl. Acud. Sci. U . S . A . 73, 45944598. Bahr, G. F. (1954). Osmium tetroxide and ruthenium tetroxide and their reactions with biologically important substances. Exp. Cell R e s . 7, 457-479. Bahr, G. F. (1955). Continued studies about the fixation with osmium tetroxide. Electron stains 1V. Exp. Cell Rev. 9, 277-285. Barnberg, E., Apell, H.-J., Dencher, N., Sperling, W., Stieve, H., and Lauger, P. (1979). Photocurrents generated by bacteriohodopsin in planar bilayer membranes. J . Mernb.

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Sobel, A., and Changeux, J.-P. (1977). Purification and characterization of the cholinergic receptor protein in its membrane-bound and detergent-soluble forms from the electric organ of Torpedo mnrmorutu. Biochem. Soc. 7run.s. 5, 51 1-514. Sone, N., Yoshida, M., Hirata, H., and Kagawa, Y. (1977). Adenosine triphosphate synthesis by electrochemical proton gradient in vesicles reconstituted from purified adenosine triphosphatase and phospholipids of thermophilic bacterium. J . B i d . Chem. 252, 2956-2960. Sorenson, M. M., and DeMeis, L. (1977). Effects of anions, pH and magnesium on calcium accumulation and release by sarcoplasmic reticulum vesicles. Biochim. Biophys. Actu 465, 220-223. Stewart, P. S., and MacLennan, D. H. (1974). Surface particles of sarcoplasmic reticulum membrane. Structural features of the adenosine triphosphatase. J . B i d . Chem. 249, 985-993. Stewart, P. S., MacLennan, D. H., and Shamoo, A. E. (1976). Isolation and characterization of the tryptic fragments of the adenosine triphosphatase of sarcoplasmic reticulum. J . B i d . Chem. 251, 712-719. Stoeckenius W. (1960). Osmium tetroxide fixation of lipids. Proc. Eio.. Reg. Conj: Electron Microsc., Devt 2, 716-720. Stoeckenius, W. (1962). Some electron microscopical observations on liquid-crystalline phases in lipid-water systems. J . Cell B i d . 12, 221-229. Sumida, M., and Tonomura, Y. (1974). Reaction mechanism of the Ca2+-dependentATPase of sarcoplasmic reticulum from skeletal muscle. J . Biochem. (Tokyo) 75, 283-297. Swann, A. C., Daniel, A,, Albers, R . W., and Koval, G. J . (1975). Interactions of lectins with (Na+ + K+)-ATPase of eel electric organ. Biochim. Biophys. Actu 401, 299-306. Sweadner, K. J., and Goldin, S. M. (1975). Reconstitution of active ion transport by the sodium and potassium ion-stimulated adenosine triphosphatase from canine brain. J . B i d . Chem. 250, 4022-4024. Szoka, F., Jr., and Papahadjopoulos, D. (1978). Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation. Proc. Null. Acad. S C ~U. . S . A . 75, 4194-4198. Tanford, C. (1978). The hydrophobic effect and the organization of living matter. Science 200, 1012-1018. Taniguchi, K., and Post, R. L. (1975). Synthesis of adenosine triphosphate and exchange between inorganic phosphate and adenosine triphosphate in sodium and potassium ion transport adenosine triphosphatase. J . Biol. Chem. 250, 3010-3018. Teissie, J . , Tocanne, J . F., and Baudras, A. (1978). A fluorescence approach of the determination of translational diffusion coefficients in phospholipid monolayer at the air-water interface. Eur. J . Biochem. 83, 77-85. Thorley-Lawson, D. A., and Green, N. M. (1973). Studies on the location and orientation of proteins in the sarcoplasmic reticulum. Eur. J . Biochem. 40, 403-413. Thorley-Lawson, D. A., and Green, N. M. (1975). Separation and characterization of tryptic fragments from the adenosine triphosphatase of sarcoplasmic reticulum. Eiir. J . Biochem. 59, 193-200. Tien, H. T. (1974). “Bilayer Lipid Membranes Theory and Practice.” Dekker, New York. Tien, H. T., and Diana, A. L. (1967a). Some physical properties of bimolecular lipid membranes produced from new lipid solutions. Nature (London) 215, 1199-1200. Tien, H. T., and Diana, A. L. (1967b). Black lipid membranes in aqueous media: the effect of salts on electrical properties. J . Colloid Interfuce Sci. 24, 287-296. Tien, H. T., and Verma, S. P. (1970). Electronic processes in bilayer lipid membranes. Nuture (London) 227, 1232- 1234.

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Yu, J., and Branton, D. (1976). Reconstitution of intramembrane particles in recombinants of erythrocyte protein Band 3 and lipid: effects of spectrin-actin association. Proc. Nail. Acad. Sci. U . S . A . 13, 3891-3895. Zingsheim, H . P., and Plattner, H. (1976). Electron microscopic methods in membrane biology. Methods Membr. B i d . 7, 1-146.

CURRENT TOPICS I N MEMBRANES A N D TRANSPORT, VOLUME

14

Th e Role of Lipids in the Functioning of a Membrane Protein: The Sarcoplasmic Reticulum Calcium Pump J . P . BENNETT Department of Experiinental Pathology University College Hospital Medical School London, England

K . A . McGILL Department of Biochemistry University of Leeds Leeds, England AND

G. B . WARREN European Moleculur Biology Laborutory Heidelberg, Federal Republic of Germany

I. Introduction . . . . . . . . . . . . . . . 11. Sarcoplasmic Reticulum . . . . . . . . . . 111. Purification of Ca-ATPase . . . . . . . . . IV. Equilibration of Lipid Pools . . . . . . . . . V. Which Lipids Support ATPase Activity? . . . . . VI. Reconstitution of Ca-ATPase into Sealed Vesicles . . VII . Only 30 Lipid Molecules Modulate Ca-ATPase Function VIII. The Composition of the Lipid Annulus Is Not the Same as the Whole Bilayer . . . . . . . . . . . IX. Lipid Asymmetry . . . . . . . . . . . . X. Distribution of Lipids across the SR Membrane . . . XI. Transbilayer Disposition of the Phospholipid Annulus . XII. Concluding Remarks . . . . . . . . . . . References . . . . . . . . . . . . . . .

Copyright @ 1980 by Academic Press, Inc. All rights of reproduction in any form reserved ISBN 012-153314-X

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I. INTRODUCTION

It is axiomatic nowadays that a biological membrane consists of a bilayer of lipid into which proteins are inserted. The principal function of the lipid bilayer is structural-to separate two aqueous compartments with a nonaqueous barrier. The characteristic proteins in a particular membrane confer the specific biological properties of that membrane. Functions of membrane proteins include the active or passive transport of particular molecules across the membrane, the transfer of hormonal messages from one compartment to another, electrical properties, and energy transduction. A membrane comprising both lipids and protein must involve some sort of specific interaction between each membrane protein and the adjacent lipids, otherwise the protein would disrupt the structural function of the lipid bilayer. The existence of such lipid-protein interactions poses a number of questions. Is there a stoichiometry in the association of lipids with a protein? Is the biological functioning of that protein dependent upon the presence of those lipids, and upon their chemical nature'? Natural membranes contain a range of lipid species, although many lipid species have singly been shown to form a structurally stable bilayer in model systems-does the protein require an interaction with certain lipids and not with others for optimal functioning? To try to answer these questions we need to choose a suitable membrane protein for experimental study and to find a way of manipulating its lipid environment. A number of methods have been developed and used to investigate lipid-protein interactions for the calcium pump of sarcoplasmic reticulum. This article shows how these methods have led to a deeper understanding of the role of lipids in the functioning of this protein, and of the structural organization of the lipids in the sarcoplasmic reticulum bilayer. In studying a single protein it is an assumption that the results obtained with it will be a paradigm of lipid-protein interactions for any membrane protein. While for other proteins the details may differ, we believe that the principles established using the calcium pump will have a general applicability.

I I. SARCOPLASMIC RETlCULUM The sarcoplasmic reticulum (SR)' is a network of closed membraneous tubules which is found in muscle cells between the myofibrils. It serves 1

Abbreviations: SR, sarcoplasmic reticulum; Ca-ATPase, (Ca2+ + Mg2+)-dependent ATP

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to regulate the level of free calcium ions in the cytoplasm, on which the interaction of the contractile proteins depends. When the muscle is stimulated the calcium stored within the SR is released into the cytoplasm, resulting in myofibril contraction. At the end of the stimulus calcium is removed from the cytoplasm and stored within the SR (Inesi, 1972). This depletion of cytoplasmic calcium is carried out by a calcium pump located within the SR membrane. This pump is ATP-, Ca2+-,and Mg2+dependent and was identified as the (Ca2+ + Mg2+)-activated ATPase (EC 3.16.13 ATP phosphohydrolase) by comparison of kinetic properties, activation energies, and the effect of inhibitors (Hasselbach, 1963). The best indication that calcium transport is mediated by the (Ca2++ MgZ+)activated ATPase (Ca-ATPase) comes from reconstitution experiments in which the purified Ca-ATPase was incorporated into lipid vesicles and ATP-dependent accumulation of calcium could be observed (see Section VI). SR is isolated in a highly purified state from skeletal muscle by differential centrifugation (e.g., MacLennan, 1970) or by density gradient or zonal centrifugation (Meissner and Fleischer, 1971; Meissner et al., 1973). The material prepared appears to consist of vesicles in electron micrographs of thin sections or negatively stained preparations (Fig. 1 ) and it is capable of ATP-dependent calcium accumulation. It is a highly specialized membrane, and around 75% of the protein consists of the CaATPase when SR preparations are examined by gel electrophoresis (MacLennan and Holland, 1975). Other protein components include a high-affinity calcium-binding protein (of about 55000 daltons) and calsequestrin (about 44000 daltons) which are both proteins capable of binding calcium ions. Ca-ATPase appears to be asymmetrically disposed in the SR membrane. When SR membranes are split through the center of the lipid bilayer by the freeze-fracture technique and examined in the electron microscope, 90% of the intramembranous particles are found on the outer (concave) half of the bilayer (Packer et al., 1974). Surface particles can be seen on the outer membrane surface by negative staining (Ikemoto et al., 1968), and these can be proteolytically removed with a loss of ATPase activity and a reduction in the molecular weight of the polypeptide asphosphohydrolase (EC 3.16.13); DOPC, dioleoylphosphatidylcholine;DMPC, dimyristoylphosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine: DSPC, distearoylphosphatidylcholine; DEPC; dielaidoylphosphatidylcholine;OPPC, 2-oleoyl-3-palrnitoylphosphatidylcholine; DOPE, dioleoylphosphatidylethanolamine;D O E , dioleoylphosphatidylglycerol; DOPS, dioleoylphosphatidylserine; DOPA, dioleoylphosphatidic acid: OMeDOPA, 0methyl-dioleoylphosphatidicacid: NAcDOPE, N-acetyl-dioleoylphosphatidylethanolamine.

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FIG. 1 . Sarcoplasmic reticulum vesicles under the electron microscope. SR was negatively stained using 3% ammonium molybdate and examined at 20,OOOx magnification. The surface particles, which are believed to contain the active site portion of the Ca-ATPase molecule, are clearly seen. The bar represents 0.1 pm. (From Thorley-Lawson and Green, 1973.)

sociated with the membrane from 115,000 to 55,000 daltons (ThorleyLawson and Green, 1973). The 55,000 molecular weight peptide which is inaccessible to trypsin is assumed to be buried within the membrane and to act as the "core" through which calcium is transported (ThorleyLawson and Green, 1973). There is some evidence that the Ca-ATPase polypeptide spans the membrane (Hidalgo and Ikemoto, 1977), and there is good reason to assume that it should since this has been demonstrated for several other ion-transporting membrane proteins such as the anion transporter ("Band 3") of erythrocytes (Bretscher, 1971) and the sodium pump (Kyte, 1975). SR does not merely interact closely with the membrane bilayer, it is crucially dependent upon phospholipids for enzyme activity. The use of phospholipases to remove phospholipids from SR leads to a loss of ATPase activity (Meissner and Fleischer, 1972); readdition of phospholipids led to a restoration of enzyme activity. The use of detergents to remove lipids also leads to inactivation (Hardwicke and Green, 1974) which must be due in part to protein denaturation since readdition of phospholipids does not restore full activity. Certain other amphipathic molecules will maintain ATPase activity in place of phospholipids, including fatty acids and lyso-lipids (Fiehn and Hasselbach, 1970) and dodecyl octaoxyethylene glycol (C l,E& which is a nonionic detergent (Dean and Tanford, 1977).

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There are several advantages of using Ca-ATPase as a model for membrane protein studies. SR can be obtained from rabbit skeletal muscle in large amounts ( H . 5 gm protein per rabbit) and Ca-ATPase is easily purified from SR (see Section 111). Although pumping of calcium can be measured only when the protein is incorporated into sealed vesicles, Ca2+-dependentATPase activity forms a convenient functional assay that can be used in unsealed membrane preparations.

111.

PURIFICATION OF Ca-ATPase

The initial problem in the purification of any membrane protein is the dispersal of the membrane into its separate protein units. A number of potential means for achieving this exist, including an extremely wide range for detergents, various phospholipases, and certain organic solvents. Although no generalized method of tackling the problem has yet been described, some degree of rationalization can be attempted. It has been suggested that all transmembrane proteins fall into one of two classes, typified by the two major proteins in the red blood cell membrane (Bretscher and Raff, 1975). The glycophorin-like “fibrous” proteins have most of their mass exposed on the noncytoplasmic side of the membrane and have a relatively small amount of protein contained within the lipid bilayer so that these proteins do not appear as intramembranous particles in freeze-fracture electron microscopy. Examples probably include, as well as glycophorin, the cell-surface histocompatibility antigens (Dobberstein et al., 1979), viral spike proteins (Garoff and Simons, 1974), and some membrane-anchored enzymes such as the aminopeptidases (Louvard et al., 1976). The Band 3-like “globular” proteins have very little protein exposed on the noncytoplasmic membrane face (where they are consequently not very chemically reactive or antigenic) but a significant proportion of the mass lies within the bilayer where it exists in a folded, globular conformation. This is the class of proteins that is visualized as intramembraneous particles, and it probably includes all transport proteins, including Ca-ATPase (Deamer and Baskin, 1969). In the case of fibrous membrane proteins, the proteins can often be completely delipidated during purification since the structure of the protein within the membrane is simple and will reform on subsequent incorporation into a lipid bilayer: for example, glycophorin contains a sequence of 22 hydrophobic amino acids which are just sufficient to form an a-helix which spans the bilayer (Furthmayr et al., 1978). Globular proteins on the other hand have quite a complicated structure within the bilayer (the best-known example is bacteriorhodopsin with seven a-he-

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lices; see Henderson and Unwin, 1975). This structure is stabilized by the lipid bilayer and delipidation tends to result in denaturation of this part of the structure with consequent nonspecific association of the hydrophobic regions and the formation of insoluble aggregates. The approach which must be used instead is to find a suitable detergent which will disrupt the lipid bilayer into solubilized lipid-protein-detergent micelles. If the protein is maintained in a lipid environment, the possibility of denaturation is minimized. The way in which different detergents interact with biological membranes has been discussed by Helenius and Simons (1975). The important parameter in micelle formation is the critical micelle concentration of the detergent (under the conditions of the experiment); this is in practical terms the highest concentration of detergent which can exist as the monomeric species in solution. At detergent concentrations higher than the critical micelle concentration, there is an equilibrium between monomers and micellar structures of associated detergent molecules. In the presence of biological membranes the micelles formed will contain both detergent molecules and membrane components. The higher the total detergent concentration is above the critical micelle concentration, the greater is the amount of detergent which will be present in these mixed micelle structures: as a result there will be a greater number of micelles each containing fewer membrane components. For detergent solubilization to be used in membrane protein separation each micelle must contain only a single protein molecule, so that the total detergent concentration must be sufficiently high for the number of micelles to exceed the number of membrane protein molecules. There are two important criteria which a detergent used in biological membrane studies must fulfill. Since the tertiary structure of a protein is maintained in part by hydrophobic interactions it is important that the detergent will not disrupt these interactions and denature the protein at the concentrations where it solubilizes the membrane. This is the case with sodium dodecyl sulfate, for example, which will denature most proteins below its critical micelle concentration and is of limited usefulness in membrane work [although a particular protein may prove detergent-resistant, e.g., sodium dodecyl sulfate is used in the Na+-K+-ATPase purification of Jorgensen (1974)l. The detergents most commonly used in membrane studies-nonionic surfactants such as the Triton series, and the bile salts-denature proteins only at concentrations well in excess of their critical micelle concentrations. The second requirement of a detergent is that it can easily be removed from the protein after purification. This is the case if the critical micelle concentration is high so that the amount of detergent present as free

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monomers is large relative to the amount bound in mixed lipid-proteindetergent micelles. When the free monomer concentration is reduced (for example, by dilution, dialysis, centrifugation, or gel exclusion chromatography) detergent will dissociate from micelles to restore the concentration of the monomeric species until the purified lipid-protein complex is essentially detergent-free. The ease of removal is also enhanced for detergents with a small micellar size. Various detergents have been used to solubilize SR. Triton X-100, a nonionic detergent widely used in membrane studies, has been used by McFarland and Inesi (1970), Ikemoto et al. (1971), and Hasselbach and Migala ( 1972). LeMaire et al. (1976) also used two other nonionic detergents, Tween 80 and dodecyl octaoxyethylene glycol monoether (C,,E,j. Deamer (1973) used lysolecithin, a modified phospholipid which has a sufficiently high critical micelle concentration to have detergent-like properties. However, the most useful detergents have proved to be the bile salts cholate and deoxycholate which readily solubilize the majority of SR protein and can afterward easily be removed (Martonosi, 1968). Purification of the Ca-ATPase from solubilized SR can be achieved by the sort of techniques used for soluble proteins, for example, ammonium acetate precipitation (MacLennan, 1970) and gel exclusion chromatography (Ikemoto et al., 1971). Warren e l al. (1974a) made use of the fact that Ca-ATPase is the only protein in SR which penetrates the bilayer: the other proteins can, for example, be removed by rigorous extraction with EDTA (Duggan and Martonosi, 1970). This means that the Ca-ATPase can be selectively extracted from solubilized SR by sucrose density gradient centrifugation. Potassium cholate (at 100 mgiml) is added to SR (at > 30 mgiml) at a ratio of 0.5 mg cholate per mg SR protein, and the mixture is centrifuged (for 16 hours at 100,000 gav)through a 20-30% ( w h ) detergent-free sucrose gradient. As the Ca-ATPase-lipid-detergent micelles enter the sucrose gradient the detergent diffuses away and the micelles reassociate to become larger and sediment faster. The other proteins do not have this ability to form membrane-like oligomeric complexes and remain at the top of the gradient. The band containing purified Ca-ATPase together with its associated lipid is removed from the bottom of the gradient and dialyzed to remove any remaining cholate. IV.

EQUILIBRATION OF LIPID POOLS

When a membrane is disrupted by detergent the micelles formed are in dynamic equilibrium-in other words there is exchange of components

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between micelles. Metcalfe and co-workers showed that if exogenous lipid is added to solubilized SR and the Ca-ATPase is isolated by sucrose density gradient centrifugation then the lipid associated with the protein is found to have the same composition as the whole lipid pool (Warren et al., 1974a). In the experiment described in Table I, differing amounts of DOPC were added to Ca-ATPase in the presence of cholate. The proportion of DOPC in the isolated lipid-protein complexes was found to be the same as the fraction of lipids in the original incubation mixture that was DOPC. The lipids surrounding each protein in the membrane have become replaced in part by the exogenous lipids. This is a conservative process; at no time is the protein delipidated with a consequent danger of denaturation. The procedure whereby lipid-protein complexes were isolated from excess lipid and detergent in the incubation mixture by sucrose density gradient centrifugation (Warren et al., 1974a,b) was called "lipid substitution" and is shown schematically in Fig. 2. The lipid substitution technique provided a means for changing the lipid environment around the native Ca-ATPase for the lipids of choice. In one step over 90% of the lipid can be replaced by a synthetic lipid (Table I): if the process is repeated >98% of the lipid surrounding the

TABLE I THE EQUILIBRATION OF EXOGENOUS DoPC ENDOGENOUS LIPID POOL^

WITH THE

Percentage of total fatty acid content DOPC-substituted Ca-ATPase Fatty acid

Ca-ATPase

16:O

26 12 14 19

18:O 18: 1

18:2 20:4 22:5

Expected Observed

16

6

I:l

1:1

5:I

20: I

15 14 8 4 7 6 3 2 61 51 85 93 8 II 2 0.5 7 8 3 3 Percentage DOPC substitution SO 53

50 50

83 82

95 92

DOPC was added to Ca-ATPase in the presence of cholate at ratios to the endogenous lipid of 1: I (two experiments), 5: I , and 20: I . The complexes obtained by the lipid substitution technique were analyzed for fatty acid content by gas-liquid chromatography and the fraction of the lipid that was DOPC was calculated. From Warren e/ a / . (1974a).

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8

, I

EOUl L l BRAT I ON OF LIPID POOLS

ATPase Endogenous lipid Synthetic lipid Detergent

DETERGENT-FREE SUCROSE GRADlENl

FIG.2. A schematic illustration of the lipid substitution process. The lipids surrounding the membrane protein equilibrate with the exogenous synthetic lipid in the presence of detergent, and the protein-lipid complexes are then separated from excess lipid and detergent by sucrose density gradient centrifugation (see text).

Ca-ATPase will consist of the exogenous lipid. Using this technique Warren er al. (1974a) showed that a single synthetic lipid (DOPC) is sufficient to support the ATPase activity and calcium accumulating activity of the Ca-ATPase. For the purposes of examining the effect of different lipids on protein function, a much simpler method for removing the detergent from a lipidprotein-detergent complex can be used. If the incubation is diluted so that the total concentration falls below the critical micelle concentration, the detergent will largely dissociate from the complexes. This is the basis The experimenof the “lipid titration” procedure (Warren et al., 1974~). tal protocol is to incubate Ca-ATPase with a large excess of test lipid in the presence of cholate (at a concentration of 5-10 mg/ml). In these experiments protein function (Ca2+-dependent ATP hydrolysis) was measured by a coupled spectrophotometric assay. When an aliquot of the incubation mixture is diluted into the assay cuvette 200-400 times, most (> 96%) of the cholate diffuses away from the micelles. This has the effect of “freezing” the lipid composition of different micelles at the moment of dilution (for at least the duration of the assay). As a result a sequential series of samples removed from an incubation

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mixture and assayed shows the time-course of equilibration of the lipid pools (Fig. 3). The rate of equilibration was found to depend upon the concentration of cholate used (Warren et al., 1974~).If the cholate concentration is 10 mg/ml or greater, equilibration takes place within a few seconds. If the cholate concentration is less than 5 mg/ml equilibration takes hours rather than minutes to occur. The interpretation that the activity changes observed in this sort of experiment are due to a change in the lipids interacting with the protein derives from a comparison of the properties of lipid titration complexes with those of Ca-ATPase associated with exogenous lipid isolated by the lipid substitution procedure (Warren ef al., 1974~). Figure 3 also shows that the cholate-mediated equilibration of lipid pools is a continuous process. After equilibration with the first lipid (DMPC) is complete and the enzyme activity has reached a stable level, the addition of a new exogenous lipid (DOPC) means further equilibration of pools and a resultant further change in enzyme activity. The fact that the enzyme activity can return to its original value makes it unlikely that the original decrease in activity was due to partial denaturation of the enzyme. One would anticipate that any detergent would allow equilibration of lipids between micelles, and could thus be used for lipid titrations. However, the ease of equilibration differs from detergent to detergent, depending upon the critical micelle concentration and the micellar structure. OMPC

I

"r

Incubation time (minutes)

FIG. 3. The time-course of lipid titration. Ca-ATPase was mixed with excess DMPC and cholate, and samples were assayed at various times for ATPase activity at 20°C. DMPC comprised -94% of the total lipid and the cholate concentration was 7.5 mg/ml. After 50 minutes an aliquot of this incubation was added to excess DOPC and cholate; in this second incubation DOPC comprised -90% of total lipid and the cholate concentration was 13.0 mg/ml (J. P. Bennett, unpublished results.)

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Triton X- 100 is reported to facilitate exchange of the phospholipids associated with Ca-ATPase for lysolecithin (Peterson et al., 1978), although it is far less adept than cholate in the exchange of diacylphosphatidylcholines (G. B. Warren, unpublished results). Octyl glucoside, a nonionic detergent with a particularly high critical micelle concentration (Helenius et al., 1979), will readily exchange phospholipids (J. P. Bennett and G. B. Warren, unpublished results). A procedure that has been validated for one phospholipid cannot automatically be used for other lipids. The lipid titration method using cholate was shown to be valid for phosphatidylcholines by comparison with lipid substitution complexes (Warren et al., 1974~);for other phospholipids Bennett et al. (1978a) developed a protocol to validate the lipid titration results which did not require large quantities of lipid (see Section V). V. WHICH LIPIDS SUPPORT ATPase ACTIVITY?

In the case of the phosphatidylcholines it was demonstrable that cholate causes complete equilibration of lipid pools, by using the lipid substitution technique (Warren et al., 1974~).This means that we can begin to answer one of the questions originally asked: can lipids modulate the enzymatic activity of the proteins? One of the best known physical properties of pure phospholipids is the phase transition temperature. Above the transition temperature the fatty acid chains flex with respect to the lipid headgroup (Levine et al., 1972); below the transition their motional freedom is restricted. The temperature of this phase transition is principally dependent on the structure of the fatty acid chains, although it is also influenced by the phospholipid headgroup. Table I1 shows how the phase transition affects the enzyme activity of the Ca-ATPase. The lipid titration method was used to measure the ATPase activities (at 37°C) supported by a number of synthetic phosphatidylcholines. The activities fall into two groups. The lipids which contained unsaturated fatty acid chains, and which show a lipid phase transition well below 37"C, have a high activity, while the lipids with only saturated fatty acid chains and relatively high transition temperatures have lower activities. The distinction between the two classes is also clear when the way in which enzyme activity varies with assay temperature is examined (Fig. 4). The DOPC-ATPase lipid titration complex is most like the native CaATPase in its behavior. The unsaturated phospholipids, such as DMPC, have a much greater variation in activity with temperature, corresponding to a higher energy of activation for the ATPase reaction.

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TABLE I1 ENZYMEACTIVITYOF Ca-ATPase I N LIPIDTITRATION COMPLEXES WITH DIFFERENT LCHOLINES” PHOSPHATIDY Transition temperature of pure lipid Lipid Dimyristoylphosphatidylcholine (DMPC) Dipalmitoylphosphatidylcholine (DPPC) Distearoylphosphatidylcholine (DSPC) Dioleoylphosphatidylcholine (DOPC) Dielaidoylphosphatidylcholine (DEPC) 2-Oleoyl-3-palmitoylphosphatidylcholine(OPPC)

Fatty acid chains

14:O

{

16:O 18:O 18: 1 cis 18: I trans 18. 6; I cis (posn 2)

(posn 3)

(“C)

ATPase activity at 37°C (pmolei minute/ mg)

23.7 41.7 58.2 -22

4.5 7.1 5.9 16. I

II

21.0

<5

23.0

Ca-ATPase was mixed with different lipids as indicated, in the presence of cholate; in each case the added lipid comprised >93% of the total lipid pool. ATPase activities were measured after the lipid pools had equilibrated (see Fig. 3). For comparison, the preparation of Ca-ATPase used in this experiment had an ATPase activity of 11.5 pmole/minute/mg at 37°C. (J. P. Bennett, unpublished results). (I

There is also evidence that the length of the fatty acid chains plays a role in the modulation of ATPase activity. Johannson e f al. (1980) have shown that lipids containing unsaturated 14-carbon chains do not support high activity despite a low phase transition temperature; however, the activity increases with the addition of agents which thicken the hydrocarbon region of the lipid bilayer. The low activity observed when Ca-ATPase is surrounded by DPPC below its transition temperature has been examined by Hidalgo et af. (1976) and by Nakamura et al. (1976). Both these studies concluded that the reduction in enzyme activity is due to a reduced rate of decomposition of the phosphorylated enzyme intermediate in the reaction mechanism (Makinose, 1973); the change of phospholipid affects to a lesser degree the rate of phosphorylation of the enzyme and the steady-state concentration of phospho-enzyme intermediate. Translocation of calcium ions from an outward-facing site on the Ca-ATPase (relative to the enzyme’s orientation in native SR) to an inward-facing site occurs subsequently to phospho-enzyme formation, and is necessary for the hydrolysis of phospho-enzyme (see, e.g., Ikemoto, 1976). This translocation of calcium is accompanied by a conformational change in the Ca-ATPase protein (Dupont and Leigh, 1978). It seems reasonable to suppose that the occurrence of this change of conformation will depend on the physical state of the

139

SARCOPLASMIC RETICULUM CALCIUM PUMP

lipids with which the protein interacts, so that when the Ca-ATPase is in a “rigid” bilayer (below the transition temperature) calcium translocation is inhibited, with a consequent reduction in the rate of phospho-enzyme decomposition and hence ATPase activity. The distribution of Ca-ATPase molecules (visualized as intramembranous particles by freeze-fracture electron microscopy) is also dependent on the physical state of the phospholipid bilayer (Kleemann and McConnell, 1976). Ca-ATPase was incorporated into bilayers consisting almost entirely of DMPC and samples were prepared for freeze-fracture electron microscopy from temperatures either above or below the lipid phase transition of DMPC (Fig. 5). Above the phase transition temperature the protein molecules appear to be randomly distributed through the membrane. Below the transition temperature the protein is seen only in certain areas, leaving extensive regions of protein-free DMPC which show the banded appearance typical of a pure saturated lipid below its phase transition.

\ oYoMPC-ATPare \

l\

\

T [“C I

1 45 40 35 30 25 32

M 34

33 (OK-’.

l? IF\, 5

35 lo3)

36

FIG. 4. Arrhenius plots for lipid titration complexes. Ca-ATPase surrounded by its native SR lipids (SR-ATPase), or by DOPC or DMPC in a lipid titration complex (DOPCATPase and DMPC-ATPase), was assayed for calcium-dependent ATPase activity over the temperature range 4-48°C. The results are expressed as an Arrhenius plot so that the slope of the lines corresponds to the energy of activation for the ATPase reaction (J. P. Bennett, unpublished results.).

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J. P. BENNETT ET AL

FIG.5 . Freeze-fracture of DMPC-ATPase vesicles. Vesicles comprising DMPC and CaATPase (at a ratio of 9:1 by weight) were quenched for freeze-fracture electron microscopy from (a) 30°C and (b) 13°C. The bar represents 0.1 pm. (From Kleeman and McConnell, 1976.)

One envisages that during the process of cooling the membrane through the phase transition (the change from fluid to rigid state takes place over a narrow temperature range for a single pure lipid) the rigid phase phospholipids form a two-dimensional "crystal" which excludes the protein: as the rigid phase increases in area the proteins are confined to a smaller area, and finally to tight clusters as seen in Fig. 5b. That this is likely to be the case is shown in experiments in which the membrane consists of a mixture of two saturated phosphatidylcholines (DMPC and DPPC) with different transition temperatures. For such a membrane the transition from all fluid to all rigid occurs over a wide temperature range, and at intermediate temperatures rigid and fluid phases coexist (Shimshick and McConnell, 1973). At such an intermediate temperature Kleemann and McConnell (1976) showed that Ca-ATPase is excluded from the rigid phase and randomly distributed in the fluid phase (Fig. 6). The clustering of Ca-ATPase molecules below the phase transition may itself affect the protein function, and it has been observed that the CaATPase in a DPPC membrane shows cooperative kinetics below the phase transition ( J . P. Bennett, unpublished results). This implies a specific interaction between the protein molecules when forced together in this way, which in turn raises the hope that conditions could be found in which the proteins would form a crystalline array in the membrane from which structural information about Ca-ATPase could be extracted by electron microscopy (Henderson and Unwin, 1975).

SARCOPLASMIC RETICULUM CALCIUM PUMP

141

In order to investigate the role of different headgroups in supporting ATPase activity, Bennett et al. (1978a) devised a method for validating the lipid titration technique for lipids that were not available in sufficient quantity for isolation and analysis of lipid-protein complexes using the lipid substitution method. A lipid titration as shown in Fig. 3 involves starting with Ca-ATPase surrounded by a lipid which supports a high activity (DOPC or SR lipids)

FIG. 6 . Freeze-fracture (DMPC + DPPC)-ATPase vesicles. Vesicles comprising an equimolar ratio of DMPC and DPPC, and CA-ATPase (at a lipid-protein ratio of 3: I by weight), were quenched for freeze-fracture electron microscopy from (a) 37"C, (b) 32"C, (c) 20°C. The bar represents 0. I pm. (From Kleeman and McConnell, 1976.)

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J. P. BENNETT ET AL.

and following the decrease in activity on mixing with a test lipid in the presence of cholate; this type of experiment can be called a “forward titration.” However, one could also start with Ca-ATPase surrounded by a lipid which supported a very low activity and observe the increase in activity: this is a “back-titration.” Bennett et al. (1978a) argued that if a forward titration and a back-titration of Ca-ATPase with a given test lipid gave the same enzyme activity then cholate must be equilibrating the test lipid with the lipid surrounding the protein. As an illustration of this technique, the lipids surrounding the CaATPase were replaced with DOPC using the lipid substitution technique. When a 70-fold molar excess of cholesterol was added to the complex obtained (DOPC-ATPase) in the presence of a high concentration of cholate the enzyme activity fell to less than 2% of that of DOPC-ATPase within 1 minute of the addition. The ATPase activity could then be completely restored by adding excess DOPC in cholate (Fig. 7). The complete reversibility of the process indicates that no irreversible inactivation of the Ca-ATPase has occurred, and that cholate is acting to allow equilibration of lipid pools. If a test lipid is used to reactivate the cholesterol-ATPase complex and the ATPase activity rises to a value that is the same as that for a

10

ATPase activity (IU/mg at 37OC)

5

DOPC-ATPose

DOPC-ATPase Cholate Cholesterol

DOPC-ATPose Cholate Cholesterol DOPC

FIG. 7. The reversibility of the back-titration procedure. A DOPC-ATPase complex (prepared by the lipid substitution technique) was incubated with an excess of cholesterol in the presence of cholate, and then more DOPC in the presence of cholate was added, as described in the text. (From Bennett et a / . , 1978a.)

143

SARCOPLASMIC RETICULUM CALCIUM PUMP

forward titration, then cholate facilitates exchange of that lipid just as it does for DOPC. If equilibration does not occur then the same lipid in cholate should not affect the ATPase activity-and this behavior was observed in the case of the cerebroside lipids ( G . B. Warren, unpublished results). By a similar argument, if cholate catalyzes only partial exchange of the test lipid then the forward and back titration procedures will not lead to the same ATPase activities. Figure 8 shows the results of forward and back titrations with seven classes of phospholipid, compared with DOPC. The lipids used all have fatty acid chains that will be fluid at 37°C and will not interfere with an analysis of headgroup specificity. All of the test lipids do lower the activity of DOPC-ATPase and raise the activity of cholesterol-ATPase to approximately the same level, so that lipid equilibration has occurred in all cases and the activities reflect the interaction of the protein with the test lipid. There is a clear correlation of enzyme activity with headgroup charge, as shown in Fig. 8. The zwitterionic dioleoylphospholipids support the highest ATPase activity while DOPA with two negative charges supports the lowest. Phospholipids with a single negative charge-DOPG and the

100

ToDOPC-ATPase activity

80

0

Forward litrotion Back titration

60 40

20 Test lipid headgroup charge

FIG.8. Phospholipid headgroup specificity for ATPase activity. Lipid titration complexes of Ca-ATPase with the indicated phospholipids were prepared by both the “forward titration” and “back titration” procedures (see text) and the ATPase activity was measured at 37°C. For lipid abbreviations, see Table 111. All lipids used had only oleoyl (18:l) fatty acid chains except cardiolipin; in this case gas-liquid chromatography analysis showed that 18:l and 18:2 fatty acid chains comprised 77% of the total so that the chains should be sufficiently fluid at 37°C not to interfere with analysis of headgroup specificity. The headgroup charge structures indicated assume that each ionizable group will be fully expressed: this will not be the case however for DOPS and DOPA under the assay conditions used. (From Bennett et al., 1978a.)

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unphysiological synthetic lipids OMeDORA and NAcDOPE-support an activity which is about 5-fold lower than DOPC, while DOPS which is zwitterionic with an additional negative charge supports an ATPase activity intermediate between that supported by zwitterionic phospholipids and those with a single negative charge. Cardiolipin has two negative charges, like DOPA, but supports an activity which is about the same as that supported by those phospholipids with a single negative charge. Cardiolipin is synthesized in vivo by condensing two molecules of phosphatidylglycerol (which has a single negative charge) so that the resulting molecule resembles two diacyl phospholipids joined by a glycerol bridge (Hirschberg and Kennedy, 1972). These results indicate that the bridge between the singly negatively charged headgroups on cardiolipin has very little effect on the activity supported by this lipid. The observation that cardiolipin, DOPG, OMeDOPA, and NAcDOPE which have structurally different headgroups all support a similar ATPase activity suggests that it is the charge structure of the headgroup that is the dominant factor in determining the phospholipid headgroup specificity of this membrane protein. Each of these lipids carries a single negative charge on each diacylglycerophosphate moiety. However, other factors must also be involved since DOPC and DOPE which are both zwitterionic support significantly different activities. VI.

RECONSTITUTION OF Ca-ATPase INTO SEALED VESICLES

The examination of the fatty acid chain and headgroup specificity of Ca-ATPase previously described considered only one functional assay: that of calcium-dependent ATPase activity. The requirements for optimum ATPase activity were that the protein should be surrounded by lipids with a zwitterionic headgroup and fatty acid chains that are in the fluid state. However, the functional requirements of SR in vivo are that the lipid environment should not only support ATPase activity but also allow accumulation of calcium. The “reconstitution” procedure first described by Racker allows one to incorporate Ca-ATPase into membrane vesicles comprising test lipid and measure the ability of these vesicles to accumulate calcium. Martonosi (1968) showed that after SR had been solubilized with deoxycholate, the detergent could be removed by simple dilution. The resulting material regained the appearance of consisting of membranous vesicles in the electron microscope and showed a partial restoration of the original calcium accumulating activity. Successful reconstitution of

SARCOPLASMIC RETICULUM CALCIUM PUMP

145

SR which has been solubilized with detergent back into vesicles into which calcium uptake can be measured was later achieved by Meissner and Fleischer ( 1973). Racker ( 1972) described a simple and reproducible reconstitution procedure whereby SR (or Ca-ATPase), solubilized with cholate, is mixed with excess lipid in cholate solution. Slow removal of the detergent by dialysis allows the lipid to form sealed membrane vesicles incorporating Ca-ATPase into which uptake of calcium could be measured. The procedure readily allows investigation of the efficiency of different lipids in these reconstituted vesicles, and the conclusions of Racker and his colleagues (Racker, 1972; Racker et d., 1975; Knowles and Racker, 1975; Knowles et al., 1975, 1976) were that maximal calcium uptake activity was seen when using total purified phospholipids from soybean, or a mixture of purified phosphatidylcholine and phosphatidylethanolamine from natural sources. Phosphatidylethanolamine alone was found to support only a reduced rate of calcium uptake, while vesicles reconstituted with phosphatidylcholine did not support calcium uptake at all. Acetylated phosphatidylethanolamine, where the amine group has been blocked, did not support calcium uptake in reconstitution experiments unless alkylamines were added to the vesicles (Knowles et al., 1975). This led Racker and his colleagues to suggest that the presence of the free amine group in the membrane was an essential prerequisite for reconstitution of the Ca-ATPase. Warren el al. (1974a) demonstrated that purified Ca-ATPase and the synthetic phospholipid DOPC were sufficient to allow measurable calcium uptake in a reconstituted system. However it was clear that in this system the competence of the reconstituted vesicles in calcium uptake was greatly dependent upon the exact conditions used. In particular Warren et al. (1980a) showed that calcium uptake was sensitive to small changes in the ratio of lipid and detergent to protein (see Fig. 9). The use of SR instead of purified Ca-ATPase allows greater latitude of experimental conditions; since exogenous lipid added in the reconstitution procedure accounts for > 99% of the total lipid, SR was used as the starting material in studies of the lipid requirements of reconstituted vesicles (Bennett et al., 1978a). A series of synthetic phospholipids were tested in the reconstitution system (Table 111); all the lipids had fluid (oleoyl) fatty acid chains since the reconstitution methodology described seems to be inadequate for lipids with saturated fatty acid chains and high transition temperatures (J. P. Bennett and G. B. Warren, unpublished results). It is apparent from these results that several different phospholipid species can meet the requirements for calcium uptake. The complex lipid composition of

146

J. P. BENNETT ET AL.

0 20 -

-

-

015 -

C a2'u pt ak e (tmoleslrninlrng) 010 005 -

-

--I

m g OOPC I m g ATPase m g tholate I m g OOPC

,

*O

*

5 0 loo 150

04

20

50 100 150

20

50 100 150

0.6

05

FIG.9. Conditions for reconstitution using DOPC-ATPase. Purified Ca-ATPase with lipids replaced by DOPC (by the lipid substitution technique) was reconstituted using DOPC and cholate in the ratios indicated, and the initial rates of calcium uptake at 25°C were determined. From Warren er ul (1980a).

TABLE I11 PROPERTIES OF MEMBRANES RECONSTITUTED FROM SARCOPLASMIC RETICULUM USING DIFFERENT LIPIDS"

Liuid Dioleoylphosphatid ylcholine (DOPC) Dioleoylphosphatidylet hanolamine (DOPE) Dioleoylphosphatid ylserine (DOPS) N-Acetyl-dioleoylphosphatidylethanolamine( N AcDOPE) Dioleoylphosphatidylgl ycerol (DOPG) 0-Methyl-dioleoylphosphatidic acid (OMeDOPA) Dioleoylphosphatidic acid (DOPA) DOPE + DOPC NAcDOPE + DOPC OMeDOPA + DOPC

Ca2+ accumulating ATPase activity activity 0.84 0 0.43 0 0. I7 0 0.24 0.68 0.05 1.20

I .55 2.03 0.37 0.32 0.32 0.38 0.15 0.06 0.45 1.01

a In each reconstitution experiment the total amount of lipid used was the same and added lipid comprised > 99% of the total; in the last three experiments equal amounts of the two lipids were used. The Ca2+accumulating and ATPase activities are in initial rates expressed in pnoleiminutelmg enzyme at 25°C. From Bennett et ul. (1978a).

SARCOPLASMIC RETICULUM CALCIUM PUMP

147

native SR is not essential either for ATPase activity o r calcium accumulation. Since four of the lipids tested supported calcium uptake there can be no absolute specificity for a particular lipid species. The inability of certain lipids (which supported ATPase activity) to support calcium accumulation was a single consequence of their inability to form intact vesicles in these experiments. The morphology in negatively stained electron micrographs of vesicles reconstituted with DOPC and DOPE are shown in Fig. 10. The reconstituted vesicles containing DOPC showed high calcium uptake activity, and in the electron micrograph they appear to be smooth intact vesicles whose membranes bound an enclosed volume; some of these may be multishelled. In contrast the reconstituted vesicles containing DOPE, which are not capable of calcium uptake, appear to consist of irregularly stacked sheets of membrane. There is no evidence of a membrane-enclosed volume into which calcium could be pumped. The nature of the reconstitution technique means that it is difficult to interpret the inability of a particular lipid to form sealed vesicles. It may reflect a basic property of that lipid, or it may reflect a methodological failure of the reconstitution technique. For example, small changes in the proportions of components in the initial incubation can significantly alter the levels of calcium accumulation by the reconstituted vesicles (Warren et al., 1980). Although workers in Metcalfe’s laboratory have been unable

FIG.10. Electron micrographs of reconstituted membranes. Membranes reconstituted using SR were negatively stained with 2% phosphotungstate and examined at 40,OOOx magnification. The lipids used were (a) DOPC, (b) DOPE, and (c) an equal mixture of DOPC and DOPE. The bars represent 0.1 p m . (From Bennett et d., 1978a.)

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J. P. B E N N E T ET AL.

to obtain successful reconstitutions with phosphatidylethanolamine from any source, Racker and co-workers have consistently had success with soybean phosphatidylethanolamine: this is presumably due to some small difference in methodology. The suggestion that a specific chemical group must be present in the phospholipids to allow the coupling of ATPase activity to calcium uptake in reconstituted membranes (Knowles et al., 1975) is clearly not borne out by the date in Table 111. Conversely the lipids which did not support calcium accumulation did not have any structural property that prevents coupling. Bennett et al. (1978a) carried out a reconstitution experiment using equal mixtures (by weight) of these lipids with DOPC: all these lipid mixtures can support measurable calcium accumulation in reconstitutions of lipid mixtures (Table 111). In the case of DOPE at least there is evidence (see Section VIII) that the DOPE molecules will interact with the Ca-ATPase protein, so that this direct interaction does not uncouple calcium uptake from ATP hydrolysis. Examination of these vesicles in the electron microscope (Fig. 10) shows that they are intact and apparently contain an enclosed volume. It seems to be this morphological requirement alone which allows the measurement of calcium uptake in reconstitution experiments. Racker and Eytan (1975) proposed that a proteolipid component of SR (MacLennan et al., 1972) acted as a “coupling factor” which might be the ionophoric component of the calcium pump (Racker, 1975): in its absence ATP hydrolysis could not be accompanied by calcium translocation. This model seems dubious because of the very small amount of proteolipid that is present in SR; Sigrist et al. (1977) found that it accounted for only 0.15% of the total protein. The molar ratio of proteo1ipid:Ca-ATPase is thus approximately I :50, which must preclude a direct stoichiometric interaction having an essential role in coupling. However, traces of proteolipid can always be detected in the preparations of purified Ca-ATPase [those of MacLennan (1970) and Warren et af. (1974a)l which have been used in reconstitution experiments so that it remains possible that the proteolipid has an indirect role in coupling-for example, by acting as the carrier for a counterion. VII.

ONLY 30 LIPID MOLECULES MODULATE Ca-ATPase FUNCTION

When Ca-ATPase is prepared using the sucrose density technique, the lipid-protein ratio in the isolated complex depends on the concentration of detergent used in the initial incubation. The higher the concentration

149

SARCOPLASMIC RETICULUM CALCIUM PUMP

of the detergent above its critical micelle concentration the more detergent micelles there will be, and as a result lipid molecules will tend to partition out of the protein-lipid-detergent micelles to form more lipiddetergent micelles. At the same time there will be an irreversible loss of enzyme activity since lipid is required for the functioning of the CaATPase. When the ATPase activity is plotted as a function of the lipidprotein ratio it is apparent that there is a critical lipid-protein ratio that represents the minimum lipid content that will support maximal activity. Figure 1 1 shows the result of this experiment, using cholate as detergent (Warren et al., 1974~).The critical lipid content required to support ATPase activity is approximately 30 mole lipid per mole protein. The figure also shows data from a similar experiment (J. P. Bennett and G. B. Warren, unpublished results) in which a different detergent, octyl glucoside (see Helenius et al., 1979), was used: the behavior observed was the same. There is evidence that 30 lipids per protein are bound to the Ca-ATPase after solubilization with other nonionic detergents: Triton X-100, Tween 80, and C,,E, (LeMaire et af., 1976). These 30 molecules of lipid per molecule Ca-ATPase, which have been called the “lipid annulus” (Warren et af., 1975), are envisaged as the first bilayer shell of lipid molecules surrounding the Ca-ATPase protein. Experiments using spin-label lipid probes have identified the same number of lipid molecules as being immobilized relative to the rest of the bilayer,

% SR - ATPase

activity 0

Cholate

o Octyl - glucoside

0 J

0

20

40

60

80

100

moles lipid I mole ATPase

FIG. 11. The dependence of ATPase activity on 1ipid:protein ratio. Ca-ATPase was incubated with detergent (either potassium cholate or octyl glucoside) at various concentrations, and centrifuged through a sucrose gradient. The complexes obtained were analyzed for 1ipid:protein ratio and ATPase activity at 37°C. [Closed symbols, cholate from Warren el ul. ( 1 9 7 4 ~ )open ; symbols, octyl glucoside (J. P. Bennett and G . B. Warren, unpublished results).]

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J. P. BENNETT ET AL.

presumably because of direct interaction with the protein (Montecucco et al., 1977; see also Nakamura and Ohnishi, 1975). Even when the protein is embedded in a bilayer containing a great excess of lipids, it is the lipid annulus which determines the protein activity. Hesketh et al. (1976) showed this using Ca-ATPase with the lipids replaced by DPPC, in various lipid-protein ratios. The physical properties of the annulus lipids (examined using spin-label techniques) were different from the physical properties of the rest of the bilayer, and it was the physical properties of the lipid annulus that appeared to modulate enzyme activity. An attempt to make data from similar experiments fit quantitatively with a theoretical prediction of physical properties of lipids associated with Ca-ATPase (using a lipid annulus model) has been unsuccessful, however (Moore et al., 1978). Spectroscopic measurements can provide an estimate of the ability of annulus lipids to diffuse into the rest of the bilayer. Hesketh et al. (1976) point out that resolution of the annulus by electron spin resonance techniques provides an upper limit for the rate of exchange of annulus lipids into the free bilayer of about lo6 sec-I; Chapman ef al. (1979) report that deuterium nuclear magnetic resonance techniques fail to resolve the two lipid components and suggest that this implies a lower limit for the annulus-bilayer exchange rate of about l O5 sec-l. Thus annulus lipids can exchange rapidly into the bilayer and appear "immobilized" only by comparison with the extremely fast diffusion rates of lipids in the free bilayer.

VIII.

THE COMPOSITION OF THE LIPID ANNULUS IS NOT THE SAME AS THE WHOLE BILAYER

Cholesterol is a lipid which was found to support an extremely low ATPase activity. However, when cholesterol is added to Ca-ATPase in an equimolar mixture with a phospholipid (at higher ratios the lipid mixture does not form a bilayer) in a lipid titration experiment, it caused no loss in ATPase activity (Warren et al., 1975). This is interpreted as meaning that cholesterol is not entering the lipid annulus and interacting with the protein in these experiments. When phospholipid-cholesterol-protein complexes are isolated by the lipid substitution technique there is a reduced ATPase activity as long as there are fewer than 30 phospholipid molecules per Ca-ATPase (Fig. 12). If phospholipid was added back to these complexes so that the final stoichiometry exceeds 30 phospholipids per Ca-ATPase, then maximal ATPase activity is regained. These experiments suggest strongly that

SARCOPLASMIC RETICULUM CALCIUM PUMP

151

Moles phospholipid I Mole ATPase FIG. 12. The ATPase activity of cholesterol- ATPase complexes. Ca-ATPase was incubated with various concentrations of cholesterol and potassium cholate, and centrifuged through a sucrose gradient. The complexes obtained were analyzed for phospholipid, cholesterol, and protein content and the ATPase activity was measured at 37°C. In every case the total lipid content (phospholipid + cholesterol) exceeded 30 mole per mole CaATPase. Open symbols are from a similar experiment on the absence of cholesterol, a s in Fig. 1 1 . (From Warren et a / . , 1975.)

cholesterol is rigorously excluded from the lipid annulus as long as there are sufficient phospholipids to complete the lipid annulus, and that cholesterol in the bilayer outside the annulus has little effect on enzyme activity. Cholesterol is not a major component of intracellular membranes such as SR in vivo, and it was a very different structure from the phospholipids. In order to find out whether there is a similar lateral segregation of different phospholipid types within the membrane Bennett et al. (1980) chose to use as a model system a binary mixture of DOPC and DOPA. These lipids differ only in their headgroups, and the differences are substantial. DOPC is zwitterionic while DOPA has two negative charges (although the second negative charge may not be fully expressed at physiological pH). They also support very different ATPase activities. The rationale was first to measure the way in which the observed ATPase activity of Ca-ATPase surrounded by just 30 phospholipid molecules depends on the proportion of DOPC and DOPA in the annulus. If all 30 annulus phospholipids have an equivalent role in the support of ATPase activity then the observed ATPase activity would be a linear function of annulus composition; however, this could not be assumed necessarily to be the case. Second, the dependence of ATPase activity on the lipid composition of a much larger pool surrounding the Ca-ATPase is measured: if the relationship between ATPase activity and lipid composition were the same as with a lipid pool consisting of only annulus lipids, then lateral segregation is not occurring. If, however, there is a

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J. P. BENNETT ET AL.

change with the pool size in the dependence of ATPase activity on phospholipid composition, this reflects lateral segregation occurring so that in the presence of excess lipids the composition of the annulus is not the same as that of the total lipid pool. In order to demonstrate the dependence of ATPase activity on the phospholipid composition in the annulus surrounding Ca-ATPase use was made of the fact that phospholipids will preferentially displace cholesterol from the lipid annulus (see previously). Ca-ATPase was prepared with its lipids replaced by cholesterol and phospholipid was added (in the presence of cholate) at a stoichiometry of 28 mole per mole Ca-ATPase. The enhancement of ATPase activity seen on addition of phospholipid (a 5-fold increase with DOPA and more than 30-fold with DOPC) is due to these phospholipids forming part of the phospholipid annulus. The ATPase activity was clearly found to be a linear function of the proportion of the two lipids in the annulus (Fig. 13). This implies that all the annulus lipids contribute equally in the modulation of enzyme activity. However, when the Ca-ATPase is embedded in a membrane containing an excess of phospholipid over and above that needed to form

o 28moles

lipid /mole ATPase

900 moles

0

20

100

80

40 60 %DOPA, 60 40

80

100 J

20

0

% DOPC

FIG. 13. ATPase activity supported by DOPC-DOPA mixtures. Phospholipid mixtures in the presence of cholate were incubated with Ca-ATPase at two different 1ipid:protein ratios and the ATPase activities were measured. Open symbols: phospholipid was added at a ratio of 28 mole/mole Ca ATPase to a cholesterol-ATPase complex prepared by the lipid substitution technique (which contained 27 mole cholesterol and 2 mole phospholipid per mole protein). Closed symbols: phospholipid was added at a ratio of 900 mole/mole CaATPase to DOPC-ATPase (which contained 38 mole phospholipid per mole protein). (From Bennett er d., 1980.)

153

SARCOPLASMIC RETICULUM CALCIUM PUMP

the annulus, the response is nonlinear. In an experiment in which the total lipid pool was approximately 900 mole phospholipid per mole CaATPase (Fig. 13) it appeared that lateral segregation must be occurring such that when 80% of the total lipid pool consists of DOPA, the lipid annulus consists principally of DOPC. Similar experiments led us to speculate that lateral segregation occurs to a lesser degree to exclude a singly negatively charged phospholipid (DOPG) from the lipid annulus, but that the protein does not distinguish between DOPC and DOPE (Fig. 14). Lateral segregation occurs most strongly to exclude from the lipid annulus just those lipids which support the lowest ATPase activity (see Fig. 8). In other words, lateral segregation acts to optimize enzyme activity in a membrane comprising a mixture of phospholipid types. In these experiments, all the phospholipids had the same (oleoyl) fatty acid chains. Warren et al. (1980b) carried out a similar experiment using lipids with different fatty acid chains: DMPC and DOPA (Fig. 15). The result is striking. A mixture of these two phospholipids supported an ATPase activity which is much higher than that supported by either phospholipid alone; it is as if the protein is responding to the elements of DOPC. Similar synergism was observed with other mixtures of phospholipids differing in both chain and headgroup. The phenomenon may be accounted for by a combination of two effects. Lipid segregation as previously discussed will mean that DMPC interacts with the Ca-ATPase protein in preference to DOPA, but at the same time the presence of

a20 . I

b

0

20

100

80

40 60 96 DOPE 60 40 % DOPC

80

100

20

0

FIG. 14. ATPase activity supported by phospholipid mixtures. The dependence of ATPase activity on phospholipid composition for (a) DOPC-DOPG mixtures and (b) DOPC-DOPE mixtures. A DOPC-ATPase complex was incubated with excess phospholipids in cholate at a ratio of about 900 mole phospholipid per mole Ca-ATPase (J. P. Bennett, unpublished results).

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J. P. BENNETT ET AL.

I 0

20

40 /a '

60

80

100

20

0

DOPA

L

100

80

60

40

%DMPC

FIG. 15. ATPase activity supported by DMPC-DOPA mixtures. The dependence of ATPase activity on phospholipid composition for DMPC-DOPA mixtures, investigated as in Fig. 14. (From Warren et a / . , 1980b.)

DOPA in the membrane with oleoyl chains (18-carbon) will tend to overcome the inhibitory effect of the shorter chains (ICcarbon) of DMPC (Johannsson et af., 1980). IX.

LIPID ASYMMETRY

There is now a substantial body of evidence that there is an asymmetric distribution of phospholipids between the two halves of the bilayer for plasma membranes from various sources (see Rothman and Lenard, 1977), although the detailed distribution of the lipids is not the same for the different membranes examined. This asymmetry is maintained by the low rate of exchange of phospholipids between the two halves of the bilayer ("flip-flop"), and its origin may lie in the mode of membrane biosynthesis (Rothman and Kennedy, 1977a; Hirata er af., 1978). Despite the apparent universality of phospholipid asymmetry in plasma membranes no role for it in cell function has been identified, although one possible physiological outcome has been noted (Zwaal et af., 1977). The way in which phospholipids are distributed across the bilayer of intracellular membranes is rather less clear. Since in cells which secrete, at the moment of secretion the inner monolayer of an intracellular membrane becomes contiguous with the external monolayer of the plasma membrane while the cytoplasmic faces of each membrane join, one might

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suppose that the lipid composition of the two cytoplasmic leaflets will resemble each other, as will those of the two luminal leaflets. If lipid asymmetry does in fact originate at biosynthesis then this relationship is necessary, since synthesis of phospholipids occurs primarily at an intracellular membrane, the endoplasmic reticulum (Jelsema and Morre, 1978), in eukaryotic cells and conservation of asymmetry during the membrane fusion event in secretion is the only way asymmetry arising from biosynthesis would be observed in the plasma membrane. However there is also the possibility that lipid asymmetry could be maintained by specific enzymes within the plasma membrane (Bretscher, 1973) in which case no such relationship of the transverse disposition of lipids across the bilayer in intracellular and plasma membranes is predicted. Published reports of measurements of the transverse disposition of phospholipids in intracellular membranes do not resolve the questions. For example, different workers using phospholipases as probes for lipid asymmetry in rat liver microsomes have variously proposed that phosphatidylethanolamine (PE) is found predominantly in the outer monolayer (Nilsson and Dallner, 1977), predominantly in the inner monolayer (Higgins and Dawson, 1977), or distributed equally between the two monolayers (Sundler et al., 1977). It is apparent that intracellular membranes are much less tractable than plasma membranes to analysis of lipid asymmetry. Rothman and Lenard (1977) have listed the experimental criteria necessary to establish lipid asymmetry. The membranes should be present as a pure preparation (no contaminating lipid from other membranes), and all the membranes should form closed vesicles with the same sidedness. It must be shown that the labeling reagent should under the conditions of the experiment neither penetrate the membrane nor lead to lysis. None of the above experiments on rat liver microsomes fulfill all these criteria. X.

DISTRIBUTION OF LIPIDS ACROSS THE SR MEMBRANE

Various chemical labeling reagents have been used to localize amino groups in SR. Hasselbach and Migala (1975) used fluorescamine, and came to the conclusion that the aminophospholipids are present mainly on the outer monolayer. However fluorescamine is by no means an ideal reagent since it decomposes rapidly, and being insoluble in water must be added to the sample as a solution in an organic solvent which may perturb the lipid bilayer. Hidalgo and Ikemoto (1977) used a stable, watersoluble, complex of fluorescamine with cycloheptaamylose which was

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shown not to disrupt vesicles (as judged by the inaccessible [14C]inulin space). They also concluded that most of the PE (the predominant aminophospholipid) was situated in the exterior half of the lipid bilayer. Vale ( 1977) used 2,4,6-trinitrobenzenesulfonate(TNBS) as a label and again concluded that the majority of PE is external; electron microscopy was used to monitor vesicle integrity after labeling. Sarzala and Michalak (1978) used both TNBS and phospholipases and concluded again that most of the PE and PS was in the outer monolayer. In a different SR preparation, believed to consist of inside out vesicles, the majority of PE was, as predicted, on the inner monolayer. PC was approximately equally distributed between the two halves of the bilayer in both cases. In an attempt to fulfill the criteria listed by Rothman and Lenard (1977) for measurement of lipid asymmetry, McGill et al. (1980) made a preparation of sealed right-side out vesicles. The rationale was that only such vesicles will be able to accumulate calcium. SR was incubated with ATP and CaCI, in the presence of potassium oxalate so that within sealed vesicles calcium oxalate was precipitated. The dense calcium oxalatecontaining vesicles were separated from leaky vesicles and inside out vesicles by sucrose density gradient centrifugation. The precipitated calcium oxalate was finally removed by allowing the calcium pump to work in reverse in a solution containing ADP and EGTA. [For methodology, see Bennett et al. (1978b). A similar procedure has been used by Bonnet et al. (19781.1 These vesicles were found to be enriched in the Ca-ATPase protein as compared with the initial SR preparation, but to have a very similar phospholipid composition. TNBS labeling (using conditions described in Rothman and Kennedy, 1977b) indicated that only half (47 5 4%) of the PE in these membranes resides in the outer monolayer. PC is also distributed approximately equally in these vesicles (5 I k 4% external measured using a phospholipid exchange protein by a method similar to that of Bloj and Zilversmit, 1976). However the small amount of glycolipid in SR (Narasimhan et al., 1974) is present only on the inner surface where it is inaccessible to galactose oxidase (using the labeling technique of Gahmberg and Hakomori, 1973). It is not clear why the sealed right-side out preparation of SR should differ from unfractionated SR in its transbilayer distribution of phospholipids. The difference cannot be explained by the rate of flip-flop of lipids between leaflets of the bilayer. McGill et al. showed that there is neglible increase in the pool of PC accessible to a phospholipid exchange protein (method of Zilversmit and Hughes, 1977) over 4 days for sealed rightside out SR, so that the half-time for flip-flop is in excess of 10 days.

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This is in contrast to rat liver microsomes where flip-flop is rapid with a half-time of 45 minutes (Zilversmit and Hughes, 1977); presumably this reflects the actively synthetic role of microsomes compared with the stability of SR as a specialized organelle.

XI.

TRANSBILAYER DISPOSITION OF THE PHOSPHOLIPID ANNULUS

The disposition of the annulus phospholipids between the two leaflets of the lipid bilayer has been investigated by Bennett et al. (1978b). It was observed that 30 lipids per Ca-ATPase protein were resistant to digestion by phospholipase D, regardless of the initial 1ipid:protein ratio. These lipids were believed to be the annulus lipids previously identified by other techniques (see Section VII). The annulus lipids will not be digested by phospholipase D because of their proximity to the Ca-ATPase protein; the reason they do not exchange into the rest of the bilayer and so become available for digestion is that phosphatidic acid (the product of phospholipase D lipolysis) will under the conditions of these experiments form a separate rigid phase in the lateral plane of the membrane (Galla and Sackmann, 1975). Phospholipase D in aqueous solution will digest the outer monolayer only of pure lipid vesicles, so that this method can be extended to determine how many of the annulus lipids reside in the outer half of the SR bilayer. A phospholipid exchange protein was used to insert into the outer monolayer only of sealed right-side out SR vesicles DOPC which was radiolabeled in the choline moiety. The proportion of these labeled lipids that are resistant to digestion by phospholipase D can be used to calculate the number of annulus lipids in the outer half of the bilayer. A representative experiment is shown in Fig. 16. When either the whole bilayer or only the outer monolayer is examined there is a pool of phospholipid which is not digested by phospholipase D. In the whole bilayer this corresponds to the 30 annulus phospholipids per Ca-ATPase protein. In the outer monolayer 15 mole lipid per mole Ca-ATPase remain undigested. This means that the annulus phospholipids are distributed approximately equally between the inner and outer leaflets of the membrane bilayer. Since the annulus phospholipids are all in contact with the penetrant hydrophobic surface of the protein, these experiments indicate that the part of the protein that penetrates the membrane must resemble a cylinder, and provide indirect evidence that the Ca-ATPase protein completely spans the bilayer.

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W

0

a I-

U

--2 4o W

. n -

a

.-

\

Whole bilayer

‘.

30

’D

+

w (51 W .’D

3

-w 10 e

Outer monolayer

0)

I

FIG. 16. Phospholipase D digestion of the lipids surrounding Ca-ATPase. The time course of disappearance of radioactivity from the lipid fraction of Ca-ATPase associated with [3H-choline]-DOPC on treatment with phospholipase D in the presence of 40 mM CaC12. Closed symbols: unsealed fragments of DOPC-ATPase complex prepared used the lipid substitution technique. Open symbols: sealed right-side out SR labeled with [3H]DOPC in the outer monolayer only. (From Bennett ef u / . , 1978b.)

XII.

CONCLUDING REMARKS

Apart from a very few exceptional examples of membrane proteins susceptible to structural analysis by physical methods (such as bacteriorhodopsin; see Henderson and Unwin, 1975), information about the structural interactions involved in the functioning of a membrane protein must be inferred from less direct biochemical approaches. In the case of the sarcoplasmic reticulum Ca-ATPase these approaches have given a fairly detailed picture of the way the protein interacts with the lipid bilayer matrix in which it resides. The experiments reviewed here have depended largely on the use of synthetic (chemically homogeneous) lipids in conjunction with mild (nondenaturing) detergents. The techniques used have included sucrose density centrifugation, simple dilution and dialysis, phospholipid exchange proteins and phospholipases as well as physical methods such as electron spin resonance spectroscopy and electron microscopy. It turns out that Ca-ATPase is critically dependent upon its lipid environment for enzyme activity. The structure of the lipids which interact with the protein is important: they should be zwitterionic with “fluid”

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fatty acid chains (which should be 16-20 carbons in length) for optimal protein function. Of the 90 or so lipids per Ca-ATPase molecule in native SR, only 30 lipid molecules-equally distributed between the halves of the bilayer-are required for maintaining and modulating the enzyme activity. The 30 “lipid annulus” phospholipids can be distinguished from the remainder of the bilayer both by physical probes and by biochemical criteria: most notably the protein can segregate into the annulus from the bilayer just those lipids which best support the protein’s function. While Ca-ATPase remains one of the best characterized membrane enzymes, a large body of work with many other examples shows that similar patterns of lipid-protein interactions occur generally (for access to the literature, see review by Sandermann, 1978). For example fluid fatty acid chains seem to be a general requirement and headgroup specificities, sometimes far more rigorous than for Ca-ATPase, are often observed. With other proteins amenable to suitable experiments a stoichiometry of lipid-protein interaction (a lipid annulus) has been identified. It is still true that our understanding of the behavior of membrane enzymes lags far behind what we know about soluble enzymes. However the efforts of the last decade, most profitably with “model” membrane systems such as sarcoplasmic reticulum, have given us sufficient factual knowledge for biochemists at least to dream of the nature of the molecular interactions within membranes that seem to be so crucial in cell function. REFERENCES Bennett, J. P., Smith, G. A , , Houslay, M. D., Hesketh, T. R . , Metcalfe, J. C., and Warren, G. B. (19784. The phospholipid headgroup specificity of an ATP-dependent calcium pump. Biochim. Biophys. Acra 513, 310-320. Bennett, J. P . , McGill, K. A., and Warren, G. B. (1978b). Transbilayer disposition of the phospholipid annulus surrounding a calcium transport protein. Nature (London) 274, 823-825. Bennett, J. P., Warren, G. B., Smith, G. A., Hesketh, T. R . , Houslay, M. D., and Metcalfe, J. C. (1980). Lateral segregation of binary phospholipid mixtures around a calcium transport protein. Submitted for publication. Bloj, B., and Zilversmit, D. B. (1976). Asymmetry and transposition rates of phosphatidylcholine in rat erythrocyte ghosts. Biochemistry 15, 1277- 1283. Bonnet, J. P . , Galante, M., Brethes, D., Dedien, J. C . , and Chevallier, J . (1978). Purification of sarcoplasmic reticulum vesicles through their loading with calcium phosphate. Arch. Biochem. Biophys. 191, 32-41. Bretscher, M. S. (1971). A major protein which spans the human erythrocyte membrane. J . Mol. B i d . 59, 351-357. Bretscher, M. S . (1973). Membrane structure: some general principles. Science 181, 622629.

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Bretscher, M. S., and Raff, M. C. (1975). Mammalian plasma membranes. Norure (London 258, 43-49. Chapman, D., Gomez-Fernandez, J. C., and Goni, F. M. (1979). Intrinsic protein-lipid interactions: physical and biochemical evidence. FEBS L e t t . 98, 21 1-223. Deamer, D. W. (1973). Isolation and characterisation of a lysolecithin-adenosine triphosphatase complex from lobster muscle microsomes. J . B i d . C h e m . 248, 5477-5485. Deamer, D. W., and Baskin, R. J . (1969). Ultrastructure of sarcoplasmic reticulum preparations. J . Cell B i d . 42, 296-307. Dean, W. L., and Tanford, C. (1977). Reactivation of lipid-depleted Ca2+-ATPaseby a nonionic detergent. J . Biol. C h e m . 252, 3551-3553. Dobberstein, B., Garoff, H., Warren, G., and Robinson, P. (1979). The cell-free synthesis and membrane insertion of mouse H2-D" histocompatibility antigen and p2-microglobd i n . Cell 17, 759-769. Duggan, P. F., and Martonosi, A. (1970). Sarcoplasmic reticulum. The permeability of sarcoplasmic reticulum membranes. J . G e n . Physiol. 56, 147- 167. Dupont, Y., and Leigh, J. B. (1978). Transient kinetics of sarcoplasmic reticulum Ca2+ + Mg2+ATPase studied by fluorescence. Nature (London) 273, 396-398. Fiehn, W., and Hasselbach, W. (1970). The effect of phospholipase A on the calcium transport and the role of unsaturated fatty acids in ATPase activity of sarcoplasmic reticulum vesicles. Eur. J . Biochem. 13, 510-514. Furthmayr, H., Galardy, R. E., Tomita, M., and Marchesi, V. T. (1978). The intramembraneous segment of human erythrocyte glycophorin A. Arch. Biochem. Biophys. 185, 21-29. Gahmberg, C. G., and Hakomori, F. (1973). External labelling of cell surface galactose and galactosamine in glycolipid and glycoproteins of human erythrocytes. J . B i d . C h e m . 248, 4311-4317. Galla, H. J . , and Sackmann, E. (1975). Chemically induced lipid phase separation in model membranes containing charged lipids: a spin label study. Biochim. Biophys. Actri 401, 509-529. Garoff, H., and Simons, K . (1974). Location of the spike glycoproteins in the Semliki Forest Virus membrane. Proc. Null. Acud. Sci. U . S . A . 71, 3988-3992. Hardwicke, P. M. D., and Green, N . M. (1974). The effect of delipidation on the adenosine triphosphatase of sarcoplasmic reticulum. Electron microscopy and physical properties. Eur. J . Biochem. 42, 183- 193. Hasselbach, W. (1963). Relaxing factor and the relaxation of muscle. f r o g . Biophys. Mol. B i d . 14, 167-222. Hasselbach, W., and Migala, A. (1972). The separation of the solubilised proteins of the sarcoplasmic reticulum on DEAE-cellulose and its modification. FEBS Lett. 26, 2024. Hasselbach, W., and Migala, A. (1975). Arrangement of proteins and lipids in the sarcoplasmic membrane. Z . Naturforsch., Teil C 30, 681-683. Helenius, A,, and Simons, K . (1975). Solubilisation of membranes by detergents. Biochim. Biophys. Acta 45, 29-79. Helenius, A., McCaslin, D. R., Fries, E., and Tanford, C. (1979). Properties of detergents. In "Biomembranes" ( S . Fleischer and L. Packer, eds.), Methods in Enzymology, Vol. 56, pp. 734-749. Academic Press, New York. Henderson, R., and Unwin, P. N . T. (1975). Three dimensional model of purple membrane obtained by electron microscopy. Norure (London) 257, 28-32. Hesketh, T. R., Smith, G. A., Houslay, M. D . , McGill, K . A., Birdsall, N. J. M., Metcalfe, J. C., and Warren, G. B. (1976). Annular lipids determine the ATPase activity of a

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calcium transport protein complexed with dipalmitoyl lecithin. Biochemistry 15, 41454151. Hidalgo, C . , and Ikemoto, N. (1977). Disposition of proteins and aminophospholipids in the sarcoplasmic reticulum membrane. J. Biol. Chem. 252, 8446-8454. Hidalgo, C., Ikemoto, N., and Gergely, J. (1976). Role of phospholipids in Ca-dependent ATPase of sarcoplasmic reticulum. Enzymatic and ESR studies with phospholipidreplaced membranes. J. Biol. Chem. 251, 4224-4232. Higgins, J. A., and Dawson, R. M. C. (1977). Asymmetry of the phospholipid bilayer of rat liver endoplasmic reticulum. Biochim. Biophys. Actu 470, 342-356. Hirata, F., Vivers, 0. H., Diliberto, E. J., and Axelrod, J. (1978). Identification and properties of two methyltransferases in conversion of phosphatidylethanolamine to phosphatidylcholine. Proc. Nurl. Acud. Sci. U . S . A . 75, 1718- 1721. Hirschberg, C. B., and Kennedy, E. P. (1972). Mechanism of the enzymatic synthesis of cardiolipin in Escherichiu coli. Proc. Nutl. Acud. Sci. U . S . A . 69, 648-651. Ikemoto, N., Sreter, F. A., and Nakamura, A. (1968). Tryptic digestion and localisation of calcium uptake and ATPase activity in fragments of sarcoplasmic reticulum. J . UItrustruct. Res. 23, 216-232. Ikemoto, N., Bhatuagar, G. M., and Gergely, J. (1971). Fractionation of solubilised sarcoplasmic reticulum. Biochem. Biophys. Res. Commun. 44, 1510- 1517. Ikemoto, N. (1976). Behavior of the Ca2+ transport sites linked with the phosphorylation reaction of ATPase purified from sarcoplasmic reticulum. J. Biol. Chem. 251, 72757277. Inesi, G. (1972). Active transport of calcium ions in sarcoplasmic membranes. Annu. Rev. Biophys. Bioeng. 1 , 191-210. Jelsema, C. L., and Morre, D. J . (1978). Distribution of phospholipid biosynthetic enzymes among cell components of rat liver. J . Biol. Chem. 253, 7960-7971. Jorgensen, P. L. (1974). Purification and characterisation of (Na+ + K+)-ATPase. Purification from the outer medulla of mammalian kidney after selective removal of membrane components by sodium dodecylsulphate. Biochim. Biophys. Acta 356, 36-52. Johannsson, A., Keighthley, C. A., Smith, G. A., Hesketh, T. R., and Metcalfe, J . C. (1980). The effect of bilayer thickness and n-alkanes on the activity of the (CaZ++ Mg2+)-dependent ATPase of sarcoplasmic reticulum. Submitted for publication. Kleemann, W., and McConnell, H. M. (1976). Interactions of proteins and cholesterol with lipids in bilayer membranes. Biochim. Biophys. Acta 419, 206-222. Knowles, A. F., and Racker, E. (1975). Properties of a reconstituted calcium pump. J. Biol. Chem. 250, 3538-3544. Knowles, A. F., Kandrach, A., Racker, E., and Khorana, H . G. (1975). Acetyl phosphatidylethanolamine in the reconstitution of ion pumps. J. Biol. Chem. 250, 1809- 1813. Knowles, A. F., Eytan, E., and Racker, E. (1976). Phospholipid-protein interactions in the Ca2+ adenosine triphosphatase of sarcoplasmic reticulum. J. Biol. Chem. 251, 5 161- 5 165. Kyte, J. (1975). Structural studies of sodium and potassium ion activated adenosine triphosphatase. The relationship between molecular structure and the mechanism of active transport. J . Biol. Chem. 250, 7443-7449. LeMaire, M., Moller, J. V., and Tanford, C. (1976). Retention of enzyme activity by detergent solubilised sarcoplasmic reticulum Ca2+-ATPase. Biochemistry 15, 23362342. Levine, Y . K., Birdsall, N. J. M., Lee, A. G., and Metcalfe, J. C. (1972). I3C Nuclear magnetic resonance relaxation measurement of synthetic lecithins and the effect of spin-labelled lipids. Biochemistry 11, 1416- 1421.

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Louvard, D., Semeriva, M., and Maroux, S. (1976). The brush-border intestinal aminopeptidase, a transmembrane protein as probed by macromolecular photolabelling. J. Mol. B i d . 106, 1023- 1035. McFarland, B. H., and Inesi, G. (1970). Studies of solubilised sarcoplasmic reticulum. Biochem. Biophys. R e s . Commun. 41, 239-243. McGill, K . A., Smith, G . A,, Plumb, R. W., and Warren, G. B. (1980). Symmetry of phosphatidylcholine and phosphatidylethanolamine distribution in a population of sarcoplasmic reticulum vesicles sealed with their cytoplasmic side outwards. Submitted for publication. MacLennan, D. H. (1970). Purification and properties of an adenosine triphosphatase from sarcoplasic reticulum. J . B i d . C h e m . 245, 4508-4518. MacLennan, D. H., and Holland, P. C. (1975). Calcium transport in sarcoplasmic reticulum. Annu. R e v . Biophys. Bioeng. 4, 377-404. MacLennan, D. H., Yip, C. C., Iles, G. H., and Seeman, P. (1972). Isolation of sarcoplasmic reticulum proteins. Cold Spring Harbor Symp. Quani. Biol. 37, 469-477. Makinose, M. (1973). Possible functional states of the enzyme of the sarcoplasmic reticulum calcium pump. FEES L e u . 37, 140- 143. Martonosi, A. (1968). Sarcoplasmic reticulum. Solubilisation of microsomal adenosine triphosphatase. J . Biol. C h e m . 243, 71-81. Meissner, G., and Fleischer, S. (1971). Caracterisation of sarcoplasmic reticulum from skeletal muscle. Biochim. Biophys. Acta 241, 356-378. Meissner, G., and Fleischer, S. (1972). The role of phospholipid in Ca2+-stimulatedATPase activity of sarcoplasmic reticulum. Biochim. Biophys. Acia 255, 19-33. Meissner, G., and Fleischer, S. (1973). Ca2+uptake in reconstituted sarcoplasmic reticulum vesicles. Biochem. Biophys. R e s . Commun. 52, 913-920. Meissner, G., Connor, G. E., and Fleischer, S. (1973). Isolation of sarcoplasmic reticulum by zonal centrifugation and purification of Ca2+-pump and Caz+-binding proteins. Biochim. Biophys. Acra 298, 246-289. Montecucco, C., Smith, G . A., Warren, G. B . , and Metcalfe, J. C. (1977). In “Structure and Function of Energy Transducing Membranes” (K. van Damm and B . F. van Gelder, eds.), pp. 187- 192. Elsevier, Amsterdam. Moore, B. M., Lentz, B. R., and Meissner, G. (1978). Effects of sarcoplasmic reticulum CaZ+-ATPaseon phospholipid bilayer fluidity: boundary lipid. Biochemistry 17, 52485255. Nakamura, M., and Ohnishi, S. (1975). Organisation of lipids in sarcoplasmic reticulum membranes and Ca2+-dependentATPase activity. J. Biochem. (Tokyo) 78, 1039- 1045. Nakamura, H . , Jilka, R. L., Boland, R., and Martonosi, A. N. (1976). Mechanism of ATP hydrolysis by sarcoplasmic reticulum and the role of phospholipids. J . Biol. C h e m . 251, 5414-5423. Narasimhan, R., Murray, R. K., and MacLennan, D. H. (1974). Presence of glycosphingolipids in the sarcoplasmic reticulum fraction of rabbit skeletal muscle. FEES Leir. 43, 23-26. Nilsson, 0.S . , and Dallner, G. (1977). Transverse asymmetry of phospholipids in subcelM a r membranes of rat liver. Biochim. Biophys. Acia 464, 453-458. Packer, L., Mehard, C. W., Meissner, G., Zahler, W. L., and Fleischer, S . (1974). The structural role of lipids in mitochondria1 and sarcoplasmic reticulum membranes. Freeze-fracture electron microscopy studies. Biochim. Biophys. Acia 363, 159- 181. Peterson, S. W., Hanna, S . , and Deamer, D. W. (1978). Comparative studies of detergent effects on the calcium adenosine triphosphatase of sarcoplasmic reticulum: protein

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isolation, lipid exchange and enzyme transport function. Arch. Biochem. Bi o p h y~ .191, 224- 232. Racker, E. (1972). Reconstitution of a calcium pump with phospholipids and a purified Ca2+-adenosinetriphosphatase from sarcoplasmic reticulum. J . B i d . Chem. 247, 81988200. Racker, E . (1975). Reconstitution, mechanism of action and control of ion pumps. Biochem. Soc. Trcins. 3, 785-802. Racker, E., and Eytan, E. (1975). A coupling factor from sarcoplasmic reticulum required for the translocation of Ca2+ions in a reconstituted Ca2+-ATPasepump. J . B i d . Chem. 250, 7533-7534. Racker, E., Knowles, A . F., and Eytan, E. (1975). Resolution and reconstitution of iontransport systems. Ann. N.Y. Accid. Sci. 264, 17-31. Rothman, J. E., and Kennedy, E. P. (1977a). Rapid transbilayer movement of newly synthesised phospholipids during membrane assembly. Proc. N a t l . Accid. Sci. U . S . A . 74, 1821-1825. Rothman, J. E., and Kennedy, E. P. (1977b). Asymmetric distribution of phospholipids in the membrane of Beicillus meguterium. J . Mol. B i d . 110, 603-618. Rothman, J. E., and Lenard, J. (1977). Membrane asymmetry. Science 195, 743-753. Sandermann, H. (1978). Regulation of membrane enzymes by lipids. Biochim. Biophys. Acta 515, 209-237. Sarzala, M. G., and Michalak, M. (1978). Studies on the heterogeneity of sarcoplasmic reticulum vesicles. Biochim. Biophys. Acici. 513, 221-235. Shimshick, E. J., and McConnell, H. M. (1973). Lateral phase separation in phospholipid membranes. Biochemisiry 12, 2351-2360. Sigrist, H., Sigrist-Nelson, K . , and Gitler, C. (1977). Single phase butanol extraction: a new tool for proteolipid isolation. Biochem. Biophys. Res. Commun. 74, 178- 184. Sundler, R., Sarcione, S. L., Alberts, A . W., and Vagelos, P. R. (1977). Evidence against phospholipid asymmetry in intracellular membranes from liver. Proc. Ncitl. Accrd. Sci. U.S.A. 74, 3350-3354. Thorley-Lawson, D. A , , and Green, N. M. (1973). Studies on the location and orientation of proteins in the sarcoplasmic reticulum. Eur. J . Biochem. 40, 403-413. Vale, M . G . P. (1977). Localisation of the amino phospholipids in sarcoplasmic reticulum membranes revealed by trinitrobenzene-sulfonateand fluorodinitrobenzene. Biochim. Biophys. Acta 471, 39-48. Warren, G. B., Toon, P. A , , Birdsall, N. J. M., Lee, A. G., and Metcalfe, J. C. (1974a). Reconstitution of a calcium pump using defined membrane components. Proc. Nciil. Acud. Sci. U.S.A. 71, 622-626. Warren, G. B., Toon, P. A,, Birdsall, N. J. M., Lee, A . G., and Metcalfe, J. C. (1974b). Complete control of the lipid environment of membrane-bound proteins: application to a calcium transport system. FEBS Lett. 41, 122-124. Warren, G. B., Toon, P. A , , Birdsall, N. J. M., Lee, A. G., and Metcalfe, J . C. (1974~). Reversible lipid titrations of the activity of pure adenosine triphosphatase-lipid complexes. Biocherni,\try 13, 5501-5507. Warren, G. B., Houslay, M. D., Metcalfe, J. C., and Birdsall, N. J . M. (1975). Cholesterol is excluded from the phospholipid annulus surrounding an active calcium transport protein. Naiure (London) 255, 684-687. Warren, G. B., Hesketh, T. R., Smith, G. A , , Metcalfe, J. C., and Bennett, J . P. (1980a). Lipid requirements for the reconstitution of an active calcium pump. Submitted for publication.

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Warren, G. B., Bennett, J. P., Smith, G . A , , Hesketh, T. R., Houslay, M. D., and Metcalfe, J . C. (1980b). Optimal function of a reconstituted calcium pump. Submitted for publication. Zilversmit, D. B., and Hughes, M. E. (1977). Extensive exchange of rat liver microsomal phospholipids. Biochim. Biophys. Acttr 469, 99- 110. Zwaal, R. F. A . , Comfurius, P., and van Deenen, L. L. M. (1977). Membrane asymmetry and blood coagulation. NOIIII.E (London) 268, 358-360.

CURRENT TOPICS I N MEMBRANES AND TRANSPORT. VOLUME

14

The Asymmetry of the Hexose Transfer Svstem in the Human Red Cell Membrane W . F . WIDDAS Department of Physiology Bedford College (University of London) London. England

I . Kinetic Asymmetry . . . . . . . . . . . . . . . . . A . Historical . . . . . . . . . . . . . . . . . . B . Substrate Facilitated Transfers . . . . . . . . . . . . C . Affinity Constants for the Hexose Transfer System . . . . . . D . Heterologous Exchanges . . . . . . . . . . . . . . E . Initial Transfer Rates in Zero-trans Experiments . . . . . . . F . Flux Measurements under Different Conditions . . . . . . . G . Asymmetry of Affinities Derived from Inhibitor Studies . . . . . H . Asymmetric Affinity of Cytochalasin B . . . . . . . . . . I . Asymmetric Proteolytic Responses . . . . . . . . . . . I1 . Kinetics of Membrane Transfers with Asymmetric Affinities . . . . . A . The Basis of Kinetic Asymmetry . . . . . . . . . . . B . Asymmetric Affinities and the Two Site Model . . . . . . . C . Redistribution of Components in the Mobile Carrier Hypothesis . . D . The Regen and Tarpley Kinetics Applied to Nontransportable Inhibitors E . Applications of Asymmetric Carrier Kinetics . . . . . . . . F . The Effects of pH and Temperature . . . . . . . . . . . 111. Morphological Asymmetry . . . . . . . . . . . . . . . A . Asymmetry in the Erythrocyte Membrane . . . . . . . . . B . Asymmetry of the Membrane Environment . . . . . . . . C . Asymmetry of the Sugar Membrane Transfer System . . ? . . IV . Implications of Asymmetry . . . . . . . . . . . . . . . A . Consequences of Asymmetry . . . . . . . . . . . . . B . Physiological Implications . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

. 166 . 166 . 167

. . . . . . .

168 170 170 172 173 177 179 . 181 . 181 . 182 . 187 . 192 . 198 . 200 . 202 . 202 . 206 . 209 . 211 . 211 . 213 215

The present state of the Carrier Hypothesis was excellently reviewed in Volume 7 of this series by LeFevre (1975) and carrier-mediated glucose transport was critically discussed in the same year by Jung (1975). A I65 Copyright @ 1980 by Academic Press. Inc . All rights of reproduction in any form reserved ISBN C-12- 153314-X

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W. F. WIDDAS

somewhat different approach to the same topic has since been given by Naftalin and Holman (1977) and readers have these and several earlier reviews to choose from to obtain an overall survey of the properties and complexities of the facilitated transfer of sugars across the human erythrocyte membrane and other sites. One of the complexities of the system in the human erythrocyte is that of asymmetry. This asymmetry of the hexose transfer system has presented itself in several ways. It is the intention in this article to review some of these and to trace their developments and to consider the implications to the concepts of facilitated transfers generally. 1.

KINETIC ASYMMETRY

By asymmetry we mean a lack of similarity of reactiveness of those parts of the hexose transfer system which may be approached from the outside or from the inside of the cell membrane. Since the transfer system can be approached effectively only by sugars which are substrates for transport or by sugar derivatives or other molecules acting as inhibitors of transfer, asymmetry shows itself as an anomaly either in the kinetics of sugar transfers or in the kinetics of inhibition of sugar transfer. A. Historical Wilbrandt (1954) first reported asymmetric behavior of the glucose transfer system in human erythrocytes. He reported that phloretin esters (particularly polyphloretin phosphate) showed a most definite preference for inhibiting glucose exit from cells as compared to the entrance. A 32Plabeled specimen could be shown not to penetrate the cells and in consequence the inhibitor could be presumed to be acting on the outside surface. On the hypothesis of enzymes being involved, one for inward transport and the other for outward transport, it was postulated that only the one involved in putward transport was inhibited. Wilbrandt pointed out however that the kinetics of inhibition could give rise to marked asymmetries even with identical enzymes cis and trans and varying with the type of inhibitor. This original observation of inhibitor asymmetry was readily confirmed by Bowyer and Widdas (1957, 1958), but these authors showed that if hexose-absorbing sites on both sides of the membrane were involved even when transfer was undirectional then competition could largely explain the differences observed. This is because in exits the outside

167

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

sugar concentration is very small and cannot competitively displace the inhibitor as can occur during an entry experiment when the outside sugar concentration is large. However Bowyer and Widdas found that inhibition of exit developed more rapidly than inhibition of entry when cells were incubated with 1fluoro-2,4-dinitrobenzene(FDNB) as shown in Fig. 1. Since this inhibitor is noncompetitive and irreversible the asymmetric effect on exits and entrance called for an alternative explanation and although the possibility of FDNB causing “internal competition” was considered, subsequent work failed to find any supporting evidence (Sen and Widdas (1962b). The idea of internal competition was however revived by Regen and Tarpley ( 1974). Asymmetric inhibitory effects of irreversible and noncompetitive inhibitors have been further studied by Batt and Schachter (1973) and these will be discussed later. B. Substrate Facilitated Transfers

Sugars acting as substrates for the membrane transfer system appear to have a facilitatory effect on such transfer and one of the earliest kinetic anomalies to be observed was the more rapid rate of glucose-glucose

’41:1 0

1

1

1

1

1

1

100 200 300 Incubation time (min)

I

400

FIG. 1, Development of inhibition of glucose entry and exit on incubation with 1.4 m M FDNB at 20.5”C in the presence of 100 m M glucose. 0, Glucose exit; X , glucose entry. Exit and entry measured at 37°C. (From Bowyer and Widdas, 1958.)

168

W. F. WIDDAS

exchange, which could be measured with radioactively labeled sugar, relative to the maximal rate of net sugar transfer determined by osmotic swelling or shrinking methods. The first hint of this was given by Britton (1957) who observed a glucose exchange rate about 4-fold the maximum rate of net transfer found by Widdas (1954; see also Britton, 1964). This early observation was confirmed by LeFevre and McGinniss ( 1960) whose experiments also emphasized the different order of magnitude for glucose-glucose exchange relative to net accumulation. The effect was analyzed in more detail by Mawe and Hempling (1965) and by Levine et L I I . (1965) whose analyses concentrated on the accelerating effect of sugars on the trans side of the membrane in measurements of the flux of radioactive sugar. The hypothesis was that, in the mobile carrier model, there was a faster movement of carriers across the membrane if they carried a substrate sugar. Although acceleration of glucose efflux was seen with glucose (or galactose) in the outside medium sorbose and fructose appeared to have a retarding effect but there may have been osmotic complications at the high concentrations used with these sugars. From the low affinity of sorbose and fructose for the hexose system they would not be expected to show either acceleration or inhibition of glucose efflux.

C. Affinity Constants for the Hexose Transfer System

The earliest attempts at determining the half-saturation constant for glucose were based on competition experiments. Widdas (1953, 1954) based his estimates on the retardation of sorbose entry by glucose acting as a competitive inhibitor whereas LeFevre (1953, 1954) used the competition between glucose and phloretin. LeFevre estimated the half-saturation constant at between 7.5 and 10 mM while Widdas's results were between 7 and 17 mM. Thus both authors agreed that the magnitude of the half-saturation constant for glucose was about 10 mM at 37°C. Sen and Widdas (1960a,b, 1962a,b) introduced the method of following exits into low outside sugar concentrations to determine the half-saturation concentration (see Fig. 2a and b). They obtained a value of 4 mM at 37"C, i.e., less than half the best estimates based on sorbose and phloretin competition. The Sen- Widdas method involves following a saturated efflux into a series of outside solutions from which an influx is progressively built up as the concentration increases. The half-saturation determined may be

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

Q

I

$-

169

LL8.4mM

.

0 . 7 m ~ J2 . 7 m d 4.6mMJ 6.5mM

1 min

12.2mM

1.01

E 0.8

v

/

b

FIG. 2. (a) Tracings of a series of records from the photoelectric apparatus during "exit" experiments at 37°C and pH 7.4. Cells equilibrated in 76 m M glucose were losing glucose into media containing glucose at the concentrations shown. The linear part of each record has been produced to cut the baseline, and the time from injection of the cells to this intersection was measured for analysis of the results. (From Sen and Widdas, 1962a.) (b) "Exit" times obtained from records such as described in (a) plotted against the concentration of glucose in the outside media. The line (drawn by eye) gives two intercepts; that on the ordinate represents the time ( t o ) which would have been taken for exit into a glucose-free medium and that on the abscissa gives the concentration of glucose into which the exit time would be twice t o . (From Sen and Widdas, 1962a.)

170

W.

F. WIDDAS

viewed as that outside concentration which engenders a half-maximal influx in opposition to the saturated efflux. Miller (1965a,b, 1968a,b) repeated estimations of the glucose affinity constants using the Sen and Widdas method and on the basis of sorbose inhibition but also showed how data from isotopic exchange experiments could be used to derive the affinity constant for exchange. Miller drew attention to the variability of the results and showed that this occurred even when the different procedures were used in the same laboratory and the divergent results were therefore ascribed to shortcomings of the kinetics of the simple carrier hypothesis. Thus at 20°C Miller found the affinity constant for glucose to be 1.8 mM by the Sen and Widdas method, 17-23 mM by the inhibition of sorbose transport, and 38 mM from a Lineweaver-Burk type plot of isotopic exchanges. D. Heterologous Exchanges

Allied to the acceleration of the sugar flux during the exchange situation (relative to the net transfers) is the observation by Miller (1968a) that the initial rate of radioactive exchange could be increased more by some sugars than others. Thus mannose and galactose in the outside medium accelerated the efflux of labeled glucose more than nonradioactive glucose itself. This result is also not to be expected from the kinetics of the simple carrier model. E. Initial Transfer Rates in Zero-trans Experiments

Zero-trans experiments involve measuring the rate of transfer of sugar from one side while there is no sugar at the other side. This is an ideal which in an entry experiment can be approached only in the initial stages but is more closely realized in exit experiments where the volume of medium can be greatly in excess of the volume of cells from which the sugar is egressing. Lacko et af. (1972b) measured the initial uptake from media of varying glucose concentrations by restricting their radioactive measurements to the first 4 seconds after adding the cells. They were able to estimate the half-saturation concentrations for this initial influx at 0 and 20°C. They compared these with the half-saturation concentrations for exchange and those determined by the Sen-Widdas exit method. In addition to the affinities they compared the maximal rates of influx with those for exit and for exchange.

171

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

They also measured exchange influx with an arbitrary inside concentration of 38 mM. Their results are summarized in Table I. The results confirmed the increased rate of exchange relative to net transfer but clearly showed that the maximal rate for initial entry was significantly less than that for exit or for entry under the arbitrary exchange condition used. Indeed the latter entry had parameters which approached those derived from the Sen and Widdas type of exit experiment which may be understood when it is appreciated that the Sen and Widdas procedure is directed to finding an influx which equals half the saturated efflux. The other finding was the confirmation of Miller's observation that the half-saturation for exchange is quite different from that for influx and remains remarkably constant over the 20°C temperature range studied. Whereas the higher rate of exchange could be explained on the basis of substrate facilitation of carrier transfers relative to transfers of unsaturated carriers the low rate of maximal initial entry relative to exit could not be so explained. This and the large value of the half-saturation for exchange required asymmetry of a more fundamental kind in the transfer process. Miller (1971) measured the parameters of glucose and galactose efflux into sugar-free solutions and compared these with the parameters from Sen and Widdas type experiments, from exchange experiments, and also from the inhibition of sorbose transfer. For the zero-trans efflux he estimated the half-saturation for glucose at 7.4 mM and for galactose at 58 mM, both substantially higher than the Sen and Widdas values but lower than for exchange. TABLE I PARAMETERS OF GLUCOSE TRANSFER UNDER DIFFERENT EXPERIMENTAL CONDITIONS DETERMINED B Y LACKO et a / . (1972b)" Concentrations Experiment type Equilibrium exchange

Temperature ("C)

Maximal flux (rnmole liter-' min-I)

Half-saturation concentration

(mM)

Out (mM)

5-37

5-37

0 20 0 20 0 20 20

22.5 264 0.18 36 12.6 159 I79

20 20 0.2 I .6 0.65 1.7 2.0

In

0

0.1-18

Exchange influx

38

0.1-37

Sen- Widdas exit

76

1-8

Initial entry

(mM)

' The experiments included equilibrium exchange, initial entry, exchange influx, and Sen-Widdas type exits.

172

W. F. WIDDAS

Karlish et NI. (1972) also measured the parameters of glucose efflux under zero-trans conditions. By suspending preincubated cells in a large volume of medium they were able to follow the loss of radioactive sugar while the outside concentration was maintained very low. For cells preloaded with 80 mM glucose their outside medium was kept to 0.16 mM. Integrating the simplified kinetic equation and solving for K , and V,,, they obtained values of K , of 25.4 -+ 3.0 mM and V,,, of 139 2 I 1 mmole liter-' min-'. These authors showed a I0-fold difference between the K , for efflux and that obtained by the Sen and Widdas procedure at the same temperature. Since the Sen and Widdas procedure gives the halfsaturation concentration for influx and the zero-trans efflux experiments give the half-saturation concentration for efflux, the results indicated asymmetry of affinities of the transfer process. Miller (1969) deduced a similar asymmetry of affinities on the two sides of the membrane from the results of Levine et al. (1965) who had measured the egress of glucose from cells loaded in the range 0-100 mM at 25°C. Their results indicated that the internal half-saturation constant was about 20 mM and Miller contrasted this with the Sen-Widdas value of about 2 mM at the same temperature.

F. Flux Measurements under Different Conditions

The slow accumulation of kinetic anomalies and the tentative sugges. gathered momentum with the tion of asymmetry by Lacko et C J ~ (1972b) republication of asymmetric carrier kinetics by Geck ( 197 1). This together with earlier statements of kinetics to be expected with asymmetric affinities by, e.g., Regen and Morgan (1964) led investigators such as Batt and Schachter (1973) and Bloch (1974) to make direct comparisons of fluxes of sugars under a variety of experimental conditions including the initial entries and zero-trans effluxes referred to previously. These studies added to the evidence for an asymmetric transfer mechanism although the results were not wholly consistent. Thus while Bloch obtained results at 7°C which confirmed those of Lacko et al. in showing the correspondence of the half-saturation constant for initial influx (and influx with exchange) with those for Sen and Widdas exits, Batt and Schachter at 15°C found the half-saturation for influx to be higher than that for efflux. However, Batt and Schachter's influx measurements were based on data collected from 15-45 seconds as opposed to the 2-5 seconds by Bloch and 4 seconds by Lacko et al. and on theoretical grounds they are unlikely to be indicative of the initial entry rates. Both groups found high half-saturation constants for ex-

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

173

change and Bloch also found a high value for zero-trans efflux. Miller (1975) has drawn attention to the difficulties in following initial uptakes of glucose even in the short times used by Bloch. This casts doubt on the significance of low initial entry rates as evidence of asymmetry. Batt and Schachter (1973) also showed the asymmetric effect on fluxes of various noncompetitive inhibitors. Thus p-chloromercuribenzene sul(DCDS), and N fonate (PCMBS), 3,3’-di-2-chloroallyldiethylstilbestrol ethylmaleimide (NEM) inhibited efflux more than influx. None of these is a simple competitive inhibitor and thus these results extend the earlier observations of Bowyer and Widdas (1958) (Fig. 1) with FDNB and of Dawson and Widdas (1964) with NEM.

G. Asymmetry of Affinities Derived from Inhibitor Studies While it is suggestive that the half-saturation concentrations derived from entry and exit flux measurement denote different affinities of the hexose system on the outside and inside of the membrane, respectively, more direct evidence of this asymmetry has come from studies with nontransportable inhibitors. Baker and Widdas (1972, 1973a) showed that 4,6-O-ethylidene-a-~glucopyranose (ethylidene glucose) was a potent inhibitor of glucose exit ( K , ca. 5 mM at 37°C) although the compound was not transported on the hexose transfer system. Thus they found that the penetration of ethylidene glucose was much slower than would be expected of a sugar with a half-saturation close to that of glucose. The entry rate did not slow as the concentration was built up in steps and the curves fitted the integrated diffusion type equation. Neither glucose nor phloretin had an inhibitory effect on ethylidene glucose entry and incubation of cells with FDNB to inhibit glucose exit 95% was without effect on the rate of penetration of ethylidene glucose. On the other hand on incubation of cells with FDNB in the presence of ethylidene glucose the development of inhibition was retarded relative to a saline control. This was the opposite to the effects of transportable sugars (Krupka, 1971) but was similar to maltose which is a nontransportable compound. Baker and Widdas also showed that ethylidene glucose inside cells could not induce transient uphill influx as is readily seen with for instance 3-O-methyl glucose, a sugar with almost identical affinity as judged by the inhibition of glucose exits. Rosenberg et a / . (1956) had found that glucose benzoate penetrated cells rapidly and was less subject to inhibition by phlorizin. They found

174

W. F. WIDDAS

the compound penetrated bovine red cells which do not have a fast facilitated transfer system like human erythrocytes and penetration was presumably made possible by the increased lipid solubility conferred on the molecule by the benzoate grouping. In ethylidene glucose the dioxane ring serves the same purpose and the oil-water partition was found to be only a little less favorable than that of glycerol. Comparable rates of penetration of ethylidene glucose were found in adult guinea pig red cells to those in human red cells and penetration by diffusion through lipid parts of the cell membrane appeared to be the most probable explanation. That ethylidene glucose can penetrate the red cell membrane slowly but independently of the hexose system brings it into a special class of reagents which allows one to examine separately the reactions occurring inside and outside the cell. Thus it is possible to preload cells with ethylidene glucose together with a transportable sugar and subsequently to spin the cells down and resuspend them in a medium free of ethylidene glucose while the exchange flux of the transportable sugar is being measured. The egress of the preloaded ethylidene glucose is very slow and for the duration of the radioactive exchange of the transportable sugar the ethylidene glucose is exerting an inhibitory effect on the inside only. Similarly with ethylidene glucose in the outside medium the short time required to take samples for measuring the radioactive exchange of a transportable sugar does not allow appreciable entry of ethylidene glucose to the cell interior thus measurements with inhibitor only on the outside are also possible. It was found (Baker and Widdas, 1973b) that ethylidene glucose inside the cells was much less effective as an inhibitor of exchange than outside. Thus 200 mM ethylidene glucose inside was no more effective than 25 mM outside. Allowing for the lower affinity of the inside for glucose the asymmetry of affinity for ethylidene glucose was estimated at 40-fold whereas that for glucose at i6”C was 10-fold. Recent estimates with purified ethylidene glucose suggest the asymmetry of this compound may be as high as 60-fold (Baker er al., 1978). Figure 3 shows the asymmetric inhibition of glucose, galactose, and 3O-methyl glucose exchanges at 20 mM and 16°C by purified ethylidene glucose. In each case the asymmetry is similar in that 200 mM ethylidene glucose inside the cells has an inhibitory effect no larger than that exerted by 25 mM outside. The intersections of the lines occur where 1/J = 1/ V,,, and the value on the abscissa corresponds to the apparent halfsaturation constant for the inhibitor. This is an “apparent” and not a true half-saturation constant for reasons which are discussed later when the asymmetric kinetics are considered. A number of related compounds were also shown by Novak and

175

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

,120

--

Inside

,100

Glucose 160

1

160 5b 50 [Et hy I ide ne glucose]

100

150

200

(m M)

FIG.3. Asymmetric inhibition of galactose, 3-O-methyl glucose, and glucose exchange by purified ethylidene glucose. Lines and points represent the means of two similar results for each sugar for 20 m M exchange at 16°C. 0 , Glucose; 0, 3-0-methyl glucose; X , galactose. (From Baker et NI., 1978.)

LeFevre (1974) to be nontransportable inhibitors of the red cell hexose transfer system. 1,2-~-Isopropy~idene-~-g~ucofuranose (isopropylidene glucose) inhibits glucose exchange with a lesser degree of asymmetry from that of ethylidene glucose (Baker et nl., 1978). The asymmetry of methyl-2,3-di-O-methyl-c~-~-glucopyranoside (trimethyl glucoside) is less still. Barnett et al. (1973a,b) studied the inhibitory properties of a number of fluoride derivatives of glucose and other hexoses. Replacement of hydroxyl groups with hydrogen alters the affinity in a variety of ways: thus it increases the affinity at C-2 but decreases the affinity at C-3 of glucose. Bonding with the transfer site was presumed to involve the hydroxyls on C-I, C-3, and possibly C-4 with a complex effect at C-6 which may have an element of hydrophobic bonding. Essentially similar conclusions had been reached by Kahlenberg and Dolansky ( I 972) by studying the effect of glucose derivatives on the stereospecific uptake of D-glucose by isolated erythrocyte membranes. Barnett et al. (1975) found that 6-0-alkyl derivatives of galactose and glucose inhibited the glucose transport system when in the outside medium. The longer chain alkyl derivatives appeared to penetrate the red cells by a glucose independent pathway at rates proportional to their

176

W. F. WIDDAS

olive oil/water partition but when on the inside did not significantly inhibit sorbose entry or glucose exit. On the other hand propyl-P-D-glucopyranoside was an effective inhibitor when inside the cells. As a result of their studies Barnett et a l . (1975) have made the interesting suggestion that the reaction with the outside and inside sites for transfer through the cell membrane involves different ends of the glucose molecule. Thus they visualize a glucose molecule, approaching from the outside, entering a reactive cleft in the transfer protein with the C-1 end of the pyranose ring leading. The transfer protein is presumed to undergo a conformational change which opens up the protein around the C-1 end of the sugar while closing it behind the C-4-C-6 end (see Fig. 4). The cleft, initially open to the outside, is, by the conformational change, transformed to an inward facing mode. Figure 4 is a representation of the model for glucose transfer proposed by Barnett et al. (1975). In illustrating the hypothesis they point out that 6-O-propyl-~-glucose(R = C3H,; R' = H in Fig. 4) can bind to the transport system in conformation A but for steric reasons is not transported. Similarly propyl-P-D-glucopyranoside (R = H; R' = C3H,) can bind to conformation B without being transported. D-Glucose can of course bind to both conformations and the conformational change brings about the effective transfer of that sugar from one side to the other. The asymmetry of the two sides of the protein cleft was also seen as the explanation for the finding of Kahlenberg and Dolansky (1972) that methyl-a- and P-D-glucopyranosides were inhibitors of glucose binding to membrane fragments although showing no competitive inhibition for Conformation A membrane protein

Conformation B

FIG. 4. Possible model for sugar transport in the human erythrocyte. 6-O-Propyl-~glucose (R = C,H,; R' = H) can bind to the transport system in conformation A but cannot be transported for steric reasons. Similarly, propyl-P-D-ghcopyranoside (R = H; R' = C,H,) can bind to conformation B but cannot be transported. D-GhCoSe can bind to both conformations and is transported by the conformational change. (Modified from Barnett et (//., 1975.)

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

177

glucose transfer in intact cells. The inhibition in fragments was seen as due to these glycosides being able to approach from the inside and bind to the transfer system in conformation B. The suggestion that glucose molecules substituted in the C-1 position act as good competitive inhibitors on the cell interior but react poorly or not at all outside while molecules with substitution on C-6 react on the outside but, like ethylidene glucose, react poorly on the inside would support their hypothesis. However, the reactivity of several nontransportable inhibitors is not so clear cut as this. Thus Baker et al. (1974, 1978) find that isopropylidene glucose with its dioxane ring structure involving C-I and C-2 of the glucose molecule nevertheless has higher affinity for the outside than inside and this may also apply to methyl-2,3di-O-methyl-a-D-glucopyranoside if allowance is made for the fact that nontransportable inhibitors may have a higher true affinity for the outside than that indicated from the inhibition of exchange (Baker et al., 1978).

H. Asymmetric Affinity of Cytochalasin B The different reactivity of the internal and external sites of the hexose transfer system is shown to a most marked degree by the inhibitor cytochalasin B. This fungal metabolite was shown by Taverna and Langdon (1973a) and by Bloch (1973) to be a powerful inhibitor of glucose transfers in the human red cell. Studying its inhibition by the Sen and Widdas exit method they found that the sugar half-saturation constant was not increased by the inhibitor which they concluded was noncompetitive. However, Lin and Spudich ( I974a) found that the high-affinity binding of cytochalasin B was reduced by about 80% in the presence of a high concentration (ca. 500 mM) of D-glucose but not L-glucose. That there was competition for exchange in the range 10-100 m M glucose was shown by Jung and Rampal (1975, 1977), and Taylor and Gagneja (1975) reported competition for exits. Basketter and Widdas (1977, 1978) showed that 3-O-methyl glucose exchange was competitively inhibited (Fig. 5) but confirmed that there was no increase of the sugar half-saturation constant in exits. This competition for exchange but not for exits was seen as evidence for a reaction between cytochalasin B and the inside hexose transferring site only. The lack of reaction with the outside site would explain the unchanged sugar half-saturation constant in exit experiments whereas the reaction with the inside site would be sufficient to give the typical competitive results for equilibrium exchanges.

178

W. F. WIDDAS

300 500 1 D O - M e t h y l glucose]

100 0

100

b-’>

FIG.5. Lineweaver-Burk type plot of 3-O-methyl glucose exchange in the range 2-40 mM at 16°C in the absence (0)and in the presence of 0.25 p M cytochalasin B ( X ) . Points are means of two experiments with similar results. (From Basketter and Widdas, 1978.)

In conforming this interpretation Deves and Krupka (1978b) have advanced more direct experimental evidence of internal competition. They used zero-trans exit experiments in the absence and presence of cytochalasin B. From an analysis of the kinetics of inhibition (with nontransportable inhibitors) Deves and Krupka (1978a) have shown that, in a plot of the reciprocal of the transfer rate against the reciprocal of the inside sugar concentration, an inhibitory effect on the outside would cause a distinct shift of the intercept on the ordinate. This was not seen but the line had a steeper slope indicating competition on the inside (Fig. 6). Thus cytochalasin B appears to show marked asymmetry with a high affinity for the hexose sites inside and little or none for those outside. The asymmetry of binding of cytochalasin B offers a reinterpretation of the interesting observation of Masiak et al. (1977) that the inhibition of hexose transfers by dione compounds is protected by glucose (150 mM) but is not protected (and may be enhanced) by cytochalasin B. Rather than assume that cytochalasin B does not react with the hexose site per se it may be suggested that the dione compounds react with the outward facing components of the hexose transfer system where cytochalasin does not compete. Taylor and Gagneja (1975) have advanced stereochemical evidence suggesting that cytochalasin B probably reacts with the hexose site and the competitive experiments are in favor of there

179

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

being mutual interference between glucose and cytochalasin B either at the same site or at closely associated sites on the inside of the membrane (Basketter and Widdas, 1978). 1. Asymmetric Proteolytic Responses

Proteolytic enzyme digestion of ghosts at first tended to confirm that the hexose transfer system was an intrinsic component of the more hydrophobic parts of the red cell membrane. Thus ghosts, following Pronase treatment which caused the loss of over 50% of membrane proteins, retained the facilitated transfer system for sugars (Jung et a/., 1973). Following mild trypsin digestion (Avruch et a/., 1973; Carter et al., 1973) which resulted in selective hydrolysis of membrane proteins in Bands 3 and 4 the ghosts still retained many of the properties characteristic of glucose transport in intact cells.

I

1

2 loo [Glucose]

3

4

5

(rnM)

FIG.6. Initial rates of glucose exit in the presence or absence of 4.2 /.LM cytochalasin B determined from the appearance of uniformly labeled ['4C]glucose in the external medium (upper and lower lines, respectively). Rates are given as mmoles liter-' min-I. Exit medium, 0.9% NaCl + 5 m M sodium phosphate, pH 7.0, 25°C. Quenching medium, cold 2 m M HgCI, and 1.25 m M KI and 2% NaCl with centrifugation at 1°C. Cell concentration 0.76%. Radioactivity determined by scintillation counting in Aquasol. (From Deves and Krupka, 1978b.)

180

W. F. WIDDAS

However, Lin and Spudich (1974a) showed that the high-affinity binding of cytochalasin B was largely lost when trypsin or Pronase was incorporated into red cell ghosts but was unaffected by these enzymes when they were in the outside medium and the cells were intact. The high-affinity binding was not only competitively displaced by high concentrations of sugars such as D-ghCOSe, as referred to in the previous section, but with a series of eight different sugars the individual displacements corresponded to their relative affinity for the hexose transfer system. Treatment with p-chloromercuribenzoate, an inhibitor of the hexose transfer system, also prevented cytochalasin B binding. The authors therefore interpreted the loss of high-affinity binding as being due to a degradation or change in the protein involved in hexose transport. Using low and high ionic strength solutions they released most of the proteins found on electrophoresis in Bands 1 , 2, 5 (Fairbanks et nl., 1971) and in Band 6, respectively, but found that this had little effect on the high-affinity binding of cytochalasin B. Low and high salt treatments do not remove Bands 3, 4.1, 4.2, or the protein designated as PAS-1 which shows up with periodic acid and Schiff reagent. Masiak and LeFevre (1977) treated the outside surface of erythrocytes with the proteolytic enzymes trypsin and a-chymotrypsin but without observing any inhibition of the hexose transfer. The incorporation of either enzyme inside erythrocyte ghosts (which were then resealed) produced a progressive reduction of hexose transfer as the incubation time increased. These authors noted the gradual loss of the spectrin band with the proteolytic treatment used but degradation products of spectrin mask possible changes in later bands and the interpretation in terms of proteins involved in the hexose transfer system was difficult. These latter two sets of experiments with proteolytic enzymes are however complementary and taken with the evidence that cytochalasin B reacts only with the internal sites of the hexose transfer system assume a clearer significance. Thus it can be seen that the hexose transfer system in the membrane is susceptible to attack by proteolytic enzymes only from the inside of the cell and this indicates either a different chemical environment or a different chemical morphology in the transfer protein itself on the two sides of the membrane. The studies with cytochalasin B and proteolytic enzymes taken with those of the nontransportable inhibitors discussed earlier combine to provide strong indications of chemical as well as kinetic asymmetry in the hexose transfer system of the human red cell.

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

181

II. KINETICS OF MEMBRANE TRANSFERS WITH ASYMMETRIC AFFINITIES

A. The Basis of Kinetic Asymmetry Rosenberg and Wilbrandt (1955) reviewing the kinetics of membrane transports involving chemical reactions either of the simple carrier type or of a carrier in cooperation with an enzyme considered the possible bases of asymmetry. They pointed out that the system could be asymmetrical in two respects as regards either reaction velocities or the equilibria. They showed that velocities for entry or exit would not necessarily correspond if the inside and outside concentrations were reversed. This aspect was further developed by Schultz (1971) who made the point that either linear or nonlinear systems acting across a composite membrane may present asymmetric fluxes when the concentration gradient is reversed. However the asymmetry of affinities was not considered by Rosenberg and Wilbrandt except as a possible means of effecting uphill transfer in energy coupled systems. Regen and Morgan (1964) removed the restriction of symmetry from the kinetics of a simple carrier transfer and opened up the possibility of asymmetric affinities at the two sides of the membrane. They derived equations for the net transfer of sugars under these general conditions and since there should be no net transfer when the inside and outside concentrations were equal they could define the relationship which must hold between the various equilibrium constants. The rate constants governing individual steps in the process were grouped to give composite constants which could be determined and these could be used to test whether simplifying assumptions of the symmetrical carrier model were in fact justified. Regen and Morgan (1964) carried out experiments on rabbit red cells with the aid of this kinetic analysis but found no evidence of asymmetry in those cells. They did not use their method of analysis on human red cells. Similar kinetics were developed by Britton (1966) and these have been further extended to develop a series of tests and rejection criteria for the simple carrier (Lieb and Stein, 1971). The kinetics are currently formulated (Lieb and Stein, 1974) in relation to a number of resistance terms of a form extensively used by Geck (1971). An interesting conclusion from these kinetic studies is that a number of carrier models with either a single or several sugar complexing sites within the membrane will, from steady-state measurements, be indistinguishable and fit generally similar kinetic expressions. Wilbrandt (1972a) in an attempt to resolve kinetic discrepancies be-

182

W. F. WIDDAS

tween carrier affinities as determined from Lineweaver-Burk treatments of zero-trans exits and from exits using the Sen-Widdas (1962a) procedure considered that the reaction rates between sugar and carrier may be finite relative to the movements of the carrier in the membrane. He also considered the reaction rates could be different inside and outside though their ratio (from which the K , was derived) was assumed to be constant. With these assumptions Wilbrandt found that the resistance terms involving the reactions on the inside came out higher than resistance terms for the outside but that taking these differences into account a more consistent value for the affinity term ( K , 5 5.2 mM) was obtained by both the Lineweaver- Burk and Sen- Widdas approach. However using an indifferent tracer in the presence of equilibrated concentrations of glucose Wilbrandt observed different values of the K , for glucose. He used D-xylose and D-arabinose as tracer sugar. He therefore suggested there may also be diffusional resistances equivalent to unstirred layers contributing to the anomaly and concluded that the diffusional resistance inside the cell must be much higher than that outside. The complication of unstirred layers had been raised by Naftalin (1971) and commented on also by Miller (1972) and by Lieb and Stein (1972b). Wilbrandt (1977) in extending and reanalyzing this study found the results to be generally in favor of an explanation in terms of structural asymmetry such as might be explained by a transfer system deep in the membrane with diffusional resistances to be overcome in approaching the transfer sites from outside or from inside. Wilbrandt's approach however, assumed that the inside and outside K,s of the transfer system were symmetrical. Regen and Tarpley (1974) applied kinetics with asymmetric affinities to various data collected from the literature. They arrived at essentially the same conclusion as Wilbrandt, namely, that any diffusional resistances involved were much greater inside the cell than outside. Edwards (1974) also argued that a carrier model with a diffusion step inside the cell would explain many features of glucose transport kinetics. This aspect will be further considered in Section II1,B when discussing the asymmetric membrane environment.

B. Asymmetric Affinities and the Two Site Model

The underlying principles involved in the asymmetric affinity assumptions can be better appreciated from the simplest treatment and will first be exemplified with the use of kinetics described by Baker and Widdas ( 1973b).

183

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

In Fig. 7 is represented a two site transfer system with reactive sites in each interface which have different affinities for sugar in the associated medium. If the half-saturations for the sites are 4 and a4, respectively, a will be the asymmetry of affinities. The probabilities that at any time a site or component will be saturated with sugar is taken to be the same as the saturation fraction of all components in that interface as given by the Michaelis-Menten relationship. The probability of unsaturation is defined as the unsaturation fraction of all components in that interface. Transfer from side 1 to side 2 is presumed to require that side I is saturated with sugar and side 2 unsaturated. These probabilities are represented by C , / ( C , + $4 and a + / ( C 1 + a+), respectively, where C , and C 2 are the sugar concentrations at side 1 and 2 . The probability that transfer will occur is proportional to a rate constant multiplied by these two probabilities. Thus Transfer 1-2

=

Vl.--.

a4

Cl

c ,+ 4

c2

+ a+

FIG.7 . The schematic basis of the Two Site Model. Each site X and Y is presumed to be i n dynamic equilibrium with the sugar in the medium at sides 1 and 2, respectively, but with different affinities. The half-saturation constants are I$ and a $ , respectively, where a is the asymmetry factor. In (A) both sites are unsaturated. In (B) site X is saturated and the sugar either returns t o side I or vectorially transfers to site Y and may dissociate into side 2 effecting a net transfer from side I to side 2. In (C) site Y is saturated and as the reverse of ( B ) may effect a net transfer from side 2 to side 1. In (D) both sites X and Y are saturated but it is assumed an intramembranous molecular exchange may occur.

184

W. F. WIDDAS

where V , is the rate constant. Similarly the probability of transfer from side 2 -+ 1 is given by Transfer 241

=

V,'

c 2

-.

4

c, + a 4 c , + 4

where V , is the rate constant. However it is also proposed that if both sites are occupied an exchange can occur between the sugar molecules occupying the respective sites. The influx and efflux of this exchange reaction would be equal and cancel out when net transfer is being considered but is included for completeness. Thus the net transfer can be expressed as [Eq. ( I ) ] Net transfer 1-2

=

V I C I . a 4+ V l . \ C I C ,- V , C , + - V,.:\C,Cz (CI + 4)(C, + 04)

(1)

where VEXis the rate constant for the exchange reaction. One of the first principles of an asymmetric affinity scheme is that there should be no further accumulation of sugar once the inside concentration builds u p to be equal to the outside concentration. In this simple treatment the condition is met if V , = a V , , thus the rate constant for transfer from the low-affinity side must be higher than that from the highaffinity side and the ratio of this difference must equal the asymmetry factor. Why this relationship should hold is not clear but one could visualize that the glucose on the high-affinity side dissociates less readily than that from the low-affinity site and that transfer through the membrane involves a vectorially directed dissociation. In the presence of a competitive inhibitor the saturation by sugar is given by C c + 4(1 + Z/4J where I and 4, are the concentration and half-saturation constant of the inhibitor. Allowing for asymmetry of inhibitor affinities and making the assumption that the inhibitor cannot cross the membrane on the hexose system nor can it exchange across the membrane for a sugar molecule then the transfer in the presence of inhibitors can be represented by [Eq.

(211

where i is the asymmetry factor for the inhibitor's affinities.

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

185

These kinetics allow an interpretation of the effects of inhibitors in two practical types of experiments, namely, the Sen- Widdas exits (Sen and Widdas, 1962a,b) and equilibrium exchanges. 1 . SEN-WIDDASEXITS The principle of the Sen-Widdas experiments is to carry out exits from cells preloaded with a high concentration of sugar either into very low sugar concentrations at various inhibitor concentrations o r into various low sugar concentrations at constant or zero inhibitor concentrations. Thus Eq. (2) can be simplified by neglecting C , in the numerator and ignoring the exchange terms to give [Eq. ( 3 ) ]

In practice the exit time found by extrapolating the linear part of the exit record to the equilibrium level is used; this is proportional to the reciprocal of the exit rate so that

where S' is the content of sugar in the cells initially. This shows that the exit time is a linear function of the outside sugar concentration C , (if the inhibitor concentration is constant or zero) and also the exit time is a linear function of Z if C , is held constant. What is not so readily understood is that the apparent half-saturation constant (the value of C , which doubles the exit time relative t o that when C , = 0) is changed only with an outside competitive inhibitor and not with an inside competitive inhibitor. This can be seen however by a closer inspection of Eq. (4). Thus the uninhibited half-saturation constant would be a value of C , equal to the outside half-saturation constant, i.e.,

c , = +.

With an outside competitive inhibitor the value of C , would need to equal +( 1 + Z1/#q) to double the exit time. However with only an inside competitive inhibitor the exit time (when C = 0) would be doubled when

c , = 4.

Thus, on the basis of these simple asymmetric kinetics, the Sen- Widdas exit technique can be visualized as giving the half-saturation of the outside site and in the presence of inhibitors will show an increase in the half-saturation for the sugar only if the inhibitor is competing for the outside site.

186

W. F. WIDDAS

2. EQUILIBRIUM EXCHANGES

In carrying out equilibrium exchanges the sugar is at equal concentrations inside and outside the cells. The flux in one direction is measured by the use of radioactive tracer sugar molecules. To study the halfsaturation for exchange such flux measurements are carried out at a variety of increasing concentrations of sugar and the reciprocal of the flux plotted against the reciprocal of the sugar concentration in a typical Lineweaver- Burk type plot. The unidirectional flux is given by

J=

[ C + 441 +

V , a + C + VExC2 I1/h>l [ C + a+(l +

12/i+J1

(5)

The rate constant for exchange is larger than that for net transfer and as C increases the net transfer term may be neglected so that

L-['

VEX

1

+(:

1

+;)I

[ + %( 1

1

+$-)I

(6)

VEX

Equations (6) and (7) show that the Lineweaver-Burk plot of uninhibited flux measurements will be largely influenced by the large half1. For the same reason it will be more saturation value, i.e., a + if a influenced by inside inhibitor (unless i is correspondingly large). However if C is maintained large while the inhibitor concentration is varied the values of the inhibitor half-saturation constants should be determinable from the point on the line where 1/J = 1 / v E X . But as pointed out by Baker et a / . (1978)for the outside acting inhibitor the intercept occurs where

*

I

- -$1

-(1

+ a + a+/C) 1 + 10 + I 2 + a$/C 1+1

1

6

when dealing with an asymmetry of ca. 10 and with values of @ ca. 2 mM and C = 20 mM. Thus the intercept value of - I may be as large as 6-fold the half-saturation constant derived from Sen- Widdas exit measurements. From an inside acting inhibitor the corresponding intercept occurs

187

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

where

I -_ ic#lI

-(I

+ a + ac#l/C)

A

a+-

a4

1

+ 10 + 1 10+ I

+

1.1

C

and is a much closer approximation to the true inhibitor affinity on the inside. Thus on the basis of these kinetics the equilibrium exchange in the absence of any inhibitor can be interpreted as giving a half-saturation value approximating that of the higher valued site (that is the inside site for human red cells with common sugars). When inhibitors are present the discrepancy observed when using exchange experiments to estimate the outside affinity of competitive inhibitors compared with the SenWiddas method also receives an interpretation. Therefore Sen- Widdas experiments will given an indication of the affinity and reactiveness of the outside site and equilibrium exchanges will give an approximation to the affinity of the inside site for transported sugars. A measure of the asymmetry factor can thus be determined from a combination of these procedures (Widdas, 1974). The reactiveness of both sites with nontransportable inhibitors may be studied by equilibrium exchange experiments but the influence of asymmetric affinities is complex and caution is required in interpreting the quantitative results. C. Redistribution of Components in the Mobile Carrier Hypothesis

The simple kinetics described by Baker and Widdas (1973b) represent a special case where transfers and inhibitions are occurring without any significant redistribution of the components as between inward facing and outward facing conformations. With the original mobile carrier hypothesis different mobilities of free and complexed carriers through the membrane (different both for inward and outward transfers) were considered but it was proposed (Widdas, 1952) that provided there were no structural restrictions the components would rapidly come to a steady state in which equal numbers of carriers would leave each interface in unit time. Thus for every different experimental situation there would be an appropriate redistribution of carriers and the kinetics could be modified by this redistribution. With asymmetric affinities at the two sides uneven mobilities of the complexed and free membrane components are a necessary provisions to ensure that sugar is not accumulated beyond the equilibrium value (Regen and Morgan, 1964).

188

W. F. WIDDAS

A hypothetical example of redistribution was given by Baker et nl. (1978) and is reproduced as Tabel 11. An asymmetric affinity factor of 10 requires that the cycle of events involved in an efflux should occur 10 times more readily than the cycle of events for an influx. An efflux cycle can be visualized as an outward movement of the complexed carrier, followed by an inward return of the free carrier. The asymmetry factor can be divided up between these two events. Thus in

TABLE I1 STEADY STATEOF AN ASYMMETRIC TRANSFER SYSTEM W I T H REDISTRIBUTION OF COMPONENTS SHOWING THE CHANGE GIVING50% INHIBITIONDUE TO A N OUTSIDE NONTRANSPORTABLE INHIBITOR" Parameters

Outside

Half-saturation constant Allotted value Sugar concentration

Inside

4

04

2 mM 2 mM

20 m M 2 mM

4

2

2

7

Components unsaturated

u = asymmetry factor u = 10 Exchange condition

state of 30 components

Uninhibited-steady Components saturated

Comment

4

20 51

Rate constants ensure equal exchanges in unit time

7

I

Total

22

8 50% Inhibition-steady

Components saturated

2

I

state of 30 components I

L

Components unsaturated

2

zy

10

Exchanges are equal but half the previous value

51 7 L

1

Components inhibited

15

Total

Note half of all components inhibited

19

Saturation outside =

L

2

11 L

+ 2( I + I/bi) = 19

I Therefore - =7.5

41

" Fraction saturated = C/(C + 4,) where 4, is the appropriate half-saturation constant. Fraction unsaturated = 4J(C + 4,). In the presence of inhibitor the denominator becomes C + +,(I + I / + , ) . The ratio of y to 1 is irrelevant in exchange conditions but affects the kinetics in other situations. Modified from Baker et u l . (1978).

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

189

the example of Table I1 the efflux of a complexed carrier has a rate constant twice that of influx whereas for the free carrier the rate constant for influx is 5-fold that for efflux. Examples can be prepared in which the ratios are 10 and 1 , 3 and 3.3, o r any other arbitrary pairs of values chosen so that their product is 10, i.e., equal to the asymmetry of affinities. The way in which redistribution affects the kinetic properties is shown by the hypothetical case of an inhibitor fixing 50% of all the components (drawn from both interfaces) on the outside. This reduces the exchange flux to half of the uninhibited value but in doing so has brought about a major redistribution of the components between inward facing and outward facing modes. In consequence of this redistribution the inhibitor is actually complexing 79% of the outside components (considered separately) and there is no simple relation between the percentage of outside components complexed and the percentage of inhibition of exchange. In this example 50% inhibition of exchange would actually require an inhibitor concentration of Z = 7.54,.This illustrates how an asymmetric transfer involving redistribution of components would, for 50% inhibition of exchange, require an outside inhibitor concentration several fold larger than that determined for 50% inhibition of exit in a Sen-Widdas experiment. Table I11 shows the distribution of components between outside and inside at different exchange concentrations and illustrates the case of an outside inhibitor which complexes only 50% of the outside components (considered separately). The asymmetry factor and ratios of the mobilities are arbitrarily taken to be the same as for Table 11. Figure 8 shows these distributions graphically and emphasizes how at low exchange concentrations the actual inhibition falls short of the 50% which might be expected from the inhibition of outside components. Taking a point close t o zero sugar concentration as the extreme example the components outside ( 16.7% in the uninhibited state) increase t o 28.6% when 50% inhibited. Of these 28.6% half are complexed with inhibitor but that still leaves 14.3% for sugar exchange relative to 16.7% in the uninhibited state. This represents a reduction of only 14.3% and corresponds to the actual inhibition of exchange. It should be noted that it is also the percentage of all the components complexed by the inhibitor. The inside components initially 83.3% are reduced to 71.4%; this is also a reduction of 14.3%. As more of the components become distributed to the outside then 50% inhibition of the outside components has a correspondingly larger effect in spite of the further redistribution of components which this brings about. This is because the proportion of components left facing

190

W. F. WIDDAS

TABLE I11 CALCULATED DISTRIBUTION OF COMPONENTS BETWEEN INSIDE A N D

EXCHANGE CONCENTRATIONS AND THE EXCHANGE WHEN 50% OF THE OUTSIDE COMPONENTS ARECOMBINED WITH ~NHIBITORa

OUTSIDE AT DIFFERENT INHIBITION OF

Exchange sugar conc ( 0 in and out

0- 0 2-2 10-10

20-20 30-30

Uninhibited distribution of components

Inhibited distribution of

Inhibition of exchange which would be observed (%)

In (9%)

out (7%)

components In Out (9%) (%I

83.3 73.3 55.6 47.6 43.9

16.7 26.7 44.4 52.4 56.1

71.4 58 48.5 41.2 28.1

28.6 42 61.5 68.8 71.9

14.3 21 31 34.4 36

a The redistribution is calculated o n the basis of 10-fold asymmetry and rate constants a s illustrated in Table 11. The half-saturation for the sugar is set at 6 = 2 m M a s is the half-saturation for the inhibitor 4, = 2 m M . Of the outside components 50% are considered t o be combined with a nontransportable inhibitor of concentration I such that for the outside components I / C + b(l + I / I $ ~ )= 1/2 or I = C + 2. Note that the percentage inhibition is equal to the percentage of all components complexed by the inhibitor, i.e., half those distributed outside in the inhibited state.

inside are reduced and the redistribution (which occurs on the development of outside inhibition) brings about a larger percentage fall on the inside. It follows that the actual inhibition is also larger. The same sort of redistribution could be brought about by increasing the inside concentration of transportable sugar while keeping the outside sugar at a low level. The effect is illustrated in Fig. 9 in which the redistribution is calculated on the basis used by Baker et nl. (1978). This illustration also assumes a 10-fold difference in the basic rate constant for saturated components relative to unsaturated components. Figure 9 shows that 50% inhibition of outside components (which would be obtained at a constant outside inhibitor concentration) would have an increasingly potent inhibitory effect on efflux as the inside concentration of transportable sugar was increased. The same pattern would apply to other levels of outside inhibition. The greater inhibitory effect of ethylidene glucose in competition with 2 mM 3-O-methyl glucose when the inside concentration was raised from 2 to 30 mM was demonstrated by Baker et al. (1978). Batt and Schachter (1973) observed differences in the kinetics of initial

191

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

influx and efflux which were most marked when the inside concentration was changed and these results also indicated effects of redistribution as well as asymmetry. They discussed an interpretation based on two carrier states the conversion from one to the other depending on intracellular glucose or other sugar. With an asymmetric system the redistribution effects complicate the interpretation of inhibitory affinities as illustrated in Tables I1 and 111 and lead to the interesting conclusion that an inhibitor acting at one side only brings about a percentage inhibition equal to the percentage of all the components which it complexes at that side and is not simply related to the percentage of components complexed in the particular interface. Redistribution of components between an inward facing and outward facing mode is inherent in the kinetic treatments of Regen and Morgan (1964). Britton (1966), Geck (1971), Lieb and Stein (1974), and Regen and Tarpley (1974) all based on the mobile carrier model. However actual translation of the components between the interfaces of the membrane is not necessarily involved. A conformational change which might be as

8oi

180

I

I

I

k 60 -+%

1Inhibiition 140

___

/*-

__ - - - - *--- - - - -- - -4 Exchange

1

;20 I

I

Inside conc (rnM)

r I I

01

0

10

20

to

30

FIG.8. Redistribution of membrane components a s a function of sugar concentration in equilibrium exchanges. Continuous lines (and left-hand scale) represent the components with an outward facing mode in the uninhibited state and when inhibited to the extent that one-half of the outward facing components are complexed by a nontransportable inhibitor. Interrupted line (and right-hand scale) represents the inhibition of exchange which would actually be observed. Note that inhibition of exchange is lowest at low sugar concentrations and is the same a s the overall percentage of components combined with inhibitor. Calculations based on the parameters used for Tables I1 and 111.

192

W.

80 -

F. WlDDAS

80 a I

L I

I

60

I % Inhibition

I - - _ - -_ _~

Efflux reduction

41

I

:!+ 140

------a-.,

,

L

L

Inside c o n c (mM)

I

FIG.9. The effect of changing the inside sugar concentration on the redistribution of membrane components. Continuous line (and left-hand scale) represent the components with an outward facing mode in the uninhibited state and when inhibited to the extent that one-half of the outward facing components are complexed by a nontransportable inhibitor. The sugar concentration on the outside is assumed to be 2 m M throughout while that to which the cells have been equilibrated varies from 2 up to 80 mM. Interrupted line (and right-hand scale) represents the inhibition of eMux actually observed. Calculations based on the parameters of Table I1 but with y = 10 x 1.

minimal as a swing of one or two hydrogen bonds (Vidaver, 1966) may be all that is required to change the sugar-reacting sites from an inward facing to an outward facing mode. The asymmetric kinetics published by the authors quoted above are described in a wealth of mathematical terms which tend to obscure the underlying mechanisms to all but the expert kineticist and the principles of redistribution of components and their effects are particularly difficult to appreciate without working through examples as illustrated here. Nevertheless some of the parameters, expressed as groups of rate constants, can be interpreted with fair precision and compared with the parameters based on the simpler kinetic treatments. In this article only the wider interpretations will be discussed and for kinetic details the reader must refer to the original articles. D. The Regen and Tarpley Kinetics Applied to Nontransportable Inhibitors

Lieb and Stein (1974) reviewed the kinetics of asymmetric carriers and showed that there was an overall consistency in the kinetics, whether

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

193

these were based on a single complex in the membrane-that is, one formed from reaction of sugar and empty carrier at either side without providing for translocation of the complex between the interfaces-or whether the kinetics were based on the more conventional two complex model in which both empty carriers and sugar-carrier complexes have kinetic terms for mobility across the membrane. Although they point out that steady-state kinetics do not enable one to distinguish between the various formulations the single complex model presents difficulties in interpretation of the reactions with nontransportable inhibitors which can react from either interface. The complexes so formed cannot be kinetically identical and one would need to postulate three complexes-one at either side when nontransportable inhibitors were involved and an internal one which was formed by transportable sugars. The two complex model on the other hand provides for mobilities in the membrane of the sugar-carrier complexes which depend upon the particular sugar involved and for nontransportable inhibitors it is plausible and convenient to set the rate constant for transfer across the membrane at zero. Thus the kinetics published earlier in the same year by Regen and Tarpley (1974) which were based on the two complex model and which provided for the case of sugars in competition are suitable for illustrating the more complex asymmetric kinetics. The kinetics provide for redistribution between the interfaces as well as asymmetry of the internal and external affinities. In the following discussion detailed reference to the individual rate constants will be omitted. In the presence of a competing sugar of concentration So and Si outside and inside the cell, respectively, the transfer of glucose (Ug) is given by

where Go G,

concentration G at outside surface = G , - U , / D , concentration in the external medium and D o is a diffusion constant for movement between the interface and bulk volume G i = concentration G at inside surface = G , + U , / D i G , = concentration in the cell and D i is the internal diffusion constant S o and S i are similarly defined for sugar S F , = activity constant for G transport = V / K , for G transport K , , = Michaelis constant for G entry K g i = Michaelis constant for G exit R , = flux ratio constant for G R , = flux ratio constant for S Bg = half-maximal constant for G exchange =

=

194

W. F. WIDDAS

(9) B s = half-maximal constant for S exchange K , , = Michaelis constant for S entry K S i = Michaelis constant for S exit

Thus the entry of glucose involves four independent parameters F,, K g o , R , , and B , [the fifth parameter K g i is related to the others through Eq. (9)]. But if unstirred layers are involved there are two further parameters D, and Di making six in all. The same four independent parameters would apply to sugar S , i.e., F,, K,,, R , , and B s so that in competitive situations the position would be more complex. Where the competitor is nontransportable R s = m. This arises since the rate constant for transfer through the membrane (and which is set at zero) is one of the terms in the denominator of R , . It follows that 1 - 1 Ngs

KsiRg

and 1 - 1 Ns, KsoR, We can therefore rewrite Eq. (8) as

ug=

Fg[Go(1 + G i / R g ) - Gi(1 + Go/Rg)I 1 + G , / K , , + G i / K g i+ G o G i / R , B g + [So/Kso(I + Gi/Rg)I + [Si/Ksi(1 + Go/Rg)I

(13)

The terms involving the nontransportable inhibitor occur only in the denominator and are enclosed in square brackets for convenience since one or the other or both terms may be zero in the various experimental conditions. For a Sen- Widdas type experiment the next exit, for the case where there is a nontransportable inhibitor only on the outside, may be written

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

195

as follows:

Fg(Go - Gi)

u, =

[2

1 + Gi/Kgi + -( 1 + Gi/Rg)

1

(14)

+ Go(l/Kgo + Gi/RgBg)

Considering G, small relative to G i the value of G,, necessary to reduce the maximal exit rate (when G, = 0) to half that value will be approximately determined by the value of G,, which doubles the value of the denominator. Thus in the absence of inhibitor the Sen-Widdas constant will be obtained when GII/K,(,--c I and G,,/R,B, fi 1/Kgi.Indeed since G i is large this last relationship, i.e., G, = R,B,/Kgi will determine the Sen- Widdas constant (Regen and Tarpley, 1974). In the presence of an outside inhibitor the value of G, necessary to double the value of the denominator must be larger so that the inhibitor terms are also compensated for. With a nontransportable inhibitor only on the inside Eq. (13) can be rewritten as Fg(G(J- Gi)

u, =

1

+ Gi/K,i +

[&] +

G, {l/Kgo + Gi/RgBg + [ ~ i / ~ g ~ s i ~ )

(15) In this case G o multiplies one of the inhibitor terms and indeed if Go/ R g fi 1 when G,, = R,B,/Kgi the denominator would be doubled without any increase in G, relative to the uninhibited value (where S i = 0). Thus an inside competitive inhibitor need not affect the Sen- Widdas constant (Basketter and Widdas, 1978). These considerations particularly apply where K,, fi R, and therefore B , fi Kgi. This type of asymmetry was deduced for the human red cell sugar transferring system by Regen and Tarpley (1974) and while applicable it follows that the Sen- Widdas constant is related to the outside K,(K,,) and the half-saturation for exchange is related to the inside K,,,(Kgi). The effect of nontransportable inhibitors on equilibrium exchange can be seen by making G, = G i = G in Eq. (13). The positive terms on the right-hand side represent influx and the negative terms efflux. Since from Eq. (9) 1 - = - +1 1 1 -+ K,,, K,i B, R, Eq. (13) simplifies to give the exchange flux JEx

196

W. F. WIDDAS

The reciprocal can be written a s 1 and where an outside inhibitor o r an inside inhibitor is present on its own the change in the slope and the intercept where l/JEx= 0 can be used to derive K,,, and KSi. However the inhibitor will appear t o have a greater affinity in the inhibition of exit compared with the inhibition of exchange and the halfsaturation concentration of the inhibitor derived from the inhibition of exit into a sugar-free medium will be smaller than that derived from inhibition of exchange. This is because the exit will appear to be half inhibited when Sv/KsV(l+ Gi/Rg) = 1 + Gi/Kgi that is the condition which will bring about a doubling of the denominator in Eq. (14) when G,, = 0. Since G i is large this will occur when So -

Rg Kgi + Gi __.

Ksv

Kgi R g + Gi

Rg Kgi

2-

thus the concentration which half inhibits exit will be smaller than K , , in proportion a s R g is smaller than Kgi. On the basis of the simpler kinetics the concentration which half inhibits exit into a sugar-free medium is a measure of the affinity for the outside component whereas on the Regen and Tarpley kinetics the socalled Michaelis constant is that derived from inhibition of exchange and the higher apparent affinity from exit measurements follows from the kinetics. This difference in interpretation affects the magnitude of the asymmetry factor to be allocated to the affinities of nontransportable inhibitors (Table IV) but does not affect the relative differences in asymmetry found for different compounds. Thus on either interpretation there is a 20-fold range in asymmetries among the three nontransportable inhibitors studied by Baker et al. (1978). Deves and Krupka (1978a) have reviewed the kinetics of inhibition in the conventional two complex model and have drawn attention t o the extra information which can be obtained from zero-trans entry and zerotrans exit experiments. The points they make can also be illustrated from the Regen and Tarpley kinetics. Thus for a nontransportable inhibitor the zero-trans entry of test sugar can be written as Fg.G v ug= (18) 1 + Gv/Kgv + [Sv/KsvI + [Si/Ksi(1 + Gv/Rg)I

197

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

TABLE IV ASYMMETRY OF PARAMETERS OF NONTRANSPORTABLE INHIBITORS~

Compound Ethylidene glucose Isopropylidene glucose Trimethyl glucoside

Apparent K ,

Asymmetry ratio

Inhibitor Inhibitor inside outside (mM (mM)

Regen and Tarpley Baker and Widdas kinetics kinetics ( K 5 >K: J (41,:4J

110 76

I35

II 59 290

10: 1 1.3: 1 0.47: 1

60:I 7.7: I 2.8: I

a The apparent K , s are obtained where the lines in plots such as Fig. 3 intercept IIJ = 1/Vmah. The intercepts correspond to K,, and K,,, in the Regen and Tarpley kinetics but the apparent K , obtained from exchange experiments with inhibitor on the outside is ca. 6-fold larger than 6 of the Baker and Widdas kinetics. The half-saturation concentration for an inhibitor in a Sen-Widdas exit experiment would be less than the Regen and Tarpley Michaelis constant K,,, by a similar factor a s explained in the text. Data from Baker et a / . (1978).

and the reciprocal flux can be written as 1 This would give a linear plot of 1/J versus l / G o in which the ordinate intercept would be increased with an inside acting inhibitor (Siterms) but not with an outside acting inhibitor. The slope would however be affected by both types of inhibitor. In a comparable approach the zero-trans exit or efflux can be shown to yield

(20) which would also be a linear plot of 1/J vs l / G i but one in which the ordinate intercept would be increased with an inhibitor acting on the outside. Deves and Krupka (1978b) point out that in showing up an inhibitor which acts only on one side of the membrane the zero-trans entry and exit approaches are the most unambiguous and they used the absence of a shift in intercept in zero-trans exit experiments with cytochalasin B to prove that that inhibitor is competitive for the internal sites in the membrane and does not react with the external sites (see Fig. 6 ) . This confirmation of the hypothesis proposed by Basketter and Widdas (1977, 1978) also marks an interesting development in the application of asymmetric carrier kinetics to problems of inhibition of hexose transfers.

198

W. F. WIDDAS

Deves and Krupka (1978a) summarizes the main results of their kinetic survey as showing that:

I . Competitive inhibitors acting at only one side of the membrane may give results (in some experiments) which are interpretable as being due to noncompetitive inhibitors but the reverse does not hold. Noncompetitive inhibitors cannot mimic competitive ones. 2. To test whether an inhibitor is competitive or not the only unambiguous single experiment is that of inhibition of exchange. 3. Zero-trans exit and entry experiments have advantages in showing up an inhibitor which competes only at one side of the membrane. 4. A combination of experiments enables one to determine whether the inhibitor is competitive and also whether it is transportable on the hexose system. E. Applications of Asymmetric Carrier Kinetics The application of the asymmetric kinetics to human red cells presents problems by virtue of the speed of the transfer process. Regen and Tarpley (1974) illustrated their original approach with data from the literature notably the anomalous findings of Miller (l968a) and the galactose data of Levine and Stein (1966). Kinetic studies designed to exploit the asymmetric kinetic approach were made on rat thymocytes by Whitesell et al. (1977a) and by Whitesell et al. (1977b). The kinetics were also used to analyze the properties of the sugar transfer process in avian erythrocytes in the same laboratory by Cheung et al. (1977). Readers should refer to the original articles for full details but it is interesting to note that emphasis was placed on the use of influx measurements into empty cells-the zero-trans influx-and also influx measurements into cells preloaded with a fixed internal concentration of sugar. These types of measurements can be more informative than the equilibrium exchange experiments alone. Thus writing Eq. (8) in the simplified form where there is no competing sugar or inhibitor gives

For the influx of sugar into empty cells G i = 0 and the equation can be put in the form of a Hanes plot (Hanes, 1932) as

199

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

the intercepts are I/F, and - K , , and the reciprocal of the slope is F,K,, = V for entry. The conditions for exchange are G , = G i = G and the equation can be written as

in the Hanes plot form. IIF, and - B , can be determined as the respective intercepts. In the influx measurements at fixed internal concentrations Whitesell e f al. (1977b) point out that the equation takes the form

with intercepts of 1/P and - H where

P

=

F g [( 1

+

?)/( + -5)] 1

and

H=

( $: / ($ &) I+-

+

Having derived F , and K , , from Eq. (22) and B , from Eq. (23), Eq. (24) allows one to evaluate the remaining parameters by making use of the relationship in Eq. (9). Thus

and

Techniques such as these are particularly powerful in analyzing the asymmetric carrier parameters in cells in which the transfers of sugar are relatively slow as in rat thymocytes and avian erythrocytes. Indeed the work with avian erythrocytes has some similarities to the original application of asymmetric kinetics to the case of rabbit erythrocytes (Regen and Morgan, 1964) in that asymmetry is not marked. In avian erythrocytes however there is a difference between aerobic and anaerobic conditions. Sugar transport is stimulated by anoxia (Wood and Morgan, 1969; Whirfield and Morgan, 1973; Cheungef al., 1977). The latter authors ascribe the effect to increased efficiency of preexisting carriers which in the aerobic state may be bound by some immobilizer or have depressed

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W. F. WIDDAS

mobility. There may also be effects on carrier affinity which shows clearer evidence of asymmetry in the aerobic state. Ginsburg (1978) has applied asymmetric carrier kinetics to the problem of galactose transfers in human erythrocytes. He collected data for zerotrans entry and exit and for equilibrium exchange. His analysis followed the lines recommended by Lieb and Stein (1974) and by Eilam and Stein (1974), but some of the data were analyzed on the basis of a pair of antiparallel carriers (Ginsburg and Stein, 1975; Eilam, 1975). Thus zerotrans entry was best resolved on the basis of an asymmetric carrier with high affinity outside and low affinity inside which accounted for 85% of the maximal transfer together with an asymmetric carrier with high affinity inside and low affinity outside which accounted for 15% of the maximal transfer. Exit and exchange could be analyzed on the basis of a single asymmetric carrier since it was shown that these measurements would not be affected by the 15% of components presumed to be arranged in an antiparallel fashion.

F. The Effects of pH and Temperature The use of influx measurements into preloaded cells has been extensively used by Lacko et al. (1972a, 1973, 1974, 1975, 1977a,b) for a different reason. By preloading cells with glucose the initial uptake from experimental solutions of low glucose concentration has the nature of an exchange uptake but under standardized conditions. The K , and the maximal uptake rate can be determined and the changes analyzed in terms of the experimental variables being studied. This approach is particularly well illustrated in their experiments on the effects of pH which they studied over the range from pH 2 to pH 11. An important consideration was that the cells were in the abnormal pH solutions only for the 5-second period of the initial uptake after which the solution was diluted by the large volume of stopping solution. Thus these experiments may be taken to refer to the effects of pH mainly on the outside components of the red cell transfer process rather than those parts more deeply situated. The results showed that there were two regions in which the transfer rate was decreased corresponding to pK, = 5.2 and pK, = 9.5, but the K , value was constant throughout. The interpretation given was that the glucose carrier if protonated or deprotonated became immobile but that the protons were not lost or gained from the glucose binding site itself. There is little effect of pH in the range 6-8 which was the region studied by exits by Sen and Widdas (1962a).

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

201

Lacko et al. (1973) used the initial uptake into preloaded cells to investigate the effect of temperature on the parameters of the initial uptake. The K , showed a marked dependence on temperature and their results confirmed the observations of Sen and Widdas (1962a) but their analysis of the change in V,,, with temperature went further. Sen and Widdas (1962a) found that V,,, had a steeper temperature coefficient at lower temperatures than at higher temperatures. The Arrhenius activation energy was of the order 20 kcal/mole at 20°C and 7- 10 kcal/mole at 37°C. Dawson and Widdas (1964) found that in fetal guinea pig red cells the K , from exits did not progressively decrease at lower temperatures. Since the Sen-Widdas exit K , could be shown to contain both a rate term for transit through the membrane as well as the association and dissociation rate terms of a true Michaelis constant Dawson and Widdas examined the possibility that a change in the rate-determining step may occur as the relative magnitudes of the three rate terms changed with temperature. Although this line of analysis went someway to explain the curvature of the Arrhenius plot of V,,, for both human and fetal guinea pig cells the results on human cells particularly the linear relation between log, K , and l/Tabs could not be explained. This point was emphasized by Bolis et 01. (1970) who confirmed the curvature of the Arrhenius plot of V,,, but interpreted the change as being due to the interplay of different heats of activation of loaded and unloaded carriers in the symmetrical carrier model with substrate facilitated mobilities of the complexed form. Lacko et al. (1973) however make the hypothesis of a definite phase transition effect round about 20°C with a sharp change in the heat of activation from 9.3 to 21.5 kcal/mole at pH 7.5. Their Arrhenius activation energies were 9.9 and 22.1 kcalhole and are comparable to the results from exits reported by Sen and Widdas (1962a). The similarity in the K , results is not surprising when it is realized that the Sen- Widdas exit technique is essentially based on finding a rate of entry which retards the exit to half its maximal value. Thus the parameter concerned is closely related to that of an entry into preloaded cells. The technique used by Lacko et al. is to look at the initial part of this entry process. The V,,, term derived by Sen-Widdas exits is however different from the V,,, derived by Lacko et al. In the latter's experiments V,,, is more akin to the V,,, for exchange apart from the fact that the glucose concentrations are very dissimilar across the membrane and thus will affect the redistribution of components between the two sides. The variation in the redistribution of components may be partly re-

202

W. F. WIDDAS

sponsible for differences between these results and the values for exchange obtained by Hankin and Stein (1972). The latter authors found no curvature in the Arrhenius plot of V,,, derived either from the exchange or from zero-trans effluxes. Their activation energies were 18 kcalhole for zero-trans effluxes and 16 kcalhole for exchange over the temperature range 5-45°C. Ginsburg and Yeroushalmy (1978) have extended the use of asymmetric carrier kinetics to study the individual parameters for galactose transfer over a range of temperatures. Their temperature range 0-25°C was somewhat limited by the techniques involved but over that range they found relatively little change in the affinity parameters which are equivalent but not identical to K,,, Kgi,and B , in the Regen and Tarpley kinetics. Thus the asymmetric affinities reported earlier (Ginsburg, 1978) are maintained over this temperature range. There was a marked temperature dependence of the transfer rate constants and the pattern was similar to that reported by Lacko et al. (1973) but suggested a further break in continuity between 5 and 15°C with much higher activation energies being required at the lower temperatures. Since the asymmetric affinities were maintained over the whole temperature range studied (0-25°C) they may be seen as a feature of the human red cell which is not peculiar to one temperature. They may therefore be assumed to apply also in vivo at 37°C.

III. MORPHOLOGICAL ASYMMETRY A. Asymmetry in the Erythrocyte Membrane

Asymmetry is recognized to be a structural feature of erythrocyte membranes affecting both lipids and proteins. Articles by Bretscher (1973), Zwaal et al. (1973), and Bretscher and Raff (1975) review the main points which for the present purposes may be summarized as follows: 1. Phospholipid molecules appear to be unevenly distributed in the red cell membrane. Thus while the lipid double layer proposed by Gorter and Grendel (1925) and Danielli and Davson (1935) is supported by X-ray evidence, enzymatic and other studies suggest that the lipids in the outermost layer tend to be phosphatidylcholine and sphingomyelin (the choline lipids) with some cholesterol whereas the amino phospholipids, phosphatidylethanolamine and phosphatidylserine, are almost wholly

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

203

situated in the inner layer. Glycolipids appear to be in the outer layer (Steck and Dawson, 1974). For a recent review see Bergelson and Barsukov (1977). 2 . Most of the lipids have two paraffin chains of variable unsaturation and a polar head group which in the water interface may have ionized groups which are charged. The majority are zwitterions but phosphatidylserine has a net negative charge, thus the asymmetric distribution of the phospholipids conveys an electrical asymmetry to the lipid bilayer. 3 . The paraffin chains of the lipids, especially where unsaturation is present, are in a liquid state with greater mobility toward the center of the bilayer. The lipid molecules also have considerable translational mobility within their particular half of the bilayer but transfer across the membrane from layer to layer-a process termed “flip-flop” by Kornberg and McConnell (1971)-is very slow. Thus whereas lipids may change position with their neighbors in less than a microsecond the flipflop motion may require hours or longer. 4. Membrane proteins may be embedded in the lipid bilayer as proposed by Singer and Nicholson (1972) so as either to traverse the bilayer completely and expose polypeptide chains at both interfaces or be partially embedded in the inner lipid membrane with free polypeptide chains approachable from the inside of the cell but not from the outside. Asymmetry of such proteins is emphasized in the review by Rothman and Lenard ( 1977). 5. Of the proteins which penetrate through the membrane one (referred to as component a by Bretscher) is a large protein with molecular weight about 100,000 and fairly globular in shape. This protein is the same as that of “Band 3” in the classification of Fairbanks et a / . (1971) and its isolation has been achieved by Furthmayr et al. (1976). It is thought that the (80 A) particles seen on freeze-fracture electron micrographs may represent molecules of this protein which among other functions may be responsible for the chloride-bicarbonate exchange function of the erythrocyte membrane (Cabantchik and Rothstein, 1974). Rothstein et a / . (1978) have drawn attention to the asymmetry of functional sites approachable from outside and inside the cell in the case of the anion transport protein. The other major protein which traverses the whole membrane is a lower molecular weight (30,000) glycoprotein which has its sugar residues exposed in the external interface. These residues include the sialic acid which gives the external surface of the erythrocyte its negative charge. The intramembranous segment of glycophorin A has been isolated by Furthmayr et a / . (1978). Both these proteins are estimated to be present in amounts sufficient

204

W. F. WIDDAS

to provide about half a million molecules per red cell and the techniques so far used would not separate minor components present in smaller quantities such as the Na-K-ATPase. 6. The proteins which partially embed in the membrane from the inner side are thought to do so by hydrophobic interaction with the amino phospholipids. Numerically the amino phospholipids are deficient relative to the choline lipids in the outer layer. Either some choline lipids must be on the inside or these partially embedded proteins fill in for the deficiency in the inner half bilayer. It may be that both factors apply. Recent x-ray studies (Pape et al., 1977) confirm the asymmetric arrangement of protein and lipid constituents and correlate with electron micrograph interpretations (Tilney and Detmers, 1975; McMillan and Luftig, 1975). 7. There has been an accumulation of experimental evidence that the Band 3 group of proteins may also include the protein responsible for glucose transport. Lin and Spudich (l974b) studied the cytochalasin B binding by ghosts from which various fractions of the membrane proteins had been released by treatment with solutions of high and low ionic strength. These experiments pointed to the involvement of Band 3, 4.1, 4.2, or the periodic acid-Schiff (PAS) sensitive material. Kasahara and Hinkle ( 1976) incorporated proteins solubilized with Triton X-100 into liposomes made from soya bean lipids and were able to show that they took up D-glucose with some of the characteristics of the hexose transfer system in intact erythrocytes. The most effective material was Band 3 protein with some material from Zones 4.1 and 4.2. I n a red cell membrane study Kahlenberg and Walker (1975) found that by crosslinking the sulfhydryl groups of Band 3 proteins there was an inhibition of glucose transfer into the membrane vesicles. This inhibition could be reversed by 2-mercaptoethanol. In a further study Zala and Kahlenberg ( 1976) used 2,3-dimethyl maleic anhydride (DMMA) to dissolve “extrinsic” proteins, i.e., those in Bands I , 2 , 2.1, 2.2, 4.1, 4.2, 5 , and most of 6. This material and the pellet left behind were tested in liposomes prepared from sonicated erythrocyte lipids and glucose uptake was promoted only by the intrinsic proteins left in the pellet. These constituted Bands 3,4.5,7, and some of 6 together with the PAS-sensitive material. Following DMMA with Triton X-100 it was possible to solubilize and remove Bands 5 , 6, and the PAS-sensitive material. The pellet left behind with Bands 3, 4.5, and 7 had enhanced sugar-transferring properties when tested in the liposomes. Band 7 is a protein of the cytoplasmic surface and for this and other reasons is thought unlikely to

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

205

be involved in the sugar transfer system (Lin and Spudich, 1974b; Kahlenberg and Walker, 1976) leaving Band 3 and Zone 4.5. The lower molecular weight proteins of Zone 4.5 were shown to induce glucose transport in further experiments by Kasahara and Hinkle (1977) and by Kahlenberg and Zala (1977). More recently Jones and Nickson (1978) have been able to incorporate red cell protein extracts into thin lipid bilayers which then showed an increase in conductance and of Dglucose permeability. The major components of their most effective extract were again Bands 3 and 4.2. That the transport protein may originally be in Band 3 has been suggested by Phutrakul and Jones (1979). What was of interest in the context of asymmetry was the finding that stable bilayers were formed only if the protein extract was added on one or the other sides but not if present on both sides. This is consistent with the view that the transmembrane proteins are themselves asymmetrical and so arrange themselves in the lipid membrane in a definite orientation. Symmetrical presentation in the lipid may lead to association with consequent instability. Van Steveninck et al. (1965) from their studies with sulfhydryl reagents concluded that there was a large asymmetry in the distribution of such groups in the membrane. Sulfhydryl groups on the outside reacted with p-chloromercuribenzene sulfonate (PCMBS) and there was some correlation with the inhibition of glucose entry. However thiol groups deeper in the membrane were also implicated in this and the work of Smith and Ellman (1973) using maleimide derivatives. The latter authors found that the reagents with highest lipid solubility inhibited glucose transfer at lowest concentrations. They considered that this supported other evidence that blockage of thiols in different membrane locations was involved in the inhibition of glucose transfer. Batt et a / . (1976) used impermeant maleimides as developed by Abbott and Schachter (1976) to label the exofacial surface of erythrocyte proteins and found labeling in four bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The major band corresponded with proteins in the 40,000 to 70,000 molecular weight range. Since the impermeant maleimides also inhibited glucose transfer there was the possibility of further identification of the hexose transfer system. In an earlier study of the same kind Taverna and Langdon ( 1973d) used D-glucosyl isothiocyanate as a covalent probe, which is also an irreversible inhibitor of glucose transport, to try and label the active site on the glucose-transferring protein. This probe was found predominantly in proteins of 70,000 and 100,000 daltons, respectively. The asymmetric orientation of Band 3 (or other protein concerned with

206

W. F. WIDDAS

glucose transfer, e.g., Zone 4.5) within the erythrocyte membrane and the hydrophobic interactions with the membrane would preclude any rotation in the plane of the membrane (Kahlenberg and Walker, 1976) and this led Kahlenberg to suggest that the transport of glucose occurred through water-filled channels formed by specific subunit aggregates of the transport protein. By progressively removing cholesterol from the lipid membrane of human erythrocytes Masiak and LeFevre (1974) found no change in the K , for glucose transport. The rate of transport was at first increased but as more cholesterol was removed the rate became inhibited. Read and McElhaney (1976) confirmed these findings and since the removal of cholesterol increased the fluidity of the lipid bilayer they argued that the absence of an increase in the rate of glucose transport was against a model for transport which involved movement of the transport protein in the lipid environment. What is clear from this brief summary is that there is an asymmetric polarization of the red cell lipid bilayer and also of the proteins traversing it. The evidence is strongly against a flip-flop mechanism involving either lipids or proteins and a mobile carrier for glucose is unlikely to be of the nature of a membrane ferryboat (Ussing, 1952). An allosteric model for membrane transport was suggested by Jardetzky (1966) and similar models have been reviewed by Singer (1974). In its simplest form such a model involves an aggregate of integral proteins with a slit or cavity large enough to admit a small molecule. The membrane molecule is presumed to be capable of adopting two different conformations with the cavity opening to one or other side of the membrane, respectively, and to contain a binding site for the transported species within the cavity. The general problem of the way in which integral proteins are incorporated into the lipid environment of the membrane and take part in transport has been further reviewed by Singer (1977). It has already been pointed out that a minimal conformational change within a transporting protein may provide the “mobile” element which is a kinetic requirement of the facilitated transfers of sugars (Vidaver, 1966).

B. Asymmetry of the Membrane Environment Besides structural asymmetry the intact red cell is peculiar in having an asymmetric environment in the majority of laboratory experiments. This is due to the high concentration of protein (hemoglobin) inside the

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

207

cell and the fact that most experiments are carried out in media free of protein. Taverna and Langdon (1973b) developed a new technique for following glucose transfer which involved incorporating glucose oxidase into red cell ghosts which were then resealed. On removal of glucose oxidase from the outside medium the consumption of oxygen measured with an oxygen electrode was shown to be dependent on the rate of penetration of glucose to the inside of the resealed ghosts. They showed (Taverna and Langdon, 1973~)that the maximum rates of entry into resealed pink ghosts was similar to that into white ghosts and also into inside-out vesicles and thus they deduced that the transport across the ghost membrane was symmetrical. However the K , for glucose was ca. 10 mM for both ghosts and vesicles which at 15.5"C corresponds more closely to the half-saturation of the inside sites of intact erythrocytes (Baker and Widdas. 1973b) and there may have been some reorganization of membrane components in preparing the ghosts. Naftalin et NI. (1974) have shown that sugars which are rapidly transported across the red cell membrane also protect red cells against osmotic hemolysis and from sugar-dependent increases in the viscosity of hemoglobin solution they deduce that glucose and other sugars can induce hemoglobin molecules to form a gel in which glucose itself is nonspecifically bound. Naftalin and Holman (1977) estimate that in the range 10100 mM glucose as much as 80% of intracellular glucose may be bound loosely to hemoglobin. The same authors have advanced a hypothesis for glucose transport incorporating the slow sorption and desorption of sugars to and from hemoglobin into the kinetics of an otherwise symmetrical membrane transport based on a gated pore model first proposed by Adair (1956) and expanded upon by Jung (1975). In this model there are recognition sites at each end of the pore which on combination with sugar induce the conformational changes on which transfer through the membrane largely depends. As in the tetramer model of Lieb and Stein (1972a) the rate constant for transfer is presumed dependent on the saturation by sugar and the rate is multiplied by the sum of fractional saturations at the two sides to give the higher rate for exchange when both sides are saturated. They advance the interesting suggestion that in this state, i.e., when the gates at both ends of the protein pore are open exchange may actually occur without a conformational change accompanying each molecule of glucose transferred. This would explain the lower activation energy for the exchange process as opposed to net transfers.

208

W. F. WIDDAS

A gated pore for glucose through which smaller molecules such as polyols (e.g., erythritol) may penetrate in a noninhibitable manner as the pore gates open and close was also proposed by Bowman and Levitt (1977). The asymmetry in the Naftalin and Holman model arises from the fact that 85% of the cell water is presumed to be bound to hemoglobin and takes up glucose only slowly. The 15% of free water therefore fills up rapidly during an entry experiment and creates a back flux which slows the entry rate relative to what might be expected if all the cell water was instantaneously available to sugar. Readers should refer to the text for a full explanation of the kinetics which involve the numerical solution of nonlinear differential equations and are not therefore immediately applicable to problems other than those illustrated by the authors. They are able to show that computer solutions predict operational parameters (K,s and Vmaxs)of the right order for the different experimental procedures for exits and entries and for exchange. One interesting example is their explanation of the asymmetry observed by Bowyer and Widdas with FDNB and illustrated in Fig. 1 . In the Naftalin and Holman model the exit of glucose is rate limited by the membrane transport system and is thus inhibited progressively as the membrane components are taken out of action by reaction with FDNB. The rate-limiting step for entry is the complexing of glucose by hemoglobin and consequently the rate of entry of glucose will not be lowered until the rate of transfer across the membrane is reduced below that for complexing by the hemoglobin. This explanation also fits in with the observations of Sen and Widdas (1962b) who showed that entry of glucose in the range 38-76 mM was more inhibited by incubation with FDNB than the initial entry from 0.7 to 38 mM. Naftalin and Holman point out that at the higher concentrations the rate of entry is again limited by transport across the membrane and consequently the inhibition is dependent on the inactivation of membrane components and is more comparable with that for inhibition of glucose exit. The sorption and desorption of sugar to the hemoglobin-bound water may well be the explanation for the apparent diffusional resistances ascribed to the inside of the cell by Wilbrandt (1972a) and by Regen and Tarpley (1974). Wilbrandt estimated that not more than 10% of the allowance he had to make for diffusional resistances could be outside the cell but that the estimated "unstirred layer" within the cell would have to be an impossible value of 0.034 cm. Regen and Tarpley (1974) also estimated the diffusional resistance inside the cell to be very much larger than that

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

209

outside and their value of l/Di = 0.062 minute cell-' ml-' was approximately half the minimal resistance for overall glucose transport. There is thus a very significant environmental factor operating inside the red cell contributing to the anomalies on which kinetic asymmetry is based.

C. Asymmetry of the Sugar Membrane Transfer System

In view of the structural asymmetry of the membrane and the asymmetry of the environment is there any need to look for asymmetry in the sugar transfer process itself? A11 the authors referred to in the previous section were drawn toward the possibility that the sugar transfer process in the membrane may in fact be symmetrical. Naftalin and Holman (1977) assumed the membrane transfer was essentially symmetrical with equal half-saturation concentrations ( K , ca. 2 mM) at the two sides. In their model the different operational K,s arise from the interplay of the rate-limiting processes for membrane transfer with those for sorption into and desorption from the hemoglobin. Wilbrandt ( 1972a) in his analysis allowing for diffusional resistances arrived at a K , of 1.95 mM at 20°C again for a symmetrical membrane transfer. Regen and Tarpley acknowledging that the kinetic analysis involved asymmetric affinities considered that these may arise due to the presence of a nontransportable inhibitor on the inside of the membrane which by competing for the carrier raises the apparent half-saturation constant for the sugar on the inside. On this basis the inherently symmetrical transfer process would have a K , determined by the outside site which would approximate the Sen- Widdas value at 20°C (1.86 mM) and be comparable with the values used by Naftalin and Holman and by Wilbrandt. Experiments which tended to show the presence of high-affinity sites on the inside of the human red cell membrane were reported by Hankin et al. (1972). Their results, which were based on the analysis of the slowing of net entry of glucose, were criticized by Foster and Jacquez (1976) but have been defended by Lieb and Stein (1977). Baker and Naftalin (1977, 1979) have also reported an experiment in which glucose exits into a constant high concentration of galactose indicated a higher internal affinity (lower K , ) than that obtained from equilibrium exchange. A symmetrical carrier in which interactions of the internal environment or redistribution of components can, under some experimental conditions, mimic the presence of low-affinity sites, might go some way to fit these results in with those that point to there being low-affinity sites on

21 0

W. F. WIDDAS

the inside. On the other hand the same factors might cause a system with low-affinity sites inside to mimic one with high affinity. However the idea of inherent symmetry whereby the hexose transfer system in the human red cell has identical sites with similar affinities facing outward and inward has the attraction that it would tend to conform with the “principle of the uniformity of nature” in postulating a similar transfer system in red cells of a variety of species. Thus asymmetry was looked for and not found in rabbit red cells (Regen and Morgan, 1964) nor is it a feature of avian erythrocytes (Cheung ef al., 1977). In avian erythrocytes there is the suggestion of preexisting “carriers” being immobilized under ordinary conditions but made available by anoxia or intracellular Ca2+ions (Carruthers and Simons, 1978) and this lends support to the possibility of there being an internal inhibitor or some other mechanism operating in human red cells which could leave the transfer process inherently symmetrical. However, the “principle of uniformity of nature” has led biologists astray in the field of muscle physiology (Huxley, 1977) and is beset with difficulties in this present context. Thus there are stereospecific differences between the sugar transfer system in rabbit and human red cells (Regen and Morgan, 1964) in respect to the handling of ketose sugars and in the response to inhibitors and such differences must be in the recognition sites or neighboring groups on the transfer protein. In beef erythrocytes although a saturable facilitated transfer system is involved in the transfer of sugars it is one with an unusually high affinity for glucose and 3-0-methyl glucose (Hoos et al., 1972). Fetal blood from a number of farm and laboratory animals have a rapid facilitated transfer of sugars similar to human red cells (Widdas, 1955). This rapid transfer is also present in blood of the newborn rabbit (Augustin et af., 1967), pig (Zeidler et nf., 1976) and dog (Lee et nf., 1976) but rapidly declines after birth. It has recently been observed that in fetal shows a similar guinea pig red cells, 4,6- 0-ethylidene-a-D-glucopyranose asymmetric inhibition of 3- O-methyl glucose exchange to that of human cells though the estimated concentrations which half inhibit the exchange are not identical (Aubby and Widdas, 1979). The small differences in inhibitor affinity could be due to the different environment of carriers in human and fetal guinea pig cells but taken with the different temperature dependence of the Sen- Widdas constant for glucose (Dawson and Widdas, 1964) it is more likely that there are small molecular differences in the transfer protein. There is no a priori reason to expect the hexose transfer system to be molecularly identical in different species. Although kinetic anomalies and kinetic asymmetries are not unambiguously in favor of a basic asymmetry in the hexose transfer mechanism

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

21 1

itself the evidence from nontransportable inhibitors is stronger and is reinforced by the demonstration that cytochalasin B reacts only with the internally situated sites of the hexose transfer system (Basketter and Widdas, 1977, 1978: Deves and Krupka. 1978a,b) and by the different susceptibility of the inside and outside sites to proteolytic enzymes (Lin and Spudich, 1974a; Masiak and LeFevre, 1977) discussed previously. Proteins which traverse the cell membrane are presumably stabilized in it by having an intramembranous structure which is in some way complementary to the asymmetrically arranged lipid environment and capable of hydrophobic interactions with it (Wickner, 1977). Looked at in the light of the overall asymmetry of the membrane involving the bulk of the lipids and proteins it would be remarkable if the components responsible for hexose transfer were not themselves asymmetrical. However, the final answer to this question of asymmetry of the membrane transfer system for sugars and some of the kinetic problems associated with it may have to wait until the molecular structure can be determined and the overall mechanism of transfer clearly described.

IV.

IMPLICATIONS OF ASYMMETRY

A. Consequences of Asymmetry

Assuming a chemical and dynamic asymmetry as previously discussed one can consider some general implications. The first consequence of asymmetry of affinities will be that the parameters of transfer, particularly the apparent K , or half-saturation constant, will vary with the type of experiment. The variation in K , will also be influenced by factors such as the sorption of sugar into the hemoglobin-bound water which may be kinetically equivalent to the diffusional resistances of unstirred layers. for entry and exit taken at their initial stages when they The V,, approach the zero-trans condition will be different principally because of the redistribution of membrane components between outward and inward facing modes coupled with the different translation "velocities" of components and sugar complexes. Indeed redistribution will largely be due to the asymmetric rate constants for the transfer of sugar complexes (and for the rearrangement of the uncomplexed components within the membrane) which are an essential corollary to the concept of asymmetric affinities if the overall system is to be nonaccumulating and not to contravene the second Law of Thermodynamics. for exchange may be interpreted as arising from the The higher V,,

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W. F. WIDDAS

greater mobilities of sugar complexes in the membrane but there are other possibilities along the lines of gated pores which may need to be examined. A pore in which the gates remain open however would not meet the requirements for uphill transfer by counterflow (Park et a/., 1956, 1968; Rosenberg and Wilbrandt, 1957; Wilbrandt, 1972b) unless the sites within the pore are given the kinetic properties of mobile carriers. Asymmetric affinities also introduce complications to the interpretation of inhibitor studies particularly in respect of the K , s obtained from inhibition of equilibrium exchange. The analysis of Deves and Krupka (1978a, suggests some new approaches to the inhibitor problems which will help in resolving the properties of inhibitors which act only at one side of the membrane. In general asymmetry of affinities brings with it a large increase in complexity of the overall framework for the facilitated transfer of sugars and its kinetic analysis. Simple treatments are still useful in describing the results of experiments in which the conditions are clearly defined and are not radically changed as between experiments. The exchange transport into red blood cells used by Lacko (Lackoer al., 1972a,b, 1973, 1974, 1975, 1977a,b, 1978a,b) may be quoted as an example. This technique has been used to study the effects of pH, temperature, alcohols, various drugs, and local anesthetics on the transport system and in general the procedure will respond to factors affecting the outward facing sites and the influx process. It may be assumed that, over the very short times involved in making the influx measurements, complications, due, for example, to the sorption of sugars into hemoglobin-bound water, are either absent or are so similar in the various experiments as to play no rate-determining part relative to the factors under investigation. The Sen and Widdas (1962a,b) procedure, like the exchange transport used by Lacko, remains useful for investigating factors affecting the outside sites of the hexose transfer system. The inhibition of the overall system can also be studied by the Sen-Widdas procedures but caution is required in interpreting inhibition in terms of its being competitive or noncompetitive. The fuller analysis of Sen-Widdas exits shows that the competitive characteristic of being able to demonstrate a n increase in the apparent half-saturation constant of the transported sugar may be given only by inhibitors which are in competition for the outside sites. Competition only for the inside sites of the hexose transfer system may give all the appearances of a noncompetitive inhibitor (Basketter and Widdas, 1978; Deves and Krupka 1978a). This surprising result is not due to asymmetry per se but considerations of asymmetry have brought it to light and of course without asymmetry of affinities it would be technically difficult to observe since the inhibitor is usually present in the outside

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medium and in a symmetrical transfer system would compete for external sites as well as for internal sites. The simple asymmetric kinetics used by Baker and Widdas (1973b) are sufficient for the interpretation of many experiments, e.g., on the inhibition of equilibrium exchanges where the concentrations are not widely varied. But if the concentrations are changed over a range where substantial redistribution of components is brought about or if a variety of entry, exit, and exchange experiments are to be analyzed then it is essential to use more sophisticated kinetic treatments which may need t o allow for diffusional resistances. Although an outline of such treatments has been given in this article reference to the original articles is recommended. B. Physiological Implications Apart from the increase in complexity of the kinetic treatments which asymmetry of affinities creates it would be interesting to consider if there were any physiological implications involved. Krupka and Deves (1979) have discussed the valve-like properties of asymmetric transfer systems. These could arise from an inherently asymmetric facilitated transfer, from a symmetrical transfer system which is asymmetrically inhibited, o r from an obligatory exchange system also inhibited asymmetrically. In all three cases the maximal rates of influx and efflux are different and it can be shown that the net transfer rate for an inwardly directed concentration gradient would be different from the transfer rate for the same gradient outwardly directed. An inhibitor acting asymmetrically outside the cell is inwardly directing in the sense that exit from the cell is inhibited more than entry and in a fluctuating medium with the sugar concentration rising and falling the level of sugar inside the cell would reach a steady state which would be higher than the case for a symmetrical and uninhibited transfer system. If the asymmetrical inhibition is exerted on the inside of the cell then the reverse situation would hold, that is, the sugar would be rapidly lost from the cell when the concentration in the medium was low but would be less rapidly accumulated when the concentration in the medium was high. Thus with a fluctuating medium concentration the steady-state level in the cell would be lower than for a cell with a symmetrical transfer system which is not inhibited. This valve-like property makes it possible to use asymmetric inhibition as a means of control of the intracellular substrate concentration where such a carrier system exists and Krupka and Deves (1979) draw attention t o the possible physiological significance of this type of mechanism.

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On the other hand if one considers a membrane transfer system delivering sugar to a cell interior under conditions of low sugar concentration one can see that utilizing enzymes inside the cell, with their high affinity for sugars, could retain a high saturation at the expense of a low saturation of the inward facing sites of the transfer protein. Indeed Coleman (1973) has argued that membrane enzymes impose a geography on the functions of the cell (see also Schrier, 1977). If the glucose concentration in the cell was close to zero and the utilizing enzymes were arranged adjacent to the transfer proteins one could visualize a vectorial movement from one to the other. However, the human red cell membrane transfers sugar so rapidly that the sugar effectively equilibrates across the cell membrane and consequently the utilizing enzymes would be in an environment of plasma-determined concentration irrespective of the affinities of the transfer protein. Teleologically one might have expected asymmetry of affinities across the rabbit red cell and not the human red cell whereas the reverse is the case. The rapid transfer of sugars across red cells is a feature not only of primate red cells but of the fetal and neonatal red cells of a considerable number of nonprimate mammals. The rapid transfer in fetal red cells may be seen as offering a distinct biological advantage in the carriage of glucose from the placenta to fetal tissues. Goodwin (1954, 1956) pointed out how the blood glucose may be approximately the same in adult and fetal animals in species like the rabbit and guinea pig but because the sugar is practically all in the plasma in the adult, whereas it is equilibrated between cells and plasma in the fetus, there is nevertheless an appreciable plasma-to-plasma gradient across the placenta. The red cells by permitting a rapid charging and discharging of sugar across their membranes during the short time (of a second or so) that they take to traverse the capillaries of the placenta and tissues will have a small buffering-type action preserving the gradient both in the placenta and between the plasma and the tissue cells. If the rapid transfer property is an adaptation primarily for fetal existence then the persistence in the adult red cells in humans and other primates may be a reflection of their evolution (Goodwin, 1954), that is, it may be an example of “biochemical” pedomorphosis. Pedomorphosis-the persistence of morphological characteristics which were embryonic or juvenile features of ancestral types-is a recognized phenomenon in primate evolution (Le Gros Clark 1959). Whether the persistence in the adult primate serves any physiological purpose such as by buffering the gradient between the blood and tissue cells has not so far been the subject of investigation. Even if it were so there seems n o advantage to be fulfilled by the asymmetry of affinities though the greater rate of maximal exit, one of the consequences of

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asymmetry, could be advantageous if blood was circulating through a capillary bed which behaved like a glucose "sink." The buffering-type action would help to maintain the gradient across the primate placenta from both the maternal and fetal sides and one naturally thinks of the primate brain, which is largely dependent on glucose metabolism, as another possible site where such a factor might apply but in the absence of experimental evidence further speculation along these lines is unwarranted. What is remarkable is that in the three or four decades over which the glucose transfer of human red cells has been extensively studied there have been no discoveries of gross abnormalities in the process or of its anomalous absence in contrast to abnormalities of several membranebound enzymes (Schrier, 1977) and proteins (Anselstetter, 1978). Bang and Orskov (1937) reported a reduced sugar permeability in the red blood cells from cases of pernicious anemia and this has been confirmed (Widdas, unpublished observations), but it was found that if allowance was made for the large volume of the cells from the pernicious anemia patients the membrane transfer was within normal limits. It would appear that the hexose transfer system in red cells is either genetically very stable or else abnormalities, if they occur, are lethal to the organism at an early stage of development. Thus in the human red cell the arrangement of components, necessary for the facilitated transfer of sugars with asymmetric affinities, serves only an obscure physiological function to the body as a whole. At present its study is therefore an example of pure research but such study opens up the possibility of getting a fuller insight into the concepts and properties of facilitated membrane transfers which may apply to other sites and to other substrates. REFERENCES Abbott, R. E., and Schachter, D. (1976). Imperrneant maleimides. Oriented probes of erythrocyte membrane proteins. J . Biol. Chem. 251, 7176-7183. Adair, G. S. (1956). A general discussion on membrane phenomena. Discuss. Furridoy Soc. 21, 285-286. Anselstetter, V. (1978). Gel electrophoresis of the human erythrocyte membrane proteins: Aberrant patterns in haematological and nonhaernatological diseases. Blut 36, 135- 144. Aubby, D. S . , and Widdas, W. F. (1979). Asymmetry in the hexose transfer system of erythrocytes from new-born guinea-pigs. J. Physiol. 293, 73P. Augustin, H . W., Rohden, L. V . , and Hacker, M. R. (1967). Uber einige eigenschaften des mono saccharid transport systems in erythrozyten neuge borenen und envachsener kaninchen. A c / o B i d . Med. Ger. 19, 723-735. Avruch, J., Price, H. D., Martin, D. B . , and Carter, J. R. (1973). Effect of low levels of trypsin on erythrocyte membranes. Biochim. Biophys. Actri 291, 494-505.

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sites of the erythrocyte anion transport protein. Review. Trends Biochem. Sci. 3, 126- 128. Schrier, S . L. (1977). Human erythrocyte membrane enzymes-current status and clinical correlates. Blood 50, 227-237. Schultz, J. S. (1971). Passive asymmetric transport through biological membranes. Biopl7y.s. J . 11, 924-943. Sen, A. K . , and Widdas, W. F. (1960a). A new method for determining the half-saturation of the facilitated transfer of glucose across the human erythrocyte membrane and for studying the effect of inhibitors. J . Physiol. (London) 152, 32P-33P. Sen, A. K . , and Widdas, W. F. (1960b). The effect of temperature and pH on the facilitated transfer of glucose across the human erythrocyte membrane. J . Physiol. (London) 152, 64P-65P. Sen, A . K., and Widdas, W. F. (1962a). Determination of the temperature and pH dependence of glucose transfer across the human erythrocyte membrane measured by glucose exit. J . Physiol. (London) 160, 392-403. Sen, A. K., and Widdas, W. F. (1962b). Variations of the parameters of glucose transfer across the human erythrocyte membrane in the presence of inhibitors of transfer. J . Physiol. (London) 160, 44-416. Singer, S. J. (1974). The molecular organization of membranes. Annu. Rers. Biochem. 43, 805-833. Singer, S . J. (1977). Thermodynamics, the structure of integral membrane proteins, and transport. J . Suprumol. Struct. 6, 313-323. Singer, S . J., and Nicolson, G. L. (1972). The fluid mosaic model of the structure of cell membranes. Science 175, 720-731. Smith, R. P. P., and Ellman, G. L. (1973). A study of the dependence of the human erythrocyte glucose transport system on membrane sulfhydryl groups. J . Memhr. Biol. 12, 177-188. Steck, T. L., and Dawson, G. (1974). Topographical distribution of complex carbohydrates in the erythrocyte membrane. J . B i d . Chem. 249, 2135-2142. Taverna, R. D., and Langdon, R. G . (1973a). Reversible association of Cytochalasin B with the human erythrocyte membrane. Inhibition of glucose transport and the stoichiometry of cytochalasin binding. Biochim. Biophys. Actcr 323, 207-219. Taverna, R. D., and Langdon, R. G. (1973b). A new method for measuring glucose translocation through biological membranes and its application to human erythrocyte ghosts. Biochim. Biophys. Acrn 298, 412-421. Taverna, R. D., and Langdon, R. G . (1973~).Glucose transport in white erythrocyte ghosts and membrane-derived vesicles. Biochim. Biophys. Acra 298, 422-428. isothiocyanate, an affinity label Taverna, R. D., and Langdon, R. G . (1973d). D-GIUCOSYI for the glucose transport proteins of the human erythrocyte membrane. Biochem. Biophys. Res. Commun. 54, 593-599. Taylor, N . F., and Gagneja, G . L. (1975). A model for the mode of action of Cytochalasin B inhibition of D-glucose transport in the human erythrocyte. Can. J . Biochem. 53, 1078- 1084. Tilney, L. G . , and Detmers, P. (1975). Actin in erythrocyte ghosts and the association with spectrin. Evidence of a non-filamentous form of these two molecules in situ. J . Cell B i d . 66, 508-520. Ussing, H. H. (1952). Some aspects of the application of tracers in permeability studies. Adv. Enzymol. 13, 21-65. Van Steveninck, J . , Weed, R. I., and Rothstein, A. (1965). Localization of erythrocyte

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membrane sulfkydryl groups essential for glucose transport. J . Gen. Physiol. 48, 617632. Vidaver, G. A. (1966). Inhibition of parallel flux and augmentation of counter flux shown by transport models not involving a mobile carrier. J . 7heor. Biol. 10, 301-306. Whitesell, R. R., Hoffman, L. H., and Regen, D. M. (1977a). Dynamic aspects of glucose transport modulation in thymocytes. J . Biol. Chem. 252, 3533-3537. Whitesell, R. R., Tarpley, H. L., and Regen, D. M. (1977b). Sugar-transport kinetics of the rat thymocyte. Arch. Biochem. Biophys. 181, 5%-602. Whitfield, C. F., and Morgan, H. E. (1973). Effect of anoxia on sugar transport in avian erythrocytes. Biochim. Biophys. Actcr 307, 181- 196. Wickner, W. T. (1977). Role of hydrophobic forces in membrane protein asymmetry. Biocliemistry 16, 254-258. Widdas, W. F. (1952). Inability of diffusion to account for placental glucose transfer in the sheep and consideration of the kinetics of a possible carrier transfer. J . Pliysiol. (London) 118, 23-39. Widdas, W. F. (1953). Kinetics of glucose transfer across the human erythrocyte membrane. J . Physiol. (London) 120, 23P-24P. Widdas, W. F. (1954). Facilitated transfer of hexoses across the human erythrocyte membrane. J . Physiol. (London) 125, 163- 180. Widdas, W. F. (1955). Hexose permeability of foetal erythrocytes. J . Physiol. (London) 127, 318-327. Widdas, W. F. (1974). Pharmacological significance of new concepts for hexose transfers in erythrocytes. I n “Drugs and Transport Processes’‘ (B. Callingham, ed.), pp. 329340. Macmillan, New York. Wilbrandt, W. (1954). Secretion and transport of non-electrolytes. S y m p . Soc. E.rp. Biol. 8, 136-162. Wilbrandt, W. (1972a). Carrier diffusion. In “Biomembranes” (F. Kreuzer and J. F. G . Slegers, eds.), Vol. 3, pp. 79-99. Plenum, New York. Wilbrandt, W. (1972b). Coupling between simultaneous movements of carrier substrates. J . Memhr. B i d . 10, 357-366. Wilbrandt, W. (1977). The asymmetry of sugar transport in the red cell membrane. f t i “Biochemistry of Membrane Transport’‘ (G. Semenza and E. Carafoli, eds.), FEBS Symposium, No. 42, pp. 204-21 1. Springer-Verlag, Berlin and New York. Wood, R. E., and Morgan, H. E. (1969). Regulation of sugar transport in avian erythrocytes. J . B i d . Chem. 244, 1451-1460. Zala, C. A , , and Kahlenberg, A . (1976). Reconstitution of D-glucose transport in vesicles composed of lipids and a partially purified protein from the human erythrocyte membrane. Biochem. Biophys. Res. Cornmirn. 72, 866-872. Ziedler, R. B., Lee, P., and Kim, H. D. (1976). Kinetics of 3-0-methyl glucose transport in red blood cells of newborn pigs. J . Gen. Pliysiol. 67, 67-80. Zwaal, R. F. A., Roelofsen, B., and Colley, C. M. (1973). Localization of red cell membrane constituents. Biochim. Biophys. Acttr 300, 159- 182.

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C U R R E N T TOPICS I N M E M B R A N E S A N D TRANSPORT. VOLUME

14

Permeation of Nucleosides. Nucleic Acid Bases. and Nucleotides in Animal Cells PETER G . W . PLAGEMANN A N D ROBERT M . WOHLHUETER Department of Microbiology University of Minnesota Minneapolis. Minnesota

I . Introduction and Technical Principles . . . . . . . . . . . I1 . Carrier Model for Facilitated Diffusion and Tests for Its Applicability to Nucleoside and Base Transport . . . . . . . . . . . . . A . Zero-trans Influx and Efflux . . . . . . . . . . . . B . Equilibrium Exchange Inward and Outward . . . . . . . . C . Infinite-trans Procedure . . . . . . . . . . . . . . D . Infinite-cis Procedure . . . . . . . . . . . . . . E . Interpretation of Experimental Data . . . . . . . . . . 111 . Uptake of Nucleosides and Purine Bases . . . . . . . . . . A . General Considerations . . . . . . . . . . . . . . B . Relationship between Transport and Metabolism Operating in Tandem C . Estimation of Zero-trans Transport Kinetic Parameters from Substrate Uptake Curves . . . . . . . . . . . . . D . Uptake into Vesicles of Mammalian Cells . . . . . . . . E . Contributions of Transport and Nonmediated Permeation to Overall Uptake . . . . . . . . . . . . . . . . IV . Properties of Nucleoside and Free Base Transport Systems . . . . A . Specificity for Natural Substrates . . . . . . . . . . . B . Transport of Substrate Analogs . . . . . . . . . . . C . Effect of Temperature . . . . . . . . . . . . . . D . Effect of pH . . . . . . . . . . . . . . . . . E . Presumptive Cell Clones Defective in Transport . . . . . . F . Comparison to Transport in Other Types of Organisms . . . . V . Transport Inhibitors and Inactivation . . . . . . . . . . . A . Effects of Sulfhydryl Reagents . . . . . . . . . . . . B . Effect of Other Nonspecific Inhibitors . . . . . . . . . C . Inhibition of Nucleoside Transport by p-Nitrobenzylthiopurine Nucleosides . . . . . . . . . . D . Heat Shock . . . . . . . . . . . . . . . . . E . Effect of Hydrolytic Enzymes . . . . . . . . . . . .

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VI. Regulation of Nucleoside and Free Base Transport and Uptake . . . . 295 VII. Permeation of Nucleotides . . . . . . . . . . . . . . . 303 VIII. Summary and Conclusions . . . . . . . . . . . . . . . 310 References . . . . . . . . . . . . . . . . . . . . 3 13

1.

INTRODUCTION AND TECHNICAL PRINCIPLES

Greater knowledge of the mechanism of permeation of nucleosides, nucleobases, and nucleotides through the cell membranes of eukaryotes is important for several reasons. First, cells of certain tissues in animals and man are deficient in the pathway for de novo synthesis of purines and thus need to take up from the circulation purines that have been synthesized and released by other body cells or derived from food (Murray, 1971). Evidence accumulated during the last 10 years has indicated that the uptake of nucleosides and nucleobases involves their transport through the plasma membrane by specific carriers and their subsequent intracellular phosphorylation (the salvage pathways). Second, many anticancer and immunosuppressive agents presently in use or under development are nucleoside, nucleotide, or nucleobase analogs and a clear understanding of their mode of entry into cells and metabolism is important in the assessment of their mode of action, efficacy, and optimal administration, and of the development of drug-resistant mutants (Sirotnak ef al., 1979). Third, radioactively labeled nucleosides and nucleic acid bases are widely used as precursors to label specifically the nucleic acids of various types of organisms or of the viruses or plasmids replicating therein as well as to assess the rates of nucleic acid synthesis. An interpretation of the rates of nucleoside and base incorporation into nucleic acids, be it R N A or DNA, depends on a clear understanding of the extent to which these rates may reflect the rates of the conversion of the extracellular substrate to intracellular nucleotides which are the direct precursors in nucleic acid synthesis. In cultured animal cells the incorporation of nucleosides and bases into the nucleotide pool seems to be the main ratedetermining step in their incorporation into nucleic acids. Alterations in either their transport or phosphorylation have been found to cause proportional changes in the rates of incorporation into nucleic acid and can occur independently of changes in the rate of nucleic acid synthesis per se. Thus great care is required in equating rates of nucleoside or base incorporation into acid-insoluble cell material with rates of nucleic acid synthesis.

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The relationship between nucleoside and base transport, phosphorylation, and other metabolism by animal cells has only been recently evaluated and only in a few cell systems (see Section I I I ) , but is important for an understanding of the uptake process as well as for measuring nucleoside and base transport rates in metabolizing cells. The question has acquired additional significance because of the finding that the incorporation rates for nucleosides, bases, and other substrates by animal cells vary as the function of the growth stage and that tumor cells often exhibit higher incorporation rates than untransformed cells (see Section VI). In fact, it has been proposed that an enhanced nutrient transport capacity may be an essential aspect of the altered growth potential of tumor cells (Holley, 1972; Pardee, 1971). Numerous studies have assessed the kinetics of uptake of radioactively labeled nucleosides and bases by cultured animal cells (see Section 111). “Uptake” here denotes the accumulation of radioactivity derived from exogenous, labeled substrate within the cell, regardless of metabolic conversion, in the same sense as used by Berlin and Oliver (1975). It is a composite phenomenon, which, in the case of nucleosides and nucleic acid bases, results from the tandem operation of a nonconcentrative transport system and of various cytoplasmic enzymes, including kinases, hydrolases, phosphoribosyltransferases, and nucleic acid polymerases. “Transport,” in contrast, here denotes only the transfer of a substance (or the translocation of a chemical substituent) across the plasma membrane in either direction, as mediated by a saturable, selective carrier. “Incorporation” denotes the appearance of radioactivity derived from substrate in a specified cellular compartment, chemical compound, or class of compounds. Uptake of nucleosides and bases by cultured cells has generally been found to be approximately linear with time for between 1 and 10 minutes and initial uptake velocities have been assumed to reflect those of transport of the substrates into the cell. Some indirect lines of evidence reviewed previously (Plagemann and Richey, 1974) supported this assumption. The prime evidence was ( I ) that uptake obeyed simple Michaelian kinetics suggestive of a single, saturable, rate-determining step in the overall uptake process: (2) that the kinetic constants for uptake by whole cells were much lower than those for the phosphorylation of the substrates in cell lysates: (3) that the intracellular steady-state concentration of free substrate appeared to be far below that in the extracellular fluid: and (4) that uptake was inhibited in an apparent competitive manner by various substances which were presumed to act on the transport step since they did not affect the intracellular metabolism of the substrate. Recent evidence, however, has indicated that, although these observa-

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tions are correct, their interpretation is more complicated and that the uptake rates estimated from time intervals of minutes (here referred to as long-term rate) reflect the intracellular accumulation of phosphorylated intermediates. Transport, on the other hand, has been found to be so rapid that intracellular steady-state concentrations of free substrate are attained within seconds, at least at concentrations below the MichaelisMenten constant of transport. Catabolic reactions which occur in most types of cells also complicate studies of transport of several nucleosides. Adenosine, deoxyadenosine, cytidine, and deoxycytidine are deaminated, and guanosine, deoxyguanosine, inosine, deoxyinosine, uridine, deoxyuridine, and thymidine are subject to phosphorolysis, the products of which may be further catabolized. Hypoxanthine and guanine, on the other hand, are converted to nucleosides by purine nucleoside phosphorylase. In animal cells these reactions occur intracellularly and the products, if not phosphorylated, are subject to exit transport. Nucleotides, in contrast, are largely retained by the cells, since the plasma membrane is relatively impermeable to most phosphorylated compounds (see Section VII). The distinction between long-term rates of nucleoside uptake and rates of transport is illustrated most strikingly by data on the uptake and metabolism of [3H]adenosine in P388 mouse leukemia cells. At an extracellular adenosine concentration of 100 p M , long-term uptake was approximately linear for at least 30 minutes (Fig. 1A). Though the uptake curve appears to extrapolate near the origin, closer inspection shows that, at the first time point (30 seconds), the intracellular concentration of radioactivity already exceeded that in the medium. Furthermore, analysis of the culture fluid (Fig. 1B) showed that adenosine disappeared from the medium 70 times more rapidly than radioactivity accumulated within the cells. By 30 minutes most of the radioactivity accumulated in the medium was in the form of inosine and hypoxanthine, a consequence of the deamination of adenosine and the phosphorolysis of the product inosine. Since all evidence indicates that deamination is solely an intracellular process (Lum et a / . , 1979), it is obvious that the rate of uptake of radioactivity by the cells from exogenous labeled adenosine represents at best 1.5% of the transport rate. In fact, it was shown that upon exposure of P388 cells to adenosine at concentrations of 100 p M and above, the intracellular rate of deamination approaches that of the transport rate, since deaminase and transport have similar Michaelis-Menten constants with respect to adenosine, while the maximum velocity of deamination (as measured in cell lysates and expressed per cell) exceeds that of transport 2- to 3-fold (Lum et al., 1979; see also Table IV). The need to sample at short intervals if one is to estimate initial

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r-B

CULTURE FLUID

TOTAL

v,, =63pmole/pl CELL H 2 0 . sec

0 60

ADENOSINE

W

z 0

10

20

3(

TIME I MIN)

FIG. I . Adenosine uptake and metabolism by P388 mouse leukemia cells. (A) A suspension of I .5 X 10' cellsiml of serum-free basal medium was supplemented (0 time) with 100 p M [3H]adenosine (3.4 cpm/pmole) and incubated at 37°C. At various times, duplicate 0.5-ml samples of suspension were centrifuged through an oil layer (see text; Section I) and the cell pellets were analyzed for radioactivity. All values are averages of the duplicate samples corrected for nonspecific substrate trapping as estimated with [14C]inulin. (B) Adenosine, inosine, and hypoxanthine in the cell-free culture fluid were separated chromatographically a s described by Lum et a / . (1979). The velocities of uptake into cell material (A) and of the disappearance of adenosine from the medium (B) were estimated graphically from the linear portions of the curves and are based on an intracellular water volume of 1.3 pl/106 cells as estimated with 3H20 (Wohlhueter et a/., 1978a). The broken line in (A) indicates the intracellular concentration of radioactivity equivalent to that in the medium. Data are similar to those reported by Lum et ( I / . (1979).

transport velocities in metabolizing cells has been emphasized previously by Berlin and Oliver (1975). The validity of this admonition, and a quantitative appreciation of "short ," is becoming increasingly clear. The data in Fig. 1, for example, reveal that a 30-second sample would grossly underestimate the adenosine transport rate. In fact, at 24°C samples at 3-5 seconds were the longest that yielded reasonably accurate estimates of initial velocities of adenosine transport in P388 cells (Lum et a / . , 1979). Even so, identification of the main rate-determining step in long-term uptake may be contingent on the concentration of exogenous substrate. For example, at extracellular nucleoside concentrations far below the Michaelis constant for transport, the rates of intracellular phosphorylation of some nucleosides and of hypoxanthine approach those of trans-

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port, whereas at concentrations comparable to or exceeding the Michaelis constant for transport, the transport rate far exceeds the phosphorylation rate (for detailed discussion see Section 111). Elimination of the ambiguities inherent in metabolizing cells is of clear advantage to transport studies. Nucleoside and purine transpmt has been studied successfully in the absence of intracellular metabolism by the use of erythrocytes or of mutant clones of cultured animal cells which are deficient in specific metabolic enzymes and by the use of cellisubstrate systems in which substrate metabolism is blocked in some other manner. For example, uridine and thymidine transport has been studied in human erythrocytes which lack enzymes for the phosphorylation and phosphorolysis of pyrimidine nucleosides (Oliver and Paterson, 1971; Lieu et a / . , 1971). The use of enzyme-deficient mutants of cultured cells for transport studies was introduced by Kessel and Shurin (1968), who studied the uptake of deoxycytidine and cytosine arabinoside in a deoxycytidine kinase-deficient line of L 12 10 mouse leukemia cells. Subsequently, mutant cell lines deficient in thymidine kinase, uridine kinase, hypoxanthinei guanine phosphoribosyltransferase and adenine phosphoribosyltransferase have been employed to study the transport of thymidine, uridine, hypoxanthineiguanine, and adenine, respectively (Schuster and Hare, 1971: Cunningham and Remo, 1973; Zylka and Plagemann, 1975; Plagemann et a / . , 1976; Wohlhueter et a / . , 1976; Alford and Barnes, 1976; Plagemann et a / . , 1978b: Witney and Taylor, 1978: Murphy et u / . , 1977). Another approach to the prevention of substrate phosphorylation has been to deplete cells of ATP by preincubation in a glucose-free medium containing KCN and iodoacetate. This approach has the advantage of general applicability, since it prevents all phosphorylation reactions (Plagemann and Erbe, 1973; Plagemann et a / . , 1976; Wohlhueter et n / . , 1978a). Such treatment renders cells somewhat more fragile osmotically (Plagemann el "/., 1976), perhaps because ATP depletion causes the aggregation of integral membrane proteins (Gazitt et a / . , 1976), and, in some cell lines, has been observed to cause a slight decrease in transport capacity (Plagemann et a / . , 1978b). Overall, however, the kinetics of transport of nucleosides and hypoxanthine in enzyme-deficient and ATPdepleted cells are comparable (Plagemann et u / . , 1978b; Wohlhueter et a / . , 1978a, 1979a; Marz et a / . , 1979). Substrate metabolism other than phosphorylation can sometimes be inhibited by treatment of cells with appropriate inhibitors. For example, adenosine deamination has been blocked by preincubation of the cells with deoxycoformycin thus permitting an accurate assessment of adenosine transport (Lum et [ I / . , 1979). A third general approach for measuring substrate transport in the ab-

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sence of metabolism is the use of natural nucleosides or nucleobases or analogs thereof that are not subject to enzymatic modification, but still possess substrate activity for the respective transport systems. This approach has been successfully employed in the case of hexose transport by the use of 3-O-methyl-~-glucose, but has been applied to the study of nucleoside transport only recently. Kessel(l978) has shown that 5’-deoxyadenosine is transported by the nucleoside transport system in L 12 10 mouse leukemia cells, but is neither phosphorylated nor deaminated by the cells. Yoshida sarcoma cells (Mulder and Harrap, 1975),golden hamster fibroblasts (Heichal et u / . , 1978, 1979), and rat uterus (Oliver, 1971) do not phosphorylate cytosine arabinoside or do so only slowly, and thus lend themselves to a study of transport of this nucleoside. Thymine likewise is metabolically inert, and uracil is converted to nucleosides and nucleotides only very slowly in many animal cells and both have been used in transport studies (Zylka and Plagemann, 1975; Plagemann et ( I / . , 1978b). Thymidine transport has been examined in primary cultures of rat hepatoma cells which phosphorylate this nucleoside to only a limited extent (Ungemach and Hegner, 1978). Studies with cells in which the substrate was not modified intracellularly clearly indicated that nucleoside, purine, and uracil transport in most animal cells is energy independent and nonconcentrative, i.e., the intracellular concentration at equilibrium equals that in the extracellular fluid. This type of transport is generally referred to as facilitated diffusion or facilitated transport. The availability of cells incapable of substrate metabolism by itself, however, does not assure success in transport measurements. Initial studies with enzyme-deficient or ATP-depleted cultured animal cells clearly indicated the great rapidity of the nucleoside and purine transport systems, but it was just this rapidity of transport which presented technical difficulties in the estimation of accurate transport velocities from substrate accumulation curves (see Section 11,E). These difficulties have been successfully solved only recently by the development of various technical improvements in measuring substrate accumulation by both cells in suspension and attached to supporting media. These technical improvements can be divided into three categories: ( 1 ) the rapid separation of cells and extracellular fluid; ( 2 ) the development of rapid mixing/ sampling procedures; and (3) the estimation of transport velocities and assessment of transport models from the entire time course of attainment of substrate transmembrane equilibrium. For studies of nucleoside and base influx, cells in suspension have been rapidly separated from the extracellular medium containing labeled

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substrate by centrifugation through or into an inert oil layer which has a density higher than that of the aqueous suspension medium, but lower than that of the cells themselves. Such a procedure has long been applied to study the distribution of substrate across the membrane of mitochondria (Werkheiser and Bartley, 1957).To study uridine influx human erythrocytes have been separated from the medium by centrifugation into dibutylphthalate and the transport rate was estimated from the rate of disappearance of radioactivity from the medium (Oliver and Paterson, 1971). To study nucleoside and base influx in suspensions of cultured animal cells (Wohlhueter et ( I / . , 1976, 1978a: Ungemach and Hegner, 1978), mouse lymphocytes (Strauss et [ I / . , 1976), and mouse lung macrophages (Pofit and Strauss, 1977) cells have been centrifuged into silicone-oil mixtures and influx measured by the appearance of radioactivity in the pelleted cells. If high-speed centrifugation is employed cells can be separated from the medium in less than 2 seconds (Wohlhueter et i l l . , 1978a). The time resolution of this approach has been further improved by use of a rapid mixing technique similar to those of the stop-flow kineticist, with which the intracellular accumulation (and metabolism) of substrate can be followed in intervals as short as 1 second (Wohlhueter et i l l . , 1976, 1978a). Fixed aliquots of a suspension of cells are rapidly mixed with a solution of radioactively labeled substrate at short time intervals by means of a hand-operated dual-syringe apparatus. Cell substrate mixtures emerging from the mixing chamber are dispensed into 12 tubes which contain an oil mixture and are mounted in a microcentrifuge. After the last sample is mixed the centrifuge is started and within 2 seconds the cells have entered the oil phase, thus terminating transport. Thus it is possible to obtain 12 time points of substrate accumulation within a time period as short as 15 seconds (see Section 111). The apparatus and methodology have been described in detail by Wohlhueter et id. ( 1978a). In substrate efflux measurements from erythrocytes preloaded with radioactive substrate a rapid separation of the medium from the cells has been achieved by filtration through Millipore filters, followed by analysis of the filtrate for residual substrate concentration (Mawe and Hempling, 1965; Lassen, 1967; Cabantchik and Ginsburg, 1977). I t has been estimated that the sampling time can be reduced with practice to 1 second (Cabantchik and Ginsburg, 1977). Cells attached to solid substrate lend themselves to another approach to the rapid removal of extracellular medium in substrate influx studies. For example, cells attached to glass coverslips have been immersed in substrate solution and then rapidly rinsed by repeated immersion in cold

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buffer rinse solutions (Hawkins and Berlin, 1969; Foster and Pardee, 1969: Sander and Pardee, 1972). In other studies monolayer cultures of animal cells in Petri plates have been used and the substrate solution and rinse fluids have been removed by rapid aspiration (Rozengurt et a / . , 1977b; Plagemann et al., 1978b). In this procedure cell monolayers can be washed free of extracellular substrate within about 6 seconds (Plagemann et u / . , 1978b). Each of these experimental approaches has certain advantages and disadvantages. Separation of cells from the substrate solution by centrifugation into an inert oil layer necessitates corrections for trapping of extracellular substrate in an aqueous layer surrounding the cells which is not removed by centrifugation through oil and which represents between 10 and 20% of the intracellular aqueous space in various types of cultured cells (Wohlhueter et a / . , 1976, 1978a). The trapped extracellular water space relative to the intracellular water space increases progressively with a decrease in cell size. Such substrate trapping has been assessed by the use of substances to which cells are impermeable or only slowly permeable such as inulin or L-glucose (see Wohlhueter et a / . , 1978a). Such corrections are not required when removing extracellular substrate by rinsing cell layers attached to dishes or coverslips with aqueous buffers, but in this procedure the possible loss of intracellular substrate during the rinsing period needs to be considered. Low-temperature rinses minimize, but do not abolish, such losses. For example, it has been calculated that Novikoff cells would be expected to lose half their intracellular thymidine pool, if it is less than 100 p M , during a 43second rinse with buffer at 4°C (Wohlhueter et a / . , 1979a). However, such losses can be completely prevented in the case of nucleosides and purines by inclusion of high concentrations of a transport inhibitor, such as dipyridamole (Persantin) (Plagemann et a / ., 1978b) or 6-([4-nitroben(nitrobenzylthioinosine) (Rozengurt zyl] thio)-9-p-~-ribofuranosylpurine et u / . , 1978) in the rinse fluid. The use of “stopper solutions” to quench transmembrane fluxes rapidly has earlier been applied to measure nucleoside transport in cells in suspension. Cass and Paterson (1972) introduced the use of 2-hydroxylnitrobenzylthioguanosine in this capacity followed by the centrifugal separation of the cells from the medium to demonstrate nucleoside accelerated exchange diffusion in human erythrocytes. Cabantchik and Ginsburg (1977) used the same inhibitor, as well as nitrobenzylthioinosine, followed by centrifugal separation of the cells from the medium and removal of residual extracellular substrate by washing in aqueous solutions containing the inhibitor to study uridine influx in human erythrocytes. A similar approach using dipyridamole has been

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employed successfully to measure uridine accumulation in uridine kinasedeficient cultured Novikoff rat hepatoma cells (Plagemann et a / . , 1978b) and of 5’-deoxyadenosine in L1210 cells (Kessel, 1978). In the latter study a solution of mercuric chloride plus sodium iodide was also used as a stopper solution. The filtration method, on the other hand, appears useful only where the experimental protocol permits measurement of radioactivity in cell-free filtrate, as in efflux studies (Cabantchik and Ginsburg, 1977; Wohlhueter et ul., 1978a). A disadvantage encountered in transport studies with cells attached to culture dishes or coverglasses is the small intracellular volume available relative to the total medium volume required to cover the cell layer. At best, the available intracellular volume represents 0.5% of the extracellular volume. In nonphosphorylating cells, therefore, the amount of substrate present intracellularly at equilibrium is severely limited and incomplete removal of extracellular substrate introduces large errors in uptake values. Because of this relatively small intracellular volume available in culture dishes some investigators failed to detect nucleoside and purine transport in cells lacking the enzymes responsible for their conversion to nucleotides (see Section 11, E). Furthermore, it is difficult to estimate accurately the intracellular volume of attached cells, knowledge of which is needed to assess the absolute intracellular concentration of substrate. These volume limitations do not apply to suspensions of cells which can be prepared at any desired density. Erythrocyte suspensions with hematocrits of up to 40% (v/v) have been used (Oliver and Paterson, 1971), in which case one can estimate substrate uptake by measuring its disappearance from the medium. Much lower cell densities (1.5-7%, v/v) have been used with suspensions of cultured cells (Wohlhueter et d., 1978a) which, though precluding measurement of substrate disappearance from medium, assures accurate direct measurement of accumulation within cells. Intracellular volumes of suspended cells can be readily estimated by exposing cells to 3 H 2 0 and then separating the cells from the extracellular fluid by centrifugation through an oil layer (Wohlhueter et a / . , 1978a). Regardless of the experimental approach employed, transport velocities have generally been estimated graphically from the initial linear phases of time courses of intracellular accumulation of substrate (influx), of appearance of substrate in extracellular fluid from preloaded cells (efflux), or of the movement across the membrane of isotopically labeled substrate at chemical equilibrium (equilibrium exchange). In general, initial entry or exit rates have been based on only few early time points. Often, because of the rapidity of nucleoside and base transport, the

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235

initial, linear phase is impractically short, especially at low extracellular substrate concentrations. These facts, together with theoretical considerations discussed in the next section, limit the accuracy of graphical estimation of initial velocities. More accurate estimates of transport velocities are obtainable by an analysis of the entire time course of attainment of transmembrane equilibrium in terms of integrated rate equations describing various transport models (Section 11). In addition, such an approach yields considerable information on the mechanism of transport per se. Though we have stressed here transport measurements in the absence of metabolism, we consider in Section I11 the relationship of transport to phosphorylation, and their operation in tandem. This topic is developed more fully by Wohlhueter and Plagemann (1980). Other reviews pertinent to nucleoside and/or purine uptake in eukaryotic cells are those by Plagemann and Richey (1974), Berlin and Oliver (1973, Hochstadt (1974), and Perdue (1978). Studies on nucleoside and base uptake by prokaryotic cells have been reviewed by Hochstadt (1974). II. CARRIER MODEL FOR FACILITATED DIFFUSION AND TESTS FOR ITS APPLICABILITY TO NUCLEOSIDE AND BASE TRANSPORT

Recent studies in which the transport of nucleoside and purine base transport by animal cells has been kinetically separated from subsequent metabolic steps are clearly compatible with the view that the movement of uridine across the human erythrocyte membrane (Cabantchik and Ginsburg, 1977) and the movement of various nucleosides and purines across the membrane of animal cells in culture (Wohlhueter et al., 1979a; Marz ei al., 1979; Lum ei al., 1979) proceeds via the simple carrier mechanism formulated mathematically by Lieb and Stein ( 1974) and Edam and Stein (1974). The model is summarized in Fig. 2 along with the various experimental protocols which have been used to test its applicability to the transport of nucleosides and purines in human erythrocytes and cultured animal cells. Edam and Stein (1974) developed unidirectional flux, initial velocity, and integrated rate equations corresponding to these experimental protocols. It should be noted that these experimental designs are strictly applicable only to cells which fail to metabolize the substrate or in which metabolism is blocked in some manner. In general, we follow the terminology of Eilam and Stein (1974).

236

PETER G . W. PLAGEMANN AND ROBERT M. WOHLHUETER

EXPERIMENTAL DESIGN =

0, s2 = 0

S2 V A R I E D (CELLS PRELOADED) s2 VARIED AS s1 IS VARIED (CELLS PRELOADED)

s2’

VARIED (CELLS PRELOADED)

S2 ’’ K

(CELLS PRELOADED)

S2 V A R I E D 0 * S1 (CELLS PRELOADED)

S2

>>

K (CELLS PRELOADEU)

S2 V A R I E D 0 *

I MEMBRANE I

S1

(CELLS

PRELOADED)

FIG.2 . The simple carrier model in various experimental configurations. Nomenclature is that of Eilam and Stein (1974) and pertains to the conditions set at the beginning of the experiment: S, = extracellular substrate concentration; S, = intracellular substrate concentration; S* = isotopically labeled substrate; K = a fundamental constant reflecting substrate-carrier affinity: C = carrier; zt = zero-trans: ee = equilibrium exchange: it = infinite-trans; ic = infinite-cis. The external face of the plasma membrane is designated face 1 , and the internal face as face 2: k , , k , , and g , and g , are the rate constants for the “movement” of the unloaded and loaded carrier, respectively.

A. Zero-trans Influx and Efflux

In the zero-trans (zt) procedure one measures the transport of a substrate from one side of the membrane (the cis side), where its concentration is varied, to the other side (the trans side) where its concentration is initially zero (Fig. 2 ) . The intracellular concentrations of nucleosides and purines in animal cells are generally very low ( < 1 pM) and in influx studies (transport from face I to face 2 ) , therefore, the initial intracellular substrate concentration is considered to be zero. Initial rates of uridine entry (v;:) into human erythrocytes and exit ( ~ 5 : )from these cells have been estimated by graphical or linear regression analysis of initial time courses of change in intracellular ( S , ) or extracellular ( S uridine concentration (Oliver and Paterson, 1971 : Cabantchik and Ginsburg, 1977). Michaelis constants for uridine influx ( K ; ; ) and efflux ( K ; : ) and the corresponding maximum velocities @and Vzi) were computed from plots of the estimated slopes as a

237

PERMEATION IN ANIMAL CELLS

function of S on the cis side. The estimated kinetic constants are summarized in Table I and are further discussed in Section II,E. Because of the rapidity of nucleoside and purine transport in most animal cells, however, it has often proved technically infeasible to obtain accurate, direct estimates of initial velocities, thus necessitating the use of integrated rate equations. The application of nonlinear equations not only permits estimation of true initial velocities when the linear portion of the curve is impractically short, but also can, theoretically at least, yield valuable information about the molecular properties of the transport system (Eilam and Stein, 1974; Wohlhueter et a / . , 1978a, I979a). Edam and Stein ( 1974) have developed integrated rate equations to describe the time course of transport beginning at zero-trans and proceeding to equilibrium. They detail a graphical method for estimating the various rate and association constants. The graphical method relies on a logarithmic transformation in which the relative errors become large as the trans substrate concentration approaches the cis concentration and statistical weighting becomes problematic. The approach has been rarely used. Wohlhueter et a / . (1978a, 1979a) and Heichal et al. (1979) have arrived at somewhat more simplified, integrated rate equations (for zero-trans in the I to 2 direction) by assuming that the exogenous substrate concentration ( S , ) is constant. This assumption holds for zero-trans influx studies if the intracellular H,O volume represents not more than about 5% of the total volume of suspension, a condition generally met in studies with suspensions of cultured animal cells. Heichal et a/. (1979) have applied the equation in a linearized form to evaluate the transport kinetics of cytosine arabinoside. Wohlhueter et a / . (1978a, 1979a) have employed the equation in exponential form to fit transport data by nonlinear regression. They write the equation in a form analogous to the equation for first-order approach to equilibrium, although implicit with respect to intracellular concentration (S'J:

f

1

(S1)

(1)

where S,J = concentration of S , at time t (Sz,o= 0); and f l and f 2 are functions of various kinetic parameters as defined by Eilam and Stein ( 1974):

f,(S)= KRoo + R12S1 + R21S1 + ST ReeIK f z ( S 1 , Sz,t) = (Rz1 + ReeSlIK)

Sz,t

(2)

TABLE I KINETICPARAMETERS FOR ZERO-TRANS, EQUILIBRIUM EXCHANGE, A N D INFINITE-CIS INFLUX ERYTHROCYTES~ Michaelis-Menten constant

(WW

Protocol zt zt ee ee ic ic

(12) (21) (12) (21) (12) (21)

400 2 122 73 2 63 1310 2 92 1280 2 142 252 ? 90b 937 2 226b

Maximum velocity (pmoleipl H,O.second)

33 8.9 130 120 14' 11.5'

?

2 k 2

5.2 0.6 20 6.1

Michaelis- Menten constant (P1Z.T)

710d N De 530V ND ND 7000

AND

EFFLUXOF U R I D I N IEN HUMAN

Maximum velocity (pmole/pl packed cells.second)

8.4d 3Ih 100 156h ND 1700

Temperature ("C) 15

25 25 25 37

Data are from Cabantchik and Ginsburg (1977), and were collected at 25°C. Sources of other data are indicated in individual footnotes, and were collected at the temperature given in the final column. * K Z and KFl values calculated from the experimentally determined ee and zt maximum velocities according to K V e e = K i12c V"' or = KF1V;:, were 231 and 1080, respectively. Equals V ; : or V ; : , respectively; calculated from the Y-intercepts of Figs. 7 and 9 of Cabantchik and Ginsburg (1977) [see Eq. @)I. From Oliver and Paterson (1971). N D = not determined. From Pickard and Paterson (1972). From Lieu et ( I / . (1971). Estimated from data i n Fig. 6 . a

'

PERMEATION IN ANIMAL CELLS

239

K is a measure of the affinity of the carrier for its substrate, and is related directly to the Michaelis-Menten constants apparent in various experimental protocols (see Table I); with zero-trans influx, for example, K?: = K ( R c J R l z ) .The R-terms are resistance factors, proportional to the time of round-trip for the carrier in one of four modes; ( I ) loaded on the inbound trip and empty on the outbound trip (Rlz);( 2 ) empty on the inbound and loaded on the outbound trip (Rzl); (3) empty in both directions (Roo);and (4) loaded in both directions (Ree). Necessarily, Roo + Re, = R l 2 + RZl. Functional symmetry of the carrier is manifest as equivalence of the various R-terms. If carrier movement is indifferent with respect to direction (idout symmetry) R,, = Rzl. If loaded carrier moves as rapidly as the unloaded carrier Re, = Roo,and for a cmpletely symmetrical carrier all R-terms are equal (and represented by R ) , and the Michaelis- Menten constants apparent in various experimental protocols are all equal to K . The R constants are the reciprocals of the corresponding maximum velocities, for example, R 12 = I/VZ,:. As t approaches 0, fz approaches 0, and as t approaches CQ, fz becomes a constant 6 t . If one assumes f z to be negligible at all t , as Wohlhueter er al. did in earlier studies (Wohlhueter et al., 1976, 1978a) for the sake of mathematical simplicity, Eq. (1) is reduced to the integrated equation for a first-order reaction: Sz,t = S , [ l - exp(-k’t)]

(4)

where k’ = pseudo-first-order rate constant = l / f l ( S l ) .In these studies initial zero-trans velocities were calculated from computed k’ values according to the first derivative of Eq. (4) at t = 0: vZ,\= k ’ S 1 . When S l 4 K?:, f z is indeed insignificant, so that Eq. (4) describes the influx of substrate under these conditions; this of course is the condition in which influx is first order with respect to substrate concentration. Subsequently, computational procedures have been developed (Wohlhueter er al., 1979a; Marz er al., 1979) to permit exact solutions of Eq. ( I ) , thus allowing least-squares fits of Eq. (1) to time courses of the approach to transmembrane equilibrium, whereby t and S , are treated as independent variables and Sz,t as dependent variable (based on the experimentally determined substrate radioactivity and intracellular HzO space).’ This non-linear, multivariable regression procedure determines the values of K , the substrate: carrier affinity constant, and of the various

’

All computations were carried out on a Hewlett-Packard (Loveland, CO) 9825A desktop computer equipped with a 9871A printer. Curve fitting programs were developed on the algorithm of Dietrich and Rothmann (1975); the authors will honor requests for program listings.

240

PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER

R-constants (and associated standard errors of estimate) best fitting a set of data comprising complete time courses at 6 or more substrate concentrations. Application of this procedure to uridine transport in uridine kinasedeficient Novikoff rat hepatoma cells is illustrated in Fig. 3 . Kinetic parameters for the transport of thymidine, uridine, adenosine, cytidine, deoxycytidine, adenine, and hypoxanthine by this and other cell lines are summarized in Table I1 and discussed further in Section II,E. The data in Fig. 3 also emphasize the technical difficulties encountered in estimating accurate uf: values by graphical or linear regression methods [see Eq. (5) in legend to Fig. 31. The curves described by the integrated zero-trans equation, Eq. ( l ) , deviated from linearity within the first few seconds of incubation at 24°C. The hazards involved in regarding these early time points as representing initial, linear substrate entry are indicated by linear regression analysis of the data (not shown). The linear regression lines fit reasonably well (correlation coefficient ru,B = 0.920.97), but do not pass through the origin-a frequent observation made by other investigators using linear regression-and the slopes of the lines underestimate the true u:; by 65-70%. The velocities estimated from such slopes yield reasonable Michaelis-Menten hyperbolae, but the estimated maximum velocities grossly underestimate V B and, consequently, Kq;.

B. Equilibrium Exchange Inward and Outward In the equilibrium exchange procedure, the substrate concentration at the two faces of the membrane is held equal and the movement of radioactively labeled substrate from one face to the other ( 1 to 2 or vice versa) is followed as a function of time and substrate concentration (Fig. 2). For the equilibrium exchange procedure, too, an integrated rate equation has been solved by Eilam and Stein (1974) for the simple carrier model. For isotope initially on side 1 of the membrane it is:

where N z , t = intracellular concentration of radioactivity at time t , which is proportional to the specific radioactivity of intracellular substrate; N z , , = intracellular concentration of radioactivity at f = co which is equal to N the concentration of radioactivity per equivalent volume of medium; S = concentration of substrate and K e e and V"" are the apparent Michaelis-Menten constants for equilibrium exchange. Equation (6) as-

241

PERMEATION IN ANIMAL CELLS 8otC

1

SI=80pM

2 10

20 TIME

20

20

40

I SEC)

FIG.3. Kinetics of zero-trans uridine transport i n uridine kinase-deficient Novikoff cells at 24°C. Samples of 448 pI of cell suspension ( I .6 x lo7 cells) were mixed in rapid succession with 61 pI of solutions of [S3H]uridine, the mixtures were centrifuged through oil layers, and the cell pellets were analyzed for radioactivity (for detailed description of methodology, see Wohlhueter et a / . , 1978a, 1979a). The final uridine concentrations were 20, 40, 80, 160, 320, 640, and 1280 p M (240 cpm/pl, irrespective of concentration) and the ambient temperature was 24°C. All values were corrected for trapping of substrate in extracellular H 2 0 space. The intracellular and extracellular water spaces in cell pellets were 13 and 1.3 pl/107 cells, respectively, i.e., 20.5 and 2.0 p1/509-pl sample, respectively. Data are from Table I by Plagemann et a / . (1978b), but have been reanalyzed by fitting Eq. (1) to the pooled data with all R parameters held equal; time ( t ) and S , were treated a s independent variables and S2,tas independent variable. The best fitting parameters were K = 261 f 12 p M and V = 25.8 f 0.5 pmole/pl cell H,O.second (= UR). The correlation coefficient ( r , 3 was 0.9918. The theoretical curves for S , = 20, 40, 80, 160, 320, and 640 pLM are illustrated in (A-F), respectively. Initial zt velocities (of:) in pmole/pl cell H20.second were calculated from the computed kinetic parameters according to the zero-trans rate equation of Eilam and Stein (1974) at S, = 0:

sumes that the volume available to substrate within the cell is negligible relative to the total volume of cell suspension, a condition that generally applies to such transport studies with animal cells (as discussed already). A similar equation, with subscripts identifying membrane face reversal, holds for equilibrium exchange in the 2 to 1 direction. Equation (6) reduces to a first-order equation by substituting V e e / ( K e " + S) = k'. Cabantchik and Ginsburg (1977) used this formulation to measure the equilibrium exchange of uridine by human erythrocytes in

TABLE I1 KINETICPARAMETERS FOR ZERO-TRANS, EQUILIBRIUM EXCHANGE, A N D INFINITE-TRANS INFLUXOF VARIW- NUCLEOSIDES A N D PURINES IN CULTURED MAMMALIAN CELLSAT 24"Ca,b Michaelis-Menten constant (zt,12) Cell line Novikoff

P388

CHO

Substrate Thymidine Uridine Deoxycytidine Cytosine arabinoside Cytidine Inosine Adenosine Hypoxanthine Adenine Uracil Thymidine Uridine Adenosine Hypoxanthine Thymidine Uridine Hypoxanthine Adenine

(ee,12)

(it,12)

(it,12)

cell H,O.second)

(PM

228 t 14(9) 250 ? 13(12) 626 t 52(4) 762 2 57(1) 2,425 f 497(4) 1 5 0 5 9(1) 103 2 8(1) 349 & 17(4) 3,300 2 524(1) 14,200 2 950(1) 125 f l O ( 1 ) 230 t 17(1) 123 ? 9(1) 445 t 31(1) 103 t S(5) 1 6 9 ? 12(1) 1,463 t 69(1) 2,109 ? SOO(1)

273 2 42(4) 241 2 22(1)

246 2 44( 1) 254 2 55(2)

7-

1 I-5

2.5

r_'

1

46 2 9 .'5 2 0.8

71 2 4 26 2 2

Ji + i; 59 2. 2

J8 t

136 ? 27(1) 551

?

85(1)

:-/

13 f E: ? 17 2 O . i 53 2 10

!! t 1.0 68 t 5

1 1 .z 1 1 164 i 5

31

--: c1:7

19 z

138 ? 14(1)

0.3

295 07 18 ?: 1.3 6.8 f 0.8

18 t 0.6

- .

0.1 1.5 t 0.3 39 f 5 3 . b i

' Data are from Plagemann et al. (1978a,b); Wohlhueter et al. (1979a,b); Marz et al. (1979): Luni P I ul. (1979) o: pr-eviousiy unpublished in the case of cytidine, inosine, and adenosine transport in Novikoff cells. Those for uridine and deoxpcytidine (PI:.iscmann ei u!., 1978a,b) were recalculated by fitting Eq. (1) to the data originally reported (see Fig. 3). Values for uridine equilibrium exchar?ge transpwi are from Fig. 4. Values for uridine infinite-trans transport are averages of data from Fig. 5 and from Fig. 8 in Plagclmann r i (11. i i978bi. * Michaelis-Menten constants apparent in zero-trans, equilibrium exchange and infinite-trans protoculs ( K l ; , ,K"'. and K\\) and the corresponding maximum velocities ( V f : , Veeand V',$). To evaluate zero-trans data, Eq. (1) was fit to combined data with Seven or eight substrate concentrations with all R-parameters held equal (see Fig. 3), so that K f : = K and U V f ; = R. Equilibrium exchangc and infinite-trans data were evaluated as described in Sections II,B and C. Values are either means of the numbcr of experiments indiraied in parentheses ? standard error (of the mean), or from single experiments where the kinetic parameters are stated ? standard error of thc csriinate (as defined in Wohlhueter et al., 1979a).

PERMEATION IN ANIMAL CELLS

243

both directions. k' was estimated by linear regression of plots of experimental data where k' = u " / S at t = 0 and ue" expresses the unidirectional flux of isotope. For equilibrium exchange experiments in the 1 to 2 direction uee was also estimated graphically from the initial rate of entry of radioactivity into the cells; both methods gave similar results. K"" and V e ewere computed by conventional plots of wee versus S, as in the zero-trans procedure. Computed values are summarized in Table I and are discussed further in Section I1,E. Wohlhueter et d.(1978a, 1979a) rearranged Eq. (6) in exponential form:

in which form V"' and K e e can be estimated directly by nonlinear regression to pooled time courses of attainment of radioactivity equilibrium at several substrate concentrations, whereby t and S are treated as independent variables and N2,t as dependent variable ( N 2 , tis based on the experimentally determined 3 H 2 0 space of the cells or on the apparent intracellular radioactivity space as t+m, N2,m;Wohlhueter, Erbe and Plagemann, previously unpublished procedure). This approach is exemplified in Fig. 4A-D for uridine equilibrium exchange in the I to 2 direction in uridine kinase-deficient Novikoff cells. The kinetic parameters are summarized in Table I 1 and discussed further in Section II,E. I n a previous study on thymidine equilibrium exchange in thymidine kinase-deficient Novikoff cells (Wohlhueter et al., 1979a) V"" and K'" (see Table 11) were estimated in a different, though statistically less defensible, manner: k' = V e e / ( K e e+ S ) was substituted in Eq. (7) and k' was computed by nonlinear regression procedures for the individual time courses of attainment of radioactivity equilibrium at each substrate concentration. V'" and Kee were evaluated by replots of k' versus S or of the product k ' S = uee versus S (Fig. 4E and F). These alternative methods of fitting data do not necessarily give identical results for K"' and V"', for in essence each weights the data differently. C. Infinite-trans Procedure

In the infinite-trans (it) procedure, one measures the movement of radiolabeled substrate at a given concentration from one face of the membrane (the cis side) to the other face (the trans side) where unlabeled substrate is present at a concentration + K of the transport system (i'e., S2+m) (Fig. 2).

244

PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER

........ .................

-

w

r 0 05

.................

O

.... 1

................... 2

3

{"

loo0

5oo

..,

"

0

S = 80 pM

$lZft:pM

,

ry,; = 0 9 9 3 6

S =1280vM OO TIME (SEC)

100

200 URlDlNE ( p M 1

FIG.4. Kinetics of uridine equilibrium exchange in uridine kinase-deficient Novikoff rat hepatoma cells. Samples of a suspension of about 3.6 x 10' cells/ml of basal medium were preincubated with 20, 40, 80, 160, 320, 640, 1280, or 2560 F M nonradioactive uridine at 37°C for 30 minutes and then brought to 24°C. Samples of 448 pI of each suspension were mixed in rapid succession with 61 ~1 of a solution of an equivalent concentration of [SH]uridine, the mixtures were centrifuged through an oil layer, and the pellets were analyzed for radioactivity. The radioactivity concentration of the final mixtures was 380 c p d p l , irrespective of concentration. All values were corrected for uridine trapped in extracellular space of cell pellets (3.4 pl). The intracellular water space was 22.5 pCLYcell pellet. Equation (7) was fitted by nonlinear regression to the pooled data; time ( t ) and S were treated as independent variables and N2,f as dependent variable. The best fitting parameters were K" = 24 I + 22 p M and V" = 34.5 ? 0.8 pmoleipl cell H,O-second. The correlation coefficient (ry.r) was 0.9936. The theoretical curves for S = 40, 80, 640, and 1280 pM are illustrated in (A-D), respectively. In addition, a single-variable version of Eq. (7), N 2 , *= N 2 . - [I-exp(-k't)] (Wohlhueter et al., 1979a) was fitted to each individual time course of attainment of radioactivity equilibrium by nonlinear regression procedures. V"' and Kee were then computed by replots of the computed k' versus S ( E ) or of the product k'S = uee versus S (Michaelis-Menten plot, F). The best fitting kinetic parameters are listed for each plot. Vee is in pmole/pI cell H,O.second. (Previously unpublished data of Plagemann and Wohlhueter.)

The rapid kinetic technique developed by Wohlhueter et ul. (1976, 1978a) is not suited to an infinite-trans protocol, nor is rapid sampling as essential as with other experimental protocols. In the infinite-trans procedure radioactivity accumulates on the trans side against a concentration gradient (generally referred to as countertransport), because the transferred radiolabeled substrate becomes extensively diluted by the high concentration of unlabeled substrate on the trans face. Due to the reduced specific radioactivity of the labeled substrate on the trans side backflow of radioactivity is initially minimal and the linearity of radioactivity move-

245

PERMEATION IN ANIMAL CELLS

ment from the cis to the trans face is prolonged. Indeed, this fact is the unique feature of the infinite-trans procedure and countertransport is generally considered one of the unequivocal criteria for establishing carrier-mediated transport of a specific substrate. Figure 5 illustrates these principles. Infinite-trans accumulation of uridine in uridine kinase-deficient Novikoff hepatoma cells was estimated by conventional sampling methods at 15-second intervals, and is compared to zero-trans influx measured with rapid sampling techniques. It should be emphasized in connection with the data in Fig. 5 that an underestimation of v;: which is inherent in linear graphical methods for estimating v;: from substrate accumulation curves would make 0; appear to be significantly lower than vl;t,, when, in fact, they are the same. Such underestimation of vf; could lead to the conclusion of carrier asymmetry. Computed V & and K & values for nucleoside transport in cultured animal cells are summarized in Table I1 and are discussed further in Section II,E. D. Infinite-cis Procedure

Two different infinite-cis (ic) protocols have been used to study nucleoside transport in animal cells. One protocol was originally developed for measuring hexose transport by Sen and Widdas (1962). It measures the net movement of substrate from one face of the membrane, where it is present at a concentration % K of the transport system, to the trans face, where the substrate concentration is varied (Fig. 2). Sen and Widdas (1962) extrapolate the initial slope of the efflux curve to its intersection with the asymptote corresponding to equilibrium, thereby defining a time (re), which relates linearly to S, and the kinetic constants apparent in infinite-cis experiments. This procedure was followed by Lieu er al. (1971) to measure the efflux of various nucleosides from human erythrocytes. Their results are summarized in Table I and discussed further in Section II,E. Cabantchik and Ginsburg (1977) applied the infinite-cis protocol to the measurement of net uridine transport in human erythrocytes in both the 1 to 2 and 2 to 1 directions. uie was estimated by measuring the initial rate of transfer of radioactivity from one side of the membrane (cis face), where radioactively labeled substrate was present at a very high concentration, to the trans side, where the concentration of labeled substrate of the same specific radioactivity was varied (Fig. 2). These investigators converted the infinite-cis net flux equation of Eilam and Stein (1974) to a linear form: SZ -- 1 - - 1 vk(net) V:; KiC 12 VZt 12

+-

246

PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER

TIME I S E C l

FIG. 5 . Infinite-trans analysis of uridine transport i n uridine kinase-deficient Novikoff rat hepatoma cells. Samples of a suspension of 2 x lo' cellsiml of basal medium were incubated with and without 10 mM unlabeled uridine at 37°C for 20 minutes and then thermally equilibrated at 25°C for at least 5 minutes. Cells from 5-ml samples of uridinepreloaded suspensions were collected by centrifugation and washed rapidly once in 10 ml cold (0°C) basal medium. The cell pellets were rapidly warmed to 25°C and then suspended in 5 ml of basal medium (at 25°C) and containing 20, 40, 80, 160, 240, 320, 480, 640, 1280, or 2560 p M [3H]uridine (430 cpm/pl, irrespective of concentration). At 15, 30, 45, and 60 seconds of incubation at 25°C duplicate 509-pI samples of each suspension were centrifuged through oil and the pellets were analyzed for radioactivity. All values are averages of duplicate samples (infinite-trans procedure). Cells from the control suspension (not preloaded with uridine) were washed and suspended to the same density in basal medium and then analyzed for uridine transport by the rapid kinetic technique as described in the legend to Fig. 3, but with the substrate concentrations indicated above (zero-trans procedure). The intracellular H,O space was 22.5 pV509-pI sample. All values were corrected for substrate trapping in extracellular space of the cell pellet (3.6 p1/509-p1 sample). The time courses of zero-trans and infinite-trans accumulation of radioactivity at S , = 20 p M are compared in (A). Equation ( I ) was fitted to the pooled zero-trans data and uf: was calculated from the computed parameters as in Fig. 3. The best fitting parameters of transport for all seven concentrations were K = 290 9 p M and V = 26 ? 3 pmole/pI cell H,O.second. Initial infinite-trans velocities ( u & ) were estimated from the initial linear portion of the uptake curves (B) and were subjected to Michaelis-Menten analysis. The best fitting parameters were K\\ = 332 87 p M and V\: = 24 ? 3 pmole/pl cell H,O.second. The broken lines indicate the intracellular radioactivity concentration equivalent to the extracellular concentration.

*

*

247

PERMEATION IN ANIMAL CELLS

In a plot of l/uf; versus S, the X-intercept yields directly - K $ and the Y-intercept is l/VSk. Corresponding relationships with 1 and 2 interchanged apply to infinite-cis in the 2 to 1 direction. Kic Kic V B , and Vgl values for uridine transport in human erythro12, cytes, computed by linear regression analysis from such plots, are summarized in Table I and are discussed further in Section II,E. In the second protocol, also referred to as "accelerated exchange diffusion" (Cass and Paterson, 1972, 1973), the unidirectional flux of substrate is measured. One measures the movement of radioactivity from the cis side of the membrane, where labeled substrate is present at a concentration * K of the transport system, to the trans side, where the concentration of unlabeled substrate is varied. For unidirectional infinitecis transport in the 1 to 2 direction the unidirectional flux equation of Eilam and Stein (1974) reduces to (Wohlhueter et al., 1978a): vlc

-

K + S, ( K / V ? ' , )+ S , / P )

(9)

The subscripts are altered accordingly for the opposite direction. These equations are not Michaelian in form, so that KY2 and K',", are not defined, and moreover, as Vq', and VZ,:approach V e e(that is, as for a carrier symmetrical with respect to the mobility of loaded and unloaded carrier) the equations degenerate into identities. Only if the movement of loaded and unloaded carrier differs, i.e., V e e # Vzt, does the presence of unlabeled substrate at the trans face cause an accelerated transfer of radioactivity from the cis face. In fact, one of the useful features of the infinitecis procedure is that it allows the unequivocal demonstration of differences between VZtand V"". For example, such differences were observed by Cass and Paterson (1972) from infinite-cis transport experiments in the 2 to 1 direction for the transport of uridine and thymidine in human erythrocytes. uic was estimated from initial rates (20 to 30 seconds) of release of radioactivity from cells preloaded with "infinitely" high concentrations of labeled substrate into the medium containing varying concentrations of the same (or an alternate) substrate in unlabeled form. These investigators observed a linear relationship between I / v & and I! S1, and calculated apparent K , and V,,, from these plots. But, as previously mentioned, these parameters probably have little meaning with respect to the simple carrier model (Fig. 2). However, if Vz' and V" are sufficiently different Eq. (9) takes on some useful attributes. A plot of u';i versus S , (or v!$ versus S l ) , for example, is hyperbolic with a horizontal asymptote Vee, with Y-intercept = V;; and X-intercept = - K . Thus, theoretically, the infinite-cis protocol can directly yield values for

248

PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER

K as well as for V;", V;:, and V e e .In practice, however, this approach may not be satisfactory (Wohlhueter et al., 1978a). First, because the concentration of labeled substrate on the cis side is infinitely high, the relative rates of transfer of radioactivity are slow, and the radioactivity on the cis face is high in comparison to that on the trans face. The unfavorable "signal-to-noise ratio" results in imprecise estimations of vie and, consequently, in unsatisfactory fits of Eq. (9). Second, the hyperbolic extrapolation to the X-axis, and hence the value of K , is quite imprecise. Furthermore, this problem of extrapolation is accentuated with decrease in difference between V z tand V". For example, whatever difference there may be between V z t and Veefor nucleoside transport in cultured animal cells seems to be too small (see Section II,E) to allow this kind of analysis (Wohlhueter rt af., 1978a). Our attempts to fit Eq. (9) to some data of Cass and Paterson (1972) for the accelerated exchange diffusion of uridine by cytidine in the 2 to 1 direction in human erythrocytes did not converge satisfactorily, even though in this instance VZ:, and V" clearly differed (Fig. 6). Only when V;: was fixed at a graphically estimated value ( 3 1 pmole/pI cell H,O*second) was a successful fit of Eq. (9) to the experimental data obtained, with K = 336 & 58 p M and V e e = 156 k 12 pmole/pl cell H,O.second.

I.

l

I

I

ry,g = O . S E J ~ I 1 I

1

0

20

40

60

S, I CYTIDINE, m M )

FIG.6. Infinite-cis transport (accelerated exchange diffusion) of uridine in human erythrocytes. Data have been calculated from Fig. 6 of Cass and Paterson (1973). Initial rates of appearance of radioactivity in the medium were determined at 25°C when cells preloaded with 6 m M [2-I4C]uridine were incubated in media containing different concentrations of nonradioactive cytidine. Attempted regression of Eq. (9) for the 2 to 1 direction whereby S , was treated as independent variable and IJ& as dependent variable did not satisfactorily converge. Only when Vg: was fixed at a value estimated graphically (31 pmoleipl cell H,O.second) was a successful fit of the equation obtained with K = 326 ? 58 p M and V e e = 156 % 12 pmole/pI cell H,O.second.

PERMEATION IN ANIMAL CELLS

249

E. Interpretation of Experimental Data The kinetic parameters reported by Cabantchik and Ginsburg (1977) (Table I) are consistent with the operation of a simple carrier mechanism in the transport of uridine in human erythrocytes. First, as required by the simple carrier model (Fig. 2), the ratios of apparent maximum velocities/Michaelis-Menten constants for the various experimental protocols were all equal (to about 0.1) within the experimental error. Second, the KiCvalues calculated from the fundamental constant K and maximum velocities of equilibrium exchange and zero-trans influx and efflux agreed well with Kic values determined experimentally (Table I, footnote b). On the basis of these kinetic parameters, Cabantchik and Ginsburg (1977) suggested that the uridine carrier of human red blood cells exhibits two types of asymmetry. First, in either direction the loaded carrier moves more rapidly than the unloaded carrier, implying that the movement of unloaded carrier is the rate-limiting step in uridine transport. Second, on the basis of the zero-trans data, they suggested that the unloaded carrier moves about four times more rapidly in the 1 to 2 direction than in the 2 to 1 direction, whereas the loaded carrier moves equally rapidly in either direction. Thus, influx would be faster than efflux. Not all experimental data seem to support the conclusion that the unloaded carrier moves more rapidly in the I to 2 direction than in the 2 to 1 direction. For example, the VZtvalues calculated from the infinitecis data of Cabantchik and Ginsburg (1977) according to Eq. (8) (see Table I, footnote c ) , are not in agreement with this conclusion. Furthermore, the V;: and VZt, for uridine transport in human erythrocytes estimated from the zero-trans and infinite-cis experiments of Paterson and co-workers differ in the opposite direction (Table I). However, zero-trans velocities estimated directly in the zero-trans procedure may be more accurate than those calculated from infinite-cis kinetic parameters. Furthermore, because of differences in experimental conditions and methodology for estimating initial velocities a comparison of data from different laboratories may not be entirely appropriate. Such differences in methodology and conditions might account for the discrepancies between the kinetic parameters (Table I) reported by Oliver and Paterson (1971), Pickard and Paterson (1972), Lieu et al. (1971), and Cabantchik and Ginsburg (1977). Nevertheless, most values are consistent with the conclusion that the loaded carrier moves faster than the unloaded carrier, as was first indicated by the data of Oliver and Paterson (1971) and Pickard and Paterson (1972). Overall, the suggested asymmetry of the nucleoside

250

PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER

carrier of human erythrocytes is similar to that for the hexose carrier of these cells (Miller, 1971; Geck, 1971; Bloch, 1974; LeFevre, 1976). The nucleoside carrier of cultured mammalian cells, on the other hand, exhibits little, if any, asymmetry. The kinetic parameters for zero-trans, equilibrium exchange and infinite-trans transport of thymidine, uridine, and adenosine in Novikoff hepatoma and P388 leukemia cells are the same within the experimental errors (Table 11). Furthermore, the fully symmetrical version of the integrated rate equation (see Eqs. 1-3) well describes the transport of thymidine and uridine, as is evident in Wohlhueter et al. (1979a) and in Fig. 3 , respectively. Similarly, Heichal et al. ( 1 979) calculated from zero-trans data of cytosine arabinoside transport in transformed hamster cells (Nil 8 HSV) that the carrier half saturates at similar substrate concentrations at the outer (250 pA4) and inner (150 pM) faces of the membrane. The kinetic parameters for the transport of various nucleosides-with the exception of cytidine-are similar for the three different cell lines represented in Table 11. The exceptional status of cytidine has not been explored further. This generalization holds also for the transport of nucleic acid bases, but with more qualifications. For example, hypoxanthine transport in CHO cells exhibited a much higher K than those observed with other cell lines (Table 11; see also Marz et al., 1979). The basis for this difference is unknown. Furthermore, except in CHO cells, the K values for adenine and uracil transport are much higher than that for hypoxanthine (or those for nucleosides). The results for adenine tabulated here do not agree with those reported by Witney and Taylor (1978), who observed two Kqe values (6- 10 p M and 80 pA4) for the transport of adenine in an adenine phosphoribosyltransferase-deficient line of CHO cells. Marz et al. (1979), on the other hand, detected only a low-affinity adenine transport system in these same cells ( K = 2-3 mM, similar to that of wild type CHO cells), as well as in an adenine phosphoribosyltransferasedeficient mouse L cell line ( K = 3058 -+ 569 pM; V = 53 4 6 pmole/pl cell H,O-second; P. Plagemann and R. Wohlheuter, unpublished data). No evidence for additional systems with higher affinity was found. The reason for this discrepancy is not known, but it may be related to the fact that in the study of Witney and Taylor (1978) transport velocities were estimated from single 15-second time points at 30°C, which may have underestimated true, initial transport velocities. Other studies pioneering the use of enzyme-deficient mutant cell lines for nucleoside and purine zero-trans transport studies are subject to the same uncertainty. Kessel and Shurin (1968) found the transport of deoxycytidine and cytosine arabinoside in deoxycytidine kinase-deficient

PERMEATION IN ANIMAL CELLS

251

L1210 murine leukemia cells to be saturable and, based on I-minute uptake time points, obtained a K ; ; = 7.5 mMat 0°C for both nucleosides. At 37"C, however, transport seemed to become saturable only above 20 mM. Nevertheless, these studies showed that deoxycytidine transport was nonconcentrative, was inhibited by various other nucleosides and by dipyridamole, but not by free bases or sugars, and that transport was not affected by inhibitors of energy production. Saturable, temperature-dependent efflux of deoxycytidine and its inhibition by dipyridamole was also demonstrated (Kessel and Hall, 1970). Similar results have recently been reported for the transport of the nonmetabolizable nucleoside, 5'deoxyadenosine in L1210 cells (Kessel, 1978). A K ; ; = 115 p M was estimated on the basis of single 18-second time points at 20"C, which were corrected for a nonsaturable uptake component. Plagemann and Erbe (1973) and Zylka and Plagemann (1975) studied the transport of uridine in ATP-depleted Novikoff cells, of hypoxanthine and guanine in hypoxanthine/guanine phosphoribosyltransferase-deficient Novikoff cells and of uracil in the same cell line, but the estimated Kqi (40, 9, and 9 F M , respectively) and V ; ; values for transport, based on 0.5- or 1-minute uptake values, were greatly underestimated due to the lack of methodology for measuring accurate initial transport velocities. Schuster and Hare (1971) examined thymidine transport in thymidine kinase-deficient BHK2, cells. Five-minute uptake values were directly proportional to the extracellular thymidine concentration, but these values probably represented equilibrium levels, and the conclusion that thymidine entry into these cells was solely by nonmediated permeation is, therefore, unwarranted. The 10-minute uptake values for thymidine at 37°C in thymidine kinase-deficient 3T3 cells measured in a study by Cunningham and Remo (1973) must have also represented equilibrium levels. Ungemach and Hegner (1978) observed two saturable processes for the accumulation of thymidine at 37°C in cultured rat hepatocytes, in which little thymidine phosphorylation occurred. Based on 30-second accumulations, values of two K ; ; of 5.3 and 480 p M were estimated. In several recent studies the transport of cytosine arabinoside has been investigated in cell lines that either fail to phosphorylate this nucleoside or do so only slowly. For example, Mulder and Harrap (1975) observed two saturable components for cytosine arabinoside and deoxycytidine accumulation in Yoshida sarcoma cells with K ; ; of 400-600 p M and 2 mM, as well as a passive diffusion component. Transport rates were estimated from uptake slopes between 15 and 120 seconds of incubation at 19°C with labeled substrate. Uptake of these nucleosides was inhibited by each other and other nucleosides in an apparent simple competitive manner. Heichal ef a / . (1978) measured cytosine arabinoside influx and

252

PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER

outward equilibrium exchange at 20°C in chemically transformed golden hamster fibroblasts (MTC) which phosphorylate the nucleoside only very slowly as compared to its influx. Influx was measured graphically from apparently linear initial 10-20 second uptake curves and yielded Ktk = 350 pM and V ; ; = 13 pmole/pl cell H,O.second. Equilibrium exchange efflux, on the other hand, involved a saturable component ( K e e = 503 p M ; V" = 21 pmole/pl cell H,O.second) and an unsaturable component ( k = O.OOS/second).Cytosine arabinoside influx was inhibited by various other nucleosides and itself inhibited the uptake of other nucleosides. Using similar methodology, Koren et a / . (1979) analyzed the kinetic parameters for cytosine arabinoside influx in B77 rat cells (Kik = 500 pM, V;; = 3.9 pmole/pI cell H,O.second). Fitting the linear form of the integrated zero-trans rate equation [Eq. ( I ) ] to data on cytosine arabinoside influx in murine sarcoma virus-transformed golden hamster cells (Nil 8 HSV) yielded KTk = 250 p M and V;; = 3 . 5 pmole/pI cell H,O.second (Heichal ez a / . , 1979). These kinetic parameters are similar to those determined for cytosine arabinoside transport in Novikoff rat hepatoma cells (see Table 11), which are based on nonlinear regression analysis of uptake curves to substrate equilibrium, although the estimated V t ; values are somewhat lower. Cytosine arabinoside is also not metabolized in rat uterus. Cytosine arabinoside uptake by this tissue was saturable, nonconcentrative and competitively inhibited by other nucleosides, and countertransport by uridine was demonstrated (Oliver, 197I). Only sheep erythrocytes homozygous for a specific gene locus ( N u ' ) possess a functional nucleoside carrier (Jarvis and Young, 1978a; see Section IV,E), but even these cells transport various nucleosides at only about 0.3% the rate observed with human erythrocytes (Young, 1978). The Michaelis constants for zero-trans influx of inosine, adenosine, and uridine ( K ? ; = 200, 130, and 470 p M , respectively; Young, 1978), on the other hand, are similar to those reported for human erythrocytes (Table I) and cultured animal cells (Table 11). Benke et a / . (1973) failed to detect significant transport of hypoxanthine in cultures of hypoxanthine/guanine phosphoribosyltransferase-deficient human fibroblasts from Lesch-Nyhan patients, but the facilitated transport of hypoxanthine has been unequivocally demonstrated in transferase-deficient Novikoff hepatoma cells, Chinese hamster lung cells, and human Lesch-Nyhan fibroblasts by Zylka and Plagemann (1975), Alford and Barnes (1976), and Murphy ez a / . (1977), respectively, by observing the countertransport of hypoxanthine in these cells. Based on 15-second uptake time points, Alford and Barnes (1976) observed two saturable transport components with K;: = 17-26 and 530-710 p M and found that transport was inhibited by p-hydroxymercuribenzoate. Epstein and Lit-

PERMEATION IN ANIMAL CELLS

253

tlefield ( 1977) reported that hypoxanthine accumulation by hypoxanthine/ guanine phosphoribosyltransferase-deficient diploid human lymphoblast lines was nonsaturable at 20 or 30°C. However, initial transport velocities were estimated from 1-minute uptake time points and the highest hypoxanthine concentration tested was 560 p M . Thus, the conclusion of these investigators that the transferase is involved in the saturable transport of hypoxanthine is not warranted. It is only the accumulation of the phosphorylated products that is transferase dependent and responsible for long-term uptake of hypoxanthine (see Section 111,A). Many of the above studies with enzyme-deficient cells clearly indicated the existence of facilitated diffusion systems for nucleosides and free bases in animal cells other than erythrocytes, but the reported kinetic parameters, with the exception of those for cytosine arabinoside transport, varied greatly and are inconsistent with values that have been obtained more recently by more rigorous analyses (Table 11). The low K:: systems detected in some of the above studies may reflect either residual phosphorylating activity of the cells or, more likely, an inadequacy in estimating true initial zero-trans transport velocities. The observation of unusually high or nonsaturable permeation components might also result from inaccurate initial velocity measurements, but more often seems to be due to the analysis of relatively long uptake time points, which measured merely the intracellular equilibrium concentrations of substrate. Necessarily for facilitated diffusion systems such equilibrium concentrations are a direct function of the extracellular concentration (see Section 111,E).

111.

UPTAKE OF NUCLEOSIDES AND PURINE BASES

A. General Considerations

The kinetic parameters for nucleoside and purine uptake (as defined earlier) in cultured Novikoff cells are much lower than those for the corresponding transport systems (compare Tables I1 and 111) (uptake data are for 37°C transport data for 24-25°C). In all reported uptake studies (Table III), uptake velocities were estimated from 0.5- to 10-minute substrate uptake time points and, with a few exceptions, similar substrate K , values were obtained for different types of cells in numerous laboratories. The exceptions were K , values for thymidine uptake of 40-50 p M for rabbit polymorphonuclear leukocytes (Taube and Berlin, 1972) and for CHO cells (Sander and Pardee, 1972). In both studies cells were propagated on glass coverslips, but uptake velocities estimated from 45-second

254

PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER

TABLE 111 KINETICPARAMETERS FOR THE UPTAKEOF NUCLEOSIDES A N D FREEBASESBY NOVIKOFF RAT HEPATOMA CELLSA N D OTHERTYPESOF ANIMAL CELLS Other types of cells

Novikoff cells"

-

Vmax

Substrate Thymidine

K, (wM) 0.4-0.5

Deoxycytidine 0.8- I .6 Deoxyadenosine 40-80 Uridine 12-16

Cytidine Adenosine

Guanosine Hypoxanthine Adenine

(pmolelpl cell H,O.second) 0.15-0.3

0.04-0.08 2-4 1-2.6

K m

(WW

Reference"

0.2- I .5

3,8,9,10,20,22,25,27, 30.31-36.47 43,56 17 2,10,30,45

4@50 400 0.7-8 4-30

1-3 2-4

8- 9 1-20

8-15 4-8

2-4 1.5-2.2

30-50

3-4.5

40 102 I- 10 3-36 400 0.2- 100

15-25 6-10

3,4,13,14,18-21.24, 26,27,29,3 1-34,37, 39,42-45,50- 5 4 3 7 44 7,16,22,32-34,38,44, 45,46,49,54,56,58 40 41 33,34,44 1,5, I I , 1 2 , 6 2 2 3 9 28 6,1 1,22,23,48,55,59

~~

a From Plagemann and Roth (1969), Plagemann (1970, 1971a,b), Plagemann and Erbe (1972, 1974a), Plagemann and Richey (1974), and Zylka and Plagemann (1975). In all instances uptake rates were estimated from 1-5 minute time points of uptake of various concentrations of radioactively labeled substrate at 37°C by cell suspensions of 1-3 x lo6 cellslml of a serum-free medium. Previously reported V,,, values expressed on a lo6 cell basis were converted for comparative purpose to pmolelpl cell H,O.second on the basis of an average cell H 2 0 space of 1.3 pVI06 Novikoff cells (Wohlhueter et al., 1978a). References: 1. Alford and Barnes (1976); 2. Barlow (1976); 3. Barlow and Ord (1975); 4. Benedetto and Casson (1974); 5. Benke et a / . (1973); 6. Berlin (1970); 7. Berlin (1973); 8. Bittlingmaier et al. (1977a); 9. Cass and Paterson (1977); 10. Cunningham and Remo (1973); I I . Dybing (1974a); 12. Dybing (1974b); 13. Eilam and Bibi (1977); 14. Eilam and Cabantchik (1977); 15. Epstein and Littlefield (1977); 16. Fleit et al. (1975); 17. Freed and Mezger-Freed (1973); 18. Goldenberg and Stein (1978); 19. Hakala et al. (1975); 20. Hand (1976); 21. Hare (1972a); 22. Harris and Whitmore (1974); 23. Hawkins and Berlin (1969); 24. Heichal et al. (1979); 25. Hopwood et a / . (1975); 26. Jiminez de Asua et a/. (1974); 27. Kunimoto et al. (1974); 28. Lassen (1967); 29. Lemkin and Hare (1973); 30. Leung and Visser (1976); 31. Loike and Horwitz (1976); 32. Lynch et al. (1977); 33. Lynch et al. (1978); 34. Mizel and Wilson (1972); 35. Myers and Feinendegen (1975); 36. Paterson et a/. (1975); 37. Paterson et a / . (1977a); 38. Paterson et al. (l977b); 39. Peters and Hausen (1971); 40. Pofit and Strauss (1977); 41. Roos and Plfeger (1972); 42. Rozengurt and Stein (1977); 43. Sander and Pardee (1972); 44. Scholtissek (1968); 45. Scholtissek (1972); 46. Schrader et al. (1972); 47. Schuster and Hare (1971); 48. Sixma et al. (1973); 49. Sixmaet al. (1976); 50. Skehel et a / . (1967); 51. Stambrook et al. (1973); 52. Steck et a / . (1969); 53. Stein and Rozengurt (1975); 54. Strauss et al. (1976); 55. Suresch et al. (1979); 56. Taube and Berlin (1972); 57. Turnheim et a / . (1978); 58. Weber and Rubin (1971); 59. Yang and Visser (1977).

PERMEATION IN ANIMAL CELLS

255

and 15-minute uptake time points, respectively. A K , of 400 p M has been reported for thymidine uptake by haploid frog cells (Freed and Mezger-Freed, 1973)and thymidine uptake by mouse spleen lymphocytes seemed to be nonsaturable (Strauss et al., 1976). Other extraordinarily high K,s are those for adenosine uptake of 40 and 120 p M in mouse macrophages (Pofit and Strauss, 1977) and guinea pig erythrocytes (Roos and Pfleger, 1972), respectively, and of 400 p M for hypoxanthine uptake by human erythrocytes (Lassen, 1967). Reported K , values for adenine uptake are extremely variable ranging from 0.2 p M for human blood platelets (Sixma et al., 1973) to 100 p M for CHO cells (Harris and Whitmore, 1974). The maximum uptake velocities reported in various studies are also highly variable, but are difficult to compare, since different units were used to express the uptake capacity of the cells. The meaning of these kinetic parameters for uptake has recently come into question, since studies on the uptake of thymidine (Wohlhueter et al., 1976, 1979a; Marz e f al., 1977a), uridine (Plagemann et al., 1978b), adenosine (Lum et al., 1979), and hypoxanthine (Marz et al., 1979) by cultured cells have clearly shown that such long-term uptake velocities pertain to the period of steady-state intracellular substrate concentrations and measure the intracellular formation of nucleotides derived from exogenous substrate. A similar conclusion is indicated by the studies on uridine uptake in various other cultured cell lines (Rozengurt et al., 1977b, 1978; Koren et al., 1978; Heichal et al., 1979). Nucleoside or nucleobase transport systems with low K,s similar to those reported for the uptake of these substrates by phosphorylating cells have not been detected in Novikoff or P388 cells. The initial velocities of transport (us:) of adenosine (Lum et al., 1979), hypoxanthine, and adenine (Marz et al., 1979), and thymidine and uridine (R. Wohlheuter and P. Plagemann, unpublished data) by ATP-depleted cells or cells deficient in the appropriate phosphorylating enzymes increase in direct proportion to intracellular substrate concentration between 0.1 and 10 PM.

6. Relationship between Transport and Metabolism Operating in Tandem

Typical results for the uptake of hypoxanthine by wild-type Novikoff rat hepatoma cells at 25°C are illustrated in Fig. 7. Free hypoxanthine accumulates intracellularly to steady-state levels within seconds; within 2.5 seconds at an extracellular concentration of 0.5 p M , and within 30 seconds with 160 p M exogenous hypoxanthine. Thereafter, the uptake of radioactivity into total cell material reflects the accumulation of nucleo-

256

PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER

SEC

SEC

MIN

MIN

TIME

FIG.7. Time courses of intracellular accumulation of labeled free hypoxanthine, inosine, and nucleotides in untreated wild-type Novikoff cells incubated at 24°C with 0.5 p M (A) or 160 p M (B) [3H]hypoxanthine. Short-term time courses of [3H]hypoxanthine uptake were measured by the rapid kinetic procedure as described in the legend to Fig. 3. For long-term uptake measurements, cell suspension and substrate solution were mixed in the same proportion as i n the rapid kinetic method, incubated at 24°C and 509-pI samples were centrifuged through oil at the indicated times. The final concentrations of [3H]hypoxanthine were 0.5 p M or 160 p M (1400 cpmipl, irrespective of concentration). The cell HZO and extracellular H,O spaces were 22.8 and 2.9 plkell pellet, respectively. Initial velocities of uptake ( v , , ) at 0.5 p M hypoxanthine ( A ) were estimated graphically from the linear portions of the short-term and long-term uptake curves. In parallel experiments duplicate samples of cells were collected by centrifugation through oil directly into an acid layer, the acid extracts were further processed and chromatographed as described by Marz et a / . (1979). Time courses of intracellular accumulation of labeled hypoxanthine ( 0 - O ) , inosine (AA), and nucleotides (A-A) were constructed on the basis of these chromatographic analyses and time courses of uptake i n t o total cell material (0-0). All values were corrected for trapping of extracellular [3H]hypoxanthine in cell pellets and acid extracts. The broken lines indicate equality between extracellular and intracellular radioactivity concentrations. (Data are from Marz et a / . , 1979.)

tides. Since these nucleotides consist of over 90% of triphosphates, phosphoribosylation of hypoxanthine seems to be the rate-determining step in the long-term formation of nucleotides from exogenous hypoxanthine. Furthermore, computer simulations have shown that. the intracellular steady-state levels of free hypoxanthine and the time courses of intra-

257

PERMEATION IN ANIMAL CELLS

cellular formation of nucleotides at both 0.5 and 160 p M exogenous hypoxanthine are consistent with the kinetic parameters for the hypoxanthine transport system and the hypoxanthine/guanine phosphoribosyltransferase operating in tandem [Fig. 8; see legend for Eqs. (10) and ( I I ) ] . Differences in uptake at low and high exogenous hypoxanthine concentrations are inherent in the fact that the Michaelis constant and maximum velocity for the transport system are at least 50 times higher than the corresponding values for the transferase, whereas the ratio V / K , for transport is somewhat lower than that for phosphorylation (Marz et al., 1979; see also Table IV). At extracellular concentrations of hy-

-Isn 6

,

\

I

#

#

prnole/pl CELL

uM

0 0

n

:' 350

c W

.rn

a

,

0 5 u M HYPOXANTHINE

.en2

H*O sec Vf 50 V'MI

a > -I

a 0 I

a v) 0

I

n

z

a a

I

a

, : '

...

PHOSPHORYLATED PRODUCTS

1 3I

L

TIME I S E C )

FIG. 8. Simulated time courses of hypoxanthine transport and phosphoribosylation. The intracellular accumulation of hypoxanthine (S,, solid line) and phosphorylated products (P,dotted line) at a given exogenous concentration of hypoxanthine (S,, broken line) were computed by numerical integration of the following rate equations: dS,ldt

=

vI2 - vZl- dP/dt

dPldt = V'"" S,/(Ke,"Z + S,)

(10)

( 1 1)

where v I 2is calculated as in Eq. (9,and u2, by an analogous efflux equation. In panel A, S , = 0.5 p M at time zero; in panel B, S , = 160 p M at time zero. For both cases the kinetic parameters for transport and phosphoribosyltransferase with respect to hypoxanthine used are those determined experimentally for Novikoff cells (see Marz et al., 1979; see also Table 11): K E Z = 2 pM, VenZ= 1 pmole/second.pI cell H,O, and for transport K ( K ? = 350 p M and V (V? = 50 pmole/second.pl cell H,O. Cellular space was taken to comprise 1% of total water space.

258

PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER

TABLE IV KINETICPARAMETERS FOR TRANSPORT A N D METABOLIC REACTIONS I N ADENOSINE UPTAKEI N P388 CELLS INVOLVED Process Adenosine (inosine) transport Adenosine kinase Adenosine deaminase Nucleoside phosphorylase HGPRT Hypoxanthine transport

Km (pM)

Vmax" (pmole/pl cell H,O.second)

130' 0.4- 6 .0' 3 5- 400'

70-100 0.5- 1 160-220 40-60 1-3 80-150

13-30d

2- 6" 45oe

All V,,, values are for P388 cells at 37°C and from Lum et a / . (1979), except those for hypoxanthineiguanine phosphoribosyltransferase (HGPRT) and hypoxanthine transport which are from Marz et a / . (1979). * From Lum et (11. (1979). Range of values observed in many different mammalian cell systems and summarized by Fox and Kelley (1978). Range of values for different mammalian species; from Agarwal et a / . (1975) and Milman et a / . (1976). From Marz et a / . (1979).

poxanthine much higher than the K , of the transferase for hypoxanthine, the rate of transport of hypoxanthine greatly exceeds the maximum velocity of intracellular phosphoribosylation, so that the concentration of free hypoxanthine in the cell approaches that in the extracellular space. At extracellular concentrations of hypoxanthine within the first-order range for both transport and phosphoribosylation ( 1 p M and below), transport is slower than phosphoribosylation. Thus at such low extracellular concentrations the steady-state intracellular concentration of free hypoxanthine attains not more than a few percent of that in the extracellular fluid. It is clear that in this concentration range, the overall uptake rate for hypoxanthine is largely determined by its rate of transport into the cell; for example, increases and decreases in transport rate alone would cause similar, although not strictly proportional, changes in the overall uptake rate, whereas an increase in the phosphoribosylation capacity of the cells would have little effect on overall uptake rate. In contrast, at hypoxanthine concentrations in excess of the K , of the transferase for hypoxanthine, the overall long-term uptake rate is strictly a function of the phosphoribosylation capacity of the cells. Similar relationships between transport and phosphorylation pertain to the uptake of various nucleosides in a number of cultured cell systems. All seem to have in common that the transport carrier: substrate affinity constants and the maximum velocities for the transport systems greatly exceed the

PERMEATION IN ANIMAL CELLS

259

corresponding values for the in s i f u phosphorylation reactions (Wohlhueter et al., 1976; Marz et al., 1977a; Plagemann et al., 1978b; Lum e f al., 1979; Koren et al., 1978; Heichal et al., 1979). Thus whether transport or phorphorylation capacity is the main determinant of overall substrate uptake may often be a function of the extracellular substrate concentration. These results support the view that the kinetic parameters for the longterm uptake of nucleosides and purines (Table 111) reflect those of the in situ phosphorylation of the substrate. It is not surprising, therefore, that the substrate specificities of the uptake processes have generally been found to be the same as those of the corresponding phosphorylation reactions (see Section IV,A). In the case of hypoxanthine, a good agreement between the kinetic parameters of hypoxanthine uptake by whole cells and those of the hypoxanthine/guanine phosphoribosyltransferase as measured in cell-free preparations has been observed (Epstein and Littlefield, 1977; Marz e f al., 1979). N o such agreement has generally been observed with all nucleosides. For uridine and thymidine, for example, the K , values for uptake by whole cells (Table 111) are about one to two orders of magnitude lower than those of the corresponding kinases for the nucleoside substrate as measured in cell-free preparations (Cleaver, 1967; &hak and Rada, 1976; Wohlhueter and Plagemann, 1980). This observation originally contributed to the erroneous conclusion that nucleoside uptake rates reflected those of the transport system (Plagemann and Richey, 1974). One possibility that might account for this discrepancy is that the in vitro analyses might not measure the kinetic properties of the kinase operative in situ (Plagemann et al., 1978b). Support for this conclusion comes from the work of Goldenberg and Stein (1978) and others to be discussed in Section VI. The interrelationship between transport and metabolism operating in tandem is even more complex for substrates that are catabolized in addition to being phosphorylated. This, for example, is the case with adenosine, which is rapidly deaminated to inosine by many types of cells (see Fig. I). Inosine, in turn, is phosphorolyzed to hypoxanthine, which is then converted to IMP. Recent studies (Lum et al., 1979) have shown that the metabolism of adenosine by P388 leukemia cells as a function of extracellular adenosine concentration is entirely consistent with the kinetic properties of the nucleoside transport system and those of the intracellular enzymes involved in adenosine metabolism (Table IV). In the case of adenosine, intracellular steady-state levels of free substrate remain very low regardless of the extracellular concentration of adenosine. This is a consequence of the facts that adenosine deaminase and the nucleoside carrier exhibit similar affinities for adenosine, and that the

260

PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER

maximum velocity for adenosine deaminatibn as measured in cell lysates is several times higher than that for adenosine transport (Table IV). In contrast to transport and deamination the maximum velocities of adenosine kinase and hypoxanthine/guanine phosphoribosyltransferase are much lower, but their affinities (for adenosine and hypoxanthine, respectively) are higher. Thus when adenosine enters the cells at a relatively low rate, either because of a low ( 1 p M or below) extracellular adenosine concentration or because transport is inhibited in some manner, most of the entering adenosine becomes directly phosphorylated. With an increase in the rate of adenosine entry, consequent to an increase in extracellular concentration, the intracellular steady-state levels of adenosine are sufficient to saturate adenosine kinase, and adenosine deamination becomes the predominant reaction. However, nucleoside phosphorylase and hypoxanthine/guanine phosphoribosyltransferase also become saturated as inosine and hypoxanthine pools swell, with the result that these products exit the cell and accumulate in the medium (see Fig. I ) . It should be emphasized that the comparable rates of nucleoside and hypoxanthine transport and phosphorylation observed at low substrate concentration (below 1-10 p1M) pertain to growing cultured cells. Thus salvage activities seem to be operating maximally in these growing cells at concentrations at which certain nucleosides and purines are normally found in blood and other body fluids (Murray, 1971; Hughes et a/., 1973; Schaer et a / . , 1978; Nattebrock and Then, 1977; Kuttesch et a!., 1978; Schrader et a/., 1978). Salvage activity, however, seems to be much lower in quiescent than growing cells, not because of lowered transport capacity, but because of lowered phosphorylation capacity. This situation is further discussed in Section VI on regulation of transport, but in the context of the present discussion it should be pointed out that, under such conditions, an increase in phosphorylating capacity alone would result in an increase in nucleoside and purine uptake, even at low substrate concentrations. Little information is available on nucleoside and purine uptake by tissue cells in the body. Cornford and Oldendorf (1975) injected various concentrations of 14C-labeledpurines and pyrimidines and their nucleosides into the common carotid artery of rats, decapitated the animals 15 seconds later, and determined the amounts of 14C in the brain. Two saturable uptake processes were defined; one for adenine ( K , = 27 p.M), subject to inhibition by hypoxanthine and one for adenosine ( K , = 18 p M ) , guanosine, inosine, and uridine, subject to mutual inhibition by these nucleosides. Uptake into the brain was equated with carrier mediated transport through the blood-brain barrier. The entry of cytosine,

PERMEATION IN ANIMAL CELLS

261

uracil, thymine, thymidine, and cytidine was considered not to be significant.

C. Estimation of Zero-trans Transport Kinetic Parameters from Substrate Uptake Curves

Comparative studies with kinase-deficient or ATP-depleted Novikoff and P388 cells, in which, when necessary, substrate metabolism other than phosphorylation was also inhibited by appropriate inhibitors, have shown that the kinetics of transport of nucleosides and purines can be approximated in some instances from uptake studies with cells in which the substrate is metabolized provided that very early initial uptake time courses are analyzed (Plagemann et al., 1978b; Marz et al., 1979; Lum et al., 1979). For example, Fig. 9 shows that Eq. (1) fits quite well to early time courses of uridine and hypoxanthine uptake by wild type Novikoff rat hepatoma cells in which both substrates are rapidly converted to phosphorylated intermediates. Fitting Eq. (1) to pooled data with seven substrate concentrations (20- 1280 pM) yielded kinetic paramters similar to those estimated for uridine and hypoxanthine transport in enzyme-deficient or ATP-depleted cells (see Table 11). Equation (I), of course, applies strictly only to substrate influx and must, therefore, be confined to early time points at which the intracellular concentration of radioactivity is still well below equilibrium levels and at which phosphorylated products do not make a large contribution. It is obvious from Fig. 9 that, within these constraints, fitting Eq. (1) to uptake time courses yields more accurate estimates of initial uptake velocities-which reflect substrate influx-than a linear graphical estimation. This is true because, as with accumulation of these substrates in nonphosphorylating cells, significant backflux and a consequent downward deviation from linearity of the uptake curve occurs within the first few seconds of incubation. The greater the phosphorylating capacity of the cells, however, the sooner does the accumulation of phosphorylated products cause the uptake curves to deviate upward from the curve described by Eq. (I), particularly at the lower substrate concentrations (see Fig. 9A and E). Thus, although possible in some situations, the estimation of initial permeation rates of substrates that are rapidly phosphorylated intracellularly by graphical methods or curve-fitting of transport rate equations is fraught with problems and must be approached, if at all, with extreme caution. A possible remedy to these problems is to develop integrated rate equations describing flux in a two-step pathway, comprising carrier-mediated

262

PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER UR I D l N E

O IN

HY POXANTHINE

1

10

$;r.;

.:

..*

_,....'

1280 IrM

,,.*'

.*"

0.

00

.

..'

20

40

TIME I S E C )

FIG.9. Kinetic analyses of uridine (A-D) and hypoxanthine (E-H) transport as estimated from early uptake time courses in untreated, wild-type Novikoff rat hepatoma cells. The experiments were conducted as described in the legend to Fig. 3, except that untreated cells were used in which both substrates are converted to phosphorylated products. Final substrate concentrations were 20, 40, 80, 160, 320, 640, and 1280 p M (280 cpmipl for [3H]uridine and 470 cpmipl for [3H]hypoxanthine, irrespective of concentration). The intracellular H 2 0 spaces and trapped HzO spaces were in (A-D) 21.9 and 2.7 plicell pellet, respectively, and in (E-H) 22 and 6.5 plicell pellet, respectively. Data are from Plagemann et a / . (1978b. Table I) and Marz er a / . (1979, Fig. 9), but reanalyzed as follows. Equation ( I ) was fitted to the pooled uptake data in each experiment with all R parameters held equal. Only four of the theoretical curves are shown for each substrate. The best fitting kinetic parameters were for uridine: K = 230 ? 8 p M and V = 15 ? 0.2 pmole/pl cell H,O.second, and for hypoxanthine: K = 474 2 30 p M and V = 77 ? 2 pmoleipl cell H,O.second. The correlation coefficients (r,,g) were 0.9889 and 0.9868, respectively.

permeation and Michaelian phosphorylation. We discuss this approach in detail elsewhere (Wohlhueter and Plagemann, 1980). So far, such flux equations have been successfully fitted to uptake time courses only where some of the kinetic parameters describing the pathway are supplied known values-too severe a limitation for our present purposes. Koren et ul. (1978) and Heichal et al. (1979) have recently attempted to estimate the kinetics of uridine transport in monolayer cultures of untransformed and transformed mouse 3T3 and Nil 8 hamster cells, respectively, by estimating initial uptake rates by linear regression analysis of four to five time points taken during the first 20-30 seconds of incubation at 20°C. The estimated kinetic parameters were similar for quiescent and serum-stimulated untransformed and Simian virus 40-trans-

PERMEATION IN ANIMAL CELLS

263

formed 3T3 cells ( K , = 220-340 p M , V,,, = 27-46 pmole/106 cells*second) and for untransformed and murine sarcoma virus transformed Nil 8 hamster cells ( K , = 400-530 p M , V,,, = 25-55 pmole/106 cells second) and much higher than those estimated from long-term uptake time courses (see Table 111). It was concluded, therefore, that these kinetic parameters are reasonable estimates of Kqg and V?; for uridine transport in these cells. Since uridine influx seems to be at least as fast in these cells as in suspensions of animal cells (Fig. 9) it seems likely, however, that 20-30 second slopes underestimated true initial transport velocities. In early uptake studies it was shown that the incorporation of extracellular nucleosides and purines into cellular nucleic acids follows simple Michaelis-Menten kinetics and that the apparent K , values were similar to those estimated for the uptake of the respective substrates into total cellular material (Plagemann and Richey, 1974). Since the uptake rates were considered to reflect those of transport, the data were interpreted to indicate substrate transport to be the rate-limiting step in the incorporation into nucleic acids. These observations are still important in relation to the use of these precursors to assess rates of nucleic acid synthesis, but the interpretation with respect to rate-limiting steps needs to be modified in light of the newer information discussed already. Although, at very low extracellular substrate concentration, influx may contribute in a major way to determining the rate of its incorporation in growing cells in culture, with increase in extracellular concentration it is certainly the conversion of these substrates to nucleotides that becomes rate-determining, rather than transport. This conclusion also pertains to the uptake and incorporation into nucleic acids of various nucleoside and purine analogs that are toxic to cells and might be employed in cancer chemotherapy (Plagemann et al., 1978a). Other aspects of the relationship between transport of nutrients and their metabolism have been reviewed and discussed by Wohlhueter and Plagemann (1980).

D. Uptake into Vesicles of Mammalian Cells

Hochstadt and her collaborators (Quinlan and Hochstadt, 1974, 1976; Li and Hochstadt, 1976a,b; Hochstadt and Quinlan, 1976; Dowd at al., 1977; Hochstadt, 1974) introduced the use of membrane vesicles from cultured animal cells for the study of nucleoside and purine permeation through membranes. Although the study of substrate permeation into membrane vesicles could be potentially very informative with respect to the mechanism of transport, the particular preparations employed by

264

PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER

these investigators are compromised by the fact that they contained, although at a reduced level, most of the enzymes present in the cytoplasm of mammalian cells, including adenosine deaminase, nucleoside phosphorylase, adenosine kinase, uridine kinase, and hypoxanthine/guanine phosphoribosyltransferase, and are also capable of some conversion of extravesicular nucleosides to intravesicular phosphorylated intermediates. Because of differences in recovery of various enzymes in vesicular preparations it was reasoned that small proportions of the total cellular content of these enzymes were associated with the plasma membrane. But, whatever the subcellular location of the enzymes, in their metabolic activities these vesicles resemble, in many respects, metabolizing whole cells and thus cannot be handled as if they were metabolically inert entities. Accumulation of radioactivity from [U-14C]uridine, -adenosine, -inosine, and -guanosine by the vesicles from a number of different cell lines (LgZ9,3T3, and BHK,,) has been found to be linear for at least several minutes. Radioactivity accumulated intravesicularly in 10-20 minutes to concentrations 5- to 20-fold higher than those in the medium. Accumulation of radioactivity bore a simple, Michaelis-Menten relation to exogenous substrate concentration, and the apparent Michaelis-Menten constants (7-12 p M for uridine, 7-19 p M for adenosine, and 35-55 p M for inosine) resemble those estimated for the uptake of nucleosides by metabolizing cells (Table 111), rather than those for nucleoside transport (Table 11). However, in contrast to intact cells which accumulate mainly nucleoside triphosphates, the intravesicular content consisted mainly of labeled ribose-I-phosphate and small amounts of labeled nucleoside monophosphates. The concentrations of free nucleosides were found regularly to be very low, well below those in the extravesicular fluid. Free hypoxanthine was not detected inside vesicles of L,,, cells after incubation with labeled inosine, whereas free hypoxanthine accumulated extravesicularly in amounts similar to the amounts of ribose-lphosphate inside (Li and Hochstadt, 1976a,b). Some free hypoxanthine, on the other hand, accumulated in vesicles of 3T3 (Quinlan and Hochstadt, 1976) and BHKzl cells (Dowd et al., 1977). On the basis of these results and the finding that a small proportion of the cell’s purine nucleoside phosphorylase activity was recovered in purified membrane preparations these investigators proposed that the main route of entry of radioactivity from uniformly labeled inosine is a kind of group translocation, catalyzed by membrane-bound purine nucleoside phosphorylase, whereby ribose- I-phosphate is transferred to the intravesicular space, while hypoxanthine is released to the outside. The finding of free hypoxanthine in vesicles of some of the cell lines was

PERMEATION IN ANIMAL CELLS

265

interpreted to indicate the additional operation in these cell lines of direct permeation of inosine with subsequent phosphorolysis. The rapidity of the nucleoside and purine transport systems of mammalian cells suggests a more plausible alternate explanation for these results, namely, that inosine entered the vesicles via the nucleoside transport system and was phosphorolyzed inside the vesicles, but that most of the intravesicular hypoxanthine was transported out of the vesicles or lost from the vesicles during their extensive washing before radioactivity analysis, whereas the labeled ribose-I-phosphate was retained because the membrane is largely impermeable to phosphorylated intermediates (see Section VII). That nucleoside metabolism rather than transport has been measured in these vesicles is best illustrated by the following data. Uridine uptake over a 20-minute period was found to be about six times higher in vesicles from growing than quiescent 3T3 cells (Quinlan and Hochstadt, 1974) just as is observed in whole cells (see Section VI). However, in studies with whole cells it has been demonstrated that this difference in uptake rates is due to differences in rates of uridine phosphorylation rather than of transport (Rozengurt et al., 1977b). An involvement of purine nucleoside phosphorylase in inosine permeation has also been postulated by Cohen and Martin (1977) on the grounds that purine nucleoside phosphorylase-deficient human fibroblasts took up only 3 1% as much [8-14C]inosineat an exogenous concentration of 100 p M as normal fibroblasts. However, this conclusion was based on a single 30-minute time point, and no difference in uptake of either [8-14C]- or [U-14C]inosine between the two types of cells was detected when the exogenous inosine concentration was 10 p M . Furthermore, cells of a purine nucleoside phosphorylase-deficient subline (NSU-1) of the mouse T cell lymphoma line S49 seem to take up inosine and guanosine unabated (Ullman et al., 1979), whereas inosine uptake by a nucleoside transport mutant (AE,) of the same cell line (see Section IV,E) is reduced at least 98% when compared to wild type S49 cells, even though the cells possess normal purine nucleoside phosphorylase levels (Cohen et al., 1979). The correlation between adenosine deaminase activity of vesicles and their capacity to accumulate radioactivity from uniformly labeled adenosine, which led Li and Hochstadt (1976b) to postulate a function of adenosine deaminase in adenosine transport, has also a more plausible, alternative explanation along the lines discussed already. Since the direct phosphorylation of adenosine is minimal in these vesicles, transported intravesicular adenosine is rapidly lost during washing of the vesicles. The major form of radioactivity deriving from it, and remaining in the cell, is ribose- 1-phosphate, the formation of which, via inosine, requires

266

PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER

the presence of intracellular adenosine deaminase. An association of adenosine deaminase with the nucleoside carrier in mammalian cell membranes has also been suggested by Agarwal and Parks (1975), but detailed studies with P388 mouse leukemia cells have shown that the deamination of adenosine by these cells can be accounted for totally by the tandem operation of nucleoside influx, intracellular deamination, and subsequent efflux of inosine (Lum et af., 1979). The suggestion that adenosine kinase may be involved in group translocation of adenosine in human blood platelets (Sixma et af., 1976) is also not supported by studies with other types of cells. Hochstadt and Quinlan (1976) have suggested that hypoxanthine enters animal cells, as bacteria, by group translocation catalyzed by membraneassociated hypoxanthine/guanine phosphoribosyltransferase. Membrane vesicles from both wild type and transferase-deficient (thioguanine-resistant) 3T3 cells accumulated little labeled hypoxanthine, but accumulation of radioactivity by vesicles from wild type cells was stimulated about 10-fold by addition of phosphoribosylpyrophosphate (PRPP). Whether phosphoribosylation of hypoxanthine was intra- or extravesicular, or occurred during passage through the membrane, however, cannot be distinguished from these data. That transferase-deficient cells have normal transport capacity has clearly been shown in studies with whole cells (see Section 11,E).

E. Contributions of Transport and Nonmediated Permeation to Overall Uptake

In the context of uptake studies the entry of nucleosides and nucleic acid bases by nonmediated permeation also needs to be considered. As we have seen, the transport of nucleosides and bases can be adequately described by rate equations for carrier-mediated permeation, without invoking nonmediated components. Yet, studies of the kinetics of longterm uptake of these substrates by metabolizing cells have invariably suggested a nonsaturable uptake component (see Fig. 10B) or at least a component with a K , two to three orders of magnitude higher than that of the high-affinity uptake system (Jacquez, 1962; Lassen, 1967; Hawkins and Berlin, 1969; Hare, 1970; Schuster and Hare, 1971; Plagemann, 1971a,b,c; Roos and Pfleger, 1972; Schrader et af., 1972; Plagemann and Erbe, 1972, 1975; Stein and Rozengurt, 1975; Zylka and Plagemann, 1975; Sixma et al., 1976; Cass and Paterson, 1977; Yang and Visser, 1977; Epstein and Littlefield, 1977; Paterson et al., 1977a,b; Heichal et af., 1978; Turnheim et af., 1978). For example, in the case of hypoxanthine

267

PERMEATION IN ANIMAL CELLS

uptake, “velocities” estimated from 2-minute uptake time points (Fig. 10A) yielded apparent biphasic uptake kinetics. There was clearly a saturable uptake component with a K , of about 4 p M (Fig. IOB, upper frame), but in the range of 20 to 1250 p M the apparent rate of uptake increased in direct proportion to the extracellular concentration of hypoxanthine (Fig. IOB, lower frame). Such relationships have generally been interpreted as reflecting substrate entry by nonmediated permeation (Roos and F’fleger, 1972; Schrader et a / . , 1972; Lassen, 1967; Plagemann, 1971a,b; Plagemann and Erbe, 1972, 1975; Paterson et a / . , 1977a,b; Schuster and Hare, 1971; Stein and Rozengurt, 1975). In some studies

B MICHAELIS-MENTEN

3 02

I

I

10

20

I

=066!007

I

30 0

I SEC I

V,,

5 (MINI

- 0 0 HYPOXANTHINE ( U M I

TIME

FIG. 10. Uptake of hypoxanthine by untreated wild-type Novikoff rat hepatoma cells as function of substrate concentration at 24°C. Short-term hypoxanthine uptake (A) was measured by the rapid kinetic technique as described in the legend to Fig. 3, except that untreated wild-type cells were used and hypoxanthine was the substrate. The [3H]hypoxanthine concentrations were 1, 2, 4, 8, 20, 40, 80, 160, 320, 640, and 1280 p M (390 cpm/pl, irrespective of Concentration). For long-term uptake measurements cell suspension and substrate solution were mixed in the same proportion as in the rapid kinetic technique ( 0 time) and after the indicated time periods equivalent samples of mixture were centrifuged through oil and the cell pellets were analyzed for radioactivity. All values were corrected for substrate trapped in extracellular space in cell pellets (4.0 11.1).The intracellular H,O space was 21.5 pl. The broken lines in (A) indicate the intracellular concentration of radioactivity equal to that in the medium at 0 time. Uptake “velocities” ( u J were estimated from 2-minute time points i n (A; long-term) and plotted as a function of hypoxanthine concentration (20-1280 p M ) in (B; lower frame) or subjected (1-20 p M ) to MichaelisMenten analysis in (B; upper frame). V,,, is expressed in pmoleipl cell H,O.second. (Previously unpublished data of Plagemann and Wohlhueter.)

268

PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER

total uptake rates were corrected for this nonsaturable component in order to estimate the contribution of transport to the overall uptake rate. However, recent studies of the uptake of cytosine (Graff et al., 1977) and of the transport of other substrates in the absence of metabolism indicate that these interpretations may have been incorrect. Cytosine is not metabolized by cultured cells, its uptake is not saturable up to concentrations of 10 mM (Zylka and Plagemann, 1979, and its influx is about two orders of magnitude slower than that of transported nucleosides and purine bases at equivalent concentrations in the first-order range for the transport system (see Table V). The first-order rate constant for cytosine influx is lower than the apparent first-order rate constant of nonsaturable permeation components for transportable and metabolizable substrates estimated from curves similar to that in Fig. 10B (lower frame). For example, at a concentration of 10 p M , cytosine enters Novikoff cells at 24°C at about 0.017 pmole/pl cell H,O.second (Zylka and Plagemann, 1975; Graff er al., 1977), whereas the rate of uptake of uridine (Plagemann, I97 la,b), adenosine (Plagemann, 197 1b), thymidine (Plagemann and Erbe, 1972), hypoxanthine, and guanine (Zylka and Plagemann, 1975; See Fig. IOB) attributed to nonsaturable uptake fell in the range of 0.04 to 0.18 pmolelpl cell H,O-second. Furthermore, the apparently nonsaturable uptake components were found to be independent of temperature between 6 and 27°C (Jacquez, 1962; Plagemann and Richey, 1974), whereas the nonsaturable permeation of cytosine, like nonmediated permeation of substances through membranes in general (Lieb and Stein, 1971), is highly temperature dependent (Graff et al., 1977). According to the formulation of Lieb and Stein (1971), the permeation coefficient (0 of a substance through a lipid bilayer membrane is a function of the solubility of the substance in membrane lipids relative to that in an aqueous medium ( Z ) , z its diffusion constant describing its movement within the membrane (Dmem)and the reciprocal of the thickness of the membrane (1): Z X Dmem

P=

1

where D,,, is a complex function of the molecular properties of the substance such as molecular weight, shape, and charge. Thus for uncharged substances with similar molecular dimensions, the lipid solubility of a substance is the main determinant of its rate of permeation. Indeed, it has been found that the rates of nonsaturable uptake of L-glucose,

* Partition coefficients have generally been designated K (Lieb and Stein, 1971). For clarity, we have used Z , since K has been reserved to designate the substrate:camer affinity constant.

269

PERMEATION IN ANIMAL CELLS

TABLE V

PARTITION COEFFICIENTS AND MOLECULAR WEIGHTSFOR L-GLUCOSE, NUCLEOSIDES, AND NUCLEIC ACIDBASESAND VELOCITIES OF THEIR ZERO-TRANS TRANSPORT AND NONMEDIATED PERMEATION AT AN EXOGENOUS CONCENTRATION OF 10 p M AND 24°C Transportb Substrate L-Glucose Cytosine 8-Azaguanine DMO Thymidine Uracil Adenosine H ypoxanthine Uridine

Molecular weight I80 Ill I50 129 242 112 267 I36 244

Z“ 0.00404 f 0.0004 0.0352 f 0.00067 0.173 2 0.0064 0.983 ? 0.078 0.0730 f 0.003 0.778 f 0.0004 0.105

0.1 15 0.185

f 0.015 f 0.009 f 0.0004

Nonsaturable permeationc

9: 0 9; (pmole/pl cell H&l.second) 0.00017 0.017 0.035 0.27

u:;/Z

0.24 0.21 0.49 0.28

1.13 0.115

2.2 I .48 0.96

” Z = partition coefficient: substrate concentration in octanoYaqueous buffer solution as determined by Graff et a / . (1977).Data are from Graff et a / . (1977)and unpublished (P. Plagemann and R. Wohlheuter). Z for 8-azaguanine and 5’,5’-dimethyl-2,4-oxazolidinedione (DMO) is at pH 6.0,all others are independent of pH between 6 and 8. S, = 10 p M (24°C).Calculated from average K and V (see Table 11). ‘ S, = 10 p M (24°C).uf: were computed by first fitting Eq. (4)to time courses of substrate accumulation to equilibrium and then calculating u;: = S , k . The uptake of 8azaguanine and DMO were determined at pH 5.8-6.0(unpublished data). Other data are from Graff ef a / . (1977),but have been corrected for temperature differences (24 versus 37°C).

cytosine, 8-azaguanine, and 5 ’ ,5’-dimethyl-2,4-oxazolidinedione are similarly dependent on their lipid solubility (see vT:/Z ratios: Table V). This finding, and other evidence discussed by Graff et al. (1977), supports the view that the main mode of entry of these substances is nonmediated permeation. Because various other nucleosides and nucleic acid bases have molecular weights and lipid solubilities similar to cytosine and 8azaguanine (Table V), one would expect all these substances to enter by nonmediated permeation at about the same rate. A resolution of these discrepancies is as follows. The apparently nonsaturable uptake component becomes significant in Michaelis-Menten plots in a range of extracellular substrate concentrations that suffice to saturate the nucleoside kinases or purine phosphoribosyltransferases (see Fig. 1OB). At these substrate concentrations the formation of nucleotides is slow relative to the transport rate and free substrate rapidly accumulates intracellularly to equilibrium levels (see, e.g., Figs. 7B and 10A).

270

PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER

Thus, measurements at 2- 10 minutes encompass these equilibrium levels, which, of course, increase in direct proportion to extracellular concentration (Fig. 10A; the total concentration of radioactivity was the same for all hypoxanthine concentrations and only its specific radioactivity was decreased by addition of unlabeled h ypoxanthine). If such steadystate accumulations, divided by time, are erroneously construed as "velocities," this "velocity" component would be directly proportional to exogenous substrate concentration and would be independent of temperature. The data in Table V indicate that, in the first-order range of substrate for transport (at 10 p M , for example), less than 2% of the total rate of thymidine, adenosine, hypoxanthine, and uridine entry can be attributed to nonmediated permeation. This proportion, of course, increases as the transport carrier becomes saturated, but, in view of the relatively high substrate : carrier affinity constants of nucleoside and base transport, it would make a large (and therefore measurable) contribution, only at very high, and certainly unphysiological, concentrations. No method is presently available to estimate accurately this entry component in cultured cells, except empirically from rates of entry of substances, such as cytosine, that enter solely by a nonsaturable process. Sheep erythrocytes, however, provide confirmation of these views. Nucleosides enter sheep erythrocytes that lack a functional nucleoside transport carrier only very slowly (Young, 1978; see Section IV,E). For example, at an extracellular concentration of 5 mM, the rate of inosine uptake by these cells was estimated to be about 0.025 pmole/pl cell H,O*second at 37"C, which is appreciably slower than the rate of cytosine permeation into cultured animal cells under equivalent conditions. That nonmediated permeation plays only a minor role in nucleoside and purine uptake is also indicated by the finding that the nucleoside transport inhibitors, nitrobenzylthiopurine riboside and nitrobenzylthioguanosine (see Section V,C), protect L5 178Y leukemia cells against the toxic effects of various nucleoside analogs that seem to enter the cells via the nucleoside carrier (Warnick et al., 1972). Transport inhibitors have been employed in attempts to dissect Michaelis-Menten curves for uptake of various nucleosides and purines into transport and nonsaturable components (Plagemann, 197la; Plagemann and Erbe, 1972; Plagemann and Richey, 1974; Cass and Paterson, 1977; Paterson et al., 1977a; Young, 1978). The residual rate of uptake in the presence of excess of inhibitor was considered to represent the nonsaturable component. Such an approach might be valid with inhibitors that specifically interact with the carrier, such as the nitrobenzylthiopurine nucleosides (Cass and Paterson, 1977), but interpretation of data with nonspecific inhibitors, such as dipyridamole and cytochalasin B, is com-

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plicated by the finding that the nonsaturable uptake of cytosine and glucose is also inhibited by these inhibitors (Graff et af., 1977). IV.

L-

PROPERTIES OF NUCLEOSIDE AND FREE BASE TRANSPORT SYSTEMS

A. Specificity for Natural Substrates

T h e zero-trans influx of uridine by human erythrocytes is inhibited in ,Ipparent competitive manner by forrnqcin €3 (Oliver and Paterson, 197 1 !, and many natural nucleosides and nucleoside analogs cause ?he acielerated exchange diffusion of uridine and thymidine (Oliver and Pa!erson, 1971; Cass and Paterson, 1972, 1973). These findings suggest that human erythrocytes possess only a single nucleoside transport system with broad substrate specificity. As pointed out already, only substrates that are transported by the same carrier are expected to cause accelerated exchange diffusion, but this test is limited to asymmetric transport systems with significant difference in the rate of movement of loaded and unloaded carrier. Although quantitative values are not available from the work of Paterson and co-workers, the data suggest that many nucleosides are transported with similar efficiency. However, purine nucleosides accelerate the efflux of uridine slightly less than do pyrimidine nucleosides (Cass and Paterson, 1973). Ribose and nucleic acid bases are not substrates for the carrier. Initial studies of the long-term uptake of nucleosides by cultured cells, on the other hand, resulted in confusion and misinterpretations. Some nucleosides were found to inhibit the uptake of a given substrate-the inhibition was mutual and apparently competitive, but the K , values as inhibitor differed significantly from the K , values as substrate. Other nucleosides showed little or no inhibition with a given substrate (Plagemann, 1971a,c; Plagemann and Erbe, 1974a; Steck et al., 1969; Hare, 1970; Cass and Paterson, 1977; Paterson et af., 1977b). These findings supported the view of the existence of several nucleoside transport systems in cultured cells; one for uridine and cytidine, one for adenosine, one for thymidine, one for inosine and guanosine, and possibly others for other deoxyribonucleosides (Plagemann and Richey, 1974). Additional support for this view were the findings that certain experimental conditions resulted in changes in uridine, but not thymidine, uptake and vice versa (see Section VI), and that certain mutant cell clones exhibited defects in uptake affecting all nucleosides in a presumptive substrate class (see Section IV,E). However, it is clear now that the uptake rates measured in these studies largely reflected the rates of intracellular accumulation of nucleo-

-

‘i

272

PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER

tides derived from extracellular substrate (see Section 111), and consequently, that the observed substrate specificities reflected those of the phosphorylating enzymes involved: uridine, thymidine and adenosine kinase, and hypoxanthine/guanine phosphoribosyltransferase, respecti~ely.~ A dissenting view on the existence of several specific or overlapping nucleoside transport systems in cells other than human erythrocytes was that of Taube and Berlin (1972) based on results with monolayer cultures of nonproliferating rabbit polymorphonuclear leukocytes. These investigators reported that uridine, adenosine, and thymidine inhibited the uptake of each other in an apparently competitive manner with apparent Kis similar to the K,s of their uptake (10-40 pIM). They concluded that all purine and pyrimidine nucleosides are transported by a single system. They also explored rather thoroughly the structural requisites for inhibition of adenosine uptake in their leukocyte system. They found that, although 2’-deoxyadenosine strongly inhibited adenosine uptake, most other structural alterations in the sugar moiety greatly reduced the inhibition. Adenine itself had no significant effect. Differences in the base moiety, on the other hand, were less effective in reducing the inhibitory potency of ribonucleosides, but were more difficult to categorize on a rational basis. From spatial considerations of the nucleosides, it was concluded that in order to accommodate the structurally distinct molecules, substrate-induced conformational changes of the carrier must occur (see also review in Berlin and Oliver, 1975). However, a quantitative comparison of inhibition of adenosine uptake-on which this conclusion was based-involved some uncertainties: (1) the reported K i values were calculated from data obtained with single concentrations of adenosine (7 pkf) and of inhibitors on the assumption of competitive inhibition: ( 2 ) relatively long uptake time points (45 seconds) were used to estimate initial uptake rates in cells in which the substrates were rapidly converted to nucleotides; and (3) adenosine was rapidly deaminated in the cells. Such uncertainties may account for the fact that the K , for adenosine uptake in uninhibited controls, as well as the K,s for uptake of other substrates tested, were much lower than the nucleoside :carrier affinity constants estimated with cultured cell lines lacking phosphorylating activity or in human red blood cells (Tables I and 11). In fact, values of uptake K , for polymorphonuclear leukocytes, except for thymidine, are similar to those for the uptake of nucleosides There is no evidence that a direct phosphorylation of inosine occurs in P388 cells or most mammalian cells (Friedmann ct ( I / . , 1969: Burke et ( I / . , 1977: Fox and Kelley, 1978) as has been reported for some types of cells (Pierre and LaPage, 1968: Schaffer er ( I / . , 1973). Recently it has been demonstrated that deoxyguanosine is phosphorylated in S49 human T lymphoma cells by deoxycytidine kinase (Gudas ef ( I / . , 1978).

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273

by other phosphorylating, cultured cells (Table III), which are now known to reflect the saturation of the phosphorylating systems (see Section 111). The same correlation holds for the K , for adenine transport estimated in these polymorphonuclear leukocytes (7 p M ; Hawkins and Berlin, 1969; Berlin, 1970). In contrast, the K , for the uptake of xanthine which is not phosphorylated by the cells was found to be 2.3 mM, thus similar to K for adenine transport in ATP-depleted or adenine phosphoribosyltransferase-deficient cultured animal cells (Table 11). More direct evidence for a single nucleoside carrier with broad substrate specificity in cultured animal cells comes from recent studies in which transport per se was measured without complications of metabolism. Two lines of evidence support this conclusion. First, various riboand deoxyribonucleosides countertransport with uridine (Fig. 1 l), deoxycytidine, and thymidine (Plagemann er a / . , 1976) in ATP-depleted and/or kinase-deficient Novikoff cells. Second, deoxycytidine transport in a deoxycytidine kinase-deficient line of L12 10 leukemia is effectively inhibited by other purine and pyrimidine nucleosides (Kessel and Shurin, 1968),and cytosine arabinoside transport is inhibited by other nucleosides in a variety of cell lines (Mulder and Harrap, 1975; Heichal et a/., 1978). Similarly, various nucleosides inhibit the transport of each other in either ATP-depleted Novikoff cells or in cells deficient in the kinase specific for the substrate whose transport is measured (Plagemann et al., 1978a,b; Wohlhueter et al., 1979a). Though such data strongly suggest a common carrier, a molecular interpretation of these inhibitions is difficult at present, since the inhibitions have been found to be of a mixed type, involving both increases in carrier-substrate affinity constant and decreases in maximum velocity (Wohlhueter er al., 1979a; Marz et al., 1979). Detailed analyses of the inhibition of thymidine transport in thymidine kinase-deficient Novikoff = 192, 395, cells by inosine, uridine, and deoxycytidine yielded Ki,intercept and 1620, and Ki,sloPe= 64, 156, and 232 p M , respectively (Wohlhueter er al., 1979a). Mixed-type inhibition might be considered unexpected for alternative substrates from analogy to enzymatic reactions, but needs to be accounted for in a n y molecular and mathematical concepts of the mechanism of facilitated transport. Similar mixed-type inhibitions have been observed for the transport of hypoxanthine by apparently alternate substrates in cultured cells (Marz et al., 1979). Nevertheless, the degree of inhibition of the transport of nucleosides by each other seems to be inversely related to their carrier-substrate affinity constants. For example, K seems to be lowest for purine nucleosides and these seem to inhibit the most, whereas the opposite seems true for deoxycytidine and cytidine (Table 11; Wohlhueter et a/.,

274

PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER

v)

j:

0-

W V

PRELOADED ( I O m M ) NONE

o- URlDlNE &

DEOXYURIDINE

& CYTlDlNE P DEOXYCYTIDINE

F GUANINE D

URACIL

60

~

OO

I0

20

30

40

50

TIME ( M I N I

FIG. I I . Countertransport of [3H]uridine in ATP-depleted Novikoff cells. Samples of a suspension of 1 x lo' ATP-depleted cells/ml of glucose-free basal medium containing 5 mM KCN and 5 mM iodoacetate were supplemented where indicated with 10 mM uridine, deoxyuridine, cytidine, deoxycytidine, guanine, or uracil and incubated at 37°C for 20 minutes. The cells were collected by centrifugation and suspended to the same density in glucose-free basal medium with KCN and iodoacetate and containing 5 p M [3H]uridine (57 cpmipmole) at 18°C. The suspensions were incubated at 18°C and duplicate I-ml samples were analyzed for radioactivity in total cell material. All points are averages of the duplicate samples. (From Plagemann et a / . , 1978b.)

1979a; Marz et al., 1979; Wohlhueter and Plagemann, unpublished data). These findings suggest that some nucleosides are transported with different efficiencies. This conclusion needs to be considered in relation to the apparent symmetry of the nucleoside transport carrier. If, indeed, loaded and empty carrier move equally rapidly, the carrier movement should be indifferent toward the particular substrate with which it is loaded. Thus, although the substrate affinity of the carrier might differ for different substrates, the maximum substrate transport velocity should be the same for all substrates carried by the carrier of a specific cell line. This requirement seems to be met by the data for the limited number of substrates examined and within the experimental errors (Table 11), but more extensive data, such as kinetic analysis of the transport of different nucleosides with a single population of cells, are required to allow un-

275

PERMEATION IN ANIMAL CELLS

equivocal conclusions on this point. It is generally observed that the maximum velocities for a single substrate may vary up to 80% among cell populations of a cell line analyzed at various times (Plagemann et al., 1978b; Wohlhueter et a l . , 1979a; Marz et al., 1979). The reason for these variations is not clear, since the transport capacity of the cells varies little with the growth stage of the cells (see Section VI), but it makes quantitative comparisons of transport rates between different batches of cells difficult. The number of transport systems involved in nucleic acid base transport is less certain. Hypoxanthine and guanine seem to be transported by the same system in Novikoff hepatoma cells, since they inhibit the transport of each other about equally (Marz et a/., 1979). The kinetic parameters for their transport in these and P388 cells are similar to those of nucleoside transport (Marz et ul., 1979; see Table 11). Hypoxanthine transport is strongly inhibited by nucleosides (Marz et al., 1979) and nucleoside transport is similarly inhibited by hypoxanthine (Fig. 12A; Plagemann et al., 1978b; Wohlhueter et a / . , 1979a). These inhibitions are

1

0.021

/K i , slope =

420 pM

-500 TIME ISEC)

0

500

I000

HYPOXANTHINE ( p M 1

FIG.12. Kinetics of inhibition of uridine transport in uridine kinase-deficient Novikoff cells by hypoxanthine. The experiment was conducted as described in the legend to Fig. 3, except that where indicated the [3H]uridine solutions were supplemented with hypoxanthine to yield final concentrations of 365 and 920 pM. The final concentrations of [3H]uridine were 20, 40, 80, 160, 320, 640, and 1280 pM (350 cpm/pI, irrespective of concentration) and the intracellular and extracellular trapped water spaces were 14.6 and I .8 plisample pellet, respectively. Equation ( 1 ) was fitted to pooled corrected uptake data for each hypoxanthine concentration with all R parameters held equal. The theoretical curves for S , = 320 p M uridine plus 0, 365, and 930 p M hypoxanthine are shown in ( A ) . The best fitting parameters for 0, 365, and 930 p M hypoxanthine were K = 284 ? 13,400 ? 16, and 649 2 47, respectively; V = 30.7 ? 0.5, 25.7 ? 1 . 1 , and 24.3 0.9, respectively. The correlation coefficients (ru,G)were 0.9873, 0.9895, and 0.9722, respectively. The kinetic parameters were replotted in (B) as described by Segel (1975). (Previously unpublished data of Plagemann and Wohlhueter.)

+

276

PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER

also of mixed type (Fig. 12B). Nucleosides also countertransport with hypoxanthine in both hypoxanthine/guanine phosphoribosyltransferasedeficient and ATP(PRPP)-depleted wild type Novikoff cells (Zylka and Plagemann, 1975). These results indicate some overlap between nucleoside and hypoxanthine transport. Nucleoside and hypoxanthine transport are also similarly inhibited by p-hydroxymercuribenzoate and dipyridamole (see Sections V , A and B). Clear differences between nucleoside and hypoxanthine transport, however, exist in their sensitivity to inhibition by nitrobenzylthionucleosides. These compounds strongly inhibit the transport of nucleosides in many animal cells ( K i about 0.1-1 nM), whereas hypoxanthine transport is not affected by concentrations much higher than those affecting nucleoside transport (see Section V,C). That nucleosides and hypoxanthine/guanine are transported by different carriers is most strongly indicated by the recent isolation of single-step mutants from S49 lymphoma cells with defects in nucleoside transport, but not purine or pyrimidine uptake (Cohen et al., 1979). These studies are discussed further in Section IV,E. Adenine, on the other hand, seems to be transported in cultured animal cells by a system different from that transporting hypoxanthine, but here, too, there seems to be some overlap with the nucleoside transport system. These conclusions were anticipated on the basis of studies on adenine uptake by various types of cells. Some studies showed that adenine uptake is little affected by relatively high concentrations of hypoxanthine, guanine, cytosine, and uracil in hepatoma cells, and that similarly, adenine had little effect on the uptake of hypoxanthine and nucleosides (Plagemann, 1971a; Zylka and Plagemann, 1975; Dybing, 1974a). Adenine uptake in Novikoff hepatoma cells was also far less inhibited by p hydroxymercuribenzoate or dipyridamole than hypoxanthine or nucleoside uptake and not significantly affected by dipyridamole in various other types of cells (see Sections V,A and B). In other studies, however, inhibition of adenine uptake by guanine and hypoxanthine was observed, as in rabbit polymorphonuclear leukocytes (Hawkins and Berlin, 1969) and human blood platelets (Sixma et al., 1973), and adenine inhibited adenosine uptake in polymorphonuclear leukocytes (Strauss et al., 1976). Nucleosides, on the other hand, strongly inhibited adenine uptake (Hawkins and Berlin, 1969; Sixma et al., 1973; Zylka and Plagemann, 1975). Recently it has been shown (Suresch et al., 1979) that folate competitively inhibits ( K i = 450 pM) the uptake of adenine by L1210 mouse leukemia cells ( K , = 21 pM), while adenine competitively inhibits ( K i = 17 pM) the uptake of folate ( K , = 100-00 pM). Because of the similarity of the apparent K , of uptake and K i of inhibition the authors suggested that folate enters the cells via the same transport system as adenine. Studies in which adenine transport per se was measured in ATP-de-

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277

pleted or adenine phosphoribosyltransferase-deficient cells have confirmed the lack of effect of other nucleic acid bases on adenine transport in Novikoff hepatoma cells, but have failed to detect any effect of thymidine or inosine on adenine transport (Marz et af., 1979). Adenine, on the other hand, inhibited the transport of uridine in ATP-depleted cells (Plagemann et af., 1978b). This effect could not have been due to the formation of adenosine from adenine, since animal cells are devoid of adenosine phosphorylase. The specificity of the carriers transporting uracil and/or thymine is equally uncertain. Uracil causes a slight countertransport of uridine (Fig. 11) and hypoxanthine (Zylka and Plagemann, 1975) and weakly inhibits uridine transport (Plagemann et al., 1978b), hypoxanthine transport (Marz et af., 1979), and thymidine transport (Wohlhueter et af., 1979a), but it is not certain that these effects are caused by uracil itself and not by some uridine formed from uracil by uridine phosphorylase. Thymine, which is not converted to thymidine in these cells, however, also slightly inhibits thymidine transport (Wohlhueter et al., 1979a) and uridine transport (Plagemann et al., 1978b). Furthermore, the uracil: carrier affinity constant is very high (Table 11), approaching the solubility of uracil in aqueous solutions. Pyrimidine transport is also distinct from nucleoside transport in that thymine transport, like hypoxanthine transport, is not inhibited by nitrobenzylthioinosine (Wohlhueter et af., 1978b). The apparent competitive nature of the inhibition of the uptake of various nucleosides and purines by each other (Plagemann and Richey , 1974) warrants special consideration, particularly in view of the fact that inhibition of transport per se by alternate substrates is of a mixed type. The apparent competitive inhibition of uptake seems to result from the fact that the maximum velocities of substrate uptake reflect those of substrate phosphorylation rather than of transport. Thus, alternate transport substrates that are not substrates for the phosphorylation reaction would be expected to inhibit substrate uptake at low substrate concentrations, due to effects on transport, while the maximum velocity of uptake should not be affected-a situation tantamount to competitive inhibition (see later Fig. 15). B. Transport of Substrate Analogs

Most nucleoside and nucleic acid base analogs tested seem to be transported by the same system(s) as their natural counterparts. This conclusion was first indicated by the finding that many nucleoside analogs when present extracellularly accelerate the efflux of uridine from human erythrocytes (Oliver and Paterson, 1971; Cass and Paterson, 1972, 1973), and

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PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER

are presumed, therefore, to permeate the membrane via the same carrier as uridine. Among the several structurally diverse analogs of uridine tested, 2‘deoxyuridine and 5-bromouridine accelerated uridine efflux to about the same extent as uridine, whereas dihydrouridine and pseudouridine were less effective. Since the C-glycosides, pseudouridine and formycin B accelerated uridine efflux, it appears that the N-glycosidic linkage is not crucial for interaction of permeant with the carrier. On the other hand, 6-azauridine and orotidine were found not to accelerate uridine efflux, indicating that the presence of charged groups on the base is not accommodated by the carrier. Cytosine arabinoside and 2’-deoxynucleosides seem to be effective substrates, but other sugar substitutions, including substitutions at either the 2‘ or 3’-hydroxyl groups of ribose, were found to reduce greatly the ability of uridine or cytidine to accelerate uridine efflux. The authors concluded that the sugar moiety is more important than the base in recognition of substrate by the carrier. Paul et al. (1975) further studied the specificity of the nucleoside carrier of human erythrocytes by determining the effect on nucleoside transport of 70 different 9-/3-~-ribofuranosylpurine derivatives containing S, 0, or N atoms at the purine C-6 position and bearing various arylalkyl substitutions. The derivatives were all strongly inhibitory and the degree of inhibition increased with increase in hydrophobicity of the molecules. Many analogs have also been found to inhibit the uptake of nucleosides and purines or of each other by cultured animal cells (Taube and Berlin, 1972; Berlin, 1970, 1973; Strauss, 1974; Zylka, 1976; Plagemann and Erbe, 1974a; Plagemann, 1976; Yang and Visser, 1977; Hakala et al., 1975; Hare and Hacker, 1972; Turnheim et al., 1978; Grunicke et al., 1975). The list of effective substances is too long to be stated here, but some of the more common inhibitory analogs are as follows: cordycepin (3’-deoxyadenosine), 6-mercaptopurine riboside, 6-methyladenosine, 6chloropurine riboside, 6-dimethyladenosine, purine riboside, puromycin, showdomycin, tubercidin, 6-thioguanosine, 6-thioinosine, 5-bromodeoxyuridine, 5-bromouridine, cytosine arabinoside, 5-fluorouridine, 5fluorodeoxyuridine, tricyclic 7-deazanucleoside, formycin B, isoguanine, thioguanine, 2-amino-6-mercaptopurine, 6-mercaptopurine. For additional compounds the reader is referred to Berlin (1970) and Berlin and Oliver (1975). Where investigated, the inhibitions of uptake were of the simple competitive type, as also observed with natural alternate substrates, but such kinetic analyses are difficult to interpret in quantitative terms because of lack of information on the rate-determining step in uptake measurements (see Section 111). Direct information on the transport of these analogs in cultured animal

PERMEATION IN ANIMAL CELLS

279

cells is rather limited. Cytosine arabinoside seems to be transported with about the same efficiency as deoxycytidine (Table 11; Mulder and Harrap, 1975; Plagemann et al., 1978a). 5’-Bromodeoxyuridine, purine riboside, 6-mercaptopurine riboside, 6-methylmercaptopurine riboside, and 2’,3’,5’-trideoxyadenosineinhibit thymidine transport in thymidine kinase-deficient Novikoff cells about as effectively as deoxyuridine or deoxyinosine (Wohlhueter ot al., 1979a). The transport of 5’-deoxyadenosine, which is neither deaminated nor phosphorylated, has been directly demonstrated in L1210 mouse leukemia cells (Kqk = 115 F M ; Kessel, 1978). These studies point to the suitability of 5’-deoxyribosides as a class of chemically inert nucleosides for transport studies. 6-([4-Nitrobenzyl]thio)9-P-~-ribofuranosylpurine(nitrobenzylthioinosine)and similar nitrobenzylthionucleosides are potent and specific inhibitors of nucleoside transport; their action will be discussed in detail in Section V,C. 2’-Deoxycoformycin seems to be transported by the nucleoside carriers of mammalian cells, since extracellular uridine and nitrobenzylthioguanine prevent the inhibition of adenosine deaminase activity in whole human erythrocytes or mouse sarcoma 180 cells by 2-deoxycoformycin (RoglerBrown et id., 1978). The drug, however, seems to have relatively low affinity for the nucleoside carrier, since it does not affect the transport of adenosine in ATP-depleted P388 cells, even at a concentration of I mM (Lum et ( I / . , 1979). Slow entry into cells probably is responsible for the relatively slow effect of 2-deoxycoformycin on adenosine deaminase activity in whole cells (Rogler-Brown et a / . , 1978; Lum et a / . , 1979). Similarly, 8-azapurine analogs, such as 8-azaguanine, 8-azaadenine, etc., do not inhibit the uptake of either adenine or hypoxanthine in Novikoff cells (Zylka, 1976). Moreover, the influx of 8-azaguanine into hypoxanthine/guanine phosphoribosyltransferase-deficient cells shows no indication of saturation up to extracellular concentrations of 5 mM (Plagemann and Zylka, unpublished data), suggesting that entry might be solely by nonmediated permeation (see Section 111,E). These results indicate that the C-8 is essential for transport activity of purines. Comparison of the effect of various analogs on the uptake of adenine by rabbit polymorphonuclear leukocytes and its phosphoribosylation in cell lysates indicate that C-2 is equally important, although C-2 substituent groups can be accommodated. Furthermore, the electronic configuration about C-9 was found to be critical for uptake by whole cells, whereas wbonding or interaction with positions 7 and 8 seem more critical to the phosphoribosyltransferase (Berlin, 1970; Berlin and Oliver, 1975). C. Effect of Temperature Arrhenius plots of the uptake of adenosine by rabbit alveolar macrophages show a break at about 25°C with a change in activation energy

280

PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER

from 27 kcal/mole below 25°C to 15.7 kcal/mole above 25°C (Berlin, 1973). Similar breaks in Arrhenius plots between 15 and 23°C have been observed for the uptake of uridine and thymidine (Plagemann and Erbe, 1975), and of hypoxanthine and guanine (Zylka and Plagemann, 1975) by Novikoff rat hepatoma cells with corresponding shifts in activation energy from 15-26 to 4-7 kcalhole. Arrhenius plots of uridine uptake by quiescent and serum-stimulated 3T3 cells exhibited a similar type of curvature (Stein and Rozengurt, 1975). These findings suggested the possibility that membrane lipid transitions might affect the movement of transport carriers: greater membrane fluidity facilitating the movement of the carrier. However, no temperature transition was observed for adenosine uptake by polymorphonuclear leukocytes, and partial inhibition of adenosine uptake by macrophages by treatment with colchicine eliminated the break in the Arrhenius curve (Berlin, 1973). More informative, however, is the failure to detect any temperature discontinuities when thymidine transport per se was measured in thymidine kinasedeficient Novikoff cells (Fig. 13). The reason for the discrepancy between transport and uptake determinations has not been elucidated, but is probably related to the fact that uptake rates can be a function of both the rate of transport or phosphorylation (see Section 111). For example, the temperature discontinuity in uptake could reflect a transition from permeation-limited uptake at low temperatures to phosphorylation-limited uptake at higher temperatures. It should be noted that the activation energy estimated for thymidine transport per se (18.3 kcalhole) is similar to those observed for substrate uptake below the transition temperature. Furthermore, relatively low substrate concentrations were employed for uptake measurements (50 p M and below), but it has become apparent that the thymidine:carrier affinity constant as measured by both the zero-trans and equilibrium exchange protocols decreases markedly with decrease in temperature (see Fig. 13; Wohlhueter et a / . , 1979a). Assessment of the temperature response of transport, therefore, requires substrate concentrations well above K . Velocities of thymidine transport measured at a low substrate concentration ( 5 pkf) also yielded a curved Arrhenius plot in conformity with a dissimilar temperature dependence of Vqi and K (Wohlhueter et a / . , 1979a). In addition, linearity of Van’t Hoff plots for the thymidine :carrier affinity constant indicated an apparent enthalpy of 9.3 kcalhole and a lack of thermal discontinuities over the temperature range 4 to 37°C. That the transition temperatures for nucleoside and purine uptake are unrelated to membrane lipid changes is also suggested by the finding that no breaks are detectable in Arrhenius plots for the nonsaturable influx of prednisolone, cytosine, and L-glucose in Novikoff cells (Graff et ul., 1977; activation energies = 20-24 kcalhole).

281

PERMEATION IN ANIMAL CELLS T E M P E R A T U R E I'C)

40

35

30

20

25

10

I5

5

A 32

I

I

33

34 lo3/ T

I

35

36

IOK-')

FIG. 13. Temperature dependence of thymidine influx and exchange. Suspensions of thymidine kinase-deficient Novikoff cells (at about 3 x lo' cells/ml basal medium) were assayed for zero-trans influx of [3H]thymidine at 800 p M ( 0 )and for isotopic exchange with cells preincubated at 1330 p M (A)as described by Wohlhueter et a / . (1979a) (see Figs. 3 and 4). Cell suspensions, substrate solutions, and apparatus were thermally equilibrated before assay at the temperature indicated on the upper abscissa. The time course of appearance of radioisotope in the cell pellets was followed with 12 samplings encompassing 39 to 234 seconds, depending on temperature. Initial velocities were computed by fitting Eq. (7) to the equilibrium exchange data or Eq. ( 1 ) to the zero-trans influx data. For details, see Wohlhueter et al. (1979a). The computed initial velocities are plotted against inverse absolute temperature. The curve is the regression line on pooled influx and exchange data and corresponds to an Arrhenius activation energy of 18.3 kcaVmole (from Wohlhueter et al., 1979a). The listed K ; ; and K'" values as a function of temperature are also from Wohlhueter et al. (1979a).

D. Effect of pH The effect of extracellular pH on the uptake or transport of nucleosides and nucleic acid bases has not been investigated extensively. One recent study (Wohlhueter et al., 1979a)has shown that the velocity of thymidine transport, when expressed on the basis of intracellular HzO space in thymidine kinase-deficient Novikoff cells, increased about 90% with increase in pH between from 6.2 to 7.5. However, the HzO space of the cells was also found to increase somewhat with pH so that the increase in thymidine transport velocity when expressed on a number of cell basis increased only 40% with increase in pH. These data not only raise the question of which dimensions are most appropriate for expressing cellular transport data, but also suggest that observed differences in velocity may perhaps reflect changes in cell volume, rather than actual pH dependence of the thymidine transporter. Conversion to absolute dimensions of per-

282

PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER

meation (pmole/second-cm2of cell surface) necessitates relating cell volume to surface area. Assuming a spherical relationship yielded values of 2.18 (at pH 6.2) and 2.25 (at pH 7.5) pmole/second*cm2for thymidine permeation (Wohlhueter et al., 1979a). It should be noted that none of the natural nucleosides have pKs within the range of pH studied. E. Presumptive Cell Clones Defective in Transport

A number of drug-resistant cell lines have been isolated whose resistance has been considered to stem from a defect in nucleoside or purine transport. Breslow and Goldsby (1969) isolated a line of Chinese hamster cells partially resistant to 5-bromodeoxyuridine and unable to take up thymidine, though it possessed as much as 40% of the thymidine kinase activity of wild-type cells. Similarly, Lynch et al. (1977) isolated a 5bromodeoxyuridine/5-fluorodeoxyuridine-resistantline of HeLa cells with normal thymidine kinase activity, but with markedly reduced capacity to take up thymidine and 5-bromodeoxyuridine. In both instances it was concluded that the drug resistance was due to a defect in thymidine/ 5-bromodeoxyuridine transport. Furthermore, the finding that these lines took up other nucleosides, such as uridine, at an unabated rate supported the view that uridine and thymidine are transported by different carriers. Freed and Mezger-Freed (1973) isolated a similar, partially 5-bromodeoxyuridine-resistant, subline of haploid frog cells with apparently normal thymidine kinase activity, but reduced ability to take up thymidine. Furthermore, the cells failed to grow in HAT medium (containing hypoxanthine, aminopterin, and thymidine), but took up uridine, cytidine, and adenine normally. This finding again suggested that thymidine is normally transported by a specific carrier which was defective in the mutant subline, so that the line was referred to as thymidine transport negative (TT-). These investigators also found that it was possible to isolate, in a single step, thymidine kinase-deficient (TK-) clones from the TT- cells, but not from wild-type cells, by exposure to an even higher concentration of 5-bromodeoxyuridine, and suggested that the TT- genotype might be an obligatory intermediate in the isolation of the TKgenotype. In mammalian cells too, it has been found impossible to obtain, in a single step, thymidine kinase-deficient mutant clones by exposure to high concentrations of 5-bromodeoxyuridine (Thompson and Baker, 1973). In a subsequent study, however, Freed and Hames (1976) observed that the TT- frog cells had lost a heat-labile thymidine kinase, which made up about 30% of the total activity of wild-type cells; they suggested that this thymidine kinase participates in transport and is responsible for the reduced uptake of thymidine and the resistance to low concentrations of 5-bromodeoxyuridine. Since it is now clear that the long-term uptake

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283

rates determined by all these investigators measure the accumulation of phosphorylated intermediates rather than transport, and that all nucleosides are transported by a single carrier in all cultured lines of animal cells investigated, the validity of the interpretation that these three similar 5-bromodeoxyuridine-resistant cell lines represent thymidine transport mutants seems doubtful. It seems more likely that the partial resistance to 5-bromodeoxyuridine and the reduced uptake of thymidine is related to some alteration in thymidine kinase activity of the cells. For example, one could envision that the heat-sensitive thymidine kinase absent from the resistant haploid frog cells is the cytoplasmic enzyme, whereas the remaining activity might represent mitochondrial thymidine kinase, not readily available for the phosphorylation of thymidine entering the cells from extracellular fluid via the plasma membrane-associated nucleoside transport system. Mammalian and chicken cells possess at least one cytoplasmic and one mitochondrial thymidine kinase which differ in kinetic, electrophoretic, and other properties (Littlefield, 1979; W. C. Leung et al., 1975). Similar uncertainties pertain to the interpretation that a line of Chinese hamster ovary cells highly resistant to 8-azaguanine possesses a defective hypoxanthine transport system (Harris and Whitmore, 1974). This line is 300 times more resistant to 8-azaguanine than wild-type cells, but only five to six times more resistant to 6-thioguanine, and exhibits unaltered sensitivity to 6-mercaptopurine and azaadenine. The kinetic properties of the hypoxanthine/guanine phosphoribosyltransferase of the cells ( K , = 1.5 pcLM) and the V,,, for hypoxanthine uptake by whole cells were reported to be about the same as in wild-type cells. Only the K , for hypoxanthine uptake by whole cells differed: 18 p M compared to 7 p M for wild-type cells. These results are difficult to interpret, but are not consistent with the existence of a hypoxanthine transport defect in the mutant cells. First, as pointed out already (Section II1,E) 8-azaguanine is, at best, a very poor substrate for the hypoxanthine transport system, whereas 6-mercaptopurine and 6-thioguanine strongly inhibit hypoxanthine uptake (Zylka and Plagemann, 1975; Zylka, 1976) and transport (Plagemann, unpublished data) and thus seem to be transported by the same carrier as hypoxanthine. Thus, hypoxanthine transport mutants ought to exhibit resistance to 6-mercaptopurine and 6-thioguanine rather than to 8-azaguanine. Second, the reported uptake K,s probably reflect the saturation of the transferase reaction and not the hypoxanthine : carrier affinity constant, which was found to be greater than 1 mM for Chinese hamster ovary cells (see Table 11). Third, a high resistance of cells to a substrate analog is difficult to explain by a slight change in uptake K , without change in V,,,, particularly if the resistance level exceeds several fold the estimated uptake K , of the line. Thus, further

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PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER

work is required to elucidate the mechanism of resistance of this line to 8-azaguanine. Convincing evidence for a nucleoside carrier defect has only been recently reported for a single-step mutant subline (AE,) of the S49 mouse T cell lymphoma line which was isolated on the basis of its resistance to adenosine in t h e presence of an adenosine deaminase inhibitor (Cohen et al., 1979). These mutant cells take up all ribo- and deoxyribonucleosides examined at less than 2% of the rate obtained with wild-type cells and exhibit cross-resistance to various toxic nucleosides, but not to toxic purines or pyrimidines. Hybrids between the mutant and wild-type S49 cells exhibit normal uptake of and sensitivity to nucleosides. It has been reported that some 5-fluorouridine-resistant clones of S49 exhibit a similar nucleoside transport defect (Ullman et a l . , 1979). The properties of these mutant cell lines are strong evidence for the existence of only a single nucleoside carrier in these cells, as well as for the existence of separate purine and pyrimidine carriers. A single gene locus also seems to control expression of a functional nucleoside carrier in sheep erythrocytes (Jarvis and Young, 1978a). Erythrocytes from most, but not all, sheep lack nucleoside transport activity, but surprisingly cells from heterozygotes also fail to take up nucleosides. Thus, the lack of nucleoside carrier activity seems to be a dominant trait and the authors suggest that the gene (Nu') may code for an inhibitor of nucleoside transport rather than for the nucleoside carrier itself. The failure of [35S]nitrobenzylthioinosineto bind to sheep erythrocytes lacking nucleoside transport activity indicates the absence of functional nucleoside binding sites in membranes of these cells (Jarvis and Young, 1978b). F. Comparison to Transport in Other Types of Organisms

Results comparable to those obtained with cultured animal cells or erythrocytes in which nucleoside and purine transport was measured uncomplicated by intracellular metabolism are not available for any other organisms. In most studies, long-term uptake of substrates into cells was measured and little distinction between transport and intracellular metabolism was possible. From analogy to the results obtained with animal cells it seems, therefore, likely that in most studies substrate uptake rates reflected the accumulation of nucleotides rather than of transport. Several studies to which this analogy probably pertains are summarized in the following paragraphs. Based on a 2-minute uptake time-points, uridine uptake in Tetrahynzena

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is saturable with a K , = 2.3 p M , and competitively inhibited by other ribo- and deoxyribonucleosides, nucleoside analogs, as well as by dipyridamole, but not by uracil or ribose (Freeman and Moner, 1976; Wolfe, 1975). Most intracellular radioactivity was found to be associated with nucleotides. The uptake of thymidine and deoxyadenosine increased during germination of Neurospora crassa conidia (Schiltz and Terry, 1970). Based on 6-minute uptake time points, the K , for thymidine and deoxyadenosine uptake were 30 and 65 p M , respectively. Since thymidine inhibited competitively the uptake of deoxyadenosine, whereas deoxyadenosine had little effect on thymidine uptake, it was concluded that two different uptake systems are involved. Magill and Magill (1973) assessed the effects of various nucleosides on the growth of an adenosine auxotroph of N . crassa. They found that growth of this mutant on adenosine was inhibited by all other nucleosides tested, but not by adenine. They concluded that the results are contrary to the existence of separate purine and pyrimidine nucleoside uptake systems. This conclusion was supported by the finding that many purine and pyrimidine nucleosides competitively inhibited the uptake of labeled adenosine by this auxotroph with Kis between 7 and 28 p M (Magill et a / . , 1974). Based on 5-minute uptake values, the K,s for adenosine and uridine uptake were estimated as 6.2 and 16 p M , respectively. Foury and Goffeau (1975) reported that treatment with cyclic AMP caused an increase in the V,,, for uridine uptake by the yeast Schizosaccharomyces pombe without affecting the uptake K , of 14 p M . These values were based on 5-minute uptake time points. Since the inhibition of energy production by the cells caused a decrease in uridine uptake it was concluded that uridine enters by active transport, but this conclusion is unwarranted, since the estimated uptake rate probably reflected the accumulation of nucleotides rather than of transport. K,s for adenosine and guanosine uptake were found to be much higher (2 and >5 mM, respectively) than that for uridine uptake. Housset and Nagy (1977) reported that the K,s for guanine phosphoribosyltransferase and adenine phosphoribosyltransferase of Schizosaccharomyces pombe (28 and 69 p M , respectively) were much higher than those for the uptake of guanine and adenine by whole cells (0.66 and 0.25 p M , respectively) and that the pH optima for the enzymes and uptake also differed greatly. They concluded these data to rule out the involvement of group translocation in purine transport. They also found that the uptake K,s for adenine and guanine were higher and the V,,, values were lower in adenine phosphoribosyltransferase-deficient and guanine phosphoribosyltransferasedeficient mutants, respectively, than in wild-type cells. These results

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again suggest that the uptake velocities measured by these investigators represented rates of accumulation of phosphorylated products rather than transport rates. Polak and Grenson (1973) studied purine and pyrimidine uptake in purine and pyrimidine-requiring mutants of Saccharomyces cerevisiae , respectively. Uptake of cytosine was competitively inhibited by hypoxanthine and adenine, and cytosine inhibited purine uptake. The Kis of inhibition were similar to the K,s of their uptake (5-15 pn/J). Uridine and uracil, on the other hand, were without effect. It was concluded that hypoxanthine, adenine, and cytosine are transported by a single carrier, whereas uracil is transported by a different carrier. The literature on nucleoside transport in bacteria, mainly Escherichia coli, is voluminous, but definitive information on the nature of the transport systems is still limited. Transport studies with E . coli and other gram-negative bacteria are complicated not only by the phosphorylation of nucleosides in the cells, but also by the rapid catabolism (deamination and phosphorolysis) of nucleosides in the periplasmic space (Beck et al., 1972; Hochstadt, 1974; Munch-Petersen and Mygind, 1976). Detailed discussion of this work is beyond the scope of the present article and the reader is referred to the review by Hochstadt (1974) and recent publications by the main groups of investigators working in the field (K. K. Leung et al., 1975; Roy-Burman and Visser, 1975; von Dippe et al., 1975; Leung and Visser, 1977; Munch-Petersen and Mygind, 1976; Mygind and Munch-Petersen, 1975; Komatsu and Tanaka, 1973; Doskotil, 1976; McKeown et al., 1976; Munch-Petersen et al., 1979). Because E . coli concentrates radioactivity from labeled nucleosides several hundred fold, nucleoside transport in bacteria has generally been considered an active process, and the operation of at least two nucleoside transport systems (nupC and nupG) has been indicated on the basis of substrate specificity and antibiotic resistance (Munch-Petersen et al., 1979). On the other hand, Rader and Hochstadt (1976) postulate that the ribose moiety of uridine and adenosine is transferred through the membrane as ribose- 1-phosphate by group translocation catalyzed by the appropriate nucleoside phosphorylases. It is thought that the nucleoside phosphorylases have a transmembrane orientation with the base release site on the external face and the ribose-I-phosphate release site on the internal face. The uptake of purines is similarly believed to occur by group translocation involving purine phosphoribosyltransferases located in the periplasmic space and PRPP as cofactor (Hochstadt-Ozer and Stadtman, 1971; Hochstadt, 1974). The uptake of pyrimidines has been postulated to proceed via a similar mechanism involving uracil phosphoribosyltransferase (Hochstadt, 1974). However, here, too, a tandem

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operation of periplasmic phosphorylases, nonconcentrative transport, and intracellular phosphoribosylation may account for the experimental data, and confirmation of the operation of the postulated group translocations is still needed. One exception to the generalization that nucleoside uptake in all eukaryotes investigated resembles that in cultured animal cells might be studies with the tapeworm Hynienolepidid cestodes. This parasite lacks a mouth and alimentary tract and nutrients enter through the outer surface. Entry of uridine has been reported to occur via an active transport system, since uptake was inhibited by iodoacetate, even though little phosphorylation occurred during 2 minutes of incubation with substrate (Page and MacInnis, 1975). Based on 2-minute uptake values, MichaelisMenten constants between 100 and 200 p M were estimated for the uptake of uridine, thymidine, adenosine, deoxyadenosine, and guanosine. These nucleosides inhibited the uptake of each other in a similar manner, whereas thymine and uracil stimulated the uptake of uridine and thymidine, but not of the purine nucleosides. It was concluded that the purine and pyrimidine nucleosides are transported by different, but overlapping, transport system.

V.

TRANSPORT INHIBITORS AND NACTIVATION

A. Effects of Sulfhydryl Reagents

The preincubation of various types of cultu ed cells with sulfhydryl reagents, such as p-hydroxymercuribenzoate and p-hydroxymercuribenzenesulfonate, causes a marked inhibition of the uptake of various nucleosides and purines (Schuster and Hare, 1970, 1971; Hare, 1975; Plagemann and Richey, 1974; Plagemann and Erbe, 1972; Zylka and Plagemann, 1975; Alford and Barnes, 1976; Barlow and Ord, 1975; Tsan and Berlin, 1971). Attainment of inhibition is concentration, time, and temperature dependent. For example, a maximum inhibition of 85-90% of uridine, thymidine, and hypoxanthine uptake was caused by 70- 100 p M p-hydroxymercuribenzoate, but only after an incubation period of about 5 minutes (Plagemann and Richey, 1974). Adenine uptake was much more resistant to inactivation by p-hydroxymercuribenzoate than nucleoside or hypoxanthine uptake (Zylka and Plagemann, 1975). Since in many of the studies it was demonstrated that the treatment had no effect on the phosphorylating activity of the cells as measured in lysates of treated cells, it was concluded that the observed inhibitions of uptake were due to an effect on substrate transport (Plagemann and Richey,

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1974). This conclusion seems correct, since p-hydroxymercuribenzoate causes a similar inhibition when transport of uridine was measured in uridine kinase-deficient Novikoff cells (Plagemann et a / ., 1978b). Although the mechanism of inhibition has not been elucidated in detail, the inhibitions are probably a consequence of an interaction of these reagents with sulfhydryl groups of membrane proteins, presumably the carrier. The effect of p-hydroxymercuribenzoate treatment on thymidine uptake is almost completely reversed after 1 hour of incubation of the cells in fresh medium containing 1 mM dithiothreitol (Plagemann and Erbe, 1972). It has been reported that the efficacy of various sulfhydryl reagents in inhibiting nucleoside uptake by cultured hamster and mouse cells is related to their hydrophobicity (Schuster and Hare, 1971; Hare, 1975). This may indicate that the affected sulfhydryl groups are deeply embedded in the membrane, but it is also possible that, in part at least, the inhibition of uptake reflects an inactivation of the respective phosphorylating enzymes. The function of sulfhydryl groups in transport is not understood, but all mechanisms considered for the inactivation of enzymes by sulfhydryl reagents (Boyer, 1959; Webb, 1966) may also apply to effects on transport. I n contrast to the results previously discussed, Eilam and Cabantchik (1976, 1977), Heichal et ul. (1978), and Bibi et al. (1978) reported that treatment of golden hamster MCT cells with p-hydroxymercuribenzene sulfonate (20 p M ) stimulated the uptake of uridine and cytosine arabinoside. On the other hand, N-ethylmaleimide was inhibitory, and p hydroxymercuribenzene sulfonate and N-ethylmaleimide caused a synergistic inhibition of cytosi,ie arabinoside uptake. On the basis of these and other similar results the authors proposed the operation of a complex carrier with four different organomercurial binding sites, one of which was postulated to represent the substrate binding site and two to be SHcontaining modifier sites. This model cannot be generalized at present and seems premature, since contrary results have been reported by other investigators with other cell lines (see above).

B. Effect of Other Nonspecific Inhibitors The uptake of nucleosides and purines by various types of animal cells is directly inhibited by numerous substances which structurally have nothing in common with each other or with the substrate whose uptake they inhibit. By direct inhibition we mean that the inhibition is very rapid (within 1 minute or less) and readily reversed by removal of the inhibitors. Dipyridamole is the classical inhibitor of this type, and its effect on

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nucleoside and hypoxanthine uptake has been demonstrated in many studies (Scholtissek, 1968: Plagemann and Roth, 1969; Plagemann, 1971a; Plagemann and Erbe, 1972, 1974a; Zylka and Plagemann, 1975; Peters and Hausen, 1971; Schrader et a / . , 1972; Koos and Pfleger, 1972; Rau and Schlotissek, 1970; Crifo et al., 1973; Kessel and Hall, 1970; Kessel and Dodd, 1972; Turnheim et a / . , 1978). Its effect on nucleoside transport was first deduced from its effect on adenosine deamination in dog erythrocytes (Kiibler and Bretschneider, 1963, 1964). The effect of dipyridamole on adenine uptake observed in various animal cells has been more variable. Adenine uptake was found to be inhibited (although to a lesser extent than nucleoside and hypoxanthine transport) in human platelets (Sixma et a / . , 1973) and in Novikoff rat hepatoma cells (Zylka and Plagemann, 1975), but little or no significant effect was detected in chicken embryo cell cultures (Schlotissek, 1968), human platelets (Rozenberg et d . , 1971), perfused guinea pig and rat heart (Kolassa et d., 1970), L1210 mouse leukemia cells (Kessel and Dodd, 1972), and various transplantable mouse ascites tumors in in vitro suspension (Henderson and Zomber, 1977). Other general inhibitors of nucleoside and purine uptake are cytochalasin B (Plagemann and Estensen, 1972; Takana et al., 1975; Plagemann et a / . , 1975b, 1978c), papaverine (Plagemann and Sheppard, 1974; Sixma et a / . , 1973, 1976; Woo et a/., 1974), theophylline (Plagemann and Sheppard, 1974; Rozengurt and Jiminez de Asua, 1973; Benedetto and Casson, 1974: Woo et al., 1974), prostaglandins (Plagemann and Sheppard, 1974; Rozengurt and Jiminez de Asua, 1973), colchicine (Mizel and Wilson, 1972; Berlin, 1973; Plagemann and Erbe, 1974b; Zylka and Plagemann, 1975), 2-mercapto- 1-/3-4-pyridethylbenzirnidazole(Skehel et al., 1967; Nakata and Bader, 1969), streptovaracin (Tan and McAuslan, 1971), phloretin and phloridzin (Lemkin and Hare, 1973), aflatoxins and sterigmatocystin (Kunimoto et al., 1974), podophyllotoxin (Loike and Horwitz, 1976), and acronycin (Dunn et al., 1973). The inhibitions of substrate uptake by cultured cells were considered to be caused by an inhibition of the transport step since, where investigated in detail, the inhibitors had little if any effect on the phosphorylation of the substrates, either in whole cells or in cell lysates (for additional details, see Plagemann and Richey, 1974). This conclusion has been confirmed by direct transport measurements for some of these inhibitors. It has been shown that dipyridamole inhibits the transport of deoxycytidine in deoxycytidine kinase-deficient L 12 10 leukemia cells (Kessel and Hall, 1970), that dipyridamole and papaverine inhibit the transport of hypoxanthine in hypoxanthine-guanine phosphoribosyltransferase-deficient Novikoff cells (Zylka and Plagemann, 1975), and that dipyridamole,

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papaverine, and cytochalasin B inhibit the transport of uridine and thymidine in kinase-deficient or ATP-depleted Novikoff cells (Plagemann et al., 1976, 1978b,c). Recent studies have shown that dipyridamole causes a mixed type of inhibition of uridine transport with a Ki,int= 12 p M and Ki,slope= 3 p M (Plagemann et al., 1978b; see Fig. 14). This finding is in contrast to the simple competitive inhibition reported earlier for the inhibitions of nucleoside and purine uptake by various of the above substances (Plagemann and Richey, 1974). This competitive inhibition can now be explained on the basis of the finding that substrate uptake rates reflect rates of phosphorylation rather than transport rates (see Section 111). These inhibitors render transport the rate-determining step in uptake, but with increase in substrate concentration the effect of inhibitor is overcome, phosphorylation again becomes rate-determining, and the same apparent

T I M E ISECI

DIPYRIDAMOLE IVM I

FIG. 14. Kinetics of inhibition of uridine transport by dipyridamole in uridine kinasedeficient Novikoff rat hepatoma cells. The experiment was conducted as described in the legend to Fig. 3, except that, where indicated, the [3H]uridine solutions were supplemented with dipyridamole to yield final concentrations of 5 and 15 p M . The final concentrations of [3H]uridine were 20, 40, 80, 160, 320, 640, and 1280 p M (400 cpm/pI, irrespective of concentration) and the intracellular and extracellular trapped water spaces were 28 and 3.7 $sample pellet, respectively. Data are from Plagemann et (11. (1978b), but have been reanalyzed by fitting Eq. ( I ) to the pooled data for each dipyridamole concentration with all R parameters held equal. The theoretical curves for S, = 80 p M uridine plus 0, 5 , and 15 pM dipyridamole are illustrated in (A). The best fitting parameters for 0, 5 , and 15 pM dipyridamole were K = 202 2 15, 598 2 24, and 648 2 101 pM,respectively; V = I I .8 2 0.3, 9.3 2 0.2, and 5.3 2 0.5 pmoleipl cell H,O.second, respectively. The correlation coefficients (r,,o) were 0.9422, 0.9893, and 0.8632, respectively. The kinetic parameters were replotted in (B) as described by Segel (1975).

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maximum uptake velocity is attained as in the absence of inhibitor. A computer simulation of the effect of a theoretical transport inhibitor on thymidine transport and phosphorylation operating in tandem is illustrated in Fig. 15 [see legend for Eqs. ( l 3 ) - ( 17)]. This subject is discussed in more detail by Wohlhueter and Plagemann (1980). The molecular basis of the inhibition of transport by these substances is not known. The finding that dipyridamole causes a decrease in maximum velocity of nucleoside transport (Fig. 14) suggests that it affects the movement of the carrier, but even this conclusion is not unequivocal, since alternate substrates have also been observed to give a mixed type of inhibition (see Section IV,A). One possibility suggested by the lipid solubility and broad specificity of the inhibitors-many inhibit hexose and phosphate uptake as well as nucleoside and base transport (Plagemann and Richey, 1974)-is that they either affect the structure of the membrane or directly interact rather nonspecifically with integral membrane proteins including transport carriers (Plagemann et al., 1977). This view is supported by the finding that dipyridamole and cytochalasin B also inhibit the nonsaturable permeation of cytosine and L-glucose (Graff et a / . , 1977). Any relationship between lipid solubility of these substances and their efficacy as transport inhibitors, however, has not been ascertained as yet. Many of the substances in question have additional toxic affects on cells, but none of these effects seem to result from or be related to an inhibition of nutrient transport by these inhibitors (Plagemann and Richey, 1974). Because of the wide range of different substances already found to inhibit the transport of various substrates it is predictable that many other substances will be found to have such effect. Thus, great caution is required in studies involving the use of radioactively labeled nucleosides and purines and other precursors to assess the effect of inhibitors on metabolism and macromolecular synthesis. Another group of substances that has been found to inhibit the uptake of nucleosides, purines, and other substrates by animal cells are organic solvents such as ethanol (Scholtissek, 1974; Plagemann and Erbe, 1974b), phenethyl alcohol (Plagemann and Roth, 1969; Plagemann, 1970; Crifo et al., 1973), and dimethyl sulfoxide (Scholtissek, 1974; Collins and Roberts, 1971). Here again the finding that the phosphorylating activity of treated cells was unaltered has led to the view that the effect on uptake is mediated at the transport step, but direct evidence is still lacking. These substances have in common the ability to inhibit substrate uptake only at concentrations approaching those that cause outright lysis of the cell. Thus, it seems likely that a transport inhibition is caused by a perturbation in membrane structure, probably due to an effect of these compounds on

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3

2

0

5

llS,

FIG. 15. Computer simulation of transport and phosphorylation operating in tandem. As a simple model we take: t

s , e s,-

P

s-P,

(13)

in which reaction "t" is a symmetrical, facilitated transport system, operating bidirectionally ( K , , <=, I ) with Michaelis-Menten parameters K'and V ' . Reaction "e" is an irreversible kinase, operating with K ; and V', and whose product S-Pis trapped within the intracellular compartment. Solution for the steady-state concentration S , in terms of these kinetic parameters gives: as;

+ bS, + c = 0

(14)

where, a = -V'Kt - VeK' - V ' S ,

b

=

V'K'S, - V'KLK' - K"V'

c

=

V'K'K",,

(15) -

V'K'S,

(16)

and

(17)

In this Lineweaver-Burk simulation the reciprocal of the velocity of the enzymatic reaction normalized to the maximal enzyme velocity (V'lu') is plotted against the reciprocal of the exogenous substrate concentration ( I i S , ) for 0.2 5 S , 5 10. K'is fixed at 225 (comparable, in pM, to K of thymidine transport): K', is fixed at 0.5 (comparable to K, of thymidine uptake); and V' is fixed arbitrarily at 0.01. The four curves represent decreasing velocities of transport (V' = I , 0.8, 0.6, 0.2) at constant K', simulating addition of a transport inhibitor which affects only transport velocity, not transport K. Data falling on these curves might be construed, fallaciously, to represent competitive inhibition of a transport system operating with K = 1, if transport were thought to be limiting, and if measurements were made over a time interval in which the rate of accumulation of S-P was equivalent to the rate of uptake, i.e., where S, was in steady-state. (Modified from Wohlhueter et al., 1978b.)

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the membrane lipids. Nevertheless, as long as the cells are not rendered stainable by trypan blue by the treatment, the transport inhibition is readily reversed by removal of these substances (Plagemann, 1970; Plagemann and Erbe, 1974b; Scholtissek, 1974).

C. Inhibition of Nucleoside Transport by p-Nitrobenzylthiopurine Nucleosides The nitrobenzylthiopurine nucleosides are of special interest as nucleoside transport inhibitors and are thus considered separately. These substances were first found to inhibit the transport of nucleosides in human erythrocytes (Paterson and Oliver, 1971; Pickard et al., 1973; Cass and Paterson, 1972, 1973; Turnheim et al., 1978). Nitrobenzylthioinosine is one of the most potent of these substances and has been studied most extensively. It binds tightly to the erythrocyte membrane (dissociation constant = 1 nM), but is not altered itself chemically. Pickard et a / . (1973) estimated that about lo4 sites on the erythrocyte membrane bind radiolabeled nitrobenzylthioinosine. It also strongly binds to a waterinsoluble residue of red blood cell ghosts that consists mainly of lipids and "Band 3" protein (Pickard and Paterson, 1976). These compounds also inhibit the uptake of various nucleosides in cultured animal cells (Paterson et a / . , 1975, 1977a,b; Cass and Paterson, 1977; Eilam and Cabantchik, 1976, 1977; Bibi e t a / . , 1978; Heichal et a/., 1978) and in phytohemagglutinin-stimulated pig (Barlow and Ord, 1975) or human lymphocytes (Fleit et a / . , 1975). Nitrobenzylthioinosine monophosphate is as effective an inhibitor as the nucleoside itself (Lynch et a / ., 1978). Nucleoside uptake is inhibited by nitrobenzylthioinosine in an apparent competitive manner. Reported apparent K i values are 0.15-0.6 nM for the inhibition of uridine uptake in golden hamster MCT cells (Eilam and Cabantchik, 1977) and 4- 10 n M for the inhibition of cytosine arabinoside uptake in HeLa cells (Cass and Paterson, 1977). As discussed already (Section 111), however, the kinetics of inhibition of uptake do not allow any conclusions as to the type of inhibition of transport per se, nor precise estimates of the affinity of the inhibitor to the carrier. Recent studies have shown that inhibition of thymidine transport per se in ATP-depleted Chinese hamster ovary cells by nitrobenzylthioinosine conforms to the pattern of simple, noncompetitive inhibition, as defined for enzyme systems (Cleland, 1967): nitrobenzylthioinosine diminishes the maximum velocity of transport without affecting the substrate: carrier affinity constant (Wohlhueter et al., 1978b). Based on the total concentration of inhibitor, which significantly overestimates that

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of free inhibitor, a K iof about 1 nM was estimated. The rate of attainment = 17 seconds) relative to of maximum inhibition of transport is slow the rate of association of nucleosides with the carrier, and thymidine partially protects the transport system against inhibition by nitrobenzylthioinosine. The dissociation constant for the inhibitor :carrier complex (K,) has been estimated as about 0.1 nM and a similar value is obtained for K i when expressed on the basis of estimated concentrations of free inhibitor. The number of nitrobenzylthioinosine binding sites has been calculated as about 60,00O/CHO cell, a value similar to that estimated from the inhibition of uridine uptake by nitrobenzylthioinosine in golden hamster MCT cells (Eilam and Cabantchik, 1977). Chinese hamster ovary cells, P388 mouse leukemia cells, HeLa cells, and mouse L cells are about equally sensitive to inhibition of thymidine transport by nitrobenzylthioinosine, whereas thymidine transport in Novikoff rat hepatoma cells is about four orders of magnitude more resistant ( K i about 2 p M ; Wohlhueter ef a l . , 1978b). The transport inhibition by nitrobenzylthioinosine and other compounds of this type is highly specific for nucleosides; uptake and transport of hypoxanthine, adenine, and thymine is not affected (Paterson and Oliver, 1971; Wohlhueter et al., 1978b). The combined results on the effect of nitrobenzylthioinosine on thymidine transport suggest that its inhibitory interaction with the nucleoside carrier of the highly sensitive cell lines is more complex than a simple inhibitor: carrier association, even though one step in the interaction must involve recognition of the inhibitor by the nucleoside binding site of the carrier. But this recognition, which presumably is very fast, seems to be followed by a slower step which leads to the tight binding of the inhibitor to the carrier resulting in maximum transport inhibition. The apparent noncompetitive nature of the inhibition of transport is explained by the fact that the inhibitor binds virtually irreversibly, i.e., the rates at which its ultimate, inhibitory effect is attained or reversed are much slower, and its affinity much greater, than the rates of substrate interaction with and affinity for the nucleoside carrier. Similar observations have been made for the inhibition of enzyme reactions by tightly binding inhibitors (Agarwal et al., 1977). Whether at the final stage of binding nitrobenzylthioinosine still occupies the nucleoside binding site is not established, but such model is consistent with experimental data and has been favored by Wohlhueter et al. (1978b). That the inhibitor remains structurally intact when bound to the carrier has been indicated by the studies of Pickard et al. (1973) with radiolabeled nitrobenzylthioinosine. The secondary step in binding does not seem to occur in Novikoff cells so that in these cells, nitrobenzylthioinosine may simply act as an alternate substrate for the carrier. The high affinity of nitrobenzylthioinosine

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for the nucleoside carrier of many cell lines makes it potentially useful in further characterization of the transport system and to affinity label the carrier. D. Heat Shock

Incubation of Novikoff rat hepatoma cells at 47.5 to 48.5"C causes a rapid irreversible decrease in the cells' capacity to take up various nucleosides without affecting their phosphorylation capacity (Plagemann, 1971a; Plagemann and Erbe, 1972). Since heat shock seemed to cause a decrease in the V,,, of uptake without causing a significant change in K , it was suggested that the effect reflects an inactivation of the carrier, but direct evidence for this conclusion is still lacking. E. Effect of Hydrolytic Enzymes

Incubation of Novikoff cells with trypsin, chymotrypsin, neuraminidase, phospholipase C, bromelain, or Pronase alone or in combination at 37°C for 15-30 minutes has no effect on the capacity of the cells to take up uridine (Plagemann, 1971a; Plagemann and Richey, 1974), and recent experiments have shown that trypsin treatment also has no effect on thymidine transport per se when measured in thymidine kinase-deficient Novikoff cells (Marz et a / . , 1978). Similarly, incubation with trypsin has no effect on nucleoside uptake by rabbit alveolar macrophages, although the transport of lysine is depressed (Tsan et a / . , 1973). These results are consistent with the view that the functional parts of the nucleoside carrier are not readily accessible in the plasma membrane to external hydrolytic enzymes. Protein and carbohydrate moieties removed from the cell surface by treatment with neuraminidase and trypsin do not seem to have any function i n transport. VI.

REGULATION OF NUCLEOSIDE AND FREE BASE TRANSPORT AND UPTAKE

The uptake capacity of cultured cells and lymphocytes for nucleosides and purines (as estimated from I - to 10-minute or even longer time points) varies greatly with the growth stage of the cells. These variations have generally been considered to reflect a regulation of the transport step. And since the changes in uptake were found to be due to alterations in the maximum velocity and not in the K , of uptake, the regulation was

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perceived as involving changes in the number of functional transport carriers in the membrane (Plagemann and Richey, 1974). Recent experiments, however, have shown that the growth stage-related changes in uptake are due to alterations in the phosphorylating capacity of the cells rather than in transport. One of the first growth stage-related variations observed was a marked decrease in the capacity of untransformed, cultured mammalian or chicken cells to take up uridine when these cells become density inhibited or quiescent due to serum starvation (Weber and Rubin, 1971; Hare, 1972a,b; Eilam and Bibi, 1977; Hale et al., 1975).This decrease in uptake seems to be specific for uridine in many types of cells and the mitogenic response of quiescent, untransformed cells or lymphocytes is associated with a rapid increase in uridine uptake capacity beginning within the first few minutes after exposure to the mitogen (Weber and Rubin, 1971; Hare, 1972a,b; Rozengurt and Jiminez de Asua, 1973; Eilam and Bibi, 1977; Kram et a / . , 1973; Kram and Tomkins, 1973; Pariser and Cunningham, 1971; Otsuka and Moskowitz, 1975; Rozengurt and Stein, 1977; Kitagawa and Andoh, 1978). In fact, this increase in uridine uptake has been considered one of the characteristics of the pleiotypic (Hersko et a / . , 1971) or coordinate (Rubin, 1976)response of these cells to mitogenic stimuli. The increased uptake in stimulated, quiescent cells has also been generally found to be specific for uridine. An increase in adenosine uptake has been observed in one study (Otsuka and Moskowitz, 1975), but not in others (Hare, 1972b; Cunningham and Pardee, 1969; Pariser and Cunningham, 1971). However, serum stimulates the uptake of adenosine in nonreplicating primary cultures of rabbit alveolar macrophages (Strauss and Berlin, 1973). In contrast to rapid stimulation of uridine uptake in mitogen-stimulated, untransformed cells, increases in thymidine and deoxycytidine uptake occur only 10-20 hours after the mitogenic stimulus of 3T3 cells, coincidental with the entry of the cells into S-phase (Cunningham and Remo, 1973). The same is true for mitogen-stimulated lymphocytes (Barlow and Ord, 1975; Barlow, 1976; Peters and Hausen, 1971), whereas uridine uptake and uridine kinase activity begin to increase within 30 minutes of stimulation (Peters and Hausen, 1971). Recent experiments with monolayer cultures of mouse 3T3 cells and mouse embryo cells (see below), however, have shown that transport of uridine is not altered during the mitogenic response to serum or epidermal or fibroblast growth factors, but only its phosphorylation (Rozengurt et al., 1977b, 1978). The time course of uptake of uridine by these cells is biphasic with an initial phase lasting not more than 4-5 seconds which is followed by a second phase reflecting the accumulation of uracil nucleo-

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tides. Only the second linear phase of uptake was found to be stimulated by the mitogens. Furthermore, the accumulation to equilibrium level of cytosine arabinoside which is only very slowly phosphorylated in quiescent 3T3 cells was found not to be significantly altered 30 minutes after addition of serum. However, the increases in uridine uptake in mitogenstimulated mammalian cells are relatively little affected by inhibitors of protein synthesis (Jiminez de Asua and Rozengurt, 1974; Stein and Rozengurt, 1975), and occur in the absence of a detectable increase in uridine kinase activity as assayed in cell lysates (Hare, 1972b; Rozengurt et al., 1978: Goldenberg and Stein, 1978). These findings initially led to the idea that the uridine carriers are cryptic in serum-starved cells and become activated by mitogens (Stein and Rozengurt, 1975), but a more plausible explanation is that the uridine kinase activity measured in cell lysates may not truly reflect the in situ uridine phosphorylating capacity of cells. This conclusion is also indicated by the finding that treatment of animal cells with inhibitors of protein synthesis may result in an increase in apparent uridine kinase activity assayed in cell lysates (Plagemann et al., 1969: &hak and Rada, 1976)and even stimulates uridine uptake in serumstarved 3T3 cells (Jiminez de Asua and Rozengurt, 1974; Jiminez de Asua et al., 1974). Some evidence has been presented which suggests the possibility that the increased uridine phosphorylation in mitogen-stimulated 3T3 cells is due to a change in affinity of uridine kinase for ATP (Goldenberg and Stein, 1978), but such alteration was not detectable in cell lysates and other possibilities have not been excluded. For example, it has been shown that partial depletion of ATP in growing 3T3 cells by incubation with 2-deoxy-~-glucosereduces long-term uridine uptake to about the same level as in quiescent cells, whereas the initial transport rate estimated from the first 30-second uptake curve is not affected (Koren et al., 1978). Upon reversal of an ATP/UTP depletion, induced either by incubation with glucosamine (Scholtissek, 1972) or KCN treatment (Plagemann and Erbe, 1973), on the other hand, uridine uptake rapidly resumes at a rate higher than that observed in untreated controls. This effect most likely is due to a decrease in intracellular concentration of UTP and CTP, reducing feedback inhibition of uridine kinase (Orengo, 1969), since preincubation of cells with high concentrations of uridine has the opposite effect (Scholtissek, 1972). The increase in long-term uptake of uridine in serum-stimulated, untransformed Nil 8 cells has also been shown to reflect an increase in uridine phosphorylation (Heichal et al., 1979). K;; and Vzj; for uridine transport calculated from initial velocities estimated from 0- to 30-second uptake time courses were not affected by serum stimulation of these cells. These results also explain the finding of Eilarn and Bibi (1977) that

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the number of nitrobenzylthioinosine-binding sites remained relatively constant at 36,00O/cell during serum starvation and stimulation of golden hamster embryo cells, whereas the V,,, for uridine uptake changed 2to 3-fold. In order to explain this discrepancy these investigators suggested that the regulation of uridine uptake is via an alteration of the turnover of the carrier rather than to changes in the number of functional carriers, but it now seems more likely that the observed changes in uptake V,,, reflected alterations in uridine phosphorylating activity. Marked changes in nucleoside and purine uptake also occur during the growth of cells that are not subject to density inhibition either when propagated in monolayer or suspension culture (Plagemann et al., 1969; Plagemann et al., 1975a, 1976; Zylka and Plagemann, 1975). Uptake rates are highest in the early exponential phase of growth and decrease progressively to a minimum in stationary phase. These fluctuations also reflect decreases in the maximum uptake velocity without change in K , , and are also due to alterations in phosphorylating capacity of the cells and not to changes in transport capacity. For example, changes in uridine uptake rates of Novikoff rat hepatoma cells as a function of the age of the culture correlate with similar changes in uridine kinase activity as measured in cell lysates, whereas the uridine transport capacity measured by rapid kinetic techniques in ATP-depleted cells remains relatively constant (Fig. 16). Similar results have been reported for thymidine (Marz et af., 1978). Another convincing example for the correlation between thymidine kinase activity of cells and thymidine uptake rates of cells comes from a comparison of wild-type L cells with a mutant line thereof, C1 139, which possesses a Herpes virus deoxypyrimidine kinase, but no cellular thymidine kinase activity. It was isolated as a thymidine kinasepositive revertant of a thymidine kinase-deficient L cell mutant after infection with ultraviolet light-inactivated Herpes simplex virus type I (Munyon et af., 1971). The regulation of thymidine kinase levels in C1 139 cells differs from that observed in wild-type cells in that the thymidine kinase activity of the cells remains relatively constant throughout the growth cycle (Lin and Munyon, 1974). The thymidine uptake rate of these cells is similarly unrelated to the growth stage of the cells (Plagemann et al., 1976), whereas in wild-type cells thymidine kinase activity and thymidine uptake by whole cells decrease simultaneously to less than 10% of the maximum when cultures approach and enter stationary phase (Lin and Munyon, 1974; Plagemann et af., 1976). Such correlation between thymidine uptake by whole cells and thymidine kinase activity measured in cell lysates has also been observed for lines of untransformed and polyoma virus-transformed hamster cells that differed in thymidine kinase activity (Hare, 1970; Schuster and Hare, 1971). This kind of correlation also pertains to changes in thymidine uptake

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v w

v

TRANSPORT

1'"

--

. E

T I M E IHOURS)

FIG. 16. Effect of culture age on uridine transport, uridine incorporation into total cell material and into acid-insoluble material, and uridine kinase activity. An exponential phase culture of Novikoff cells was diluted with growth medium to about 2.5 x lo5 cellsiml. The culture was incubated at 37°C on a gyrotary shaker and monitored for cell density (O...O). At the indicated times aliquots were taken from the culture and analyzed as follows: ( I ) I x lo8 cells were collected by centrifugation and depleted of ATP at a cell density of 1 x 10' cellsiml. One aliquot was analyzed for the initial rate of uridine transport. Samples of 448 pl of the cell suspension were mixed in rapid succession with 61 pl of [3H]uridine (3.5 cpm/pmole; final concentration = 80 phi'). The mixtures were centrifuged through oil layers and the cell pellets were analyzed for radioactivity. All values were corrected for trapping of substrate in the extracellular H 2 0 space. Initial velocities of uridine transport were estimated graphically from initial approximately linear portions ( 5 - 10 seconds) of the uptake curves (V-V); ( 2 ) I x 10' cells were collected by centrifugation and suspended to 2 x 10' cellsiml of basal medium containing 5 pLM [3H]uridine (80 cpm/pmole). After 5 minutes of incubation at 3 7 T , duplicate I-ml samples were analyzed for radioactivity in total cell and acid-insoluble material (B-B) as described by Plagemann and Roth material (0-0) (1969); ( 3 ) cell sap equivalent to 5 x 10' cellsiml was prepared and analyzed for uridine kinase activity (0-0) as described by Plagemann and Roth (1969) with a uridine concentration of 400 p M . (Data are from Marz et u/., 1978.)

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(Plagemann et al., 1974, 1975a; Adams, 1969a,b; Hopwood et al., 1975; Paterson et al., 1975) and thymidine kinase activity (Littlefield, 1966; Adams, 1969a,b) during the cell cycle. Both increase during the S phase of the cell cycle of synchronized cultures of various types of cells and decrease during G2. As pointed out already, thymidine and deoxycytidine uptake in stimulated lymphocytes also increase during S phase, that is, at the same time that thymidine kinase activity becomes induced in these cells (Barlow, 1976; Barlow and Ord, 1975; Munch-Peterson and Trysted, 1977). In contrast to the results with thymidine, uridine uptake increases continuously through the cell cycle (Sander and Pardee, 1972; Plagemann et a / . , 1975a; Hale et a/., 1975; Stambrook and Sisken, 1972a,b; Stambrook et a/., 1973), although the increases in synchronized Chinese hamster V79 cells during a single cycle seem to be unexplainably high (5- to 10-fold; Stambrook and Sisken, 1972a,b; Stambrook et al., 1973). It seems likely that all these changes in nucleoside uptake rates reflect alteration in the uridine-phosphorylating capacity of the cells, even though in hamster V79 cells the uridine kinase activity seemed to increase only 2- to 3-fold during the cell cycle (Stambrook and Sisken, 1972a,b). So far, no alterations in nucleoside and purine transport capacity during the cell cycle have been reported. The nucleoside and purine uptake capacity of cells also has been shown to decrease markedly during incubation of cultured cells with inhibitors of protein or RNA synthesis (Dybing, 1974a,b,c; Plagemann et a / . , 1975a; Zylka and Plagemann, 1975; Alford and Barnes, 1977). In one recent study, however, it has now been demonstrated that this decrease is not due to a change in transport (Marz et a / . , 1978). The transport of uridine and thymidine in Novikoff cells, as measured by rapid kinetic techniques in ATP-depleted cells, was found to remain unaltered during 6 hours of incubation with 25 pg cycloheximide or 1.25 pg actinomycin D/ml, whereas the long-term uptake rate for these nucleosides decreased 5070% during the same time period. The thymidine kinase activity of the cells decreased similarly. Combined, the growth and inhibitor studies indicate that, contrary to previous conclusions based on uptake studies, the nucleoside and probably the purine carriers turn over only slowly, if at all. Phosphorylation, rather than membrane-associated transport seems to be the potentially important regulatory step in nucleoside uptake. There is also no experimental evidence for other modes of regulation of these carriers, such as by cyclic AMP, suggestions to the contrary from many studies notwithstanding. For example, the increase in uridine uptake in mitogen-stimulated cells correlates with a decrease in intra-

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cellular cyclic AMP levels (Rozengurt and Jiminez de Asua, 1973; Kram and Tomkins, 1973; Kram et al., 1973; Jiminez de Asua and Rozengurt, 1974; Jiminez de Asua et al., 1974; Hare, 1972a,b). And furthermore, the increase in uridine uptake induced by mitogen is inhibited by stimulating cyclic AMP production by treatment of the cells with prostaglandin El, theophylline, or dibutyryl cyclic AMP (Rozengurt and Jiminez de Asua, 1973; Kram et al., 1973). On the other hand, results on the direct effect of cyclic nucleotides on nucleoside and purine uptake are contradictory. In some studies incubation of cells with high concentrations of cyclic AMP or dibutyryl cyclic AMP were found to cause a decrease in rates of uridine or thymidine uptake (Hauschka et al., 1972; Kram et al., 1973; Lingwood and Thomas, 1974) or hypoxanthine uptake (Alford and Barnes, 1977), whereas in another study a n increase in thymidine uptake was observed (Roller et al., 1974). In still another study (Taylor-Papadimitriou et al., 1975) incubation of mouse L cells with cyclic AMP or AMP slightly stimulated uridine uptake, whereas incubation with the cyclic AMP phosphodiesterase inhibitor R020- 1274 lowered uridine uptake concomitantly with an increase in intracellular cyclic AMP concentration. Regardless, these changes represent long-term effects becoming apparent only after hours of incubation with cyclic nucleotides and are certainly secondary in nature. Exposure of several lines of cultured mammalian cells to 1 mM dibutyryl cyclic AMP had no immediate effect on the uptake of uridine and thymidine (Benedetto and Casson, 1974; Sheppard and Plagemann, 1975). Furthermore, changes in intracellular cyclic AMP level induced in these cells by treatment with papaverine, prostaglandin El, or isoproterenol did not correlate with the inhibition of uridine or hypoxanthine uptake caused by some of these substances (Sheppard and Plagemann, 1975). Similarly, isoproterenol-induced increases in intracellular cyclic AMP in frog skeletal muscle had no effect on nucleoside uptake by the tissue (Woo et al., 1974). Recent studies have shown that increased cyclic AMP levels induced by such treatments in CHO cells are without effect on the capacity of the cells to transport uridine, thymidine, adenosine, hypoxanthine, and adenine as measured by rapid kinetic techniques in sublines deficient in the appropriate phosphorylating enzymes (Wohlhueter et al., 1979b). Treatment of mouse lymphoma PI798 with cortisol also caused a decrease in uridine uptake, but again this effect seems to be secondary in nature and not targeted on uridine transport, since long-term uptake rates were measured and a 2- to 3-hour incubation with cortisol was required to effect the inhibition (Stevens et al., 1973). The prolonged treatment of mammalian cells with cytochalasin B has been found to cause a decrease in thymidine uptake (Everhart and Rubin,

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1974: Brownstein et al., 1975; Plagemann et al., 1975b), but to what extent this effect was on thymidine transport or phosphorylation is not known. It has also been reported that the infection of BHK cells with Herpes simplex virus type I or I1 results in increased thymidine uptake (Bittlingmaier et al., 1977a,b), and that infection of BHK or chicken embryo cells with various enveloped viruses results in increases in uptake of uridine, cytidine, and guanosine, but not of adenosine (Hammer et al., 1976). These increases in uptake are certainly due to changes in kinase activities rather than in transport, since they correlated with increases in the corresponding kinase activities in the cells. In contrast, infection of L cells with Newcastle disease viruses resulted in a decrease in thymidine uptake (Hand, 1976). Transformation of cultured animal cells to tumor cells has little, if a n y , effect on nucleoside uptake of the cells. Uridine and thymidine uptake was about the same in cultures of untransformed and murine sarcoma virus-transformed mouse embryo cells (Hatanaka er ul., 1969), only slightly higher in pol yoma virus-transformed than untransformed hamster cells (Foster and Pardee, 1969; Isselbacher, 1972), and only slightly higher in Rous sarcoma virus-transformed than untransformed chicken embryo cells (Bader er al., 1976), or about the same in both types of cells (Hale er al., 1975). Recent studies have shown that the Kf', for uridine transport was about the same (200-500 p M ) for untransformed mouse 3T3 cells and Nil 8 hamster cells and their simian virus 40-transformed counterparts (Koren er al., 1978; Heichal er al., 1979). Initial transport velocities were estimated by linear regression from the first 30-second uridine uptake curves. The V ; ; was about twice as high for transformed as untransformed 3T3 cells, but the opposite was true for the hamster cells. Yang and Visser (1977) found K,s for hypoxanthine and adenine uptake several times higher in Kirsten sarcoma virus-transformed NRK cells (30 and 92 p M , respectively) than in untransformed NRK cells (7 and 22 p M , respectively), whereas the K , for guanine uptake was about the same for both types of cells. Tsan and Berlin (1971) reported that the internalization of 30-50% of the plasma membrane of rabbit alveolar macrophages or polymorphonuclear neutrophils during phagocytosis of latex particles had no effect on the capacity of the cells to take up adenosine or adenine. On the other hand, when the cells were pretreated with colchicine or vinblastin, phagocytosis resulted in a marked decrease in adenine uptake (Ukena and Berlin, 1972). These data were interpreted to indicate that the transport carrier is normally retained in the plasma membrane during phagocytosis, probably because the membrane represents a mosaic of geographically separate regions in which transport or phagocytic sites predominate. The

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effects of vinblastin and colchicine suggested the involvement of microtubules in the maintenance of these regions. These conclusions, however, are valid only in so far as the 45-second time points of uptake determined by these investigators were a true measure of initial transport rates. The same question arises with respect to the study of Pofit and Strauss (1977), who reported that rabbit alveolar macrophages took up adenosine two to three times more rapidly when attached to coverslips than when in suspension. The question was raised whether this difference might be related to a change in cell surface area and microvilli content when the cells attach to an inert surface. In contrast, the V ; ; for uridine transport in ATP-depleted CHO cells was about the same whether transport was measured in suspended cells or after propagation in monolayer culture (Plagemann e t a / . , 1978b). Animal cells in culture are not dependent on an external source of purines or pyrimidines, since the pathways of d e novo synthesis operate fast enough to support maximum cell replication. However, the cells can be made completely dependent on external purines and pyrimidines by blockage of the de noi'o synthetic pathways, as, for example, by treatment with methotrexate. Cells treated in this manner require hypoxanthine, thymidine, and glycine for growth (Hakala and Taylor, 1959). It has been shown for many types of cells that the salvage capacity (transport plus phosphorylation) of cells is sufficiently high to allow maximal cell replication. If hypoxanthine or thymidine are available only in limiting concentrations the replication of methotrexate-treated Novikoff rat hepatoma cells occurs at the maximum rate until most of the substrate in the medium has been utilized and then ceases (Marz et [ I / . , 1977b). Upon limitation of the rate of transport of either hypoxanthine or thymidine by the presence of a transport inhibitor (dipyridamole), on the other hand, the rate of cell replication varies in direct proportion to the rate of substrate transport into the cell. A similar growth rate limitation of methotrexate-treated L5 178Y mouse lymphoma cells has been reported to occur in the presence of p-nitrobenzylthioinosine(Warnick et [ I / . , 1972). These somewhat artificial experimental situations clearly illustrate the potential of regulation of cell replication by a limitation in influx of an essential nutrilite, but whether such limitation may also occur under physiological conditions with respect to nucleosides or purines is uncertain. VII.

PERMEATION OF NUCLEOTIDES

Nucleotides permeate the plasma membrane of most animal cells only very slowly, if at all, although exceptions to this rule seem to exist (see

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below). Limited permeability of nucleotides is explained by the absence of transport systems for most nucleotides and the high negative charge of the nucleotide phosphate groups under physiological conditions, which lowers the solubility of the nucleotides in membrane lipids and thus their nonmediated permeation through membranes [see Eq. (12)]. Cyclic nucleotides seem to be the main exception to normal limited membrane permeability. Davoren and Sutherland (1963) were first to discover that much of the cyclic AMP produced by pigeon erythrocytes in response to treatment with epinephrine is released into the extracellular medium. After 120 minutes of incubation the extracellular amounts of cyclic AMP exceeded the intracellular concentration. Release seemed to occur against a concentration gradient, since the presence of extracellular cyclic AMP had no significant effect on the release of newly formed cyclic AMP. Cyclic AMP release was temperature dependent and inhibited by probenecid. These results were confirmed and extended by King and Mayer (1974). Cyclic AMP release was also found to be inhibited by colchicine, vinblastin, papaverine, and several other compounds. The possibility was suggested that the integrity of microtubules was required for cyclic AMP extrusion, but other investigators have failed to confirm these effects of microtubular agents with other types of cells (Rindler et a / . , 1978). The release of cyclic AMP has also been demonstrated in rat adipose tissue after treatment with the lipolytic hormones, epinephrine and glucagon (Zumstein et a / . , 1974), in cultures of isoproterenol-treated C6 mouse glioma cells (Penit et a / . , 1974; Doore et m l . , 1975; Rindler ef ul., 1978), in untransformed and simian virus 40-transformed human fibroblasts in culture after treatment with prostaglandin El or isoproterenol (Kelly and Butcher, 1974; Chlapowski et ( I / . , 1978; Rindler et a/., 1978; Kelly et ul., 1978), in cultured human sinovial cells after treatment with prostaglandin E l (Closek et [ I / . , 1973, in glucagon or catecholaminetreated perfused rat liver (Park et al., 1972; Kuster et al., 1973), in isoproterenol-treated perfused rat heart (O’Brien and Strange, 1975), and in adrenaline-, histamine-, or isoproterenol-stimulated isolated rat superior cervical ganglia (Cramer and Lindl, 1974). Release from epinephrinestimulated adipose tissue was detectable only in the presence of the phosphodiesterase inhibitor theophylline and reduced by addition of insulin (Zumstein ef ul., 1974). Insulin also counteracted the effect of glucagon and catecholamines on cyclic AMP release in perfused rat liver (Park et u / . , 1972). The infusion of human volunteers with /3-adrenergic agents (isoproterenol, or epinephrine-norepinephrine plus phentolamine) caused a marked increase in plasma cyclic AMP concentration, whereas infusion with a-adrenergic agents (epinephrine or norepinephrine plus propranolol) increased the plasma cyclic GMP level (Ball et al., 1972).

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Similarly, injection of humans with parathyroid hormone caused an increase in cyclic AMP production in the liver and its excretion into urine concomitant with an increase in plasma cyclic AMP levels (Kaminsky et a / . , 19701, and infusion of glucagon had a similar effect on liver cyclic AMP production and plasma cyclic AMP levels (Liljenquist et al., 1974). In all of these studies significant release of cyclic AMP was detectable only after the production of cyclic AMP was stimulated by exposure to appropriate hormones. However, cyclic nucleotide release may also play a significant physiological role in cell replication. In synchronized cultures of Novikoff rat hepatoma cells minimum and maximum intracellular concentrations of cyclic AMP were observed in M and G1, respectively, whereas the opposite was the case for cyclic GMP (Zeilig and Goldberg, 1977). These fluctuations in cyclic nucleotide content during the cell cycle could, in part, be accounted for by their release into the culture fluid. On the other hand, the continuous production and release of cyclic AMP in untransformed and simian virus 40-transformed WI-38 during 48 hours after exposure to 1 pg prostaglandin/ml of culture medium had no effect on the growth of the cells (Chlapowski et al., 1978). The release of cyclic AMP from hormonally stimulated cultured cells and avian erythrocytes has been found to be markedly inhibited by treatment of the cells with a number of inhibitors of energy production (Doore et a / . , 1975; Rindler et a / . , 1978). Decreased release correlated with decreases in intracellular ATP content and occurred in the absence of a significant change in intracellular cyclic AMP level. A similarly energy-dependent process has also been demonstrated to be responsible for the efflux of a tricyclic 7-deazapurine nucleoside monophosphate from mouse L cells (Plagemann and Erbe, 1977). This system exhibits many other properties similar to those described for cyclic AMP release in that efflux was inhibited by extracellular probenecid and papaverine. Efflux was also inhibited by theophylline, dipyridamole, phenethyl alcohol, and p-hydroxymercuribenzoate, but not by cyclic AMP, AMP, or adenosine. Release was saturable and highly temperature dependent ( Q = 3-4; 17-37°C) and seemed to occur against a concentration gradient. Thus, it seems likely that tricyclic 7-deazapurine nucleoside monophosphate functions as a substrate for the cyclic nucleotide release mecha0.14 pmole/pl cell nism. Release, however, was rather slow (V,,, H,O.second, at 500 p M , 37°C) in comparison with the rate of transport of nucleosides and similar to the rate of nonmediated permeation of nucleosides through the membrane. Overall, the results are consistent with the view that the release mechanism represents a unidirectional, ATP-dependent, active efflux transport system. Specific efflux systems for cyclic nucleotides are widespread in nature.

-

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The classical system is that of Dictyostelium discoideurn, for which cyclic AMP plays an important role as a chemotactic agent in differentiation (Loomis, 1975: Gerisch and Malchow, 1976). However, cyclic AMP (Saier et ul., 1975) and cyclic GMP (Shibuya et al., 1976) are also released from bacteria. In fact, the release of cyclic GMP from E. coli is so efficient that the intracellular concentration of cyclic GMP remains below detectable levels. The function of the release mechanism in bacteria and in mammalian cells is uncertain. In mammalian cells a particular hormone response involving cyclic nucleotides is limited by the desensitization of the adenyl cyclase to the hormone, perhaps due to the internalization of cell membrane surface receptors (for reviews, see Goldstein et al., 1979; Catt et al., 1979), but the release of cyclic nucleotides might be a contributing factor in the regulation of the magnitude and duration of the hormone response. Efflux could contribute to lowering the intracellular cyclic AMP levels and would presumably operate in concert with the degradation of cyclic nucleotides by cyclic nucleotide phosphodiesterases. The contribution of each of these potential controlling factors is difficult to assess experimentally, since various inhibitors of the phosphodiesterases also block cyclic nucleotide efflux. However, release of cyclic nucleotides from bacteria and animal cells might also play an informational function in transmitting the generated signal from cell to cell. Further work is required to assess the concentrative effectiveness, substrate specificity, distribution, and other properties of the cyclic nucleotide extrusion system of animal cells. Not all animal cells may possess such a transport system. For example, human HeLa and HEp-2 cells did not release significant amounts of tricyclic 7-deazapurine nucleoside monophosphate, whereas all rodent cells investigated were active (Plagemann and Erbe, 1977). This difference is not related to the species of origin of the cells, since hormone-stimulated human fibroblasts released cyclic AMP (Rindler et al., 1978). In the body, the kidney and liver seem to be primary sites of cyclic AMP metabolism (see above: see also Broadus, 1977). In contrast to these results, Holman (1978) presented evidence for the facilitated diffusion of cyclic AMP in resealed, pink human erythrocyte ghosts. Entry of cyclic AMP was saturable and not concentrative at 30°C with Kq: = 4.4 mM and Vi; = 4300 pmole/pl intravesicular H,O.second. The system seemed asymmetrical since the kinetic parameters for exit transport were much lower (K;; = 0.5 mM, V;t, = 470 pmoleipl intravesicular H,O*second). The authors, however, state some reservations about these kinetic parameters related to the presumed leakiness of these ghost vesicles. Anyhow, this system could not be considered a facilitated

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transport system favoring exit, in spite of the differences in entry and exit kinetic parameters, since V / K was about the same for influx and efflux and at physiological concentrations of cyclic AMP, say 1 p M , therefore, the rate of exit and entry would be about the same (about 1 pmole/pl intravesicular H,O.second). Unidirectionality of the cyclic AMP transport system is supported by many other studies in which a detectable uptake of cyclic AMP by cells could not be demonstrated. Lack of cyclic AMP uptake was first reported for pigeon erythrocytes (Davoren and Sutherland, 1963; King and Mayer, 1974),and later confirmed for various cell culture systems (Schroder and Plagemann, 1971; Kaukel and Hilz, 1972; Hsie et a / . , 1975; Granner et a / ., 1975). Radioactivity from extracellular 3H-labeled cyclic AMP was found to be rapidly accumulated by cells, but mainly in the form of adenine nucleotides. N o significant amounts of labeled cyclic AMP were detected intracellularly and all evidence indicates that the radioactivity from cyclic AMP entered cells in the form of adenosine, which was formed by the sequential extracellular or cell surface action of cyclic AMP phosphodiesterase and 5’-nucleotidase. Hydrolysis of cyclic AMP was found to be mainly due to cyclic phosphodiesterase contributed by the serum present in the cell culture medium (Schroder and Plagemann, 1971; Kaukel and Hilz, 1972), since little radioactivity was incorporated from 3H-labeled cyclic AMP when cells were suspended in a serum-free basal medium (Schroder and Plagemann, 1971). The serum cyclic AMP phosphodiesterase can be inactivated by prolonged heating of the serum at 56°C (Cohen and Plunkett, 1975). On the other hand, evidence has been presented which suggests that cyclic nucleotide phosphodiesterase located on the external cell surface might be involved in the degradation of cyclic AMP in rat skeletal muscle (Woo and Manery, 1973). The possible uptake and metabolism of cyclic AMP in tissues of whole animals, however, is not entirely clear. Levine et a / . (1969) reported that perfusion of isolated rat liver with 14C-labeled cyclic AMP resulted in rapid appearance of intact labeled cyclic AMP in bile and concluded, therefore, that cyclic AMP must be capable of permeation through the hepatic cell membrane. Intravenous injection of rats with 3H-labeled cyclic AMP also resulted in rapid appearance of radioactivity in bile, but the labeled component(s) was not identified (Strange and Percy-Robb, 1975). Gorin and Brenner (1976), however, found 3Hlabeled cyclic AMP to become rapidly degraded after intravenous injection into rats or in perfused liver. Radioactivity accumulated in tissues, but as degradation products of cyclic AMP. Perfusion of rat heart or incubation of ovaries with 3H- or 32P-labeledcyclic AMP also resulted in rapid degradation, with accumulation of radioactive products in tissues,

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but with little accumulation of labeled cyclic AMP itself (Rosberg ef ul., 1975). Tissue accumulation was inhibited by adenosine and the authors concluded that cyclic AMP degradation occurred extracellularly. On the other hand, perfusion of isolated rat kidney resulted in the appearance of intact cyclic AMP in urine, a process which was inhibited by methyl xanthine and probenecid, indicating transtubular transport (Coulson and Bowman, 1974). The evidence that dibutyryl cyclic AMP enters cells intact is more positive, but difficult to demonstrate unequivocally. Entry into the cells is believed to exert its effects on cyclic AMP levels and cyclic AMPdependent cell functions. High extracellular concentrations of dibutyryl cyclic AMP have been reported to cause increases in intracellular cyclic AMP concentration, but such increases are difficult to demonstrate experimentally, since intracellular cyclic AMP concentrations are generally at least two orders of magnitude lower than the extracellular concentrations of dibutyryl cyclic AMP administered and any dibutyryl cyclic AMP remaining associated with the cells after washing will give a positive result in the cyclic AMP assay. Radioactivity from [3H]dibutyryl cyclic AMP was found to accumulate in various types of cultured rat hepatoma and HeLa cells, but only relatively slowly (Kaukel and Hilz, 1972; Kaukel et al., 1972; Granner et al., 1975; van Rijn et al., 1974; Hsie et al., 1975; Plagemann, unpublished data). At best, 1% of the total radioactivity from [3H]dibutyryl cyclic AMP became cell-associated after several hours of incubation at 37°C. Nevertheless, although much of the intracellular radioactivity was found to be associated with adenine nucleotides or purine nucleosides, significant amounts of labeled dibutyryl- or monobutyrylcyclic AMP were recovered in cell extracts. From these studies it has generally been concluded that at least some of the dibutyryl cyclic AMP or of its monobutyryl derivatives enters cells intact and is then degraded intracellularly. Exact quantitation of this process was not possible due to the rapid degradation of the cyclic nucleotide. Degradation of dibutyryl cyclic AMP in cell-free lysates has been demonstrated, but is rather complex involving several pathways (Kaukel and Hilz, 1972; Kaukel et a / . , 1972; O'Neill et al., 1975; Plagemann, unpublished data). The products of [3H]dibutyryl cyclic AMP metabolism either in whole cells or cell lysates include N6- and 02-monobutyryl cyclic AMP, cyclic AMP, AMP, dibutyryl and N6- and 02-monobutyryl AMP, adenosine, inosine, and hypoxanthine. Radioactivity from [3H]dibutyryl cyclic AMP accumulated in rabbit kidney cortex tubules, but again mostly in the form of inosine and hypoxanthine (Boumendil-Podevin and Podevin, 1977). Other nucleotides also seem to enter animal cells slowly or are released slowly by certain cells. For example, Plunkett et al. (1974) and Cohen

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and Plunkett ( 1975) have presented evidence that cytosine arabinoside monophosphate and cyclic cytosine arabinoside monophosphate enter cultured L cells intact. The same seems to be true for 2’,3’-dideoxy AMP (Plunkett and Cohen, 1975). These nucleotides are resistant to deamination and cause the death of cultured tumor cells more effectively than their nucleoside counterparts which are deaminated. The 3Hand 32Pfrom [3H]cytosine arabin~side-[~~P]monophosphate were found to be incorporated at similar rates into the acid-soluble nucleotide pool and into R N A and DNA, and the labeled components in RNA and DNA were identified as cytosine arabinoside nucleotides. As in the case of dibutyryl cyclic AMP, however, uptake of radioactivity was very slow and not substantial, only 0.01% of the exogenous radioactivity of cytosine arabinoside monophosphate became cell associated in 3 hours at 37°C. Evidence has also been presented that ATP is specifically taken up, chemically intact, by liver and kidney (Chaudry et a/., 1976) and by rat soleus muscle (Chaudry and Could, 1970) in v i m , and that ATP is released from exercising human forearm (Forrester, 1972) and rat motor nerve terminals (Silinsky and Hubbard, 1973). In contrast, a significant release of isotopically labeled nucleotides could not be detected in cultured animal cells (Plagemann and Erbe, 1977). The mechanism of slow permeation of nucleotides through the plasma membrane of certain types of animal cells is uncertain. Because of their low lipid solubility the diffusion of nucleotides through the lipid phase of the membrane is probably negligible. Permeation might be via the cyclic AMP transport system, but more likely through aqueous channels. For example, many cultured cells can be rendered artificially and reversibly permeable to nucleotides by incubation with 0.5 mM ATP at pH 7.8-8.4 (Rozengurt et a / . , 1977a; Rozengurt and Heppel, 1979) or in hypertonic solutions (Castellot et a / . , 1978). Untransformed cultured cells do not respond to treatment with ATP (Rozengurt et al., 1977a), but the nucleotide permeability of rat peritoneal mast cells is increased by exposure to ATP concentrations as low as 1-3 p M (Cockcroft and Gomperts, 1979). Extracellular ATP also enhances the ion permeability of cells and causes a n increase in their volume (Stewart et a / . , 1969; Rorive and Kleinzeller, 1972; Trams, 1974; Heppel and Makan, 1977). Recently, it has been shown that a decrease in intracellular ATP of 3T6 and Simian virus 40-transformed 3T3 cells by incubation with various inhibitors of energy production enhances the nucleotide permeability of these cells induced by extracellular ATP (Rozengurt and Heppel, 1979). A decrease in intracellular ATP also enhances the permeability of Chinese hamster ovary cells to colchicine and various other drugs and indirect evidence was presented suggesting that for the maintenance of a low membrane

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permeability a surface glycoprotein must be in a phosphorylated state (See et a / . , 1974; Juliano et a / . , 1976; Carlsen et al., 1976, 1977). These results have led to the hypothesis that the presence and/or the size of aqueous channels in the plasma membrane is regulated by a transmembrane protein which may be either phosphorylated at the inner or outer surface with opposite effects on membrane permeability toward hydrophilic substances: phosphorylation of the protein at the inner surface decreasing permeability, phosphorylation at the outer surface increasing it (Rozengurt and Heppel, 1979). Both effects would be reversed by the action of phosphoprotein phosphatases. It seems likely that this sort of regulation of membrane permeability would occur primarily at the inner surface under physiological conditions, because of the limited availability of ATP in extracellular fluids.

VIII.

SUMMARY AND CONCLUSIONS

The purine and purine nucleoside salvage pathways play an important role in animal physiology, since certain tissues including erythrocytes, leukocytes, bone marrow, and intestinal tract are deficient in the pathway for the de n o w synthesis of purines and thus rely entirely on purines synthesized and released by other body cells (Murray, 1971). The liver seems to be the primary source of preformed purines in the body (Murray, 1971).The salvage pathways consist of the tandem operation of facilitated diffusion (transport) and intracellular phosphorylation, but it is clear now that purine salvage is mainly regulated at the level of phosphorylation rather than of transport. Regulation of phosphorylation involves turnover and resynthesis of the phosphorylating enzymes as well as feedback inhibition of the enzymes or availability of cosubstrates. This is also true for the pyrimidine nucleoside salvage pathways, even though the physiological significance of these pathways is still uncertain. A comprehensive description of the physiological role of nucleoside and base salvage in the whole organism remains an important research goal. Clearly such a description must include a characterization of the transport apparatus present in the cells of solid tissues and of the operational relationship of that apparatus to the appropriate phosphorylating enzymes. That the salvage pathway may play a specific role in certain cells or states of differentiation is suggested by the very widespread distribution of these pathways among both eukaryotic and prokaryotic organisms. Thymidine kinase is of special interest in this connection, since its presence in animal cells is confined to the S phase of the cell cycle. Its activity seems to be lost during G , and M and reappears during

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S or late GI. The significance of this fluctuation is not clear. However, even most animal and prokaryotic DNA virsues carry genes for this and other kinases. In contrast, the nucleoside and purine transport carriers of animal cells seem to exhibit little turnover, are not affected by the growth stage of the cells, nor subject to regulation by substances such as cyclic nucleotides or hormones. The transport of nucleosides and purines is inhibited by an unusual array of unrelated and structurally complex substances, but apparently in a rather nonspecific manner, since the transport of other substrates as well as nonmediated permeation of substances through the membrane also seem to be generally affected. These inhibitors may act by some kind of general interaction with integral membrane proteins, including transport carriers, probably as a consequence of their hydrophobic nature. The Michaelis-Menten constants (0.2-4 mM) and maximum velocities of the nucleoside and purine transport systems of animal cells are at least one order of magnitude higher than those for the intracellular phosphorylation reactions. Thus, at extracellular concentrations that permit the saturation of the intracellular phosphorylation reactions (generally 50 p M or higher), substrate influx greatly exceeds the capacity of the cells to phosphorylate the substrate, which rapidly accumulates intracellularly to a steady-state concentration approaching that in the extracellular fluid. When growing cells are exposed to physiological concentrations of substrate (5 p M and below), on the other hand, the intracellular phosphorylation of substrate in many instances almost keeps pace with substrate influx and the intracellular free substrate attains only a very low steadystate level. Under these conditions the salvage pathway operates with maximum efficiency. However, salvage capacity is very low, even at low extracellular substrate concentration, in nongrowing, quiescent cells, not because of a low transport capacity, but because of low rates of phosphorylation. Whether this low phosphorylating activity is due primarily to a deficiency in phosphorylating enzyme, feedback inhibition, lack of cosubstrate, or altered kinetic properties of the enzymes has been investigated only in a few instances and might vary with the salvage system. Nevertheless, the mitogenic stimulation of quiescent animal cells results in a rapid specific stimulation of the phosphorylation of uridine without affecting the ability of the cells to transport nucleosides. This stimulation, therefore, results in a markedly increased capacity of the cells to salvage uridine from the extracellular fluid. The physiological significance of this stimulation is uncertain. Analogs of nucleosides and free bases generally seem to enter cells via the same carrier transporting their natural counterparts, although often

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PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER

at a lower rate. The salvage uptake of many of these analogs is of interest in cancer chemotherapy-many are used in such capacity and others may have this potential. But here again the conversion of the analogs to phosphorylated intermediates, which are usually the toxic principles, is generally limited at the phosphorylation rather than the transport step. Some analogs, such as %azaguanine, seem to enter solely by a nonsaturable process. That their influx is rapid enough to allow maximum toxicity casts doubt on the notion that the efficacy of toxic purine or nucleoside analogs may be limited by their entry into cells. However, cell mutants have been isolated which are resistant to various nucleoside analogs due to a nucleoside transport defect. The properties of transport carriers for nucleosides and purines in cultured animal cells are consistent with the simplest model imaginable and suggest complete symmetry of function. Results with human erythrocytes, on the other hand, suggest the operation of an asymmetric carrier for nucleosides in that the loaded carrier seems to move more rapidly in either direction than the unloaded carrier and the unloaded carrier moves more rapidly from the extracellular to the intracellular face of the plasma membrane than in the opposite direction. Nevertheless, the studies with both erythrocytes and cultured animal cells have shown that all natural ribo- and deoxyribonucleosides are transported by a single carrier, although possibly with somewhat different efficiency. Hypoxanthine, adenine, and uracil are transported by systems with significantly different properties, but there are several overlapping features with the nucleoside carrier, so that the number of different carriers functioning in the transport of nucleosides and free bases cannot be stated at present. Cytosine seems to enter the cells only by a nonsaturable process. In contrast to the extensive kinetic characterization of nucleoside transport, documented here, little is known about the molecular mechanism of the transport carriers. Purification of carrier proteins, physical and chemical characterization of them, and their reconstitution in artificial membranes is a task of the future. The mutual inhibition observed among nucleosides and purines seems to involve changes in both the effective affinity constant ( K ) and maximum velocity-an observation not yet accounted for in mechanistic terms. The inhibition observed with a wide variety of unrelated, nonspecific inhibitors is also of the mixed type. These results suggest that there is an interdependence between ligand binding and carrier movement (where movement connotes some macromolecular movement, if only a conformational shift). The functional portion of the carrier seems to be deeply imbedded in the membrane, since the removal of surface proteins and carbohydrates by treatment of the cells with various hydrolytic enzymes has no effect on their capacity to trans-

31 3

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port nucleosides, purines, and other substrates. The strong inhibition of nucleoside and purine transport by sulfhydryl reagents indicates that SH groups play a functional role in transport. The strong ( K , = 0.1 nM) and specific binding of p-nitrobenzylthiopurine nucleosides to the nucleoside carrier raises the hope that these substances might be useful in tagging the carrier and thus making practicable its eventual isolation and characterization. Nucleotides, except for cyclic AMP, pass the plasma membrane of animal cells only very slowly or not at all. Low permeability is explained by the absence of nucleotide transport systems in these cells and the low lipid solubility of nucleotides. Some evidence suggests that the permeation of nucleotides, when it occurs, proceeds via aqueous channels in the plasma membrane and that the size or number of such channels might be regulated by a transmembrane protein whose regulatory function in turn is determined by being phosphorylated either on the inner or outer surface of the membrane. On the other hand, most animal cells seem to possess an active transport system for the extrusion of cyclic AMP and possibly other cyclic nucleotides. ACKNOWLEDGMENTS We wish to thank John Erbe and Jill Myers for excellent technical assistance in the reported experimental work and Cheryl Thull for the competent typing of the manuscript. The experimental work was supported by USPHS research grant GM 24468. R.M.W. was supported by USPHS training grant CA 09138.

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inhibition of nucleoside transport by cultured Novikoff rat hepatoma cells. J. Cell Biol. 55, 179-175. Plagemann, P. G. W., and Richey, D. P. (1974). Transport of nucleosides, nucleic acid bases, choline and glucose by animal cells in culture. Biochim. Biophys. Acta 344, 263-305. Plagemann, P. G. W., and Roth, M. F. (1969). Permeation as the rate-limiting step in the phosphorylation of uridine and choline and their incorporation into macromolecules by Novikoff hepatoma cells. Competitive inhibition by phenethyl alcohol, Persantin and adenosine. Biochemistvy 8, 4782-4789. Plagemann, P. G. W., and Sheppard, J. R. (1974). Competitive inhibition of the transport of nucleosides, hypoxanthine, choline and deoxyglucose by theophylline, papaverine and prostaglandins. Biochem. Biophys. Res. Commun. 56, 869-875. Plagemann, P. G. W., Ward, G. A,, Mahy, B. W. J., and Korbecki, M. (1969). Relationship between uridine kinase activity and rate of incorporation of uridine into acid-soluble pool and into RNA during growth cycle of rat hepatoma cells. J. Cell. Physiol. 73, 233-249. Plagemann, P. G. W., Richey, D. P., Zylka, J . M., and Erbe, 3. (1974). Thymidine transport by Novikoff rat hepatoma cells synchronized by double hydroxyurea treatment. Exp. Cell Res. 83, 303-310. Plagemann, P. G. W., Richey, D. P., Zylka, J . M., and Erbe, J. (197Sa). Cell cycle and growth stage-dependent changes in the transport of nucleosides, hypoxanthine, choline, and deoxyglucose in cultured Novikoff rat hepatoma cells. J. Cell Biol. 64, 29-41. Plagemann, P. G. W., Zylka, J . H., Erbe, J., and Estensen, R. D. (197Sb). Membrane effects of cytochalasin B. Competitive inhibition of facilitated difision processes in rat hepatoma cells and other cell lines and effect on formation of functional transport sites. J . Membr. Biol. 23, 77-90. Plagemann, P. G. W., Marz, R., and Erbe, J. (1976). Transport and countertransport of thymidine in ATP depleted and thymidine kinase-deficient Novikoff rat hepatoma and mouse L cells: Evidence for a high K, facilitated diffusion system with wide nucleoside specificity. J . Cell. Physiol. 89, I - 18. Plagemann, P. G. W., Graff, J. C., and Wohlhueter, R. M. (1977). Binding of [3H] cytochalasin B and its relationship to inhibition of hexose transport in Novikoff rat hepatoma cells. J. Biol. Chem. 252, 4191-4201. Plagemann, P. G. W., Marz, R., and Wohlhueter, R. M. (1978a). Transport and metabolism into cultured Novikoff rat hepof deoxycytidine and I-~-~-arabinofuranosylcytosine atoma cells, relationship to phosphorylation, and regulation of triphosphate synthesis. Cancer Res. 38, 978-989. Plagemann, P. G. W., Marz, R., and Wohlhueter, R. M. (l978b). Uridine transport in Novikoff rat hepatoma cells and other cell lines and its relationship to uridine phosphorylation and phosphorolysis. J. Cell. Physiol. 97, 49-72. Plagemann, P. G. W., Wohlhueter, R. M., Graff, J. C., and Marz, R. (1978~).Inhibition of carrier-mediated and non-mediated permeation processes by cytochalasin B. In “Cytochalasins-Biochemical and Cell Biological Aspects” (S. W. Tanenbaum, ed.), pp. 445-473. ElseviedNorth-Holland Biomed. Press, Amsterdam. Plunkett, W., and Cohen, S. S . (1975). Two approaches that increase the activity of analogs of adenine nucleosides in animal cells. Cancer Res. 35, 1547-1554. Plunkett, W., Lapi, L., Ortiz, P. J., and Cohen, S. S. (1974). Penetrations of mouse fibroblasts by the 5‘-phosphate of 9-P-D-arabinofuranosyl adenine and incorporation of the nucleotide into DNA. Proc. Nail. Acad. Sci. U . S . A . 71, 73-77.

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Pofit, J. T., and Strauss, P. R. (1977). Membrane transport by macrophages in suspension and adherent to glass. J . Cell. Physiol. 92, 249-256. Polak, A., and Grenson, M. (1973). Evidence for a common transport system for cytosine, adenine and hypoxanthine in Saccharomyces cerevisiae and Candida albicans. Eur. J . Biochem. 32, 276-282. Quinlan, D. C., and Hochstadt, J. (1974). An altered rate of uridine transport in membrane vesicles isolated from growing and quiescent mouse 3T3 fibroblast cells. Proc. Natl. Acad. Sci. U . S . A . 71, 5000-5003. Quinlan, D. C., and Hochstadt, J. (1976). Group translocation of the ribose moiety of inosine by vesicles of plasma membrane from 3T3 cells transformed by Simian virus 40. J . Biol. Chem. 251, 344-354. Rader, R. L., and Hochstadt, J. (1976). Regulation of purine utilization in bacteria. VII. Involvement of membrane-associated nucleoside phosphorylases in the uptake and the base mediated loss of the ribose moiety of nucleosides by Salmonella typhimurium membrane vesicles. J . Bacteriol. 128, 290-301. Rau, J., and Scholtissek, C. (1970). Inhibitoren der Nucleosidphosphorylierung in vivo. Z . Naturforsch. Teil B 25, 292-299. Rindler, M. J., Bashor, M. M., Spitzer, N., and Saier, M. H. (1978). Regulation of adenosine 3’ : 5’-monophosphate efflux from animal cells. J . Biol. Chem. 253, 543 1-5436. Rogler-Brown, T., Agarwal, R. P., and Parks, R. E., Jr. (1978). Tight binding inhibitors. VI. Interactions of deoxycoformycin and adenosine deaminase in intact human erythrocytes and sarcoma 180 cells. Biochem. Pharmacol. 27, 2289-2296. Roller, B., Hirai, K., and Defendi, V. (1974). Effect of CAMP on nucleoside metabolism. I. Effect on thymidine transport and incorporation in monkey cells (CV-I). J . Cell. Physiol. 83, 163- 176. Roos, H., and Pfleger, K. (1972). Kinetics of adenosine uptake by erythrocytes, and the influence of dipyridamole. Mol. Pharmacol. 8, 417-425. Rorive, G., and Kleinzeller, A. (1972). The effect of ATP and Ca++ on the cell volume in isolated kidney tubules. Biochim. Biophys. Acta 274, 226-239. Rosberg, S., Selstam, G., and Isaksson, 0. (1975). Characterization of the metabolism of exogenous cyclic AMP by perfused rat heart and incubated prepubertal ovary. Acta Physiol. Scand. 94, 522-535. Roy-Burman, S., and Visser, D. W. (1975). Transport of purines and deoxyadenosine in Escherichia coli. J . Biol. Chem. 250, 9270-9275. Rozenberg, M. C., Ledwidge, C. M., Wilcken, D. E . L., and McKeon, M. (1971). The inhibition of adenosine phosphorylation in platelets by dipyridamole. J . Lab. Clin. Invest. 77, 88-96. Rozengurt, E., and Heppel, L. A. (1979). Reciprocal control of membrane permeability of transformed cultures of mouse L cell lines by external and internal pH. J . Biol. Chem. 254, 708-714. Rozengurt, E., and Jiminez de Asua, L. (1973). Role of cyclic 3’:5’-adenosine monophosphate in the early transport changes induced by serum and insulin in quiescent fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 70, 3609-3612. Rozengurt, E., and Stein, W. D. (1977). Regulation of uridine uptake by serum and insulin in density-inhibited 3T3 cells. Biochim. Biophys. Acta 464, 417-432. Rozengurt, E., Heppel, L. A., and Friedburg, I. (1977a). Effect of exogenous ATP on the permeability properties of transformed cultures of mouse cell lines. J . Biol. Chem. 252, 4584-4590. Rozengurt, E., Stein, W. D., and Wigglesworth, N. M. (1977b). Uptake of nucleosides in density-inhibited cultures of 3T3 cells. Nature (London) 267, 442-444.

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CURRENT TOPICS I N MEMBRANES A N D TRANSPORT, VOLUME

14

Transmembrane Transport of Small Peptides D. M. MATTHEWS Department of Experimental Chemical Pathology The Vincent Square Laboratories of Westminster Hospitul London, England AND

J . W . PAYNE Depurtment of Botany, Science Laboratories University of Durham Durham, England

I. General Introduction . . . . . . . . . . . . . . . . . 11. Peptide Transport in Animal Small Intestine . . . . . . . . . . A. Introductory Considerations . . . . . . . . . . . . . B. Intralumen and Brush Border Hydrolysis of Proteins and Peptides . . C. Active Uptake of Small Peptides by the Absorptive Cells . . . . . D. Effects of Na+ Replacement on Intestinal Uptake of Small Peptides . E . Effects of Peptides on Intestinal Transport of Naf and Water . . . F. Effects of Peptides on Electrical Potential Difference across the Small Intestine . . . . . . . . . . . . . . . . . G. Influence of Molecular Structure on Uptake and Hydrolysis of Peptides by the Small Intestine . . . . . . . . . . . . . . . H . Maximum Size of Peptide Taken up by the Absorptive Cells . . . . I. Independence of Mucosal Uptake of Peptides and Amino Acids . . . J . Competition for Mucosal Uptake between Peptides . . . . . . K. The Possibility of Multiple Peptide Uptake Systems in the Small Intestine . . . . . . . . . . . . . . . . . L. Relative Rates of Absorption of Peptides and the Equivalent Free Amino Acids . . . . . . . . . . . . . . . . . . M. Kinetics of Intestinal Absorption of Peptides . . . . . . . . N. Sites of Maximal Absorption of Peptides and Amino Acids along the Length of the Small Intestine . . . . . . . . . . . . . 0. Effects of Dietary Alterations and Small Intestinal Disease on Absorption of Peptides and Amino Acids . . . . . . . . . . . . . P. Mucosal Uptake of Peptides and Amino Acids in Developing Animals . Q. Intracellular Hydrolysis of Small Peptides . . . . . . . . .

Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN U 12- I533 14-X

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V.

VI. VII.

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R. Possible Mechanisms of Peptide Absorption . . . . . . . . . S. Quantitative Importance of Mucosal Peptide Uptake in Protein Absorption . . . . . . . . . . . . . . . . T. Entry of Amino Acids and Peptides into the Blood . . . . . . . U. Absorption of Biologically Active Peptides . . . . . . . . . Peptide Transport in Animal Tissues Other Than the Small Intestine . . . Peptide Transport in Microorganisms . . . . . . . . . . . . A. Introductory Considerations . . . . . . . . . . . . . B. General Features of Nutritional Utilization of Peptides by Microorganisms . . . . . . . . . . . . . . . . C. Scope of the Topic: Transport in Bacteria, Yeasts andOtherFungi, Algae, and Lichens . . . . . . . . . . . . . . . . . . D. Nature of the Microbial Cell Surface and Membrane Location of Peptide Permeases . . . . . . . . . . . . . . . . E. Defining Characteristics of Peptide Transport . . . . . . . . F. Methods for Studying Peptide Transport: Applicability and Limitations G. Peptide Transport in Bacteria . . . . . . . . . . . . . H. Peptide Transport in Other Microorganisms . . . . . . . . . I . Related Topics . . . . . . . . . . . . . . . . . Peptide Transport in Higher Plants . . . . . . . . . . . . . A. Introductory Considerations . . . . . . . . . . . . . B. Foliar Absorption of Peptides by SarraceniaJlava . . . . . . . C. Peptide Transport by the Scutellum of Germinating Barley . . . . Possible Physiological Advantages of Transmembrane Transport of Small Peptides . . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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GENERAL INTRODUCTION

The concept of transport of small peptides into the absorptive cells of the small intestine was implicit in Cohnheim’s recognition at the turn of the century that “erepsin” was essentially an intracellular enzyme (Matthews, 1977a), and the first satisfactory evidence for transport of intact dipeptides across the intestinal wall using small intestine was obtained more than 25 years ago (Agar er al., 1953). Since that time ample evidence has been obtained for active transport of di- and tripeptides into the absorptive cells of the small intestine on a substantial scale, and there is also enough information to leave no doubt that mediated transmembrane transport of small peptides occurs elsewhere in the animal body. In addition, it has been known for many years that mediated transmembrane transport of small peptides occurs in bacteria, and it has now been shown to occur in other types of microorganism. Finally, transmembrane transport of small peptides was probably shown as long ago as 1964 in a

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carnivorous plant and has recently been shown in two separate laboratories in another higher plant-germinating barley-so that the process appears to be a phenomenon of very wide biological distribution, though there are examples of organisms which apparently do not take up intact peptides. This review will deal with transmembrane transport of small peptides in animals, in microorganisms, and in higher plants. The mechanisms by which proteins or very large peptides such as insulin cross epithelial barriers are excluded from the scope of the present article, there being no reasonable doubt that they are fundamentally different from the mechanisms largely responsible for transmembrane transport of small peptides of no more than a few amino acid residues. Certain general topics such as discussion of the possible “reasons” why transmembrane transport of small peptides occurs in addition to that of free amino acids, and its possible physiological advantages in relation to the transport of free amino acids, will be dealt with at the end of the article (Section VI). Throughout the article, standard three-letter abbreviations for amino acids and amino acid residues will be used. In the case of D-amino acids and D-amino acid residues, the appropriate prefix will be used. In the case of L-amino acids and L-amino acid residues, which are referred to much more frequently, the prefix L- will be omitted.

I!. PEPTIDE TRANSPORT IN ANIMAL SMALL INTESTINE

A. Introductory Considerations

This topic has been reviewed fairly recently in very full detail (Matthews, 1975a,b; Matthews and Adibi, 1976), so that in the present article, no attempt will be made to refer to every publication in the area. References will be mainly to key papers, and special emphasis will be placed on points that are still debatable. For readers who are not already familiar with current views on the absorption of protein digestion products in animals, it should be said at the outset that though it is now generally believed that di- and tripeptides enter the absorptive cells of the animal small intestine, in addition to free amino acids, it is not the current view that peptides enter the portal blood on any substantial scale, as sometimes claimed in the past (see Matthews, 1975a,b, 1977a). The current orthodoxy is that with a small number of exceptions (Section 11, T) small peptides taken up by the absorptive cells undergo complete hydrolysis within these cells, entering the portal blood as free amino acids. Yet some workers in the field, and the author of this section (D.M.M.) is among them, are still not satisfied that the question of the extent of entry

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of peptides into the portal blood is satisfactorily resolved. This is a remarkable situation after a series of investigations spanning about a century.

6. lntralumen and Brush Border Hydrolysis of Proteins and Peptides

The intralumen phase of protein digestion is accomplished by the gastrins (Taylor, 1968) and the proteolytic enzymes of the pancreas (Keller, 1968). The action of the pancreatic proteases is of predominant importance; in the absence of gastric digestion, neither protein digestion nor absorption is noticeably impaired. Following the intralumen phase of digestion, there is partial hydrolysis of many small peptides by the aminooligopeptidases of the brush border (microvillous region) of the small intestinal mucosa. [Digestion by pancreatic proteases adsorbed to the brush border may also continue in this region (Peters et al., 1972).] A number of analyses have been made of the protein-derived nitrogenous material in the lumen of the small intestine 15 minutes to 3 hours after protein meals in man, dog, and the rat (see Matthews, 1975a,b), and it has been found to be a complex mixture containing peptides and free amino acids in which, according to most investigators, peptides predominate. There are few estimates of the mean chain length of the peptide fraction. Chen et al. (1962) estimated that 1 hour after feeding casein or zein to rats, the mean chain length in the peptide fraction was about three amino acid residues, but after gelatin about six residues. Adibi and Mercer (1973), who fed 50 gm of bovine serum albumin to man, referred to evidence suggesting that the peptide fraction is composed largely of small peptides of two to four amino acid residues. More investigation is required in this area. It is believed that the final stage of peptide hydrolysis, prior to further hydrolysis within the absorptive cells, is a function of the peptidases of the brush border of the absorptive cells, which are believed to be capable of hydrolysis of peptides of up to about 10 amino acid residues. This hydrolysis proceeds sequentially from the NH,-terminal end of the peptide, i .e., the enzymes responsible are amino-oligopeptidases. These brush border peptidases (Peters, 1975; Matthews, 1975b; Gray and Santiago, 1977; Kania et d . , 1977; Noren et d . , 1977) are distinct from those of the cytosol of the absorptive cells. They are more active against some small peptides than others, and appear to be more active against tripeptides than dipeptides. Most of the peptidase activity of the absorptive cells against tripeptides is located in the brush border region, whereas

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most of the activity against dipeptides is located in the cytosol. Several groups have purified brush border peptidases from the intestine of the hog, the rat, and the rabbit. For example, Kim and his colleagues (Kim and Brophy , 1976; Kim, 1977) have purified two amino-oligopeptidases from the intestinal brush border of the rat; they are both glycoproteins, very similar in many respects, with an apparent molecular weight of 280,000. Gray and colleagues (Wojnarowska and Gray, 1975; Gray and Santiago, 1977; Kania et al., 1977) have also purified two peptidases from the brush border of rat small intestinal mucosa. These enzymes are not identical with those described by Kim's group, but like them they are glycoproteins of high molecular weight. The major enzyme found (oligopeptidase 11) showed broad specificity for peptides containing neutral or basic amino acid residues. Both affinity and rate of hydrolysis were greatly enhanced when the substrate consisted of a peptide containing an amino acid residue with a lipophilic side-chain (such as Leu) at the NH2terminus; affinity was also increased by the presence of a third or a fourth amino acid residue. Peptidase activity against dipeptides of proline has been reported to be absent from the intestinal brush border of the rat and of man (Fujita et al., 1972; Kim et ul., 1972). C. Active Uptake of Small Peptides by the Absorptive Cells Following uptake by the absorptive cells of the small intestine, most small peptides are so rapidly hydrolyzed by the peptidases of the cytosol of these cells that peptide uptake by small intestine in v i m is represented only by an increase in the constituent amino acids of the peptide taken up (Matthews, 1975a,b). Consequently, though it was shown by Newey and Smyth (1962) and by Cheng et al. (1971) that intestinal peptide uptake was apparently inhibited by anoxia and by metabolic inhibitors there was difficulty in providing more positive evidence that mucosal uptake of peptides was in fact an active process, i.e., that it could take place against an electrochemical gradient. This difficulty was overcome after Payne (1972a) had pointed out that the dipeptide Gly-Sar, which is exceptionally resistant to hydrolysis (and may be regarded as Gly-Gly with a methyl group in place of the H normally attached to the N of the peptide bond), was accumulated intact by bacteria. This led Addison et al. (1972) to study uptake of Gly-Sar by rings of everted hamster small intestine in v i m ; these authors showed that during an incubation period of 20 minutes this dipeptide could be accumulated intact, apparently against an electrochemical gradient, by the tissue. Its accumulation was greatly inhibited by anoxia, cyanide, and 2,4-dinitrophenol (DNP), and also by replacement of medium Na' (Section 11, D). (In spite of the unusual resistance

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of Gly-Sar to hydrolysis by the intestinal peptidases, some free Gly and free Sar did appear in the tissue, in addition to the unhydrolyzed peptide.) Following this demonstration, it was shown that the tripeptide Gly-SarSar was handled similarly by hamster small intestine, apparently undergoing active uptake (Addison et al., 1975a), but that uptake of the tetrapeptide Gly-Sar-Sar-Sar was negligibly small, and did not appear to be the result of a mediated process. Gly-Sar and Gly-Sar-Sar are not peptides commonly derived from dietary protein; however, p-Ala-His (carnosine) frequently occurs in a carnivorous or omnivorous diet, being found as such in meat. Evidence for transport against an apparent electrochemical gradient has also been provided for this dipeptide, which is even more resistant to hydrolysis by hamster small intestine than Gly-Sar (Matthews et al., 1974). It may be noted that it has not yet been proved that any of the peptides which are accumulated intact by hamster small intestine are present in the tissue in the free form, though there is no particular reason to suppose that they are not. Nor has the possibility that their accumulation is the result of adsorption at the surface of the tissue been formally excluded, though the ability of the peptides under discussion to undergo transmural transport by sacs of everted hamster small intestine (Matthews et al., 1974; Addison et al., 1975a) makes such a possibility unlikely. A second hydrolysis-resistant tripeptide, P-Ala-Gly-Gly, was taken up intact by hamster small intestine in vifro (Addison et a l . , 1975a). Its uptake was greatly reduced by anoxia, metabolic inhibitors, and replacement of medium Na'; however, its concentration in the whole tissue could not be shown to exceed the concentration in the incubation medium, though its concentration within the absorptive cells may well have done so. This tripeptide is probably also actively transported, but its transport may be unusually poor because of an exceptionally low affinity for the uptake mechanism. The fact that many di- and tripeptides which are rapidly hydrolyzed by the cytosol peptidases of the absorptive cells, and cannot be found intact in small intestinal tissue incubated with them in vitro, apparently compete with Gly-Sar and Gly-Sar-Sar for mucosal uptake (Matthews, 1975a,b) suggests that these peptides also share the same active uptake mechanism. D. Effects of Na+ Replacement on Intestinal Uptake of Small Peptides

Rubino et al. (1971) investigated the effect of replacement of medium Na+ by choline on influx of Gly from Gly-Pro into rabbit ileal mucosa in vitro. This influx is almost certainly the result of uptake of intact Gly-

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Pro. They found, working at low substrate concentrations at which a high-affinity influx mechanism (Section 11, M) was predominant, that the effect of Na' replacement was to reduce influx, decreasing V,,, without altering K t . This contrasted with the effect of Na+ replacement on influx of free Gly, which was to cause an increase in K t without change in V,,,. An appreciable influx of Gly from Gly-Pro, not attributable to simple diffusion, remained after Na' replacement. Na+ replacement by Tris or by K+ abolished the ability of hamster jejunum t o concentrate Gly-Sar and Gly-Sar-Sar in vitro (Addison er al., 1972, 1975a), and Na' replacement abolished transmural transport of Tyr-Gly and Tyr-D-Ala across rat small intestine in vitro (though it had no effect on transmural transport of these peptides across the rectal wall (Heading et al., 1978). On the other hand, the small number of experiments so far carried out in vivo have not shown peptide uptake by the small intestinal mucosa to be Na+ dependent under the experimental conditions used. At a high concentration (267 mM) in the rat, absorption of Gly-Gly from the lumen of the jejunum was not significantly reduced by replacement of intralumen Na+ by mannitol (Matthews et al., 1969). This did not seem incompatible with the apparent Na+ dependence of peptide uptake in vitro, since mucosal uptake of neutral amino acids, which is generally agreed to be Na+ dependent, is little affected by replacement of lumen Na+ at high substrate concentrations. However, Cheeseman and Parsons (1974) reported that uptake of Gly-Leu (10 mM) by the small intestine of the frog Rana pipiens in vivo was unaffected by replacement of intralumen Na+ by K+, whereas uptake of Gly and Leu from the equivalent mixture of free amino acids was inhibited. The suggestion has been made (Cheeseman and Parsons, 1976) that mucosal peptide uptake may be Na+ dependent only when it is concentrative; when transport is proceeding down an electrochemical gradient (as it probably was in the experiments of Cheeseman and Parsons with GlyLeu, since this peptide is probably very rapidly hydrolyzed by the intracellular peptidases of the absorptive cells) and it is possible that no metabolic energy is necessary for uptake, possibly there is no requirement for Na+ in transport. Against this, it must be remembered that the work of Rubino er al. (1971), being carried out under conditions of influx, almost certainly involved only "downhill" transport into the absorptive cells-yet Na+ dependence was observed. The need for further investigation in this area will be obvious. The possible complexity of the situation is indicated by the observation by Cheeseman and Parsons (1976) that exit from the absorptive cells of Gly and Leu taken up from the intestinal lumen as intact Gly-Leu is reduced by removal of Na+ from the vascular perfusate, and by a subsequent communication by Cheese-

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man (1977), who reported that uptake of P-Ala-His by rings of everted frog small intestine in vitvo was not concentrative and was unaffected by Naf replacement; nevertheless, its steady-state transfer in vivo was reduced by Na+ replacement. Cheeseman suggested that Na+ might have a role in the transport of peptide across the intestinal epithelium, but at a later stage than that of uptake.

E. Effects of Peptides on Intestinal Transport of Na+ and Water

Hellier et al. (1973), studying the absorption of Gly-Gly, Gly-Ala, and the equivalent free amino acids by jejunal perfusion in man, showed that the dipeptides, like the amino acids, stimulated absorption of Na' and water. Silk et al. (1975a) reported the effects of Gly-Ala and its constituent amino acids on water and electrolyte (NaCl) absorption over a wide range of concentrations of peptide and amino acids, using a similar technique. On the assumption that solute and water are absorbed as an isoosmotic solution (as infused) a constant relationship was found between water absorption and total solute absorption in the case of the free amino acids. On the same assumption, a similar constant relationship was found between water absorption and total solute absorption in the case of the peptide, but only if it were postulated that at the higher peptide concentrations (40, 80, and 140 mM) nearly all the peptide was taken up intact by the absorptive cells, exerting its stimulating effect on absorption of electrolytes and water before hydrolysis t o free amino acids took place. In a later report from the same group (Fairclough et al., 1977a), however, it was concluded that analyses of the relation of net solute and water absorption could not be used t o yield reliable information about the form in which peptides enter the absorptive cells. This change in outlook was because they found that though maltose is generally agreed to be hydrolyzed before absorption, maltose and glucose gave results analogous to those obtained with the peptide Gly-Ala and its constituent amino acids-not different results as expected. By altering the osmolality of glucosehaline solution perfused through human jejunum they showed that the assumption that the absorbate was always isotonic with plasma was not correct; it was found that when the lumen fluid was hypertonic to plasma, so was the absorbate. It was concluded that the previous results with the dipeptide need not be interpreted as showing that at higher concentrations this was almost entirely transported intact. More likely, it underwent some brush border hydrolysis in addition to uptake of intact peptide, this hydrolysis leading to the absorption of a hypertonic solution.

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F. Effects of Peptides on Electrical Potential Difference across the Small Intestine Kohn et a / . (1968) first reported that di- and tripeptides (Gly-Gly, GlyAla, and Gly-Gly-Gly), like free amino acids, increased the transmural potential difference in rat small intestine in vitvo. Caspary (1973) reported that this increase was greater for Gly-Gly than for the equivalent Gly. At 20 minutes from the start of the experiment, the potential difference across rat intestine was almost twice as great with Gly-Gly (15 mM) as with the equivalent free Gly (30 mM). G. Influence of Molecular Structure on Uptake and Hydrolysis of Peptides by the Small Intestine As explained in Section 11, I, intestinal mucosal uptake of peptides and uptake of free amino acids are independent processes. In most investigations it has been found that uptake of amino acids does not influence that of peptides; there is also much additional evidence for the independence of amino acid and peptide uptake. No systematic investigation of the structural requirements for peptide transport by the small intestine has yet been made. Most of the observations which can be recorded are the results of studies of uptake by rat or hamster small intestine in vitvo. 1. AMINO-TERMINAL GROUP Substitution of this group reduces or abolishes affinity for transport. Methylation of the NH,-terminal group, in Sar-Gly, resulted in poor uptake and slow hydrolysis, intact peptide being found in the intracellular fluid of the intestinal tissue (Burston et a / . , 1972). Sar-Gly also failed to cause inhibition of uptake of Gly-Sar-Sar (Addison et al., 1975b). Analogous conversion of a primary to a secondary amino group, in Pro-Gly, was associated with failure to inhibit uptake of Gly-Pro (Rubino et ul., 1971). However, Pro-Gly did inhibit uptake of Gly-Sar-Sar, though less strongly than Gly-Pro (Addison et ul., 1975b), and Pro-Gly inhibited uptake of Gly-Leu (Das and Radhakrishnan, 1975). Acetylation of the NH,-terminal group, in N-acetyl-Gly-Gly, resulted in failure to inhibit uptake of Gly-Pro (Rubino et al., 1971) and uptake of p-Ala-His (Addison et a / . , 1974), though N-acetyl-Gly-Gly did cause weak inhibition of uptake of Gly-Sar-Sar (Addison et a / ., 1975b). N-Benzyloxycarbonyl-Gly-Leu inhibited uptake of Gly-Leu, but only weakly (Das and Radhakrishnan, 1975). Substitution of the NH,-terminal group as in N,N-di-( 1-deoxy-2-

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ketosy1)-Gly-Leu (fructosyl-Gly-Leu), a product of the Maillard reaction of Gly-Leu with glucose, produced a peptide which was apparently unabsorbed by rats in vivo, in addition to being resistant to enzymic hydrolysis by leucine aminopeptidase (Amaya-F. et a / ., 1976).The unabsorbability of Maillard peptides and amino acids may be of nutritional significance.

2. CARBOXYL-TERMINAL GROUP Amidation of this group greatly reduces affinity for peptide transport. In Gly-GlyNH,, it resulted in failure to inhibit uptake of P-Ala-His and Gly-Sar-Sar (Addison et a/., 1974, 1975b), though Gly-Gly-GlyNH, did cause weak inhibition of uptake of Gly-Sar-Sar (Addison et a / . , 1975b). Pro-Leu-GlyNH,, in which both terminal groups are substituted, did not inhibit uptake of Gly-Sar-Sar (Addison et a/., 1975b). Asp-PheOMe (a peptide sweetening agent) did inhibit uptake of Gly-Sar-Sar, but less strongly so than Asp-Phe (Addison et a / . , 1975b).

3. PEPTIDEBOND(S) The presence of a peptide bond or bonds makes peptides unacceptable to the amino acid transport systems of the small intestine. Elongation of the peptide backbone by insertion of an additional carbon atom between the NH,-terminal group and the peptide bond, in P-Ala-His, did not prevent active transport but led to slow hydrolysis (Matthews et a / ., 1974). Uptake of p-Ala-His was inhibited by “ordinary” dipeptides made up of a-amino acids (Addison et a/., 1974), and P-Ala-His, like “ordinary” dipeptides, inhibited uptake of Gly-Sar-Sar (Addison et al., 1975b). However, P-Asp-Gly was poorly taken up and did not inhibit uptake of Gly-Sar-Sar, besides being slowly hydrolyzed (Addison et a / ., 1975b). Uptake of P-Ala-Gly-Gly was poor, though there was evidence suggesting that it was actively transported into the absorptive cells (Section 11, C ) and its hydrolysis was slow. It inhibited uptake of Gly-Sar-Sar (Addison et a/., 1975b), and its uptake was inhibited by Gly-Gly, Gly-Gly-Gly, and Gly-Sar-Sar (Addison et a/., 1974, 1975a). A peptide bond involving a p-amino group, which elongates the peptide backbone by insertion of an additional carbon atom between the COOH-terminal group and the peptide bond did not, in His-P-Ala, prevent this peptide from inhibiting uptake of Gly-Sar-Sar (Addison et a/., 1975b), and Gly-p-Ala was a weak inhibitor of uptake of Gly-Leu (Das and Radhakrishnan, 1975). A ylinkage, placing two additional carbon atoms between the NH,-terminal group and the peptide bond, apparently leads to both poor transport and slow hydrolysis. y-Glu-Glu was poorly taken up and slowly hydrolyzed

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(Burston et af., 1972). Gly-y-aminobutyric acid inhibited uptake of GlyLeu, but only weakly (Das and Radhakrishnan, 1975). y-Glu-Cys-Gly (glutathione) was poorly transported, failed to inhibit uptake of Gly-SarSar, and was slowly hydrolyzed (Evered and Wass, 1970; Addison et al., I975b). Methylation of the nitrogen of peptide bonds, in Gly-Sar and Gly-SarSar, was compatible with active transport but led to slow hydrolysis (Addison et af., 1972, 1975a). Gly-Sar-Sar-Sar, however, was very poorly transported, as well as very slowly hydrolyzed (Addison et al., 1975a). Substitution of the peptide bond as in Gly-Pro is compatible with carriermediated uptake (Rubino et al., 1971; Addison et af., 1975b; Das and Radhakrishnan, 1975), though it leads to slow hydrolysis (Addison et af., 1974). In Pro-Hyp, not only is the peptide bond substituted, but the terminal amino group is represented by an imino group. This peptide was a weak inhibitor of uptake of p-Ala-His by hamster jejunum (Addison et al., 1974); however, it did not appear to be actively transported by guinea pig small intestine, and was also very slowly hydrolyzed (Hueckel and Rogers, 1972). The question of the maximum size of peptide taken up by the absorptive cells of the small intestinal mucosa is discussed in Section 11, H . Both di- and tripeptides are taken up, though tripeptides probably to a lesser extent than dipeptides, owing to their greater susceptibility to brush border hydrolysis.

SPECIFICITY 4. STEREOCHEMICAL Peptide transport in the small intestine is stereochemically specific, like that of free amino acids (Wiseman, 1974); peptides made up of Lamino acid residues are favored by the peptide transport system(s), though peptides containing or made up of D-amino acid residues are probably not entirely without affinity for peptide transport. Peptides containing D-amino acid residues are poorly transported and slowly hydrolyzed. Burston et al. (1972) first showed the stereochemical specificity of intestinal peptide uptake, observing that a dipeptide containing a D-amino acid residue, Gly-D-Val, was very poorly taken up by rings of everted rat ileum in vitvo. They also noted that the intact peptide as well as its constituent amino acids appeared in the intracellular fluid (ICF) of the intestinal tissue, an indication of unusually slow hydrolysis. Cheeseman and Smyth (1973) reported that D-Leu-Gly was taken up by rat small intestine in vitro with very little hydrolysis. Its uptake, though relatively slow, appeared to be the result of a carrier-mediated process, since it was saturable and inhibited by Leu-Ala. Pro, Met, p-Ala, and D-glucose

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had no effect on uptake of D-Leu-Gly. The observation that Gly-D-Leu, like Gly-Leu, inhibits uptake of Gly-Sar-Sar, though less strongly (Addison et NI., 1975b), provides further evidence that dipeptides containing D-amino acid residues have some affinity for the intestinal peptide carrier system(s) utilized by peptides made up of L-amino acid residues. However, Das and Radhakrishnan (1975) reported that Gly-D-Leu did not cause significant inhibition of Gly-Leu uptake by monkey small intestine in tiitvo even at a molar ratio of 20: 1. The most systematic investigation of the intestinal absorption and hydrolysis of dipeptides containing D-amino acid residues was carried out by Asatoor et al. (1973), who studied absorption from the lumen of tied loops of rat small intestine in vivo and hydrolysis by mucosal homogenates. An example of their findings is that Ala-Phe was absorbed from the jejunum at about 200 times the rate of D-Ala-D-Phe, and Leu-Leu at about 24 times the rate of D-Leu-D-Leu. D-L and L-D isomers were absorbed at rates intermediate between those of L-L and D-D isomers. A positive correlation was found between the rates of absorption of the dipeptides studied and their rates of hydrolysis by homogenates of jejunal mucosa. A point of particular interest was that absorption of dipeptides containing D-amino acid residues (unlike that of ordinary peptides, Section 11, N) was much more rapid (2-20 times) from the ileum than from the jejunum. The ileum also showed a greater capacity to hydrolyze such dipeptides than the jejunum. In the case of Gly and several D-amino acids and dipeptides containing D-amino acid residues, an inverse correlation was found between the rate of jejunal absorption of the substrate and its molecular weight, and it was suggested that simple diffusion played an important part in absorption of these compounds from the jejunum. N o such correlation was found in the ileum. As far as the authors know, no investigation of the effect of incorporation of D-amino acid residues into tripeptides on their intestinal absorption has yet been made. It is probable that it would be similar to the effect on the absorption of dipeptides. 5. AMINO ACID SIDE-CHAINS The influence of the structure of the amino acid side-chains on peptide transport, and that of their charge, have not yet been adequately investigated. It is probable that with peptides, as with amino acids, lipophilic side-chains increase apparent affinity for uptake (Rubino e f nl., 1971; Caspary, 1973; Adibi and Soleimanpour, 1974; Das and Radhakrishnan, 1975).The possible influence of charge will be returned to in a subsequent Section (11, K) when discussing the question of whether or not there are multiple peptide uptake systems in the small intestine.

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H. Maximum Size of Peptide Taken up by the Absorptive Cells

It is probable that dipeptides are taken up by the absorptive cells of the small intestine to a large extent, tripeptides to a lesser extent, and tetrapeptides only to a small extent, possibly hardly at all. Note the deliberate vagueness of the wording-the present inability to quantitate the proportions of protein-derived nitrogen taken up by the intestinal mucosa as free amino acids and as various sizes of peptide is one of the most unsatisfactory features of the study of peptide absorption at the time of writing. Not long ago, the author of this section (D.M.M.) might have written that it was probable that there was no significant mediated uptake of tetra- or higher peptides, but a recent report (see following) has provided evidence for uptake by the absorptive cells of at least one tetrapeptide-and it must be remembered that there are some 160,000 possible protein-derived tetrapeptides (taking the number of amino acids derived from protein to be 20, that is 204 = 160,000). The particular tetrapeptide studied in the report quoted is therefore hardly likely to be the only tetrapeptide which might be taken up intact. Early work on the maximum size of peptide taken up by the absorptive cells has already been summarized in detail (see Matthews, 1975a,b), and this summary will not be repeated here in full. Briefly, the conclusion was reached that Gly-Gly and Gly-Gly-Gly could be taken up intact by rat small intestine, but that Gly-Gly-Gly-Gly and higher peptides of Gly up to hexaglycine were not taken up intact, undergoing brush border hydrolysis to Gly-Gly-Gly, Gly-Gly, and free Gly before uptake by the absorptive cells (Matthews et NI., 1968; Peters et al., 1972). The work with the series Gly-Sar, Gly-Sar-Sar, and Gly-Sar-Sar-Sar, already referred to (Section 11, C), showed that in contrast to the di- and tripeptide, the tetrapeptide was not actively taken up by hamster jejunum. This supported the idea that di- and tripeptides, but not higher peptides, entered the absorptive cells-though the possibility exists that this series of sarcosyl peptides is not representative, since the conformation of these peptides might be limited by the methylation of their peptide bonds. More recently, three very thorough investigations of the maximum size of peptides taken up by the absorptive cells have been reported. Adibi and Morse (1977) studied the absorption of tetraglycine, pentaglycine, and hexaglycine from human jejunum. They concluded that whereas diglycine and triglycine were taken up intact by the absorptive cells, there was no evidence for uptake of intact tetraglycine or the two higher peptides. The disappearance of tetraglycine from the jejunum appeared to be principally the result of hydrolysis by brush border oligopeptidases with uptake of the products of hydrolysis, and the rate-limiting step in

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the uptake of free and peptide-bound glycine from tetraglycine and the higher peptides was hydrolysis of these peptides to absorbable productse.g., triglycine, diglycine, and free glycine. The work in man was backed up by an investigation of peptide uptake by rings of everted rat small intestine in i ! i t r o . Intact diglycine and triglycine appeared in the rings, but no intact tetraglycine was found. Smithson and Gray (1977) investigated intestinal handling of Gly-Leu-Gly-Gly by rat jejunum in t,itro and in viw. They concluded that this tetrapeptide was completely hydrolyzed by the peptidases of the brush border, and did not utilize the mechanisms(s) for uptake of intact peptides. However, unexpected and extremely interesting results were obtained by Chung et ul. (1979), who studied absorption of Leu-Gly-Gly-Gly from the jejunum of the rat in tjiiv. They concluded that though this peptide underwent some brush border hydrolysis, a large proportion, about 50%, was taken up as intact tetrapeptide. The most important piece of evidence for this conclusion was that a high concentration of Ala, which completely inhibited uptake of Leu from a mixture of free amino acids, inhibited uptake of Leu from Leu-Gly-Gly-Gly by only about 50%. Even more remarkable was the observation that though Leu-Gly, Leu-Gly-Gly, and Gly-Pro appeared to share a common transport system, Gly-Pro did not inhibit uptake of LeuGly-Gly-Gly-suggesting that the system for uptake of the tetrapeptide was independent of the system(s) for uptake of di- and tripeptides. In the scutellum of germinating barley (Section V, C), in yeast (Section IV, H), and in Escherichici coli (Section IV, G) dipeptides and larger oligopeptides appear to share the same uptake system, though in E. coli there is also an uptake system specific for dipeptides. Recently, Burston, Taylor, and Matthews ( 1979) studied intestinal handling of Leu-Gly-Gly-Gly and Ala-Gly-Gly-Gly by hamster and rat jejunum in i,itro. Unlike Chung et ul. (1979), they could find no evidence for uptake of intact tetrapeptides. I. Independence of Mucosal Uptake of Peptides and Amino Acids

For some years there has been general agreement that mucosal uptake of peptides and uptake of free amino acids are independent processes, and there is now so much evidence pointing to this conclusion that it is hardly disputable. It is true that amino acids can inhibit total uptake of amino acids from some peptides-but this inhibition is probably largely the result of inhibition of uptake of free amino acids released by partial brush border hydrolysis of the peptides-not of uptake of intact peptides by the mucosal cells. To obtain clear-cut results in experiments on com-

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petition for mucosal uptake between amino acids and peptides, it is desirable to use peptides which undergo little or no brush border hydrolysis. Rubino et a / . (1971) showed that influx of Gly-Pro into rabbit ileum in vitro was inhibited by several other di- and tripeptides, but either not inhibited, or only slightly inhibited, by free amino acids. Furthermore, Gly-Pro did not affect influx of free Gly or free Pro. Using the hydrolysisresistant peptides Gly-Sar and Gly-Sar-Sar, it was shown that uptake of these peptides by hamster jejunum in vitro was inhibited by other diand tripeptides but not by free amino acids (Addison et al., 1972, 1975b). Comparable results were obtained with the same preparation using the hydrolysis-resistant dipeptide p-Ala-His (Addison et al., 1974). Das and Radhakrishnan (1974) reported that influx of Gly-Leu into strips of monkey small intestine in vitro was unaffected by free amino acids but was inhibited by several dipeptides, and Sigrist-Nelson found the same result using vesicles from the brush-border membrane of rat small intestine (Wacker and Semenza, 1977). Adibi and his colleagues (Adibi and Soleimanpour, 1974; Adibi et al., 1975) have demonstrated the independence of amino acid and peptide uptake by jejunal perfusion in man using similar principles, and so has Cook (1974a). Another method of demonstrating the independence of peptide and amino acid uptake is to saturate mediated transport of amino acid(s) and show that peptides can still be taken up. Thus Adibi (1971) blocked uptake of free Leu from the human jejunum with a high concentration of Ile; under these conditions large quantities of peptide-bound Leu could still be taken up from Gly-Leu. Cheeseman and Parsons (1974) saturated the uptake mechanism(s) of the small intestine of R . pipiens for free Gly and free Leu, and found that addition of Gly-Leu produced a large increase in absorption of both Gly and Leu. Crampton et a / . (1973) showed an analogous phenomenon with Met and Met-Met in the rat, and Cook (1973) did so with Gly and Gly-Gly usingjejunal perfusion in man. Another very important line of evidence showing the independence of mucosal uptake of peptides and amino acids comes from investigations of the genetic amino acid transport defects of Hartnup disease and cystinuria. These have been fully described elsewhere (Matthews, 1975a,b), and will not be recapitulated here at length. Briefly, in Hartnup disease there is a congenital defect of intestinal and renal uptake of free neutral amino acids. In 1969 it was first shown by Milne and colleagues that this defect did not apply to mucosal uptake of peptides containing or composed of the affected amino acids; even a dipeptide, Phe-Phe, composed entirely of an affected amino acid was relatively well absorbed, whereas absorption of free Phe was very poor (Navab and Asatoor, 1970; Asatoor

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et a / . , 1970; Tarlow et a / . , 1972; Leonard et a/., 1976). Findings analogous to those in Hartnup disease have been obtained in cystinuria, in which there is an absorptive defect for lysine, arginine, ornithine, and cystine; as in Hartnup disease, peptides containing the affected amino acids, including a peptide composed entirely of an affected amino acid, LysLys, were relatively well absorbed (Hellier et a/., 1970, 1972; Asatoor et a/., 1971, 1972a; Silk et a / . , 1975~).Presumably retention of the ability of the small intestine to take up peptides containing the affected amino acids is largely responsible for maintaining protein nutrition in patients with the absorptive defects of Hartnup disease and cystinuria. Further evidence supporting the concept of the independence of mucosal uptake of amino acids and peptides comes from the observations that in the rat and hamster the sites of maximal uptake of peptide and amino acids along the length of the small intestine are not the same (Section 11, N), that the effects of dietary alterations on mucosal uptake of peptides and amino acids are different (Section 11, 0),that the patterns of development of amino acid and peptide uptake in the fetal and neonatal rabbit are distinct (Section 11, P), and that the kinetic responses to Na' replacement of amino acid uptake and of peptide uptake have been reported to be different (Section 11, D). A report by Radhakrishnan (1977) does suggest that some inhibitory effect of amino acids on the uptake of peptides is demonstrable in monkey small intestine.

J. Competition for Mucosal Uptake between Peptides

A large number of experiments have now been carried out on competition for mucosal uptake between various di- and tripeptides. Most of these have been described in detail by Matthews (1975b), and will not be described again at length in the present article. Some key references are as follows: Rubino et a / . (1971); Addison et a / . (1974, 1975b); Das and Radhakrishnan (1974, 1975). In several cases, the kinetics of the inhibitory effects have been shown to be competitive (Rubino et a / . , 1971; Adibi and Soleimanpour, 1974; Das and Radhakrishnan, 1975; Sleisenger et al., 1976). The impression given by the work is that a very large number of di- and tripeptides share the same mucosal uptake system or systems. The possibility that there are in fact multiple peptide uptake systems in the small intestine, as there are for amino acids, will be discussed in the next Section (11, K).

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K. The Possibility of Multiple Peptide Uptake Systems in the Small Intestine The surveys of competition for mucosal uptake between peptides referred to in the previous Section (11, J) showed that so many di- and tripeptides were inhibitors of uptake of each other that it seemed quite possible that there was only one peptide uptake system in mammalian small intestine, and Das and Radhakrishnan (1975) stated their support for this hypothesis. Yet over the last few years, evidence has accumulated which makes it difficult to maintain such a hypothesis with complete confidence. Edwards (1970), in an abstract, was the first to suggest that there might be more than one mucosal peptide uptake system in animal small intestine; he suggested that two carriers might exist, utilized by leucyl and palanyl dipeptides, respectively. The work of Rubino et al. (1971) (Section 11, M) on kinetics of influx of Gly-Pro suggested that there might be more than one uptake system for this dipeptide. Addison et al. (1974) observed that whereas uptake of p-Ala-His by hamster jejunum was inhibited by equimolar ( 5 mM) concentrations of several peptides of neutral amino acids, it was not inhibited by equimolar Glu-Glu or Lys-Lys-nor did equimolar Glu-Glu or Lys-Lys significantly inhibit uptake of each other. These authors made the tentative suggestion, probably unwisely in view of the very tenuous evidence, that as with amino acids, acidic and basic peptides might be taken up by separate systems from those for neutral peptides. Lane ef al. (1975) studied absorption of Gly-Pro and Pro-Gly did not from the jejunum of the rat, and found that Pro-Gly (40 d) inhibit absorption of Gly-Pro (10 mM); however, Gly-Pro (40 mM) did produce a 40% inhibition of absorption of Pro-Gly (10 mM). Taking into consideration the observation of Rubino et al. (1971) that Pro-Gly did not inhibit influx of Gly-Pro, as well as their own evidence, these authors suggested that Gly-Pro and Pro-Gly might be taken up largely by separate systems-though they might also share the same system to some extent. On the other hand, Addison et al. (1975b) showed that both Pro-Gly and Gly-Pro were inhibitors of uptake of Gly-Sar-Sar by hamster jejunum in vitvo, suggesting a common system for uptake of all these peptides. Another piece of evidence bearing on the question of the existence of multiple peptide uptake systems came from a study of the effect of a high concentration of Gly-Gly (100 mM) on absorption from human jejunum of a partial enzymic hydrolysate of casein consisting of about 50% oligopeptides and 50% amino acids (Fairclough et al., 1977b). Gly-Gly caused significant inhibition of uptake of only two or three amino acids (Ser and Glu/Gln) from the hydrolysate; in other words the inhibitory

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effects of Gly-Gly on peptide uptake from the mixture of peptides in the hydrolysate were so unexpectedly weak that the authors were compelled to consider the possibility that some of these peptides might be taken up by a system or systems not available to Gly-Gly; however, since this work was carried out it has become apparent that Gly-Gly is an exceptionally weak inhibitor of peptide uptake not only in man and animal intestine but in both microorganisms (Section IV) and in germinating barley (Section V, C). Gupta and Edwards (1976), as the result of an investigation of absorption from proximal small intestine of the rat in vivo, and taking into account Edwards’ previous work (Edwards, 1970), have claimed that at least three dipeptide uptake systems are identifiable, representative members of the different peptide transport groups being p-Ala-His, Leu-Gly , and Pro-Hyp. For example, they found that Leu-Gly and p-Ala-His had no inhibitory effect on uptake of Pro-Hyp at 10 times equimolar concentration (inhibitor 100 mM, substrate 10 mM), while uptake of Pro-Hyp was inhibited by other prolyl peptides, Pro-Gly and Pro-Leu. In view of the inhibition of uptake by hamster jejunum of p-Ala-His by Pro-Hyp (Addison e? al., 1974) and several other di- and tripeptides of ordinary structure, the inhibition of uptake by monkey jejunum of Gly-Leu by Pro-Gly (Das and Radhakrishnan, 1975), and the inhibition of uptake by hamster jejunum of Gly-Sar-Sar by Pro-Gly (Addison et al., 1975b), it appears that the separate peptide systems described by Gupta and Edwards may not always be as clearly distinguishable as the work of these authors would suggest. For a considerable time, work has been going on in the laboratory of one of us (D.M.M.) on the apparently simple problem of whether GlySar and Glu-Glu are taken up by hamster jejunum in vitro by the same carrier or carriers, or whether one of the peptides is taken up in part by a carrier unavailable to the other. Many early experiments on this problem have been described by Burston e? al. (1977). More recent experiments have now been carried out using more refined techniques (Matthews e? al., 1979). Like the earlier experiments, they have been done at pH 5 , to minimize brush border hydrolysis of Glu-Glu, and they have been carried out under conditions of influx, using 14C-labeledpeptides. The principle has been to study influx of one of the peptides in the presence of a range of concentrations of the other, and to extrapolate the effect of the inhibitor peptide on uptake of the inhibited peptide to infinitely high concentrations of inhibitor (this extrapolation may be carried out as in Sleisenger et al., 1976) using a plot described by Preston e? al. (1974), or some similar plot. It has been found that infinitely high concentrations of Glu-Glu can completely inhibit mediated uptake of Gly-

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Sar, and that infinitely high concentrations of Gly-Sar can completely inhibit mediated uptake of Glu-Glu; the inhibitory effects appear t o be competitive. Thus it seems that neither Gly-Sar nor Glu-Glu is taken up by a system unavailable to the other peptide; they may well be taken up by a single system, though the possibility that they share more than one uptake system has not been excluded. Similar findings have now been obtained in our laboratory with Gly-Sar and Lys-Lys. This suggests that neutral, acidic, and basic peptides all share the same uptake system(s). Of course, the question of the possible existence of multiple peptide uptake systems in the small intestine is not confined to the possibility that there may be more than one dipeptide uptake system. In E . coli (Section IV, H) it has been shown that separate dipeptide and oligopeptide uptake systems are identifiable. By analogy, the gut might contain separate systems for dipeptides and for higher peptides. An approach to this problem was made by Sleisenger et af. (1976) who studied influx of the dipeptide Gly-Sar and the tripeptide Gly-Sar-Sar into hamster small intestine in vitro. No evidence was obtained that either peptide was taken up by a system unavailable to the other; the results showed clearly that both peptides shared the same uptake system or systems. The surprising report of Chung et al. (1979) that mucosal uptake of a tetrapeptide was independent of that of di- and tripeptides has already been referred to (Section 11, H). To summarize, it does seem possible that there are multiple peptide uptake systems in mammalian small intestine-but to characterize these adequately is likely t o require many more years of work. The problem of the possible existence of multiple peptide uptake systems is certainly not one which can be solved by a small number of simple experiments.

L. Relative Rates of Absorption of Peptides and the Equivalent Free Amino Acids The reports in 1968 from independent laboratories in Great Britain (Craft et al., 1968; Matthews et a[., 1968) and the United States (Adibi and Phillips, 1968) that di- and tripeptides could be absorbed more rapidly than the equivalent free amino acids in both the human subject and in the rat aroused surprise and even incredulity at the time. Now that it is generally recognized that peptide uptake by the intestinal mucosa is distinct from amino acid uptake and has different kinetic characteristics (Section 11, M) the findings no longer seem particularly surprising, and it should be unnecessary to labor the point that peptides are frequently

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found to be absorbed more rapidly than the equivalent amino acids (solutions of peptides and amino acids are said to be equivalent when the peptide solution would, on complete hydrolysis, yield the amino acid solution). The phenomenon of more rapid absorption of peptides than the equivalent amino acids has been reviewed in great detail by Matthews (1975b); this article refers to 22 papers describing the phenomenon in man, rat, hamster, guinea pig, rabbit, ferret, and frog, including omnivorous, herbivorous, and carnivorous animals. Representative examples of these papers, in addition to those quoted at the beginning of this section, include Adibi (1971), Cheng et a / . (1971), Lis et a / . (1971), Burston et a / . (1972), Cook (1972), Smirnova and Ugolev (1972), Crampton et a / . (1973), Silk et a / . (1973a), Cheeseman and Parsons (1974), and Adibi et a / . (1975). An example of the phenomenon is illustrated in Fig. 1, which is based on data obtained by Matthews et a / . (1969) in the rat in vivo. When an equimolar mixture of Gly and Met is absorbed, absorption of Gly is inhibited by Met, since these amino acids share a common carrier for mucosal uptake for which Met has the higher affinity. When, on the other hand, the equivalent Gly-Met is absorbed, competition between the constituent amino acids is almost entirely avoided, and total absorption of Gly and Met is considerably greater than from the equivalent mixture of amino acids. The fact that absorption of Gly from the peptide is slightly less than that of Met is almost certainly due to the fact that this peptide does undergo some brush border hydrolysis, so that total uptake, though largely the result of peptide transport, is augmented to some extent by uptake of free amino acids released in the brush border-where of course they compete for uptake. It is important to notice that the superior absorption of the peptide is not simply the result of avoidance of the competition for transport occurring between the amino acids in the equivalent amino acid mixture-for absorption of Met from the peptide is substantially greater than that of equimolar Met when present in the intestinal lumen on its own. More rapid absorption of amino acids from peptides than from the equivalent free amino acids is not invariably found. In some experiments, the two solutions are absorbed at approximately equal rates. In most instances, the phenomenon becomes increasingly prominent as substrate concentrations are raised. On the other hand, examples have been described of peptides (a-Glu-Glu, Lys-Lys, p-Ala-His) which are absorbed more rapidly than the equivalent amino acids only at low concentrations and not at higher ones (Burston et a/., 1972; Matthews et a / . , 1974). For example, p-Ala-His was taken up more rapidly by hamster jejunum in vitt-o than the equivalent amino acids at 1 mM but at about the same rate

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5

4

3 Q

+ ul

n

U c

C

' 2

E U

1

Met

G ~ Y

A

Gly + M e t

Gly - M e t

B

C

FIG.1 . Rates of absorption of Gly and Met from (A) Gly alone, Met alone; (B)equimolar mixture of Gly and Met; (C) equivalent dipeptide Gly-Met. (Based on data of Matthews et a / . , 1969; reproduced from Matthews, 1977a.)

at 5 and 20 m M (Matthews et al., 1974). A few examples have also been reported of peptides being absorbed more slowly than the equivalent amino acids (Matthews, 1975b). It has also been found that partial hydrolysates of proteins, consisting largely of oligopeptides with a proportion of free amino acids, are absorbed more rapidly than the equivalent mixtures of free amino acids, and that amino acids that are absorbed particularly slowly from the free amino acids, including lysine and the dicarboxylic amino acids, are absorbed relatively rapidly from the partial hydrolysates (Section 11, S).

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M. Kinetics of Intestinal Absorption of Peptides Not many investigations have yet been made of the kinetics of mucosal uptake of peptides, but in cases in which they have been carried out, it has been found that mediated uptake conforms t o Michaelis- Menten kinetics. Early work on absorption of peptides of Gly from rat jejunum in vivo (Matthews et a / . , 1968) suggested that Gly-Gly and Gly-Gly-Gly had a lower apparent affinity for uptake than Gly, and a higher V,,,, and that faster absorption of Met from Gly-Met than from equimolar Met was the than free Met (Matthews et al., result of Gly-Met having a higher V,, 1969). An investigation of 5-minute uptake of Met and Met-Met from rings of everted rat ileum (Cheng et al., 1971) showed that both these processes were saturable, having rather similar K t values. Rubino et al. (1971) studied influx of ['4C]Gly(Gly-Pro) into rabbit ileal mucosa in vifro, and plotted influx against influx/concentration (the Hofstee plot). The plot was obviously biphasic, and could be interpreted as the result of two saturable mechanisms, each conforming t o MichaelisMenten kinetics. One mechanism had a high apparent affinity for GlyPro and a low maximal transport velocity ( K , 0.93 mM, V,,, 0.01 pmole min-' cm-2) while the other had a low apparent affinity and a high maximal velocity ( K , 57 mM, V,,, 0.07 pmole min-' cm-'). Adibi and Soleimanpour (1974) investigated the kinetics of absorption of Gly-Gly, Gly-Leu, and the constituent amino acids from human jejunum, with the following results: K t Gly, 43 m M , Leu 20 mM, Gly-Gly 43 mM, Gly-Leu 27 mM; V,, Gly, 40 pmole cm-' min-', Leu, 40, GlyGly, 55, Gly-Leu, 41. It was suggested that as with neutral amino acids, the presence of a lipophilic side-chain in a dipeptide might increase affinity for transport, and the experiments of Das and Radhakrishnan (1975) on competition for uptake between dipeptides support this view. Adibi and Soleimanpour further suggested that peptide transport might be characterized by higher V,, values than those for free amino acids. Matthews et al. (1974) investigated the kinetics of influx of p-Ala-His 2.7 pmole gm into hamster jejunum in vitro. K , was 9.4 mM and V,, wet wt-' min-'. Nutzenadel and Scriver (1976) estimated kinetic constants for the same peptide with rat intestine in vitro, obtaining a very similar K t (10.2 mM). V,, was 17.7 pmole ml intracellular fluid-' 15 min-' (say about 0.8 pmole gm wet wt-' min-', assuming a n I C F of 70% of total wet wt). The lower V,, is compatible with the observation that rat small intestine in vitro takes up amino acids and peptides t o a lesser extent, on a weight basis, than that of the hamster (Matthews, 1977a) but it is almost certainly also lowered by the experiments not being done under conditions of influx.

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Das and Radhakrishnan (1975) studied the kinetics of influx of ['4ClGly(Gly-Leu) and ['4C]Leu(Gly-Leu) into monkey (Macaca radiata) jejunum in t'itro. For [14C]Gly(Gly-Leu)K t was 4.0 mM and Vmax was 0.5 pmole gm-' min-'; for ['4C]Leu(Gly-Leu) K t was 4.0 mM but V,,, was 1.4 pmole gm-' min-'. Influx into human jejunal mucosa was also estimated under similar conditions. For ['4C]Gly(Gly-Leu) K t was 2.5 mM and V,, was 1.4 pmole gm-' min-', and for ['4C]Leu(Gly-Leu) K t was 2.5 mM and V,,, was 2.2 pmole gm-' min-'. The higher V,,, values for ['4ClLeu(Gly-Leu) than for ['4C]Gly(Gly-Leu) were associated with a higher uptake of Leu than of Gly. This higher uptake of Leu may have been the result of an element of brush border hydrolysis accompanied by uptake of free Leu, but the reasons for it are not entirely clear (see Matthews, 1975b, Section X, A). Recently, Radhakrishnan (1977) has reported an additional mode of uptake of Gly-Leu in monkey small intestine with a K t of about 30 mM, and has also reported the existence of two saturable uptake systems for Gly-Gly with K t values of 5 and 30 mM, in addition to significant uptake, at high concentrations, by simple diffusion. The kinetics of influx of Gly-Sar and Gly-Sar-Sar into rings of everted hamster jejunum in v i m were investigated by Sleisenger et af. (1976), who obtained the following values: Gly-Sar, K t 7.6 mM, V,,, 2.7 pmole gm-' min-'; Gly-Sar-Sar, K t 12.6 mM, V,,, 1.9 pmole gm-' min-'. Thus the tripeptide had a lower affinity than the dipeptide for what appeared to be a common uptake mechanism and a slightly lower V,,,. In this study, the possible effect of the unstirred layer on observed K t values was taken into account, and it was estimated that true K t values might be some 1-2 m M lower than the uncorrected values reported above. Reanalysis of the data of this article, using a more sophisticated technique (Matthews et al., 1979), showed that a small element of simple diffusion in uptake of the two peptides had been overlooked, and that when this was allowed for, the kinetic constants became slightly lower than those originally published; thus revised values (uncorrected for the unstirred layer) for Gly-Sar were K t 5.3 mM and V,,, 1.8 pmole gm-' min-'. Unfortunately many of the figures given are not usefully comparable, having been obtained under a variety of experimental conditions with different animal species. N o systematic study of the kinetics of mucosal uptake of a range of different di- and tripeptides has yet been undertaken.

N. Sites of Maximal Absorption of Peptides and Amino Acids along the Length of the Small Intestine

In the rat and hamster at least, the site of maximal absorption of most peptides seems to be more proximal than that of most free amino acids.

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Lis et al. (1972a) observed that in the rat in vivo, the site of maximal absorption of a pancreatic hydrolysate of casein consisting mainly of oligopeptides with a mean chain length of two to three amino acid residues was in the proximal third of the small intestine, whereas that of the equivalent amino acid mixture was in the distal third. An investigation of the absorption of Met, Met-Met, Gly, and Gly-Gly in the rat in vivo (Crampton et al., 1973) showed that absorption of Met-Met was maximal in the proximal half of the small intestine, whereas absorption of Met was maximal in the distal half. The sites of maximal absorption of GlyGly and of Gly were similar, in the proximal half of the small intestine near its mid-point; however, in the two most proximal sites absorption of Gly fell off markedly, whereas that of Gly-Gly was not much less than at the site of maximal absorption. Lane et al. (1975) obtained different results with Gly-Pro, Pro-Gly, and the equivalent mixture of Gly and Pro in the rat in vivo; the rates of absorption of both peptides were similar in the proximal and in the distal thirds of the small intestine, as were the rates of absorption of the amino acid mixture. In both sites, absorption of Gly-Pro (10 mM) was more rapid than that of the amino acids, whereas that of Pro-Gly (10 mM) was less rapid. However, according to Heading et al. (1977) both free Gly and Gly-Pro were absorbed more rapidly in the jejunum of the rat in vivo than in the ileum. Rubino and Guandalini (1977) have reported results in the small intestine of the newborn rabbit in vitro which are similar to the findings in the rat by Lis et a / . (1972a) and Crampton et al. (1973): they found that uptake of Gly and Phe was maximal in the ileum while uptake of Gly-Pro and Gly-Phe was maximal in the jejunum. Recently, Schedl et a / . (1979a) investigated the kinetics of influx of Gly, Leu, and Gly-Sar into rings of everted proximal and distal hamster small intestine over the concentration range 0.1- 100 mM. At all concentrations studied, influx of the peptide was more rapid in the proximal intestine than in the distal intestine, and influx of the amino acids more rapid in the distal small intestine than in the proximal small intestine. Das and Radhakrishnan (1974) studied the kinetics of influx of Gly-Leu and of free Leu into monkey small intestine at four sites along the length of the intestine. From the K t and V,,, values obtained, they concluded that at low concentrations maximal uptake of both the peptide and the amino acid would occur in a site about one-third of the length of the small intestine measured distally from the pylorus. In man, also, results have been different from those obtained in the rat. Adibi (1971) found that in man absorption of Gly-Gly and Gly-Leu was greatest in the jejunum, and so was that of the constituent amino acids. Silk et a / . (1974a) found that absorption of Gly-Ala was approximately equal in

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jejunum and ileum, whereas absorption of Gly and Ala was greater in the jejunum than in the ileum. The results available at the time of writing suggest not only that more systematic and extensive investigations are required, but that there may be species differences in the sites of maximal absorption of amino acids along the length of the small intestine: probably in man the site of maximal absorption of amino acids is more proximal than in the rat or hamster. It is noteworthy that in several investigations in man (Adibi, 1971; Silk ef d., 1974aj and in the rat (Crampton ef a / . , 1973; Lane ef N/., 1975: Silk ef al., 1976) lumen appearance of free amino acids during peptide absorption has been greater in the ileum, suggesting either greater brush border hydrolysis of peptides in the ileum than the jejunum, or greater back-diffusion of amino acids from an intracellular site of peptide hydrolysis, or both. 0. Effects of Dietary Alterations and Small Intestinal Disease on Absorption of Peptides and Amino Acids

The effects of dietary alterations on the absorption of peptides and on the absorption of free amino acids are not the same. Lis et a / . (1972b) studied absorption of Met and Met-Met from the small intestine of the rat in v i t v . Absorption of Met was increased by short-term (10 days) restriction of dietary intake, and by short-term feeding of a high-protein or high-Met diet, but these alterations did not affect absorption of MetMet. Long-term (41 days) feeding of an almost protein-free diet reduced absorption of Met to half the control value, but had no effect on absorption of Met-Met. A difference was also found in the effect of long-term protein deprivation in the rat on absorption of a partial hydrolysate of casein consisting largely of oligopeptides and the equivalent amino acid mixture (Lis et a / . , 1972a). There was a decrease in absorption of the amino acid mixture in the middle and distal parts of the small intestine, but no change in absorption of the partial hydrolysate in any part of the small intestine. Cook (1974b, 1977) reported that in man acute bacterial infections increased absorption of Gly and His from the jejunum, possibly as the result of decreased dietary intake, but did not alter absorption of GlyGly . Fogel et ( I / . (1975) reported than in man protein-calorie malnutrition reduced jejunal absorption of Leu without affecting that of Gly-Leu. In celiac sprue, dipeptide absorption has been reported to be less severely affected than that of free amino acids (Silk et a / . , 1974b; Adibi et a / . , 1974).

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Recently, Schedl et al. (1979b) studied the effect of reduced dietary intake and starvation on the kinetics of uptake of Leu and Gly-Sar by hamster jejunum and ileum in vim.The main effect was a reduction in V,,, for both substrates, compatible with a reduction in the number of mediated transport sites for the amino acid and the peptide. In general, peptide absorption appears to be more resistant to adverse conditions than that of free amino acids. Besides supporting the concept of the independence of mucosal uptake of peptide and amino acids, the changes which have been outlined suggest that the effects of dietary alterations on amino acid absorption are on the uptake mechanisms for amino acids rather than on the mechanisms by which they leave the absorptive cells. If the effects were on the exit mechanisms for amino acids, amino acid and peptide absorption would be expected to be affected similarly. Asatoor et al. (1972b) reported that pyridoxine deficiency in the rat depressed absorption of amino acids to about half normal values, but that absorption of dipeptides was depressed to a smaller extent.

P. Mucosal Uptake of Peptides and Amino Acids in Developing Animals

Rubino and Guandalini ( 1977) have reported some remarkably interesting results from experiments on peptide and amino acid influx into the jejunum of developing rabbits. Uptake was studied from the twenty-fifth day of gestation (about a week before birth) until 50 days of postnatal age. Throughout this period, uptake of Gly showed no significant change. Uptake of Gly-Pro, however, showed a striking perinatal peak, at the apex of which uptake of Gly-Pro was about 10 times as rapid as that of free Gly. Jejunal V,,, for Gly-Pro was about 13 times greater in the newborn animal than in the adult. In the adult animal, Gly-Pro was still taken up somewhat more rapidly than free Gly, though the difference was much smaller than in the newborn. A perinatal peak was also found in the uptake of Gly-Phe; about the time of birth influx of this peptide was substantially more rapid than that of free Phe. Uptake of Phe, on the other hand, was most rapid in the fetus, less rapid in the newborn, and least rapid in the adult. The differences in the developmental pattern for peptide and amino acid uptake provide yet more evidence for the independence of mucosal uptake of these substrates (Section 11, I). The reason for the extraordinary rapidity of uptake of Gly-Pro in relation to that of Gly in the newborn animal remains a matter for conjecture. The

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authors comment on the possible relevance of their observations to the preparation of artificial diets for premature and newborn infants. Zeman and Fratzke (1977) studied the effects of maternal protein deprivation in newborn rats. Absorption of casein, amino acids, and intact Gly-Leu was all reduced in these animals. However, there was an increase in retention of intact Gly-Leu by the absorptive cells. This was attributed to reduced Gly-Leu dipeptidase activity associated with the prenatal protein deprivation.

Q. lntracellular Hydrolysis of Small Peptides Information concerning the intracellular hydrolysis of small peptides will be found in Lindberg et al. (1975), Peters (1979, Matthews (1975b), Radhakrishnan (1977), Kim (1977), and Josefsson et a f . (1977). It has been estimated that about 90% of the peptidase activity of the small intestinal mucosa against dipeptides is located in the cytosol of the absorptive cells. The cytosol also contains activity against tripeptides, though less than that in the brush border. It is probable that cytosol peptidase activity is largely (Kim et al., 1974) though perhaps not exclusively (Chung et al., 1979) confined to di- and tripeptides. As in the brush border, peptides are hydrolyzed in the cytosol from the NH,-terminal end. Most di- and tripeptides can probably be hydrolyzed by both the brush border peptidases and those of the cytosol, though in man and the rat dipeptides containing proline cannot be hydrolyzed in the brush border (Section 11, B). There appear to be many peptidases in the cytosol of the absorptive cells, some with a very broad range of activity. Noren et a / . (1971) purified a dipeptide from pig small intestine which hydrolyzed Gly-Leu and many other dipeptides. Das and Radhakrishnan (1973) purified from monkey small intestine what they termed a “master dipeptidase” with very high activity and a very wide range of substrate specificity; it was capable of hydrolyzing Gly-Leu and also most of the 400 possible dipeptides containing the 20 amino acids found in proteins, though Gly-Gly, Gly-Pro, Gly-His, and some dipeptides containing basic or acidic amino acids were not substrates for this enzyme, and are probably hydrolyzed by separate enzymes (Radhakrishnan, 1977). Schiller et al. (1977) have recently reported the isolation and characterization from the cytosol of rat intestinal mucosa of four distinct peptidases with molecular weights ranging from 58,000 to 113,000, and different substrate specificities. They believe it is premature to conclude that any one peptidase is of predominant importance in intracellular hydrolysis of

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peptides of dietary origin. More than one group has emphasized that the peptidases of the cytosol of the intestinal mucosa appear to occur in multiple forms with overlapping specificities (Peters et a / . , 1972; Kim et a / . , 1972). Clearly there is much work still to be done on isolation and characterization of the activities of the peptidases of the cytosol of the absorptive cells. As Section 11, T will show, the total intestinal (brush border plus cytosol) peptidase activity against certain peptides is inadequate for complete hydrolysis of the substrates, at least at high substrate loads. This applies, for example, to Gly-Gly dipeptidase, carnosinase, and the enzymes responsible for hydrolysis of small peptides of proline and hydroxyproline. A point which should be mentioned in connection with the interpretation of the results of investigations of the handling of peptides by small intestine in vitro is tha,t Silk and Kim (1976) have reported that large quantities of peptidases, mainly originating from the cytosol, are released during incubation in vitro of rat small intestine and cause hydrolysis of peptides in the incubation medium. This contrasts with the situation in I ~ W , in which release of mucosal peptidases into the intestinal lumen is believed to be small.

R. Possible Mechanisms of Peptide Absorption

Over the last 10 years, it has repeatedly been suggested that some of the phenomena characteristic of peptide absorption, in particular the more rapid absorption of peptides than amino acids, avoidance of competition for mucosal uptake between amino acids when peptides are absorbed, and the independence of mucosal uptake of peptides and amino acids, might be due not to transmembrane transport of intact peptides but to some alternative transport mechanism (Matthews et ( I / . , 1969; Rubino et a / . , 1971; Ugolev, 1972; Matthews, 1972, 1975a,b, 1977b; Ugolev et a / . , 1977). Three possible transport schemes have been considered. In scheme A , a brush border peptidase functions also as a carrier," taking up peptides on the luminal side of the plasma membrane of the absorptive cells and releasing amino acids on the intracellular side. I n scheme B, peptides undergo hydrolysis in the brush border and the constituent amino acids are taken up without release in free solution by special carriers which are available only to amino acids released by the brush border peptidases. In scheme C , peptides undergo transmembrane transport by one or more peptide-specific transport mechanisms, undergoing hydrolysis subsequent to the peptide transport mechanism( s), probably in the cytosol of the absorptive cells. The pros and cons of I'

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these schemes have been argued at length in a previous review (Matthews, 1975b) and since no new relevant evidence has become available since the previous review was written, the arguments will not be repeated here. Neither scheme A nor scheme B has any positive evidence in its favor, and both encounter certain objections; furthermore, evidence for peptide transport schemes of this type has not been described in any form of life. Scheme C , on the other hand, will readily explain all the known phenomena of peptide absorption, and has in its favor the ability of the intestine to accumulate peptides which are resistant to hydrolysis. It is a reasonable working hypothesis (Adibi, 1971) that peptide absorption is the result of only three main processes: (1) intraluminal hydrolysis followed by uptake of free amino acids; (2) brush border hydrolysis followed by uptake of free amino acids; and (3) peptide transport into the absorptive cells, followed in most cases by intracellular hydrolysis, but in some cases resulting in entry of intact peptides into the portal and even the peripheral blood. To this it might be added that the mechanisms for uptake of free amino acids may serve to some extent as “recapture” mechanisms for amino acids which have diffused back from an intracellular site of peptide hydrolysis (Silk et al., 197%; Heading ez al., 1977). A question of major interest is that of the relative importance of the processes previously listed in the absorption of different peptides. It is generally thought that intraluminal hydrolysis of most small peptides is relatively slight, so the main question is that of the relative importance of brush border hydrolysis with uptake of free amino acids and uptake of intact peptides. It is likely that the relative importance of these two processes varies widely from one peptide to another-at one extreme there are peptides that are predominantly hydrolyzed in the brush border, and, at the other, peptides such as dipeptides of Pro, p-Ala-His, and (under experimental conditions) Gly-Sar and Gly-Sar-Sar which undergo little if any hydrolysis in the brush border, absorption being entirely or almost entirely the result of uptake of intact peptide. The information we have suggests that brush border hydrolysis is more important with tripeptides than with dipeptides, and more important in the ileum than in the jejunum. The presence of a lipophilic amino acid side-chain at the NH,-terminus may increase affinity for brush border hydrolysis (Section 11, B)-but the presence of lipophilic side-chains probably also increases affinity for peptide transport (Section 11, M)-so that the overall effect of such side-chains is uncertain. The relative importance of brush border hydrolysis and uptake of intact peptides might also be related to the load of peptides presented for absorption. At a high load, peptides normally hydrolyzed largely in the brush border might “break through” the brush

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border peptidase barrier to gain access to the peptide uptake mechanism(s) of the absorptive cells on a more extensive scale. This suggestion is purely speculative. Some attempts have been made to quantitate the relative importance of the various modes of peptide absorption for individual peptides. Adibi (1971), using intestinal perfusion in man, concluded that in the jejunum Gly-Gly was almost exclusively absorbed as an intact peptide, while jejunal absorption of Gly-Leu (20 mM) was about 90% the result of uptake of intact peptide, brush border hydrolysis of this dipeptide being slight and intraluminal hydrolysis of very minor importance. These conclusions were based on experiments in which uptake of free amino acids was blocked by saturating the amino acid uptake systems. Experiments in which the activity of the brush border peptidases was reduced to insignificant levels by reducing intrajejunal pH (Fogel and Adibi, 1974) were compatible with the conclusion that absorption of Gly-Gly and GlyLeu was very largely the result of mucosal uptake of intact peptide. Cheeseman and Parsons (1974) using the principle of saturating the uptake mechanisms for free amino acids, estimated that in the small intestine of the frog R . pipiens, 60% of absorption of Gly-Leu ( 5 mM) was the result of uptake of intact peptide. It can also be calculated from the data of Crampton et a / . (1973) that between one-half and two-thirds of Met-Met (100 mM) is taken up intact in the proximal small intestine of the rat, and something less than one-half in the distal small intestine. Silk er a / . (197%) estimated from the results of investigation in cystinuria (Section 11, I) that in the normal human subject about two-thirds of Arg-Leu ( 1 mM) would be absorbed into the small intestinal mucosa as such and one-third in the form of free amino acids. Wiseman (1977) has reported the result of a novel approach to the problem of the relative importance of brush border versus intracellular hydrolysis of dipeptides, using hamster small intestine in virro. It is based on the observation that amino acids inhibit active transport of D-glucose, the mechanism for D-glucose transport being at a superficial site apparently in the brush border. Wiseman argued that peptides which are either more or less potent in inhibiting glucose transport than the equivalent amino acids probably pass beyond the site of active transport of glucose before they undergo complete hydrolysis, whereas peptides which have the same effect as the equivalent amino acids are probably hydrolyzed at a site superficial to that of active transport of the sugar. On the basis of his results, obtained with amino acids and peptide concentrations ranging up to 20 mM, he suggested that Ala-Gly, Gly-Gly, Val-Val, AlaVal, Val-Ala, and Pro-Gly were mainly hydrolyzed deep to the transport mechanism for D-glucose (presumably within the cell) whereas Ala-Ala,

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Leu-Leu, Gly-Ala, Ala-Leu, Leu-Ala, and Gly-Pro were mainly hydrolyzed superficial to the transport mechanism for D-glucose, possibly at the surface of the cell. Though we do not know the subcellular location of dipeptidase activity against peptides of Pro in the hamster, this activity is believed to be absent from the brush border in the rat and in man (Section 11, B) so that the attribution of a superficial site of hydrolysis to Gly-Pro in the hamster is of interest. Some other attempts have been made to arrive at qualitative estimates of the relative importance of brush border hydrolysis with uptake of free amino acids and peptide uptake followed by intracellular hydrolysis in peptide absorption by comparing rates of mucosal hydrolysis of peptides in vitro with rates of lumen disappearance in vivo. By this means, Silk et ul. (1976) concluded that in the rat Gly-Phe was taken up predominantly intact, whereas brush border hydrolysis played an important part in the absorption of Phe-Gly. By similar means, Arvanitakis et al. (1976) concluded that a predominant part in the absorption of Pro-Gly-Gly was played by uptake of the intact tripeptide, whereas brush border hydrolysis of Leu-Gly-Gly played a predominant part in absorption of this tripeptide, the main products of brush border hydrolysis, free Leu and Gly-Gly, then undergoing mucosal uptake. N o direct information is available about the nature of the molecular mechanisms involved in mediated peptide transport. The possible role of Meister’s y-glutamyl cycle in the membrane transport of peptides and amino acids is reviewed by Meister et a / . (1977). It has recently been shown that a simple diffusion plays an appreciable part in the uptake of two dipeptides, Gly-Sar and Glu-Glu, by hamster small intestine in vitro (Matthews et a / . , 1979), as it does in the uptake of Gly and of Leu in similar circumstances (Schedl et a/., 1979). This role is probably small at physiological concentrations, but should not be ignored when analyzing the kinetics of influx. If it is ignored, especially at high concentrations, linearizing plots such as those of Lineweaver and Burk, of Hanes, and of Hofstee may be obviously biphasic, giving the possibly false impression that more than one mediated mechanism is involved in substrate uptake. On general grounds, everyone supposes that simple diffusion must play some role in the intestinal absorption in vivo of small water-soluble molecules with a molecular weight of, say, up to 200-300. However, no one knows the quantitative importance of this mechanism of absorption. Studies in the inborn absorptive defects of Hartnup disease and cystinuria (Section 11, I), and in glucose-galactose malabsorption (Fairclough et a/., 1978), suggest that in intact man simple diffusion does not play more than a minor role in the intestinal absorption of amino acids and sugars.

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S. Quantitative Importance of Mucosal Peptide Uptake in Protein Absorption One of the most difficult questions in the study of the absorption of protein digestion products is that of the quantitative importance of mucosal peptide uptake, as opposed to uptake of free amino acids. It can hardly be expected that investigations of the absorptive behavior of individual peptides will go very far toward providing an answer to this. These investigations do suggest, however, that peptide uptake plays a substantial part in the overall process of protein absorption, and the idea that peptide uptake is an important mode of absorption of protein digestion products-possibly the predominant mode-is supported by other evidence. The findings in Hartnup disease and cystinuria (Section 11, 1) provide good evidence that peptide uptake must be of considerable quantitative importance in man. In Hartnup disease, in particular, there is malabsorption of most, if not all, neutral amino acids including essential onesyet protein nutrition is almost normally maintained and clinical evidence of malabsorption is not common. The patients probably have to rely mainly on retention of their ability to take up peptides to maintain their nutritional status. Experimentally, there is evidence (Nasset, 1965; Coulson and Hernandez, 1970; Nixon and Mawer, 1970a,b), summarized by Matthews (1975a,b), that the pattern of absorption of amino acids from protein meals is very different from that of their absorption from mixtures of amino acids simulating proteins, reflecting the composition of the protein much more closely. The differences are probably largely the result of mucosal peptide uptake, and their existence suggests the importance of this process. Crampton et al. (1971) studied absorption of partial hydrolysates of several proteins, consisting mainly of oligopeptides, from the jejunum of the rat, and compared their rates of absorption with those of the equivalent amino acid mixtures. In all cases, total absorption of aNHzN from the partial hydrolysates was more rapid than from the amino acid mixtures, indicating the importance of peptide uptake from hydrolysates probably bearing some resemblance to the normal products of intraluminal digestion. Silk et al. (1973b, 1975b) carried out similar experiments by jejunal perfusion in man, using partial hydrolysates of casein and the equivalent amino acid mixtures. Not only was total absorption of a-NH,N more rapid from the partial hydrolysates than from the amino acid mixtures, but it was found that a number of amino acids which were relatively slowly absorbed from the amino acid mixtures, including Phe, Ala, Ser, His, Gly, Lys, Glu, and Asp, were more rapidly absorbed from

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the partial hydrolysates, and the great variation in percentage absorption of individual amino acids from the mixtures was much reduced when the hydrolysates were perfused. Fairclough (1977) extended the work in man to two more proteins. A partial hydrolysate of fish protein and the equivalent amino acid mixture gave results broadly similar to those obtained in the previous (casein) experiments. A partial hydrolysate of lactalbumin and the equivalent amino acid mixture gave the most striking results obtained, 13 of 16 amino acids being more rapidly absorbed from the partial hydrolysate than from the amino acid mixture (Fig. 2). Marrs et a / . (1975) found no significant difference in the increments in concentrations of amino acids in human peripheral plasma after rather small oral doses (ca. 10 gm) of a partial hydrolysate of casein and the equivalent amino acid mixture, but Silk (1977) reported that after oral administration in man of a partial hydrolysate of fish protein and the equivalent mixture of amino acids, the rise in a-amino nitrogen in peripheral plasma was greater after the partial hydrolysate than after the equivalent amino acid mixture, a probable explanation being more rapid absorption of the partial hydrolysate. The observations previously described might have important nutritional and clinical implications (Matthews, 1975a,b; Matthews and Payne, 1975a). It should be added that the results obtained in the type of experiment previously described obviously vary with the protein on

0 AMINO ACID MIXTURE HYDROLYSATE

p

MET LEU ILE ARG PRO VAL ALA LYS PHE GLU TYR GLY ASP THR SER HIS NS NS NS NS NS NS NS NSc0.005 NS cO.01 NS NS ~0.01<0.005<0.01

FIG. 2. Absorption of individual amino acids from a partial hydrolysate of lactalbumin containing oligopeptides and from the equivalent amino acid mixture by human jejunum. Each solution contained 70 mM a-NH,N. The horizontal lines across the columns indicate 1 SEM, and the significance of differences in absorption of each amino acid from the two solutions is indicated. N S = not statistically significant. (Reproduced from Fairclough, 1977.)

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which they are based, and may well vary according to the mode of partial hydrolysis employed. Fairclough er L i I . (197713) tried to assess the importance of peptide uptake from a partial hydrolysate of casein by human jejunum by attempting to block peptide uptake with a high concentration of Gly-Gly (100 mM), on the assumption that there might be only one peptide uptake system and if this could be saturated by Gly-Gly, an estimate of the extent of peptide uptake from the hydrolysate could be made. In fact, the attempt was unsuccessful, the inhibitory effect of Gly-Gly being very slight. This led the authors to consider the possibility that some peptides were taken up by a system or systems unavailable to Gly-Gly (Section 11, K). T. Entry of Amino Acids and Peptides into the Blood

While it is generally believed that the bulk of the products of protein digestion enter the portal blood in the form of free amino acids, there is no lack of examples of small peptides that are known to enter the blood intact, especially when the intraluminal load is high. These include GlyGly, p-Ala-His (carnosine), p-Ala-MeHis (anserine), peptides of Pro and Hyp, and (when administered) peptides containing D-amino acid residues (for references, see Matthews, 1975a,b). (Nothing is known of the mechanism(s) by which such peptides leave the absorptive cells.) There is also some evidence suggesting the possibility that small peptides may enter the portal blood on a larger scale than generally realized. Gardner (1975) showed that during absorption of a pancreatic hydrolysate of casein by an isolated preparation of perfused rat intestine, about one-third of the amino acids undergoing transmural transport to the serosal side of the gut did so in peptide-bound form. Sleisenger ef cil. (1977) studied the absorption of a partial hydrolysate of casein and the equivalent amino acid mixture in the guinea pig; the results, though hard to interpret, were at least compatible with the conclusion that a substantial proportion of the hydrolysate entered the portal blood in peptide-bound form. There is a need for further investigation in this area. Possibly casein is an anomalous substrate. Though the subject of transmembrane transport of macromolecules is outside the scope of this review, it should be noted that it is generally accepted that large quantities of intact protein are absorbed by the newborn animal, and Warshaw et a / . (1974) reported that the pinocytotic mechanism responsible may persist on a small scale in adult life, since they found that during the absorption of bovine serum albumin in the

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adult rat about 2% enters the blood and lymph in macromolecular form. Hemmings (e.g., Hemmings et al., 1977) maintains that macromolecular breakdown products of protein enter the blood of the rat on a massive scale.

U. Absorption of Biologically Active Peptides Very little is known about the mechanisms of absorption of biologically active peptides, in spite of the theoretical and clinical interest of this subject. Large polypeptides might be absorbed by the mechanisms responsible for absorption of whole protein. Some biologically active peptides are lipid soluble, and might be absorbed by diffusion through membrane lipid. Small lipid-insoluble biologically active peptides, such as thyrotropin-releasing factor, pyroGlu-His-ProNH,, might be absorbed by diffusion through aqueous areas in the absorptive membrane. Most small biologically active peptides are probably unsuitable for mediated transport by the mechanism(s) responsible for mucosal uptake of small peptides of dietary origin-but there appears to be at least one exception to this-the antibiotic cephalexin. The subject is more fully discussed by Matthews (1975a,b). 111.

PEPTIDE TRANSPORT IN ANIMAL TISSUES OTHER THAN THE SMALL INTESTINE

Peptide transport in the animal body is not confined to the small intestine, though we know fewer of the details of uptake of small peptides in organs other than the gut. In the kidney, some small peptides, including Gly-Pro and peptides of Hyp, are actively secreted by the renal tubules, while others are reabsorbed (Matthews and Payne, 1975b). Furthermore, the renal tubules can secrete thyrotropin-releasing factor (pyroGlu-His-ProNH,) (Leppaluoto et al., 1972), a peptide that would appear to be unsuitable for carriermediated transport by the small intestine (Section 11, G), and is indeed only very poorly absorbed from the gut (Ormston, 1972). Some years ago an active, Na+-dependent mechanism was described in rat and mouse brain which concentrates p-Ala-His and p-Ala-MeHis. This mechanism probably handles other di- and tripeptides which do not contain a p-amino acid, but is little affected by free amino acids (Abraham et a / . , 1964; Yamaguchi et al., 1970). The observations of Abraham et (11. probably provided the first evidence for concentrative transport of small

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peptides by an animal tissue, though the authors did not emphasize this point. Though glutathione is poorly taken up by the small intestine (Section 11, G), oxidized glutathione has been reported to be actively transported out of human erythrocytes (Srivastava and Beutler, 1969; Srivastava, 1977). More recently, glutathione has been reported to be taken up intact by ascites hepatoma cells, line AH-I30 (Inoue et al., 1977). Nutzenadel and Scriver (1976) investigated uptake of p-Ala-His by rat intestine, kidney slices, and diaphragm in vitro. Evidence for mediated Naf-dependent uptake of this peptide was obtained with all three tissues; uptake was inhibited by other di- and tripeptides but less affected by free amino acids. Burston et al. (1977) investigated uptake of dipeptides (GlyGly, Gly-Sar, and p-Ala-His) by liver and kidney slices from the hamster, and by Ehrlich ascites carcinoma cells: evidence for uptake by a saturable energy-dependent mediated mechanism was obtained in all three cases. The peptides appeared intact in liver and kidney slices and in the carcinoma cells, though there appeared to be some intracellular hydrolysis. In contrast to the findings of Nutzenadel and Scriver, replacement of medium Na' by K+ had no effect on uptake of p-Ala-His by either kidney or liver. Uptake of Gly-Gly was accompanied by an increase in free Gly in the carcinoma cells. Uptake of p-Ala-His by these cells was strongly inhibited by Gly-Sar. Replacement of medium Na' by K+ had no effect on uptake of p-Ala-His by tissues or cells. The ability of Ehrlich ascites carcinoma cells to take up intact Gly-Gly (as well as a-Glu-Glu and probably Gly-Gly-Gly) was reported by Christensen and Rafn (1952) many years ago. Peptides have important but poorly understood effects on the growth of animal cells in culture; these are discussed by Matthews and Payne (1975a). In some cases these effects appear to be due to direct utilization in nutrition after intracellular hydrolysis, and there are reports that certain cell lines can take up dipeptides intact. Grahl-Nielsen et ul. (1974) reported that cultures of cell line RPMI No. 2402, originating in a carcinoma of the small intestine of the hamster, could take up a series of homopeptides of Lys from di-Lys to deca-Lys, hydrolyzing them intracellularly. The ability to take up a peptide as large as deca-Lys on an appreciable scale appears unusual for any organism. Contrary to an impression which may still persist that parenteral administration of mixtures of small peptides is an inefficient and wasteful process associated with heavy urinary losses, such mixtures are well utilized (Matthews and Payne, 1975a). Adibi and colleagues (Adibi and Krzysik, 1977; Adibi et al., 1977; Adibi, 1977; Krzysik and Adibi, 1977) have recently made a detailed study of the fate of intravenously admin-

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istered dipeptides-Gly-Leu, Gly-Gly, and Gly-Sar-in the rat. They found that the peptides were rapidly cleared from the plasma, and that they were as effective as the corresponding free amino acids in enriching tissue amino acid pools. Plasma clearance appeared to be mainly the result of uptake by tissue cells followed by intracellular hydrolysis. GlySar was found intact in liver, muscle, intestinal mucosa, and renal cortex. After nephrectomy, Gly-Gly was also found intact in liver, muscle, and intestinal mucosa, and Gly-Leu was found intact in muscle. The maximal rates of hydrolysis of Gly-Leu and Gly-Gly were much greater in kidney and intestine than in muscle, liver, or blood; however, when hydrolytic activities were calculated on the basis of total tissue weights, the total activities of liver and muscle were comparable to those of kidney and intestine.

IV.

PEPTIDE TRANSPORT I N MICROORGANISMS

A. Introductory Considerations

Over the last few years peptide transport in bacteria has been reviewed fairly regularly (Payne and Gilvarg, 1971; Sussman and Gilvarg, 1971; Barak and Gilvarg, 1975a; Payne, 1975a, 1976, 1977). In several instances discussion of the topic in prokaryotes (single cells without intracellular compartments) has been placed alongside reviews of the same topic in eukaryotes (cells which contain internal organelles) (Elliott and O'Connor, 1972, 1977; Payne, 1975a; Matthews and Payne, 1975a). In toto, these reviews carry an extensive bibliography of the subject, especially of the early literature. A forthcoming book deals with the topic in the broader context of the transport and utilization of nitrogen sources by microorganisms (Payne, 1980a). Consequently, in this section attention will be directed mainly to recent developments, e.g., improved methodology, regulation of uptake, energetics, and microorganisms given little coverage previously, e.g., yeasts. B. General Features of Nutritional Utilization of Peptides by Microorganisms

The microbial world comprises an immense diversity of species, ranging from the simpler prokaryotes such as E . coli to large eukaryotic organisms such as the algae and fungi. This feature should be remembered when generalized conclusions are being made, for there is no reason to

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assume that the properties of the much-studied E . coli are representative of those to be found throughout the whole of microbial “E. cology.” This caution can be applied to peptide utilization by microorganisms. Although it is no doubt generally true that peptides are nutritionally valuable to microorganisms (see reviews in Matthews and Payne, 1975a; Payne, 1976) this is not universally so. For species able to use peptides, these substrates provide preformed amino acids for protein synthesis, and they can act as sources of nitrogen, sulfur, carbon, and energy. However, no doubt some microbial species lack the means to utilize peptides because they have adapted to environments generally devoid of these substrates; instead they may have the ability to fix nitrogen, perform photosynthesis, utilize less common carbon and nitrogen sources, etc. There is no evidence that in microorganisms any protein cleavage products possess special nutritional properties such as vitamin-like activity, although in early studies this view was entertained for many years because of the apparent unique stimulatory activity of certain peptides (strepogenin) on the growth of some fastidious bacterial species (Stueptococci, Lactohcrcilli). However, this effect was finally attributed to interactions between the constituents of the complex growth media that differentially affected the absorption and utilization of peptides and amino acids (Kihara and Snell, 1960; see review in Matthews and Payne, 1975a). When peptides act mainly to provide free amino acids for protein synthesis it is often observed with fastidious bacteria that are naturally auxotrophic [i.e., have an absolute requirement for an exogenous nutrient(~)] for a variety of amino acids, and with some laboratory-derived auxotrophic mutants, that the growth yield and/or rates are different for amino acids and the equivalent peptides. Growth rates can differ when uptake of one substrate is insufficient to satisfy the requirements of protein synthesis. Growth yields can differ when substrate uptake exceeds growth needs and the excess is metabolized. It is clear therefore that the transport step can markedly influence the overall nutritional response. Different conditions are likely to obtain when peptides act mainly as C or N sources. Conditions of C - or N-limitation commonly lead to derepressed synthesis of general transport systems for amino acids and possibly even peptides (see Sections IV, G, 5 and H, I ) , and also to derepressed peptidase synthesis and secretion (Wagner et N I . , 1972; Litchfield and Prescott, 1970; Payne, 1972b; Drucker, 1975; see reviews in Cohen, 1980; Law, 1980; Payne, 1975b, 1980b). In the feast and famine existence of microorganisms in nature such growth limitation will be the norm. For example, the average generation time for E . coli in the gut is

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calculated to be about 20 hours (Savage, 1977), while it can reach about 20 minutes during culture in vitro, and differences such as this may be possible for many species. This feature should be borne in mind when attempts are made to relate the demonstrated biological activities of bacteria grown in batch culture to the activities operative during growth in vivo; any differences could well influence the nutritional fate of peptides. More relevant studies on peptide utilization, using microorganisms growing in chemostat culture, have not yet been reported. C. Scope of the Topic: Transport in Bacteria, Yeasts and Other Fungi, Algae, and Lichens

Previous reviews have considered mainly bacteria and to a lesser extent various fungi. This approach reflects the information presently available for microorganisms as a whole. However, for the first time, algae and lichens are also discussed because there is some indirect information suggesting that they may take up peptides, and no doubt direct studies will be carried out in the near future. D. Nature of the Microbial Cell Surface and Membrane Location of Peptide Permeases

To permit their autonomous existence in varied environments microorganisms possess strong, shape-determining surface layers. These outer structures help protect the organism, but also form an initial barrier to the passage of some substrates to the underlying cytoplasmic membrane, which is the main location of the specific substrate transport systems. The surface structures vary enormously among microorganisms, and presumably this variation could cause marked differences in peptide uptake even if the underlying peptide permeases were to be identical in all species. However, it is beyond the scope of this article to survey microbial envelope structures in general, and consideration is confined to those prokaryotic species in which peptide transport has been studied most intensively. These are members of the Gram-negative Enterobucteriaceae, Pseudomonadaceae, and Bacteroidaceae, and the Gram-positive Streptococcaceae and Lactobacillaceae . The Gram-negative cell envelope comprises (Costerton et al., 1974; Braun and Hantke, 1974; Braun, 1978) an outer membrane (OM), an underlying peptidoglycan layer (which together constitute the cell wall), and between this and the cytoplasmic or inner membrane (IM) the periplasmic space. The cell wall exerts a sieving function, first noted in E .

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coli from the exclusion of “large” oligopeptides and subsequently demonstrated for other molecules of similar size; current studies indicate that “the sieve” may actually be outer membrane pores used by hydrophilic molecules to traverse the cell wall (this topic is considered in detail in Section IV, G, 3, g). If analogous pores are found to occur in other microorganisms they could minimize the effects of the diverse surface structures on peptide uptake. The Gram-positive cell wall lacks an outer membrane but it has a thicker integument of peptidoglycan and unique teichoicheichuronic acid components (Braun and Hantke, 1974; Rogers et ul., 1978). The typical periplasmic space is absent, although an apparently analogous membranewall interlayer has been described in Staphylococci (Giesbrecht et ul., 1977). How these structures affect peptides during their passage across the envelope to the permease is not known. It is possible that the crosslinked peptidoglycan might act as a sieve, and although early reports (Scherrer et ul., 1971; Scherrer and Gerhardt, 1971) indicated that large molecules could penetrate the cell wall, in a more recent report Scherrer et al. (1977) question whether their earlier results are applicable to whole cells in vivo. The periplasmic space, which under commonly used experimental conditions comprises about 20-40% of the total cell volume and contains about 5% of the cell protein (Stock et al., 1977), is the specific location of the binding-protein components of many permeases (Oxender and Quay, 1976). Specifically, binding proteins are the recognition components of those permeases that are energized via ATP, and as peptide permeases belong to this class (see Section IV, G, 4) peptide-binding proteins may exist, although none has yet been identified. It is not known whether binding proteins occur predominantly as peripheral proteins loosely attached to the inner membrane permeases, or in a free form able to move substrates across the periplasmic space. This uncertainty is further compounded by not knowing whether pore structures reach only to the exterior of the periplasmic space or extend across it to the cytoplasmic membrane. Finally, when considering the influence of the cell envelope on peptide uptake it should not be forgotten that its composition, and thus its properties, are not constant. Specific changes in the protein composition of the inner and outer membranes take place in response to particular nutrient shifts (Braun, 1978). Various growth limitations, and cessation of protein synthesis in particular, produce gross compositional alterations (Rothfield and Pearlman-Kothencz, 1969; Shockman et ul., 1974; Ellwood and Tempest, 1969), which in some cases have been shown to produce marked changes in cell permeability (Leive, 1974). As imbalanced growth

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of this type will be normal for bacteria in v i w , it may well be that transport characteristics established for exponential-phase cells may differ somewhat from those of the organism in its natural environment (see also Section IV, B). E. Defining Characteristics of Peptide Transport

Having considered the surface anatomy of microorganisms, it is appropriate here to be more exact about our use of the term peptide transport. If cell-envelope peptidases can cleave peptides so that their released amino acid residues compete for uptake with exogenously supplied free amino acid then the peptide can be utilized but it does not undergo transport. Thus, peptide transport describes the circumstances when either a peptide is taken up intact, or its free amino acid residues are accumulated without the possibility of them mixing with exogenous amino acids. Possible mechanisms compatible with this definition were considered in Section 11, R. Intact peptide uptake has been described in several microbial studies, but intracellular peptidase activity is usually so great that intact peptides cannot be detected in the cytoplasm making it difficult to exclude the possible involvement of a transport system coupled to a membrane peptidase (see Section 11, R). F. Methods for Studying Peptide Transport: Applicability and Limitations Substrate transport in microorganisms is usually studied in the following manner. Organisms are incubated with a labeled (e.g., 14C, 3H, 32P) substrate for appropriate times, separated from the incubation medium by rapid filtration, and washed, and the residual radioactivity associated with the cells on the filter is determined by scintillation counting, and taken as a measure of substrate uptake. However, this protocol gives rise to fundamental difficulty for studies of peptide transport, because the radiolabeled substrates are generally not available; only about half a dozen labeled dipeptides and no higher peptides have ever been produced commercially. A few investigators have themselves synthesized particular radiolabeled peptides, and useful results have been obtained, but it has invariably required that general conclusions have had to be made by extrapolation from results with but a few substrates. Thus, it has come about that alternative methodology has been evolved; in the main this has allowed only indirect assays of peptide transport, but it is this approach that has provided most of the results discussed here. However,

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in the last year, with the development of direct assays based on fluorescent labeling of substrates, a new definitive phase of study has been entered. These various methods are summarized in the following sections. 1 . USE OF A v * ~ . rACID o AUXOTROPHS a . Moniroring Growrh Response. In order for an amino acid auxotroph to grow it must be supplied with its required amino acid, which can be provided in the form of a peptide. Furthermore, if the auxotroph lacks cell-envelope peptidases then its ability to grow upon peptides is an indication that the nutrient substrates are being transported (see Section IV, E) However, the method is of limited value, assessment of transport being only indirect and growth not being solely dependent on the transport step. It is a relatively insensitive technique, each assay requiring approximately 1 pmole of peptide at a concentration of about 2 mM. Each incubation can take about 12-36 hours, with growth being monitored usually by change in turbidity of the culture, although plate techniques can also be used. In principle it can be applied to many microorganisms, e.g., bacteria, yeasts, but it is only possible to obtain certain types of general information, such as the N- and C-terminal requirements, size restriction, etc. of peptide permeases; the method, and the types of results obtained from its use, have been considered in more detail in previous reviews (Payne and Gilvarg, 1971, 1978; Sussman and Gilvarg, 1971; Barak and Gilvarg, 1975a; Payne, 1975a, 1976, 1977).The procedure is particularly useful when interest centers not simply on transport per se but also on the relative utilizability of amino acids and peptides, i.e., growth yield and growth rate, or when peptide toxicity is under investigation. It can only be used satisfactorily under conditions permitting growth, and thus experiments into energy coupling, pH, and cation dependence, etc. cannot be performed. b. Monitoring Enzyme Synthesis. Instead of measuring peptide-dependent growth, i.e., cell multiplication over many generations, as in the above method, here the peptide-dependent synthesis of one enzyme is assayed. Conditions for the induced or derepressed synthesis of a particular enzyme, e.g., P-galactosidase, alkaline phosphatase, are established in the amino acid auxotroph just prior to addition of the peptide to the medium; uptake of the peptide then supplies the required nutrient and permits synthesis of the enzyme. The rate and extent of induced enzyme synthesis is conveniently measured using enzyme substrates that yield colored or fluorescent products. This technique (Bell er ul., 1977) suffers from many of the disadvantages of method a above that are inherent in the requirement for a “growth environment”, however it is much more

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sensitive and requires only a few minutes incubation. The results obtainable are similar to those from the growth assay but, in addition, by progressively lowering the peptide concentration until its rate of uptake just begins to slow enzyme synthesis a “limit peptide concentration” can be determined. This important parameter, “the limit concentration,” analogous to that described for amino acids by Ames (1964), is the external concentration that just permits normal growth in the absence of endogenous biosynthesis of the specified nutrient. c. Monitoring Protein Synthesis. This is a radioactive incorporation method that uses an auxotroph requiring two amino acids (Payne and Bell, 1977a). One amino acid is supplied in free radioactive form, and the other in peptide linkage. Protein synthesis is dependent on the simultaneous availability of both nutrients. The rate and amount of total protein synthesis is measured from incorporation of labeled amino acid, the extent of which is, in turn, dependent on the uptake of the peptide. The method is extremely sensitive, and rapid, and in general it has the same pros and cons as method h . Both these latter two methods have been used only with E. coli. 2. USE OF RADIOACTIVELY LABELED PEPTIDES The principles of this method were outlined in the introduction to this section. The early literature upon the use of radioactively labeled peptides was discussed by Payne and Gilvarg (1971). Since that report, the following labeled peptides have been used to study transport in the indicated microorganisms: E. coli, [‘T]Gly-Gly (De Felice et al., 1973; Cowell, 1974; Neuhaus et a/., 1977) and [‘4C]~-Ala-[’4C]~-Ala (Neuhaus et a/., 1977); S. fyphimurium, Ala-Pr~-[’~C]Gly(Jackson et al., 1976) and [‘4C]Gly-Pro (Yang et a / ., 1977); Group N Streptococci, Gly-[’4C]Leu (Law, 1978); Ps. pudita, Gly-Gly-[’4C]Ala (Cascieri and Mallette, 1976); Saccharomyces cerevisiae, Met-Met-[I4C]Met and [3H]acetyl-Met-MetMet (Becker and Naider, 1977); Neurospora crmsa, G l y - L e ~ - [ ~ H ] T y r (Wolfinbarger and Marzluf, 1975b). In most of the studies, chloramphenicol has been used to prevent protein synthesis; otherwise isotope is continuously incorporated into protein. When protein synthesis is inhibited, it is assumed that uptake into the intracellular pool can be followed. However, recent studies (Payne and Bell, 1977b,c, 1979) have shown a fundamental flaw in this assumption because, with E. coli (and Sulmonella typhimurium, J. W. Payne, unpublished observations) at least, the transported peptides are rapidly hydrolyzed in the cytoplasm and the released amino acid residues can undergo immediate and rapid exodus from the bacterium at the sume time as further peptide is being taken up.

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Thus, under these circumstances, measurement of mere counts in the bacteria would underestimate both the rate and extent of peptide transport. A survey of the literature indicates that this feature probably occurred during several earlier studies using radiolabeled peptides and its significance generally went unnoticed (see Section IV, G, 6), although Cowell (1974), measuring uptake of counts from [14C]Gly-Glyinto chloramphenicol-treated E. coli, noted that uptake failed to plateau out and suggested some form of metabolism might be responsible. Providing the possibility of amino acid exodus is taken into account, the method has many obvious advantages: it is direct, sensitive, is not limited to auxotrophs, and incubations can be performed under any (nongrowth) conditions. The main difficulty arises from the need to synthesize the radioactively labeled substrates.

3. FLUORESCENT LABELING OF PEPTIDES

a . Dansyl Method. This approach (Payne and Bell, 1977b,c, 1979) is also direct, sensitive, and in principle applicable to any microbial species incubated under any conditions. Thus, a microorganism is incubated with one or more peptide substrates and periodically samples are filtered to remove the microorganisms. Samples of the filtrates are treated with dansyl chloride to fluorescently label the peptides, which are separated by two-dimensional thin-layer chromatography; the fluorescent intensities of the derivatives on the chromatograms are then measured, thereby providing quantitation of the peptides in the medium. Peptide uptake is therefore measured by quantitating substrate removal from the medium. In a complementary manner, the pool contents of the microorganisms retained on the filters are extracted, dansylated, and chromatographed, allowing the absorbed substrates (and their cleavage products) to be quantitated; however, this approach is really useful only in those cases in which intact peptide is accumulated. Use of this method allows measurements to be made of the individual rates at which each peptide is taken up from a complex peptide mixture. It has been applied successfully not only with bacteria but also with yeast (Section IV, H, 1) and plants (Section V). The exodus of amino acids that accompanies peptide uptake was first noted using this method, and it has also proved most valuable for distinguishing a peptide transport mutant from a peptidase-deficient strain (J. W. Payne, unpublished observations) b. Fluorescarnine Method. Fluorescamine (fluoram) reacts with primary amino groups at pH 8-9 to form intensely fluorescent products. However, because the pKbs of the N-terminal a-amino groups of a peptide and an amino acid are different, lowering the pH to about 6.2 pre-

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vents reaction with amino acids while still allowing peptides to be labeled (Perrett et a / . , 1975). Furthermore, neither the reagent itself nor the products it forms with ammonia or water are fluorescent. These properties have allowed the development of a simple, direct assay for peptide transport (Nisbet and Payne, 1979). Thus, as in the dansyl method, incubation media containing peptides are sampled periodically and freed of microorganisms by filtration. The filtrates are reacted with fluorescamine and from the resultant fluorescence of the solutions the extent to which the peptides were taken up by the microorganisms during the incubation can be determined. This method complements the dansyl procedure and is ideal for quantitatively surveying peptide transport by different microorganisms under varied incubation conditions. G. Peptide Transport in Bacteria

1. ON T H E OCCURRENCE OF SEPARATE TRANSPORT SYSTEMS FOR AMINO ACIDS,DIPEPTIDES, A N D OLIGOPEPTIDES Although long familiar to animal physiologists, the “concept of selective permeation was looked upon with suspicion by many microbiologists” even in the early 1950s (Cohen and Monod, 1957). However, the critical exposition of the defining characteristics of bacterial permeases [i.e., specific permeation systems that mediate the entry (exodus) into bacteria of organic nutrients] changed the outlook of many bacteriologists. Thus, when about this time workers reconsidered early reports showing that the relative nutritional utilizability of peptides and their equivalent amino acid mixtures were frequently different in microorganisms, they had no reservations about interpreting the data in terms of separate routes of absorption; these early results on bacterial nutrition, strepogenin, etc., and the developments in their interpretation have been considered previously in detail (Matthews and Payne, 1975a), and elsewhere (Payne and Gilvarg, 1971; Payne, 1976). Subsequently these interpretations were given experimental validation by the isolation of mutants that failed to transport certain free amino acids but retained the ability of the parent organism to transport the same amino acids in peptide form (Kessel and Lubin, 1962, 1963; Levine and Simmonds, 1960; Peters et u / . , 1953; Guardiola and Iaccarino, 1971),and by studies with radioactive substrates that indicated peptides were taken up faster and more extensively than amino acids and that there was a general lack of competition between the two classes (Leach and Snell, 1959, 1960; Kessel and Lubin, 1963; Mayshak et a/., 1966; Pittman et al., 1967; Yoder et NI., 1965a,b).

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The reciprocal class of mutant, deficient in peptide transport, was first reported by Payne (1968); the strain retained normal ability to transport amino acids and dipeptides but was defective in oligopeptide uptake, and this revealed the separate nature of the di- and oligopeptide systems in E . coli. Subsequently, similar mutants have been characterized in various strains of E. coli (Barak and Gilvarg, 1974; De Felice et a / . , 1973), and Salmonella typhimurium (Ames et a / . , 1973; Jackson et ul., 1976). In E. coli, mutants defective in the dipeptide permease (Dpp-; or Dpt-, dipeptide transport) have also been isolated (De Felice et al., 1973; Vonder Haar and Umbarger, 1972). How widespread is the occurrence of both di- and oligopeptide transport systems in bacteria remains to be established. In Streptococci, some strains apparently have both, whereas others may have but one (Law, 1977, 1978), and in S . aureus evidence from use of the toxic dipeptide analog, bacilysin, is most readily explained by assuming the existence of a single peptide uptake system (Kenig and Abraham, 1976; Kenig et a/., 1976). 2 . PEPTIDETRANSPORT MUTANTSA N D PEPTIDEPERMEASES

THE

GENETICS OF

That oligopeptide transport is mediated by a system(s) distinct from the one(s) involved in dipeptide uptake has been clearly established for several bacterial species. This feature is implied by the minimal competition that occurs between di- and oligopeptides during uptake by E. coli (Payne, 1968, 1976; Payne and Gilvarg, 1971; Barak and Gilvarg, 1975a; Diddens et ml., 1976; Payne and Bell, 1979), by Salmonella typhimurium (Ames et al., 1973; Jackson et a / . , 1976; Yang et d . , 1977), and by strains of Pseudomonas (Cascieri and Mallette, 1976), and has been clearly shown by isolation of mutants specifically defective in oligopeptide uptake in E. coli (Payne, 1968; Barak and Gilvarg, 1974; De Felice et a/., 1973; Becker and Naider, 1974; Diddens et a / . , 1976) and in S . typhimlrrium (Ames et UI., 1973; Jackson et nl., 1976). Most of the oligopeptide transport mutants isolated have been selected on the basis of their resistance to the toxicity of triornithine (they are variously referred to as TOR, triornithine resistance: Opp-, oligopeptide permease; or Opt-, oligopeptide transport); simultaneously they have been found to acquire cross-resistance to many other toxic oligopeptides (Barak and Gilvarg, 1974; Ames et ul., 1973; De Felice et ul., 1973; Diddens et a/., 1976), and appropriate auxotrophic strains have become unable to grow upon previously utilizable oligopeptides. Selection on the basis of resistance to any of a number of the other toxic oligopeptides has, in a reciprocal

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manner, been found to confer resistance to triornithine and to the remaining inhibitory oligopeptides. Although the biochemical defect responsible for the TOR phenotype is unknown, the gene(s) involved has been mapped by two groups independently (Barak and Gilvarg, 1974; De Felice et ul., 1973) and is located at 27 minutes on the latest E . coli linkage map (Bachmann et a/., 1976). In E. coli the frequency of spontaneous TOR mutants (selected by resistance to triornithine) is remarkably high, being about one in 5 x lo4 in a number of tested strains (Barak and Gilvarg, 1974; Gilvarg and Levin, 1972). No explanation is yet available for this high frequency. A further interesting characteristic of these spontaneous TOR strains is that they all appear to be deletion mutants, as judged from complete inability to obtain revertants. In fact, various unrelated reports seem to indicate that the region around 27 minutes may be a “hyperactive” deletion region. This feature, coupled perhaps with the fact that genes coding for the attachment sites for various colicins and phages have genetic loci close to 27 minutes (Bachmann er ul., 1976) (and resistance to which would be advantageous) may explain why a great many unselected strains of E. coli possess the TOR character (J. W. Payne and C. Gilvarg, unpublished observations). Although all tested oligopeptides can use the transport system previously discussed, it was noted early (Payne, 1968) that certain oligopeptides could (with diminished ability) still support growth of TOR auxotrophs. Definitive studies, in several strains of E. coli (Barak and Gilvarg, 1975b; Naider and Becker, 1975) and S . typhimurium (Jackson et a/., 1976), have led to the specific proposal that additional oligopeptide transport system(s) may exist. This system(s) can transport certain peptides, e.g., Leu-Leu-Leu and Met-Met-Met, at rates adequate to support normal growth in auxotrophic TOR strains, although most peptides fail to support any growth in such strains. Furthermore, Thr-Thr-Thr, which is toxic to E. coli K12 but not to E. coli W, inhibits TOR strains of the former and supports growth of threonine-requiring TOR strains of the latter (Barak and Gilvarg, 1975b); a mutant resistant to Thr-Thr-Thr had an impaired growth response to trileucine. Using the dansyl technique, it has been found (J. W. Payne, unpublished observations) that many oligopeptides are in fact taken up by TOR strains, but at diminished rates that are inadequate to support typical auxotrophic growth (see calculation, Section IV, G 3 ) . Further studies are required to characterize this secondary uptake system(s). Because dipeptides are transported not only by a specific dipeptide permease(s) but also (to a lesser extent) by the oligopeptide system,

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direct selection for mutants defective in dipeptide uptake (Dpp-, dipeptide permease, or Dpt-, dipeptide transport) is usually unsuccessful. Nevertheless, Vonder Haar and Umbarger ( 1972) apparently isolated such a strain from E. coli using resistance to Gly-Leu as a selection method. De Felice et al. (1973) started with an E. coli TOR strain and more easily selected a Dpp- substrain from it. They reported the map location for the gene as between opp (27 minutes) and the pro C marker (9 minutes), however, dpp is shown located at 5 minutes in the latest E . coli linkage map (Bachmann et al., 1976), a discrepancy that is not explicable simply by the recent decision to represent the chromosome by a 100-minute, instead of a 90-minute, map. In fact, P. E. Hartman (1978 personal communication) states that the information on which the 5-minute locus was assigned has proved incorrect, and that consequently the only mapping data are those of De Felice et al. (1973). In addition, it appears that there is more than one possible interpretation of these data and about all one can say with certainty is that it (dpp) is somewhere within the first 20 minutes of the map. Again the biochemical defect responsible for the mutation is unknown.

SPECIFICITIES OF PEPTIDE PERMEASES 3. SUBSTRATE A more detailed treatment of this subject can be found in several of the reviews cited earlier (Payne, 1975a, 1976; Barak and Gilvarg, 1975a). Most of the results are based on growth studies with auxotrophs, a feature that in the light of recent studies calls first for a cautionary comment in evaluating the results. Let us consider the rate at which its required amino acid must be made available to an auxotroph in order to support typical exponential growth in batch culture. Assume the amino acid is used only for protein synthesis. Consider a culture containing (say) the equivalent of 1 mg dry weight of bacteria. Assume (not unreasonably) 50%, i.e., 500 pg, is protein. Let, e.g., Ser (MW 100) comprise 5% of the protein; therefore, the amount of Ser in protein = 5/100 x 1/ 100 X 500 = 0.25 pmole, and this amount is required for the culture to double in mass in (say) 60 minutes. Therefore, an average rate of uptake of 4 nmole Ser (2 nmole Ser-Ser, 1.3 nmole Ser-Ser-Ser etc.) per minute per mg dry wt is required to achieve this doubling. Now, in E. coli and S. typhimurium it has been found (Payne and Bell, 1977a,b, 1979; Cowell, 1974; De Felice et al., 1973; Jackson et al., 1976; Yang et al., 1977) that typical rates of peptide uptake are 2-25 nmole per minute per mg dry wt, and these rates remain constant until uptake is complete (see Sections IV, G, 5 and 6). Thus, for some peptides it appears that merely halving

-

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the rate of uptake might decrease auxotrophic growth rate, while inability to support any growth may reflect only a lower and not necessarily a zero transport rate. u. N-Terminal a-Amino Group. In early studies, in which derivatization of this group was shown to impair nutritional utilizability of peptides, it was not clarified whether the change primarily affected uptake or intracellular hydrolysis. Gilvarg and Katchalski (1965) resolved this point by showing that intracellular peptidases could cleave a-N-acetylated peptides although the peptides were not utilized; thus, it was inferred that uptake was impaired. Payne (1971, 1974) used a variety of peptides in which the a-amino group was absent, acylated, or alkylated to show that E . coli utilizes mono N-substituted peptides providing the positive charge is retained; thus, N-monoalkyl derivatives are utilized but not N-dialkyl or N-acyl. This conclusion applies to both dipeptides and oligopeptides. Similar conclusions have been reached for various strains of E. coli (Becker and Naider, 1974), S . typhimurium (Jackson el ul., 1976), Streptococci (Law, 1977, 1978), and Pseudomonas (Cascieri and Mallette, 1976; Miller and Becker, 1978). b. C-Terminul a-Curboxyl Group. For many species it is found that nutritional utilizability is retained for oligopeptides devoid of the terminal carboxyl group (Payne and Gilvarg, 1968a; Payne, 1973), or having it variously substituted (Becker and Naider, 1974; Ames et a/., 1973; Fickel and Gilvarg, 1973; Cascieri and Mallette, 1976; Allen et al., 1978; Ringrose, 1980). However, the effects of such changes may not be negligible, for competitively the derivatives are often inferior to the parent compound, and in one tested case (J. W. Payne, unpublished observations) the rate of uptake of trialanine methyl ester was decreased over 50-fold relative to trialanine. The dipeptide permease apparently has a stricter requirement than the oligopeptide permease toward the carboxyl group. Thus, although dipeptides lacking this group (Payne and Gilvarg, 1968a; Payne, 1973) or having it variously derivatized (Fickel and Gilvarg, 1973; Ames et al., 1973; Cascieri and Mallette, 1976; Law, 1978; Hirshfield and Price, 1975) are utilized, their uptake is very markedly affected by oligopeptides and they are not utilized by TOR mutants, implying that they use the oligopeptide system. However, earlier suggestions that the carboxyl group is essential for the dipeptide permease are not supported by the finding that alaphosphin (Allen et al., 1978) (Ala-Ala in which the carboxyl is replaced by a phosphoryl group) still inhibits TOR strains ofE. coli (P. S. Ringrose, personal communication). c. Peptide Bonds. The peptide permeases require normal (protein-de-

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rived) a-linked peptides. This conclusion derives exclusively from results of growth studies, early references to which were cited in Payne (1976). Recently, the conclusion has been extended to include the Streptococci (Law, 1978), Pseudomonas putidci (Cascieri and Mallette, 1976), and Salmonella typhimurium (Yang et cil., 1977), although not all species known to possess peptide permeases have yet been tested. When the peptide-bond nitrogen is methylated the substituted peptides are transported by E . coli (Payne, 1972a), but at rates slower than the parent peptides (Payne, 1977). The substituted bonds are markedly more resistant to peptidase action, and consequently the residues forming the bond are not nutritionally available although other residues in the peptides can be utilized (Payne, 1972a). Fluorescent labeling has been used to show accumulation of these peptides intact in the intracellular pool of E. coli (Payne and Bell, 1979). A nonhydrolyzable analog of the dipeptide Gly-Leu, which contains a thiomethylene group substituted for the peptide linkage, was kindly provided by Dr. J. A. Yankeelov (Fok and Yankeelov, 1977) and tested for uptake by E . coli using fluorescent labeling. N o uptake of the analog [ ( S ) 2-(S-cysteaminyl)-4-methylpentoicacid] was detected and neither did it inhibit uptake of Gly-Leu when present at up to a 100-fold excess (J. W. Payne, unpublished observations). The analog, which conserves both charge and conformation of the parent compound, binds to and competitively inhibits the action of several peptidases (Fok and Yankeelov, 1977). d . a-Hydrogen A t o m . Peptides without the hydrogen atom on an acarbon are rapidly absorbed by Lucrobucillus cusei (Young et al., 1964). Thus Gly-Aib-Ala (Aib is a-aminoisobutyric acid, in which the a-hydrogen is replaced by a methyl group), and Gly-Ccl-Ala and Gly-Ccl-Val (Ccl is cycloleucine, 1-amino-cyclopentane- 1-carboxylic acid) are all transported and the C-terminal bond at least undergoes intracellular cleavage. Uptake of Gly-Aib-Ala was reported to be competitively inhibited by Gly-Gly-Gly (Smith et al., 1970). e . Srereochemical Specificity. Di- and oligopeptide permeases both show marked, but not complete, stereospecificity for peptides formed from L-amino acid residues. Although auxotrophic growth tests have been used, they are particularly unsuitable for such studies because even if the substrates were to be transported, peptide bonds containing Dresidues are generally resistant to peptidases and the D-amino acids themselves frequently cannot be utilized. Meaningful results therefore require direct studies using radiolabeled substrates or fluorescent labeling, or competition studies.

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For dipeptides, early studies indicated that uptake of radiolabeled GlyGly and Leu-Gly by E . coli was not inhibited by any of a number of dipeptides containing D-residues (Levine and Simmonds, 1962; Kessel and Lubin, 19631, and similar conclusions have been reached for various Lactobacilli (Leach and Snell, 1960; Kihara e f al., 1961; Shelton and Nutter, 1964: Yoder et al., 1965a,b), Streptococci (Law, 1977. 1978), Pseudomonads (Cascieri and Mallette, 1976), and Salmonella typhimurium (Yang et a/., 1977). [14C]~-Ala-[14C]-~-Ala uptake by E . coli (Neuhaus et a / ., 1977) was less than 0.1% the rate of that typically found for dipeptides (J. W. Payne, unpublished observations). Fluorescent labeling studies were used for the following peptides and uptake was either undetectable or less than 1% the rate for the LL form: the LD, DL,DD forms of Ala-Ala, Val-Val, and Leu-Leu; D-Ala-Gly, Gly-D-Ala, D-Leu-Gly, and Gly-D-Leu (J. W. Payne, unpublished observations). For oligopeptides, Becker and Naider (1974) reported that of the eight stereoisomers of Met-Met-Met, only the LLL and LLD forms were utilized by a methionine auxotroph of E . coli. Although growth of the parent on the DDL form was minimal a substrain was obtained that could utilize it and, as a TOR mutant of this substrain failed to grow on this isomer, the authors concluded that it probably entered via the oligopeptide transport system. Shankman et a / . (1962) reported that L-Val-L-Val-D-Val was taken up by lactobacilli although D-substituents at other positions prevented uptake. Effects of the positional substitution of D-residues on the antibacterial action of some tri-, tetra-, and penta-phosphonopeptides was reported by Allen et a / . (1978). Using fluorescent labeling techniques, rates of uptake by E . coli for the following peptides were determined (J. W. Payne, unpublished observations): uptake of D-Leu-Gly-Gly and DAla-Gly-Gly was undetectable although the L-isomers were rapidly transported; L-Ala-L-Ala-D-Alawas taken up at about 40% the rate of the LLL form whereas uptake of LDL and DDD was not detectable; L-Val-L-ValD-Val was taken up at about 20% the rate of the LLL form, but uptake of DDD was not detectable; Gly-Gly-D-Leu was transported at about 25% the rate of the L-isomer. Most interestingly, in these last three cases LAla-D-Ala, L-Val-D-Val, and Gly-D-Leu were found to be present intact in the cell, but as tripeptide accumulation continued these dipeptides underwent selective exodus together with the cleaved N-terminal residues. It should be noted that the dipeptides themselves cannot be accumulated. The relevance of these observations in distinguishing between the processes of uptake and exodus remains to be explored. In summary, substituting one or more D-residues prevents dipeptide uptake. For tripeptides, uptake of the LLD form occurs albeit at decreased

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rates, and this result is compatible with the lack of specificity of the oligopeptide permease toward the C-terminus (Payne and Gilvarg, I968a); D-residue substitution at other positions effectively prevents transport. f. Amino Acid Side-Chains. Results from competition studies, and use of transport mutants, indicate that bacterial peptide permeases possess no strict requirements toward the side-chains of a peptide although the nature of the side-chains does affect the kinetic characteristics of transport. Thus, for dipeptides, inhibitions of transport during auxotrophic utilization have been reported for many species (Payne, 1968, 1975a; Barak and Gilvarg, 1975a; Jackson et al., 1976; Cascieri and Mallette, 1976; Miller and Becker, 1978; Law, 1977, 1978); similarly, many dipeptides inhibit uptake of individual radioactively labeled dipeptides (Levine and Simmonds, 1962; Kessel and Lubin, 1963; Leach and Snell, 1960; Yang et al., 1977; Law, 1978), and the same conclusion has been reached using fluorescent labeling techniques (Payne and Bell, 1979). In addition, dipeptides with “unnatural” and derivatized side chains are absorbed via the permeases (Ames et al., 1973; Kenig and Abraham, 1976; Alper and Ames, 1978; Ringrose, 1980). Finally, Dpp mutants (selected as resistant to a specific dipeptide) fail to utilize a range of dipeptides, and show decreased transport of some radioactively labeled dipeptides (Kessel and Lubin, 1963; De Felice et al., 1973; Kenig et a / . , 1976). Similar results are found with oligopeptides. Competitive inhibitions of transport during nutritional utilization have been found in various species (Payne, 1968, 1975a; Payne and Gilvarg, 1971; Barak and Gilvarg, 1974, 1975a; Ames et ul., 1973; Jackson et a / . , 1976; Cascieri and Mallette, 1976; Miller and Becker, 1978; Diddens et al., 1976). Using radioactive and fluorescent-labeling techniques, competition between many different oligopeptides for uptake has been shown (Smith et al., 1970; Jackson et al., 1976; Payne and Bell, 1977a, 1979). In addition, oligopeptides with “unnatural” and derivatized side chains are absorbed via the oligopeptide permeases (Payne, 1968; Payne and Gilvarg, 1971; Ames et a / . , 1973; Diddens et al., 1976; Alper and Ames, 1978; Ringrose, 1980). Finally, TOR mutants show diminished utilization and decreased uptake of all oligopeptides tested, (Payne, 1968; De Felice et a/., 1973; Jackson et a/., 1976; Barak and Gilvarg, 1974, 1975b; Naider and Becker, 1975; Becker and Naider, 1974; Payne and Bell, 1977a,b). The additional mode of oligopeptide uptake described in TOR strains by Barak and Gilvarg (1975b) and by Naider and Becker (1979, may, when it is investigated in greater depth using radioactive and fluorescent techniques, turn out to have broader substrate specificity than their results from growth studies might indicate.

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In conclusion, it should be noted that the simple ability of a vast spectrum of peptides to be transported by a single permease should not conceal the fact that, as in animal small intestine (Section 11, J), the different side chains have a pronounced effect upon the binding affinities ( K J and rates of uptake (Vmax) (De Felice et a / . , 1973; Jackson et a / . , 1976; Yang et a / . , 1977; Cowell, 1974; Payne and Bell, 1977a,b, 1979), and the differences in affinity for transport can be reflected in their varied competitive abilities (Leach and Snell, 1959, 1960; Kessel and Lubin, 1963; Payne and Gilvarg, 1971). g . Peptide Size. The observation of a size restriction upon peptide uptake by E . coli was first noted by Gilvarg and Katchalski (1965). The exclusion limit was quantified in a later study (Payne and Gilvarg, 1968b), in which it was argued that it was a sieve-like property of the outer cell envelope that prevented uptake rather than steric hindrance at the permease. The occurrence of this effect in other bacterial species and its relevance to peptide utilization was reviewed earlier (Matthews and Payne, 1975a). Since that time, no experiments designed to investigate steric requirements of the permeases have been reported, and no circumstantial evidence relating to this feature has been noted. In contrast, many studies have endorsed the conclusion that a cell-envelope component acts as a sieve, excluding not only peptides but also many other types of molecules having molecular weights about 600 and above (Decad and Nikaido, 1976; Nikaido, 1976; Zimmerman and Rosselet, 1977). In addition, it has been suggested that passage of the smaller hydrophilic substances through the cell wall to the inner membrane is via membrane channels or pores, termed porins (Nakae, 1976a; Nikaido e f a / . , 1977a). Intensive efforts are being made to try and identify the proteins that constitute these pores (Nakae, 1976b; Nikaido et a / . , 1977b; van Alphen et a / . , 1978). Braun (1978) has recently reviewed this area. It should be noted that the exclusion limit previously mentioned can affect only oligopeptide uptake and not that of ordinary dipeptides, because the limit is larger than can be achieved by a combination of any two protein amino acids, although a derivatized dipeptide might be excluded if the substituent(s) were sufficiently large. 4. ENERGY COUPLING TO PEPTIDE TRANSPORT

Peptides are taken up by bacteria by an energy-dependent, mediated transport process. Two arguments may be advanced for this assertion. First, there is a large body of information, much of it fragmentary and indirect and relating to many species, which taken in toto is convincing. Thus, uptake requires a metabolizable energy source, is inhibited by

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conventional “energy poisons,” e.g., dinitrophenol, azide, and shows a specific temperature dependence, etc. ; this material has been summarized and referenced earlier (Payne, 1975a, 1976). Second, specific investigations of the topic in E . coli (Cowell, 1974; Payne and Bell, 1979) and S. typhimurium (Yang et a / . , 1977) have recently provided definitive evidence with these organisms. The most acceptable experimental criterion for an active transport process is ability to accumulate a substrate in an unmodified form against its electrochemical gradient. For peptides, the presence of highly active intracellular peptidases normally makes it impossible to establish this feature as it also does in animal small intestine (Section 11, C) and in barley (Section V , C). However, the following experimental tactics allow this difficulty to be overcome: (1) manipulation of growth or incubation conditions to lower peptidase activity, (2) use of peptidase-deficient mutants, and (3) use of peptides resistant to intracellular peptidases. Relevant to (1) is evidence that the peptidase activity of E . coli is varied by the growth phase and by particular nutrient limitations (Simmonds, 1972; Hermsdorf and Simmonds, 1980; Payne, 1972b; Miller, 1975a), which allows accumulation of certain intact dipeptides (Levine and Simmonds, 1962), although this is the least satisfactory of the three approaches. Mutants with one or more defective peptidases have been characterized in E . coli (Kessel and Lubin, 1963; Latil et al., 1976; Sussman and Gilvarg, 1970; Miller and Schwartz, 1980) and in S. typhirnuriurn (McHugh and Miller, 1974; Miller, 1975a,b; Miller and MacKinnon, 1974; Kirsh et a / . , 19781, and used very successfully to demonstrate extensive accumulation of intact peptides that are the normal substrates of these peptidases (Kessel and Lubin, 1963; Jackson et a / . , 1976; Yang et a/., 1977). Finally, triornithine and glycylsarcosine are examples of peptidaseresistant substrates that can be accumulated to high intracellular levels (Payne and Bell, 1979). In most of these instances it has also been shown that the intact peptides were transported by the systems normally responsible for the normal bulk peptide uptake. Investigations of the energization of active peptide transport have followed along the lines established for amino acid uptake, which broadly speaking are as follows. Uptake of some amino acids is linked closely to a supply of phosphate bond energy (ATP) while others are energized by a proton motive force (pmf, previously often called “an energized membrane”) (Berger, 1973); the former systems involve periplasmic binding proteins, are inactivated by osmotic shock treatment, and are nonfunctional in cytoplasmic membrane vesicles, while the latter do not have binding proteins and are functional in shocked cells and membrane vesicles (Berger and Heppel, 1974). For the former systems, ATP may be

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supplied by substrate level phosphorylation (glycolysis) and by oxidative phosphorylation. For the latter systems, the pmf, Ap, arising from primary active extrusion of H', is an electrochemical gradient made up of a membrane potential component A+ and a pH gradient ApH, thus: Ap = A+ - ApH; either component may provide the main driving force for transport depending on the substrate and the conditions, especially the external pH (Ramos and Kaback, 1977a,b). Furthermore, there is evidence that the A+ component of the proton motive force may also contribute to the efficiency of energy coupling in the shock-sensitive systems which apparently require more than just ATP (Lieberman and Hong, 1976a,b, 1977; Plate, 1976). The possible involvement of a mode of energization involving reducing equivalents (Garcia-Sancho et al., 1977), or operation of the y-glutamyl cycle (Meister et ul., 1977, Meister, 1980) has yet to be assessed. This area has recently been reviewed (Konings, 1977; Haddock and Hamilton, 1977; Harold, 1977; Anraku, 1980; Hamilton and Booth, 1980). For peptides, Cowell's (1974) studies indicated that the uptake system for diglycine in E . coli was shock sensitive, absent from vesicles, and energized by ATP. Recently, the ATP dependence has been extended to the transport of a variety of other dipeptides and also to oligopeptides (Payne and Bell, 1979). Although ATP-dependent systems usually are associated with periplasmic binding proteins, no report of a peptide-binding protein has appeared, although several workers have looked for them in the osmotic shock fluids in which these proteins are normally found. Although these results may truly reflect a feature unique to the peptide permeases, it should nevertheless be noted that all attempts to detect peptide-binding proteins have used di- or triglycine which have the lowest permease affinities of any of the natural peptides tested (J. W. Payne, unpublished observations). The mechanism by which energy is coupled to peptide transport is now a little clearer, but many question remain. For example, at which stage(s), i.e., external binding, translocation, intracellular release, is energy required? Is approximately the same quantity of energy required to transport a di-, tri-, tetrapeptide molecule (Section VI)? How do neutral, positive-, and negative-charged peptides differ in their energy requirements for uptake? Maintenance of constant net charge distribution across the membrane during uptake of peptides of varied charges will involve accompanying counterion (H+, K+, OH-) migrations in either direction as necessary (symport or antiport). How will these ion:peptide stoichiometries vary with pH over the growth range pH 8-5.5, given that ApH (the chemical difference in proton concentrations across the membrane) will

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change (Ramos and Kaback, 1977a,c), and titration of the terminal a amino group of the peptide over this pH range will alter the nature of the substrate (Payne, 1977)? These aspects could be studied with a fluorescent assay using peptidase-deficient cells starved for energy (Berger, 1973), for in this system the extent of peptide uptake as a function of supplied energy could in principle be determined. Another dimension is added to the energy-transport equation when the hydrolysis of the peptide is considered. Several workers have suggested that the free energy released on peptide bond hydrolysis might be used to help fuel transport (Section VI), although this has not been formally quantified and related to the energy requirements of transport. When hydrolysis is intracellular, and clearly subsequent to the transport event, the liberated energy can only indirectly offset the energy consumed during translocation [although, surprisingly, it has repeatedly been claimed that energy is actually required for proteolysis in vivo (Pine, 1980; Goldberg and St. John, 1976)l. However, for a membrane-bound, peptidaselinked permease this energy might conceivably be channeled more directly into transport. Special attention should be paid to the change in proton-binding capacity that accompanies the hydrolysis of peptide substrates to amino acids at about neutral pH (Payne, 1977). Thus the pKb of the a-amino group of the N-terminal residue of a peptide is generally lower than the pKb for this group in the free amino acid, and as these pKs occur commonly in the range pH 5.5-8.5, aminopeptidase action will liberate products with a greater tendency than the substrate to bind protons. Furthermore, hydrolysis of the neutral peptide bond produces amino and carboxyl groups that carry a partial positive, and a negative charge, respectively; in effect, this increases the relative H+ content of the solution, and compensatory changes must occur whether or not these protons have a special fate and function. Finally, the amino acid exodus that can accompany peptide uptake and hydrolysis must be considered. Whether this exodus requires energy, or whether it can be coupled to the counterflow of other amino acids or peptides (Brock and Wooley, 1964; Quay ef al., 1977), is unknown.

5. REGULATION OF PEPTIDETRANSPORT Much is now known about the ways in which amino acid transport is regulated in bacteria. Recently, information relevant to the possibility of peptide transport regulation has also been obtained. Because both transport modes supply the intracellular amino acid pool it is not unlikely that they may be regulated by interrelated mechanisms. Accordingly they will be considered together. Many bacteria absorb each protein amino acid

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through several transport systems. It is frequently found that of the several available permeases one system of high affinity is specific for a particular amino acid, and the other systems, generally with lower affinity, are shared by other amino acids with similar structures. The specific systems generally appear to be constitutive and thus only their uctit2ify can be regulated. In contrast, synthesis of the general systems is not fully expressed at all times, and thus both their concenrration in the membrane and their activity are under metabolic control. Various mechanisms have been described for regulating the activity of amino acid permeases (Ring 1970; Oxender, 1972; Oxender er a/., 1980). In some organisms, intracellular amino acids or some of their metabolites provide negative feedback signals causing inhibition of their own uptake and of other amino acids (transinhibition) (Langheinrich and Ring, 1976). These authors further suggested that charged tRNA species might be involved in positive control of transport in Streptomyces hydrogenuns, being responsible for the enhanced uptake of one amino acid caused by the presence of another (transstimulation). In contrast, Oxender and colleagues (Quay et a/., 1975; Quay and Oxender, 1976) have argued that charged tRNA molecules repress amino acid uptake in E . coli. Regulating amino acid transport through an effect on the biosynthesis of the protein components of the permeases has been described in various forms. A mechanism of induction either by amino acids themselves or their metabolites has been claimed (Ring, 1970; Rosenfeld and Feigelson, 1969; Fan et al., 1972). However, most interestingly, changes in transport capacity occur in the absence of exogenous amino acids and in response to nutritional alterations. These changes are best explained by repression/ derepression of general amino acid permeases that function primarily to provide substrates for carbon (and nitrogen) requirements rather than directly for protein synthesis. Thus, changes in active transport capacity can vary with the growth phase (Langheinrich and Ring, 1976) and in response to particular nutrient limitations (Alim and Ring, 1976). Some time ago, while discussing the general aromatic amino acid permease of S . typhimurium Ames (1972) suggested that cyclic AMP may be the regulator involved in the derepression of these systems, and recently her husband (Alper and Ames, 1978) and others (Ring et al., 1977a,b) have provided substantial evidence for this view. It is known that levels of cAMP fluctuate in bacteria (Rickenberg, 1974; Pastan and Adhya, 1976). Alper and Ames (1978) consider that the cellular concentration of cAMP increases in response to carbon starvation yielding in turn a CAMP-CRP complex (CRP is cAMP receptor protein), that will interact in a graded manner with different promoters, thus bringing about activation of their respective operons. In support of this thesis are observations that carbon

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limitation, and addition of exogenous CAMP, both apparently derepress many different permeases, including general amino acid systems (Alper and Ames, 1978; Ring et a/., 1977a,b). When an assessment is made of the applicability of these ideas to the problem of controlling peptide uptake, the following conclusions can be drawn. First, the permutated sequences of small peptides are so numerous as to make it impossible for a commensurate number of specific peptide permeases to exist, and, in fact, usually only one or two systems are found. These systems are constitutive; certainly they are present in organisms that have not been exposed to exogenous peptides. How then might the activity of these permeases be regulated? Being constitutive, it may be presumed that they are normally present in a fully functional state, and thus we need be concerned only with ways to switch them off. Feedback regulation by intact peptides seems improbable, if only because their rapid intracellular cleavage prevents them accumulating inside the cells to a significant level. It is also difficult to envisage how the freed amino acid residues might collectively inhibit peptide uptake. One dismisses the idea that just a limited number of amino acids, having attained appropriate internal concentrations, might effect such control because this could deny entry to peptides containing other amino acids that the organism was still in need of (Payne and Bell, 1977~).When considering this feature it is pertinent to note that with 20 protein amino acids, a n y one residue will occur in about 10, 14, and 18% of all di-, tri-, and tetrapeptides, respectively. At the level of tRNA species one can speculate about a possible negative form of control; thus, the presence of a certain overall concentration of any or all tRNA species in an uncharged form might maintain the transport system operative, but as the pool amino acid level rises as a result of peptide uptake, more of the tRNA species would become charged and peptide uptake would be shut off through a deficiency of unloaded tRNA. It should be remembered here that the regulation of amino acid uptake by tRNA has been interpreted as an effect primarily of increasing the differential rate of synthesis of transport components relative to total cellular proteins (Quay and Oxender, 1976). No experimental information is available about any of the above mechanisms and it may be that no means exist by which bacteria can regulate the ingress of peptides. If this is so, then it would appear essential that the means for selective amino acid exodus be available in order to maintain optimal pool levels. In fact, experimental evidence that such exodus does accompany peptide uptake has recently been obtained in E . coli (Payne and Bell 1977b,c, 1979). It is conceivable that particular amino acid permeases that apparently function primarily for exodus could be involved here (Quay et a l . , 1977); these systems may indeed be

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activated as a result of peptide uptake whereas others that function primarily for amino acid uptake might be subjected to feedback inhibition. These possibilities may call for a reevaluation of certain data on the influence of peptides on amino acid transport (see, e.g., discussions in Payne and Bell, 1977c; Kiritani and Ohnishi, 1977). Finally, it is known that exogenous peptides can induce the synthesis of extracellular peptidases in some species (reviewed in Law, 1980; Hermsdorf and Simmonds, 1980; Payne, 1980) and the possibility of induced synthesis of peptide permeases should not yet be dismissed, for it is certain that neither growth tests nor radioactive assay methods could be used with any certainty to detect increased transport capacity in cells grown on peptides. Indirect evidence has appeared for derepression of peptide permease in E. coli via a CAMP-CRP mechanism (Alper and Ames, 1978). 6.

INTERRELATIONS BETWEEN

UPTAKE, EXODUS, A N D METABOLISM

This topic was not considered in any earlier review and yet it is now apparent that it is of extreme importance, and will form a major research area in the future. In E. coli, uptake of defined peptides normally proceeds continuously and to completion, and concurrently most of the intracellularly freed amino acid residues undergo exodus (Payne and Bell, 1979). Subsequent scrutiny of the early literature has provided several examples, mainly with studies using radioactively labeled peptides, for the exodus phenomenon in E. coli and other species (Leach and Snell, 1960; Young et al., 1964; Pittman et al., 1967; Shelton and Nutter, 1974; Levine and Simmonds, 1962) however, it was hardly noted at the time and its significance certainly not appreciated. In some instances, failure to allow for the effect has almost certainly led to a dramatic underestimate of the rates of peptide transport (Young et al., 1964) while apparent anomalies in absorption, such as the apparent differences in the uptake of Gly-[14C]Alaand [14C]Gly-Ala(Leach and Snell, 1960), may well be explicable by a greater exodus of one moiety (glycine) than the other. In addition, auxotroph growth curves showing equal rates of growth on all members of some homologous peptide series (Gilvarg and Katchalski, 1965; Payne, 1968) need reevaluation, after the finding (Payne and Bell, 1979; J. W. Payne unpublished observations) that in such tests complete peptide uptake may occur very quickly, and that for much of the measured growth period the organisms are actually taking up and utilizing the effluxed amino acid residues. Not all peptides show equivalent exodus of the freed residues; there is little exodus of Glu, Asp, Asn, Gln, Trp, or Ser, although their peptides

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are well absorbed and hydrolyzed, and the other constituent moieties undergo efflux. This feature may occur when the pool has a larger than average capacity for certain residues, but also, and perhaps more importantly, when the residues are rapidly metabolized. This latter case apparently occurs for most of the above cited residues which, after their uptake as peptides, are not found in commensurate amounts in the intracellular pool. Another consequence of metabolism is the exodus of further amino acids that are not constituents of the transported peptide; for example, Gly (and Thr) exodus accompanies threonyl peptide transport (perhaps as a result of threonine aldolase activity), and valyl-peptide uptake causes the exodus of proline also (Payne and Bell, 1977b). It is likely therefore that transport of defined peptides may well affect both the activities and the synthesis of not only the permeases for the constituent amino acid residues but also enzymes in the biosynthetic pathways of the corresponding amino acids: there are already several examples of coordinate regulation of amino acid transport and synthesis (Oxender et al., 1980). Furthermore, once regulation at these levels is effected, then continued peptide uptake could lead to involvement of enzymes for the catabolism of the amino acids, and perhaps finally to extensive changes in general metabolism. Studies of these possibilities will require use of amino acid auxotrophs lacking key biosynthetic enzymes and also amino acid permease and regulatory mutants. One thing that is clear from the results so far is that in future studies of peptide transport and utilization cognizance must be taken of these varied interactions. Finally, it should be emphasized that the exodus observed when E . coli (and Salmonella typhimurium, J. W. Payne, unpublished observations) is incubated with just one or two defined peptides may not occur markedly in its natural habitat (Savage, 1977), because in the gut the spectrum of peptides prnduced by digestive hydrolysis of proteins should permit a more balanced uptake of amino acids, matching the requirements of the growing organism. H. Peptide Transport in Other Microorganisms 1. PEPTIDE TRANSPORT I N YEAST

In the preparation of this section extensive use was made of a recent review by Becker and Naider (1980), the main workers in this area. The yeasts are a group of unicellular eukaryotic microorganisms that occupy a position between the simple prokaryotes such as E . coli, and complex mammalian organs like the intestine. Specifically, they are a

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class of fungi in which unicellular forms predominate, and budding is the usual form of vegetative reproduction. Although the yeasts comprise a heterogeneous collection of organisms, most biochemical and physiological studies have been carried out using the one species Snccharomyces ceret,isiae. Many amino acid auxotrophs of S. cerevisiae are available: this coupled to the fact that extracellular peptidases are not produced by the species (or by Candida albicans) (Becker et ul., 1973; Marder et al., 1977; Naider et al., 1974) has allowed peptide transport to be studied using growth response, analogously to the procedures with bacteria. In fact, the exact locations of the yeast peptidases that hydrolyze transported peptides for nutritional use are not clearly established. Some are apparently cytoplasmic while others occur in organelles such as the vacuole; different peptides may therefore require hydrolysis at separate sites before their constituent amino acids are made nutritionally available, and for some this may involve crossing not only the cytoplasmic but also the vacuolar membrane (Frey and Rohm, 1977; Wolf and Holzer, 1980). Certainly compartmentalization of amino acids is recognized (Wiemken and Durr, 1974; Wipf and Leisinger, 1977; Eddy, 1980). This possibility of compartmentalization should therefore be borne in mind when growth studies are used as an index of peptide transport. a . Distinction between Amino Acid and Peptide Transport. Early nutritional studies showed that yeasts responded differently to amino acids and to peptides and provided indirect evidence for separate transport modes. Direct evidence came with the observation that a lysine auxotroph of S. cerevisiae could grow on the free amino acid but not on lysine peptides, although it possessed enzymes able to hydrolyze the peptides (Marder et ul., 1977). Becker and Naider (1980) refer to unpublished work in which they have isolated an apparent transport mutant of S. cerevisiae that can grow on Leu but not on Leu peptides, and in a reciprocal manner mutants deficient in general and specific amino acid permeases can transport peptides normally (Nisbet and Payne, 1979). b. A Single Di-, Oligopeptide Transport System. Unlike the situation in E. coli, it appears that a single system mediates both di- and oligopeptide transport in S. cerevisiae. Thus, both di- and trileucine inhibit growth of a methionine auxotroph on di- or trimethionine (Marder et ul., 1977), high dimethionine concentrations inhibit uptake of radioactive trimethionine (Becker and Naider, 1977), mutual competitive inhibition of transport between various di- and oligopeptides has been shown using direct fluorescent assays of uptake (Nisbet and Payne, 1979), and a single step mutant that loses both di- and oligopeptide uptake has been reported (unpublished observations, cited in Becker and Naider, 1980). This mu-

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tant may be similar to that isolated as a bacilysin-resistant strain from C. albicans (Kenig and Abraham, 1976). c. Structural Specificities of Peptide Trunsport Systems. No unambiguous conclusions can yet be drawn for the N-terminal a-amino group, although it seems that the requirements for this group are not as strict as with bacteria. Thus, Dunn and Dittmer (1951) reported that a-acetylation lowered the toxicity of certain peptide analogs for S. cerevisiae, while Jones ( 1977) reported that benzyloxycarbonyl (cbz) dileucine supported growth of 15 strains of this organism. More definitive tests (Naider e f ul., 1974; Marder et a / . , 1977) have indicated that the acetyl and cbz derivatives of some di- and tripeptides were utilized by auxotrophs while others were not. Becker and Naider (1977) reported that [3H]acetyl-trimethionine was taken up by S. cerevisiae, and uptake was inhibited by trimethionine, however, the converse competition could not be demonstrated and it remains a possibility that separate systems are involved. Varied results have been found for the terminal a-carboxyl group. In S. cerevisiue esterification of this group does not prevent utilization; the methyl esters of tri, tetra-, and pentamethionine are utilized, albeit after a prolonged lag phase, and trimethionine methyl ester is as effective as the free peptide in preventing uptake of labeled trimethionine (Naider et ml., 1974; Becker and Naider, 1977). In contrast, the same methyl esters were not utilized by a strain of C. albicans (Lichliter et a / . , 1976). Peptides with substituted peptide bonds such as in Gly-Sar, Gly-SarSar, and Gly-Sar-Sar-Sar are taken up intact by S. cerevisiae, and competition studies indicate that they enter by the same system as normal peptides (Nisbet and Payne, 1979). Similar requirements for stereochemical specificity are found as with E. coli. Thus, S. cereiisiae utilized only the LLL and LLD forms of MetMet-Met methyl ester, even though cell extracts could hydrolyze all of the eight diastereomers except the DDD form (Becker and Naider, 1980). The LLD methyl ester also inhibited uptake of radioactively labeled trimethionine, although less well than the LLL analog (Becker and Naider, 1977). Application of fluorescent methods has been particularly useful in studying amino acid side chains, and wide differences in uptake rates have been noted for a variety of peptides tested at a substrate concentration of 0.1 M .For example, relative to Ala, = 100 (i.e., mean initial rate of uptake, 2 . 2 nmole min-' mg-l dry wt) the following rates were found: Ala,, (70-IOO), Met, (50-70), Gly, (
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From these examples, assessment can be made of the extent to which changes in the composition and sequence of a peptide affect its rate of uptake. Results of earlier studies on this topic using ,auxotrophic growth responses are difficult to interpret because hydrolysis by peptidases may have been affected at least as much as transport. Thus, several lysine auxotrophs of S . cerevisiae and of C . albicans failed to utilize lysine homopeptides although mixed lysine peptides were utilized (Becker and Naider, 1977; Lichliter et al., 1976) (compare with previous transport rates and introduction to Section IV, 3). Competition studies indicate that peptides containing hydrophobic side chains are good competitors while those with Gly or Lys residues are poor (Marder et al., 1977). Peptides containing unnatural and substituted side chains can apparently be accumulated via the peptide permease (Kenig and Abraham, 1976; Kenig et al., 1976: Becker and Naider, 1980). No firm conclusions are yet available concerning size limit. Because the structure of the cell envelope in yeast is quite different from that in bacteria, different exclusion limits might be expected. There is evidence that passive diffusion into the yeast envelope is limited in a sieve-like manner (Scherrer et al., 1977; Arnold and Lacy, 1977). With respect to peptide penetration into S . cerevisiae little can be said, for strain differences are found in the utilization of different sized peptides. Thus, strain G 1333 utilizes pentamethionine (Naider et al., 1974) while others do not (Becker and Naider, 1980), and strain ZI-2D is unusual in that tetra- or pentapeptides are not apparently utilized nor do they compete with shorter chain peptides (Marder et a/., 1977). When considering size it should be noted that mating factors (hormones controlling sexual conjugation between haploid cells) in various yeasts are simple oligopeptides about a dozen residues long (Stotzler et al., 1977; Masui et al., 1977). d . Energy Coupling to Peptide Transport. The active transport of certain amino acids by yeast is energized by a H+ symport mechanism (Seaston et al., 1973; Eddy, 1980). Becker and Naider (1977) reported that peptide uptake by S. cerevisiae required an energy source, and was inhibited by various poisons, e.g., azide, DNP, and KCN, but their evidence that ATP was not involved, i.e., lack of inhibition by arsenate, is ambiguous because the incubation mixtures included phosphate which can prevent the inhibitory action of arsenate. None of these studies provided evidence for an active peptide transport process because peptides were not accumulated intact. However, evidence for this feature has been obtained using sarcosine peptides (Nisbet and Payne, 1979). e . Regulation of Peptide Transport. Few relevant studies have been performed. No evidence relating to the induction of peptide transport systems is available. Uptake is faster in cells grown on a poor nitrogen

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source (e.g., Pro, Glu) (Becker and Naider, 1977; Nisbet and Payne, 1979), and it is possible that NH4+ may repress peptide transport in a manner similar to that found for amino acid uptake (Grenson and Hou, 1972; Woodward and Cirillo, 1977; Eddy, 1980), Unlike the situation in E . coli (Section IV, G, 6 ) , exodus of released amino acid residues does not immediately follow peptide uptake, perhaps because more extensive accumulation of the cleaved amino acid residues can occur (Nisbet and Payne, 1979). The intracellular locations of these accumulated amino acid residues has not been established but it is likely that the bulk is in the vacuole.

2. PEPTIDE TRANSPORT IN OTHER FUNGI To date, investigations have been confined to the Ascomycete fungus Neurospora crassa and have been carried out almost exclusively by Wolfinbarger who has recently reviewed the area (Wolfinbarger, 1980). At present, general conclusions must be based on limited studies, mostly using auxotrophic growth tests. In this organism these are complicated by the presence of extracellular peptidases, and transport of peptides per se is adjudged positive only if auxotrophic growth on a peptide is not affected by an amino acid that blocks transport (and thus auxotroph utilization) of the required free amino acid. Compartmentalization is also a potential difficulty in this organism (Weiss and Davis, 1977). Using the growth test approach, tripeptide uptake was demonstrated but dipeptides were utilized only following extracellular hydrolysis (Wolfinbarger and Marzluf, 1974). Uptake of intact oligopeptide has not been demonstrated and involvement of a peptidase-linked uptake system cannot be excluded. In a later study (Wolfinbarger and Marzluf, 1975b), uptake of Gly-Le~-[~H]Tyr was described; kinetic constants were, K t = 3.4 X low5M and V,,, = 2 nmole min-' mg-' dry wt, values that are comparable to those found in yeast but considerably lower in affinity than usually found in bacteria. Although a number of tripeptides inhibited uptake of the radiolabeled tripeptide, acetyl-Gly-Leu-Tyr did not compete, perhaps indicating a requirement for a free amino terminus. In a certain mutant strain, ability to utilize leucine peptides was lost and uptake of the labeled tripeptide was decreased by over 90%; this mutant is therefore a presumptive opp- strain. Although it was expected from molecular sieving studies of Neurospora cell walls that large peptides might be transported, a detailed study (Wolfinbarger and Marzluf, 1975a) revealed a comparable cut-off size to that found with E . coli. However, experiments with mutants (0s-1) having highly porous walls led the authors to suggest that the size restriction

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occurred at the oligopeptide transport system rather than the cell wall. In later unpublished work (Wolfinbarger, 1980) it was found that when peptides were supplied both as sole nitrogen source and as a source of required amino acid the large (excluded) peptides could specifically induce secretion of peptidase/protease activity that facilitated their cleavage; the smaller peptides that could be transported directly did not induce this secretion. Interestingly, in the glt-r mutant both large and small peptides caused secretion of proteolytic activity. Drucker and co-workers (Cohen and Drucker, 1977) have extensively studied protease regulation and secretion in N . crassa.

3 . PEPTIDE TRANSPORT I N ALGAEA N D LICHENS

N o reports have appeared of direct studies of peptide transport in these organisms. However, fluorescent assays should be entirely feasible, especially with simple algae such as Anabena and Chlorella. A few defined peptides have been shown to be utilized nutritionally by simple algae, but the form and manner in which they were absorbed were not established. More reports testify to the converse situation, i.e., appearance of peptides and certain other nitrogenous compounds in the medium during growth of algae on more simple nitrogen sources (Fogg, 1962; Taha and Elregai, 1962; Stewart, 1963; Whitton, 1965; Millbank, 1974), although the way in which they appear (direct efflux?) has not been established. These reports are of possible relevance to lichen growth in which it appears that the transfer of nitrogen from the algal symbiont to its fungal associate may be mainly in the form of peptides (Millbank, 1974, 1976). The general topic of transport and utilization of nitrogen sources by algae and lichens has recently been reviewed by Stewart (1980). 1. Related Topics

1 . PEPTIDES AS SUBSTRATES FOR FUNDAMENTAL STUDIESOF MICROBIAL TRANSPORT SYSTEMS

Much of the research into the transport of peptides in microorganisms has been motivated by a desire to determine the nutritional significance of the process for different groups of organisms. However, when one considers peptide transport in microorganisms, it can now be argued that the characteristics so far established, and the chemical versatility inherent in peptides, makes peptide transport a system par excellence for fundamental investigations of the mechanisms of active transport and this applies not only to microorganisms but also to animals and higher plants.

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For example, for what other permease can one so easily modify the substrate so as to vary its size, net charge, spatial distribution of charge, isoelectric point, hydrophobicity, hydrophilicity , etc. and assess how such changes affect the kinetic parameters, energy dependence, and regulatory control of the process? It is our hope, therefore, that with satisfactory techniques (i.e., fluorescent labeling, ion exchange chromatography) now available for directly assaying peptide transport, the system(s) will be studied by an increasing number of workers in many areas of investigation.

2. THE“SMUGGLIN” CONCEPT When it was found that bacterial peptide permeases lacked specificity toward the side-chains and carboxyl terminus of their substrates (Section IV, G, 3), it was suggested that if other moieties were attached at these sites they might be carried into organisms via the peptide permeases and that such uptake could have great potential for the design of antibacterial agents (Payne, 1972~).The same might apply to cytotoxic agents in animal tumors. When the moiety attached is itself intrinsically impermeant the resultant complex is called a “smugglin” (Matthews and Payne, 1975b). Almost simultaneously, two groups experimentally verified that smugglins could be transported into E . coli and S. ryphimurium via the oligopeptide permease (Fickel and Gilvarg, 1973; Ames et al., 1973). Ames et al. (1973) referred to the process as illicit transport. Subsequently, many other workers have described peptide mimetics that are actively transported by peptide permeases into bacteria and yeasts (Diddens et a l . , 1976; Kenig and Abraham, 1976; Kenig et al., 1976; Lichliter et al., 1976; Scannell and Pruess, 1974; Allen et al., 1978; Alper and Ames, 1978); a number of these smugglins had antibiotic activity. However, most exciting has been the commercial exploitation of the concept, which has led to the rational design of a new class of antibiotics (Allen et al;, 1978). However, somewhat soberingly, it has recently been pointed out (Alper and Ames, 1978) that nature always recognizes a good idea first and that a great many natural antibiotics subvert peptide (and other) permeases involved in entry of nutrients into organisms. The first comprehensive review of this topic has been prepared by Ringrose (1980). : THEIRINTERACTIONS WITH MICROBIAL 3. “NONPROTEIN PEPTIDES” MEMBRANESA N D CELLS

Finally, it should be reiterated that the peptides considered previously have in the main been typical a-linked protein cleavage products. However, a spectrum of naturally occurring nonprotein peptides are known:

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ones containing D-residues, nonprotein amino acids, peptide and ester linkages, conjugated peptides, etc., and peptide chemists could no doubt continue synthesizing such novel compounds forever. In general, the functions of the natural products are unknown, although many of the better characterized ones affect the permeability properties of membranes and cells, and a common characteristic is ability to transfer ions into cells and vesicles. However, these effects are achieved by peptides with hydrophobic exteriors “dissolving” in the membrane and acting as carriers, or by formation of transmembrane channels with amphipathic peptides; in no case is there any evidence that these classes of peptides can use peptide permeases, and indeed their structures seem to preclude this possibility. Discussion of nonprotein peptides has appeared in other reviews (Matthews and Payne, 1975b; Payne, 1976; Ringrose, 1980; Stephenson and Ratledge, 1980).

V.

PEPTIDE TRANSPORT IN HIGHER PLANTS

A. Introductory Considerations

It has been known for many years that some higher plants are capable of obtaining their nitrogen from organic sources, and that it is in organic forms that nitrogen is translocated through the organism (Bollard, 1966). Bollard (1966) studied the utilization of a wide range of organic nitrogenous compounds by the floating water plant Spirodella oligorrhiza and showed that it could utilize several amino acids and dipeptides as sole sources of nitrogen, though the means by which the plant took up these compounds was not studied. Thus the peptides might have been taken up intact, or first hydrolyzed and then taken up in the form of free amino acids. Similarly, Salonen and Simola (1977) showed that some amino acids and dipeptides could be utilized as nitrogen sources for the callus of Atropa belludonna, but did not determine whether or not the peptides were transported as such into the cells. Nevertheless, transmembrane transport of small peptides has been demonstrated in higher plantsprobably in the carnivorous plant Sarracenia flavu and more recently and certainly in the scutellum of germinating barley, Hordeum vulgare. The detailed work in barley suggested that active transport of peptides may occur in many types of germinating seed, and indeed, recent studies (C. F. Higgins and J. W. Payne, unpublished observations) have provided evidence (accumulation of Gly-Sar against a concentration gradient) for similar systems in germinating wheat, oats, sorghum, and corn (Zea maize).

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B. Foliar Absorption of Peptides by Sarracenia flava

Plummer and Kethley (1964) studied uptake of a variety of nutrients by the pitcher-leaves of the carnivorous plant S . flava. They showed that free amino acids were taken up by the leaves, and also investigated foliar uptake of three dipeptides, DL-Ala-DL-Asp, DL-Ala-DL-Leu, and DL-AlaDL-Met. When these peptides were introduced into the pitcher, they were not hydrolyzed within the liquor it contained, but taken up intact by the leaf, subsequently undergoing hydrolysis within the leaf tissue, so that what was found in the tissue was a mixture of the peptide taken up and an increase in its constituent amino acids. DL-Ala-DL-Leu was taken up more rapidly than the other two peptides. This peptide also remained intact longer within the leaf than the other peptides, being detectable in the leaf for as long as 4-5 days. The details of the mechanism of foliar uptake of peptides were not studied. Plummer and Kethley were probably fortunate in their use of peptides containing D-amino acid residues, which are resistant to hydrolysis. Had they used peptides made up of L-amino acids, it is unlikely that they would have detected intact peptides in the leaves. C. Peptide Transport by the Scutellum of Germinating Barley 1. DEMONSTRATION OF PEPTIDE TRANSPORT BY T H E SCUTELLUM

The demonstration of active uptake of small peptides by the scutellum of germinating barley resulted from consideration of what was already known of the distribution and actions of proteases and peptidases in the germinating barley grain, providing a good example of the importance of integrating studies of hydrolysis and transport of nutrients (Matthews, 1977b). It was already known that during the germination of barley, the endopeptidases and carboxypeptidases of the endosperm (storage tissue) were likely to hydrolyze the proteins of the endosperm into a mixture of small peptides and amino acids, and that the scutellum, the absorptive organ responsible for transferring digestion products to the seedling, contained high activities of peptidases capable of hydrolyzing small peptides (Enari and Mikola, 1977; Sopanen et al., 1977). This led Mikola and Kolehmainen (1972) to make the prescient suggestion that small peptides as well as free amino acids might be taken up by the scutellum and hydrolyzed in this organ. As a result, two groups, Burston et al. (1977) and Higgins and Payne (1977a), tested the ability of the scutellum to take up small peptides, and showed that uptake of intact peptides did take place, and that it was the result of an active transport mechanism.

399

TRANSMEMBRANE TRANSPORT OF SMALL PEPTIDES

The anatomical details of the germinating barley grain, including the scutellum, are shown in Fig. 3. 2. ACTIVETRANSPORT OF PEPTIDES BY

THE

SCUTELLUM

As in the case of the small intestine (Section 11, C) the most convincing evidence for active transport of peptides by the scutellum of germinating barley is the ability of the organ to take up hydrolysis-resistant sarcosyl peptides against an apparent concentration gradient. Burston et al. (1977) and Sopanen et al. (1977) showed that Gly-Sar and Gly-Sar-Sar could be concentrated by isolated scutella incubated in solutions of these peptides in vituo, as did Higgins and Payne (1977a, 1978a). The possibility of adsorption to the surface of the tissue was ruled out by Higgins and Payne (1977a), who showed that Gly-Sar was translocated from the scutellum to the roots and shoots of the plant. The concentration gradients attainable with Gly-Sar were high: thus Sopanen et al. (1977) found an apparent tissue water:medium concentration gradient of 32: 1 after 9 hours incubation at 30°C in Gly-Sar ( 2 M ) .Higgins and Payne (1978~)have also shown that L-Leu-D-Leu, which, like Gly-Sar, is relatively resistant to hydrolysis, is another peptide which is apparently accumulated by the

aleurone layer

atarchy endosperm (major food store)

intermediate layer

acute1 lum

Structure of barley grain

FIG.3. The grain of germinating barley: diagrammatic.

400

D. M. MATTHEWS AND J. W. PAYNE

scutellum against a concentration gradient. Confirmatory evidence for active uptake of Gly-Sar has been provided by the finding that its uptake is greatly reduced by anoxia and a variety of metabolic inhibitors (Higgins and Payne, 1977b; Sopanen et NI., 1978). Sopanen et al. (1978) have shown that scutellar uptake of Gly-Gly, a peptide which, like most small peptides, is too rapidly hydrolyzed by the tissue to be detected in it in the intact form, is also greatly inhibited by anoxia and metabolic inhibitors. Many other small peptides apparently share the same uptake system as that utilized by Gly-Sar and Gly-Gly (Section V, C, 6 ) , so that they, too, are probably taken up by an active mechanism. 3. EFFECTOF IONS A N D pH

ON

PEPTIDE UPTAKE BY

THE

SCUTELLUM

Uptake of peptides by the scutellum of germinating barley is not Na+ dependent; removal of Na+ from the incubation medium or its replacement by K+ had no effect on uptake of Gly-Sar (Sopanen et al., 1977; Higgins and Payne, 1977b) nor did removal of Na+ affect uptake of GlyGly (Sopanen et al., 1978). Very high concentrations of NaCl or KCl (ca. 100 mM) had a moderate inhibitory on uptake of Gly-Gly, but similar concentrations of MgClz or CaCI, had no significant effect (Sopanen e f al., 1978). Higgins and Payne (1977b) found a pH optimum of 3.8 for uptake of Gly-Sar, and Sopanen et al. (1978) found a pH optimum of about 4.5 for Gly-Gly. These pH optima may appear surprisingly low, but in fact a pH optimum of about 4 is in good agreement with the apparent pH of the endosperm during germination, and the pH optimum of endosperm endopeptidase activity. Higgins and Payne (1977b) showed that uptake of Gly-Sar was equally good in citrate and phosphate buffers, but that acetate had a strong inhibitory effect on uptake. They suggested that this might be due to undissociated acetic acid molecules conducting protons across the plasma membrane, and that an H+ ion gradient may be involved in the energization of peptide transport in the scutellum, as it apparently is in many other active transport systems in plants and microorganisms. 4. STEREOSPECIFICITY OF PEPTIDE UPTAKEBY

THE

SCUTELLUM

As yet, little is known about the structural requirements for peptide transport by the scutellum of germinating barley, but Higgins and Payne ( 1978a,c) have established the stereochemical specificity of the process, and made some important observations about the features of this specificity. They found that many di- and tripeptides containing one or more D-amino acid residues were taken up by the scutellum, but the replacement of an L-amino acid residue in a peptide by the corresponding D-stereoisomer decreased apparent affinity for transport, reducing its

401

TRANSMEMBRANE TRANSPORT OF SMALL PEPTIDES

rate. Substitution of a second D-residue reduced apparent affinity for transport still further. Substitution of D-residues at the COOH-terminus of the peptide had a greater inhibitory effect on transport than substitution at the NH,-terminus. As in animal small intestine (Section 11, G) peptides containing D-amino acid residues competed for transport with those made up of L-amino acid residues and with Gly-Sar, apparently sharing the same transport system. In most cases, the peptides studied were not found intact within the scutellum, but among the evidence for their uptake in intact form was the very striking observation that D-Ala and D-Leu, which were not taken up at all in the free form, could be rapidly taken up when presented as certain peptides. This ruled out the possibility that uptake of amino acids from these peptides was the result of their hydrolysis followed by uptake of amino acids from free solution. 5 . MAXIMUM SIZEOF PEPTIDESTAKENUP

BY THE

SCUTELLUM

The two groups who initiated investigations of peptide transport by the scutellum used different strains of barley and slightly different experimental conditions. Burston et al., used H . vulgare L. cv. Himalaya, incubating at 30°C and Higgins and Payne used H. vulgare var. Maris Otter, Winter, incubating at 25°C. However, though both groups found that the scutellum accumulated Gly-Sar and Gly-Sar-Sar to a higher concentration than that in the incubation medium neither group found that there was appreciable uptake of Gly-Sar-Sar-Sar. This tended to give the impression that the scutellum might be limited to uptake of di- and tripeptides, though Sopanen et al. (1977) did show that tetra-Gly had a moderate inhibitory effect on uptake of Gly-Sar, suggesting that the tetrapeptide had some affinity for transport, though less than that of diand tri-Gly, which were much more powerful inhibitors. Higgins and Payne (1978a) found that of the series di-, tri-, tetra-, and penta-Gly only the di- and tripeptides were taken up. In spite of this they found that all peptides in the series di-, tri-, tetra-, and penta-Ala were rapidly taken up, at several times the rate of equimolar free Ala, uptake of tetra-Ala being the most rapid. This shows that the scutellum has the ability to transport certain peptides of up to at least five amino acid residues. 6. INDEPENDENCE OF SCUTELLAR UPTAKEOF PEPTIDES AND AMINO FOR UPTAKEBETWEEN PEPTIDES ACIDSA N D COMPETITION

As in most if not all organisms, peptide uptake by the scutellum of germinating barley is independent of that of free amino acids. Burston et al. (1977) and Sopanen et al. (1977) showed that Gly had no inhibitory

402

D.

M. MATTHEWS AND J.

W. PAYNE

effect on uptake of Gly-Sar. Higgins and Payne (1978b) tested the effect of a number of free amino acids on uptake of Gly-Sar, Gly-Sar-Sar, AlaAla, and Gly-Ile, but no inhibitory effect was found. Sopanen el al. (1978) found no inhibitory effect of Gly or Leu on uptake of Gly-Gly. Many experiments have now shown that peptides do compete among themselves for scutellar uptake. Gly-Gly , Gly-Gly-Gly, and Gly-Gly-GlyGly all inhibited uptake of Gly-Sar (Burston et al., 1977; Sopanen et al., 1977). Higgins and Payne (1978b) carried out a survey of competitive effects among many small peptides, the results suggesting that di-, tri-, tetra-, and pentapeptides all shared a common transport system. Moreover, they showed by extrapolation of their data to infinitely high concentrations of inhibitors that di-Ala and tri-Ala could cause total inhibition of uptake of Gly-Sar and Gly-Sar-Sar, showing that neither Gly-Sar nor Gly-Sar-Sar was transported, wholly or in part, by a system unavailable to the inhibitor peptides. Sopanen et al. (1978) tested the inhibitory effect of 10 dipeptides of widely different structure, including peptides containing basic or acidic amino acid residues and dipeptides of Pro, on uptake of Gly-Gly; inhibition was shown in all cases. They also showed that inhibition of uptake of Gly-Gly by Gly-Sar was competitive, and that Gly-Sar could cause total inhibition of mediated uptake of GlyGly at infinitely high inhibitor concentrations, showing that no Gly-Gly was taken up by a system unavailable to Gly-Sar. To summarize, the impression given by these investigations is that peptides of two to five amino acid residues share a common active uptake mechanism by the scutellum. So far, no evidence has been published to suggest the existence of more than one peptide uptake system in the scutellum, but there is certainly a possibility that more than one exists (T. Sopanen, 1978 personal communcation). OF PEPTIDE UPTAKEBY 7. KINETICS

THE

SCUTELLUM

As in animal small intestine (Section 11, L) and certain microorganisms (Section IV), uptake of peptides by the scutellum of germinating barley may be more rapid, in some cases much more rapid, than that of free amino acids. For example, Burston et al. (1977) showed that uptake of Gly-Gly (40 mM) was about twice as rapid as that of equimolar Gly. At medium concentrations of 1 mM, di-, tri-, tetra-, and penta-Ala were all taken up at several times the rate of equimolar free Ala, di- and tri-Gly were taken up respectively at two or more than three times the rate of uptake of equimolar Gly, and Gly-Ile was taken up at about seven times the rate of Ile (Higgins and Payne, 1978a). This phenomenon suggests the quantitative importance of peptide uptake by the scutellum, as opposed to uptake of free amino acids.

TRANSMEMBRANE TRANSPORT OF SMALL PEPTIDES

403

It was shown by Sopanen e f al., (1977) (at 30°C and pH 5.2), after correction for a very minor nonmediated component in uptake, that uptake of Gly-Sar by the scutellum conformed to Michaelis-Menten 1.0 pmole gm wet wt-' kinetics, apparent K , being 9.6 mM and V,, min-'. Higgins and Payne (1977b), working at 25°C and pH 4.2, also found that Gly-Sar was taken up by a saturable process. This became fully saturated at about 8 mM. Sopanen et al. found (at 30°C and pH 4 . 9 , and after correction for the nonmediated component in uptake, that uptake of Gly-Gly conformed to Michaelis- Menten kinetics, apparent K t being 2.3 mM and V,,, 0.7 pmole gm wet wt-' min-'. It may be of interest that the figure obtained for K t for Gly-Sar in barley scutellum in vitro by Sopanen et ul. (1977) is very similar to that obtained by Sleisenger et al. (1976) for hamster jejunum in vitro (Section 11, M), and that the scutellum can take up this peptide at a maximal rate, at 30"C, which is roughly onethird of its maximal rate of uptake by hamster intestine at 37°C. In this connection, it should be borne in mind that, unlike the intestine, the scutellum is an organ without villi which increase its area.

8. CONCLUDING REMARKS The isolated scutellum of germinating barley is a particularly useful preparation with which to study peptide transport. The experimental techniques required are simple. The preparation continues to take up peptides at a constant rate for 1-2 hours after the start of incubation, and results are far more reproducible than those with preparations of small intestine in vitro. Finally, the scutellum has the advantage that unlike the intestine, it is possible to study peptide transport in relative isolation, without the complication of simultaneous uptake of large amounts of free amino acids released at the cell surface and in the incubation medium. That the characterized peptide transport system of barley may actually play the role in vivo that has been speculated for it is supported by the recent finding (C. F. Higgins and J. W. Payne, unpublished observations) that a large pool of small peptides does indeed occur in the endosperm during the early stages of germination when the storage proteins are being hydrolyzed prior to their transfer into the embryo. VI. POSSIBLE PHYSIOLOGICAL ADVANTAGES OF TRANSMEMBRANE TRANSPORT OF SMALL PEPTIDES

Since some readers, especially animal physiologists, may have had the impression, for most of their lives, that small peptides are not transported across biological membranes at all, and that only free amino acids

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D. M. MATTHEWS AND J. W. PAYNE

undergo transmembrane transport, this discussion might be started by asking the rhetorical question: why should small peptides not undergo mediated transmembrane transport? There is no obvious reason why not-and the evidence that they do so in many forms of life is now overwhelming. A molecular mechanism of the type responsible for active transport of free amino acids should surely, with appropriate modification, be capable of active transport of small peptides also. There may be certain advantages to the organism in the ability to take up peptides (Elliott and O'Connor, 1977): several have been suggested. The authors do not necessarily agree with all the suggestions mentioned. One advantage may lie in the conservation of metabolic energy. A substantial proportion of the metabolic energy utilized by the cell is believed to be expended in transport. Reduction of this expenditure of energy by peptide transport in place of amino acid transport might be of little significance in animals, in which the main organ taking up peptides is the small intestine, since this forms only a small part of the total body massbut in unicellular organisms, conservation of metabolic energy by peptide uptake might be much more important. Peptide uptake, as opposed to the uptake of free amino acids, might reduce the energy requirement for transport in more than one way. First, if peptides entering the cell are rapidly hydrolyzed (as they appear to be in most instances in bacteria and in mammalian gut) the peptide concentration within the cell may be maintained at a lower level than that outside it. In such a case, peptides could, in principle at least, enter the cell by a process of facilitated diffusion, no expenditure of metabolic energy being required. Second, even if active transport is normally involved in peptide uptake, as is probable, theoretical considerations would suggest (neglecting the factor of electrical potential) that it should need no more energy to transport one molecule of a small peptide, containing two, three, or more amino acid residues, than to transport a single molecule of a free amino acid. If the effects of electrical potential are considered, it should be borne in mind that a free amino acid, if it bears a net negative charge, must go against an electrical gradient to reach the cell interior if this is negatively charged in relation to the environment, as in the absorptive cells of the small intestine. In some peptides, containing both negatively and positively charged amino acid residues, this effect would be minimized or avoided. Finally, Parsons (1972) and others have suggested that the free energy released in hydrolysis of small peptides, which is not negligible, might be utilized in some way in driving peptide transport. N o scheme describing how this might be done is known to us. A second advantage of the ability of organisms to take up peptides is nutritional. In the case of certain bacteria, this is indisputable; some

TRANSMEMBRANE TRANSPORT OF SMALL PEPTIDES

405

bacteria grow poorly if at all if supplied only with free amino acids, whereas if peptides are added, growth is excellent. Part at least of the explanation of this phenomenon, which is fully discussed by Matthews and Payne (1975a), is the avoidance of competition for uptake between amino acids which occurs when they are presented in peptide form. The nutritional value of “peptones” in culture media for certain pathogenic bacteria has been recognized for nearly a century, though for about 70 years it remained an empirical observation. In animals, the nutritional importance of the ability of the intestinal mucosa to take up small peptides cannot yet be regarded as established beyond all possible doubt (except in the human amino acid transport defects of Hartnup disease and cystinuria), but there are reasons for suspecting that peptide uptake may be of major nutritional significance, and it is known that the ability of the small intestine to take up small peptides gives this organ a much greater transport capacity for protein digestion products than it would possess if it took up only free amino acids. As shown in Section 11, S, the intestinal absorption of free amino acids is a grossly uneven process, some amino acids being absorbed much more slowly than others. When mixtures of peptides and amino acids are absorbed, all amino acids are likely to be absorbed to an approximately equal extent in a given time, according to their proportions in the protein fed. This would be expected to lead to more simultaneous presentation to the tissues, possibly resulting in more effective protein synthesis. If this hypothesis (Matthews and Payne, 1975a) is correct, it could account for reports of superior growth in animals fed whole proteins or partial hydrolysates of proteins rather than the equivalent mixtures of free amino acids. What is not yet at all clear is the reason for the ability of animal tissues such as skeletal muscle to take up peptides by mediated transport (Section 111), for it is believed that most animal tissues do not normally encounter peptides except at low concentrations which are probably of no nutritional importance. It is generally agreed that amino acids circulate in the peripheral blood almost entirely in the free form. Were they to circulate as small peptides, the possibility of simple selective uptake of individual amino acids by a tissue from the bloodstream might be lost. The question of why amino acids are so often observed to be taken up in peptide form many times more rapidly than in the free form in representatives of all the main forms of life-animals, a higher plant, and microorganisms-is one for which no adequate answer can yet be suggested. It has been suggested that in the intestine at least most peptides enter cells down a concentration gradient. This may be a minor contributing factor in accelerating peptide entry. It certainly cannot be more

TABLE I COMPARISON OF CHARACTERISTICS OF PEPTIDE UPTAKEBY ANIMALS (SMALL INTESTINE), A HIGHER PLANT(BARLEY SCUTELLUM), A YEAST(S. cerevisiae), A N D BACTERIA (E. coli)

Relation to amino acid transport systems Independent Uptake can be faster Uptake can be more extensive Number of transport systems

Energetics Active transport Na+ dependent

Maximum size of transportable peptide (approx. number of residues) Structural requirements for optimal transport Free terminal NH2 group Free terminal COOH group

a-peptide bonds Stereochemical preference for L-form Amino acid side chains

Peptidase activity at cell surface

Animals

Plants

Yeast

Bacteria

Yes Yes Yes Probably one main system. Possibly others

Yes Yes n.d. Probably one main system. Possibly others

Yes n.d.n n.d. Possibly a single system

Yes Yes Yes Separate di- and oligo peptide systems

Yes Yes

Yes No. Possibly H+ linked

Yes n.d.

No. Dependence o n

5b

phosphate bond energy 5-6'

3

High specificity High specificity

Low specificity Medium specificity

Medium specificity Low specificity

High specificity Yes n.d. but lipophilic chain increases affinity Present for most peptides

n.d. Yes Low specificity

n.d. Yes Low specificity

Negligible

Probably not present

%.d. = not determined. bUptake of peptides of more than five residues has not been studied. 'Uptake is limited by "sieving effect" of outer cell wall.

Yes

High specificity High specificity for di-, low specificity for oligopeptides High specificity Yes Low specificity

Not present for E. coli but can be for some other bacteria

TRANSMEMBRANE TRANSPORT OF SMALL PEPTIDES

407

than a contributing factor in this situation since hydrolysis-resistant peptides which are concentrated in the cell may be taken up much more rapidly than the equivalent free amino acids in both animal intestine and microorganisms. For example, uptake of P-Ala-His by hamster jejunum (Section 11, C) may occur at about three times the rate of uptake of the equivalent free amino acids (Matthews ef nl., 1974).

VII.

CONCLUDING REMARKS

The bulk of the work described in the present article has been carried out over the last 10 years. During this time it has become clear that active transmembrane transport of small peptides is a mechanism of very wide distribution in nature and one which must be of major biological importance. Table I summarizes the main characteristics of peptide transport in animal intestine, the scutellum of germinating barley, a yeast, and in E . coli. While the authors believe that many of the main features of transport of small peptides in several very different organisms have now been established, a great deal of work remains to be done before we have an understanding of this process and its biological significance. The field is one in which many more investigators might usefully be involved. At this stage, any review of the field can only be inconclusive in parts, and must have many deficiences. The reader can hardly help sharing the feelings attributed to Hadrian in a letter nearly 2000 years old: “But these reports, so artlessly detailed, add to my store of documents without aiding me in the least to render a final verdict” (Yourcenar, 1978).

ACKNOWLEDGMENTS D. M. M. gratefully acknowledges financial or other support for work on peptide transport by himself and his sabbatical and other collaborators from the Medical Research Council of Great Britain, the Westminster Hospital Special Trustees, the Variety Club of Great Britain, the Macy Foundation, the Finnish Cultural Foundation and the Emil Aaltonen Foundation of Finland, the Wellcome Research Laboratories, the British Industrial Biological Research Association, Syntex of California, Roche Products Limited, Byk Gulden Pharmazeutika, and COFAG (Comite des Fabricants d’Acide Glutamique). J . W. P. is indebted to the following collaborators and students for their invaluable contributions toward t h e results of his work described here: G. Bell, C. F. Higgins, T. M. Nisbet, and G. M. Payne. These studies have been supported by grants from the Science Research Council, the Royal Society, and the Smith Kline and French Foundation. Both authors are grateful to their wives for assistance and tolerance. We are grateful for much invaluable secretarial assistance, in particular to that of Mrs. C. E. Angelo, who was responsible for the bulk of the secretarial work.

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Wiemken, A,, and Durr, M. (1974). Characterization of amino acid pools in the vacuolar compartment of Strcchriromycrs cere,i.sitie. Arch. Microhiol. 101, 45-57. Wipf, B., and Leisinger, T . ( 1977). Compartmentation of arginine biosynthesis in Scrcchtrrotnyces cerevisicre. FEMS Lett. 2, 239-242. Wiseman, G. ( 1974). Absorption of protein digestion products. I n “Intestinal Absorption” ( D . H . Smyth, ed.), Biomembranes, Vol. 4A, pp. 363-481. Plenum, New York. Wiseman, G . ( 1977). Site of intestinal dipeptide hydrolysis. J . Phy.tiol. (London) 273, 731743. Wojnarowska, F., and Gray, G. M. (1975). Intestinal surface peptide hydrolases: identification and characterization of three enzymes from rat brush border. Biochiw. Biophys. Actci 403, 147-160. Wolf, D., and Holzer, H., (1980). Proteolysis in yeast. In “Microorganisms and Nitrogen Sources” ( J . W. Payne, ed.), pp. 431-458. Wiley, New York. Wolfinbarger, L. ( 1980). Transport and utilization of peptides by fungi. I n “Microorganisms and Nitrogen Sources” ( J . W. Payne, ed.), pp. 281-300. Wiley, New York. Wolfinbarger, L., and Marzluf, G. A. (1974). Peptide utilization by amino acid auxotrophs of Neuro.sporci crcr.s.sci. J . Bercreriol. 119, 371-378. Wolfinbarger, L., and Marzluf, G. A. (1975a). Size restriction on utilization of peptides by amino acid auxotrophs of Neurosporrr crassa. J . Btrcteriol. 122, 949-956. Wolfinbarger, L . , and Marzluf, G. A. (1975b). Specificity and regulation ofpeptide transport in Nerrrosportr cci~sci.Arch. Biochem. Biophys. 171, 637-644. Woodward, J . R., and Cirillo, V. P. (1977). Amino acid transport and metabolism in nitrogen-starved cells of Sercrherrotnyces ceretlisicie. J . Brrcteriol. 130, 714-723. Yamaguchi, T., Yamaguchi, M., and Lajtha, A. (1970). Inhibition of dipeptide transport in mouse brain slices. J . Nrurol. Sci. 10, 323-329.

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Yang, S . L., Becker, J . M., and Naider, F. (1977). Transport of (IT) Gly-Pro in a proline peptidase mutant of Scilrnonellci typhirnuriurn. Biochim. BiophyJ. Acta 471, 135- 144. Yoder, 0. C., Beamer, K . C., Cipolloni, P. B., and Shelton, D. C. (1965a). Kinetic study of L-valine and glycyl-L-valine uptake by Leuconostoc mesenteroides. Arch. Biochern. BiophyJ. 110, 336-343. Yoder, 0. C., Beamer, K . C., and Shelton, D. C. (1965b). Stuctural and stereochemical specificity in transport systems for glycine, valine and their dipeptides in Leuconostoc rnesenteroideJ. Fed. Proc. Fed. A m . Soc. Exp. Biol. 24, 352. Young, E. A . , Bowen, D. O., and Diehl, J . F. (1964). Transport studies with peptides containing unnatural amino acids. Biochern. Biophys. Res. Cornmun. 14, 250-255. Yourcenar, M. (1978). “Memoirs of Hadrian,” p. 25. Penguin Books, London. Zeman, F. J., and Fratzke, M . L. (1977). Protein, dipeptide, and amino acid absorption in the young of protein-deprived rats. Pedicitr. Re5. 11, 972-977. Zimmerman, W., and Rosselet, A . (1977). Function of the outer membrane of Escherichiu coli a s a permeability barrier to beta-lactam antibiotics. Antirnicrob. Agents Chernotlier. 12. 368-378.

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C U R R E N T TOPICS IN MEMBRANES A N D TRANSPORT, VOLUME

14

Characteristics of Epit he Iial Transport in Insect Malpighian Tubules S . H . P. MADDRELL Agricultural Research Couricil Uriit of Imvrtebrate Chemistry arid Physiology Departmerit of Zoology, Uriiversity of Cambridge Cambridge, Eriglarid

I . The Route of Water Transport . . . . . . . . . Transepithelial Potential Differences in Malpighian Tubules 11. The Passive Epithelial Permeability of Malpighian Tubules 111. Correlation of Structure with Function . . . . . . IV. Regulatory Properties of Malpighian Tubules . . . . A. Hormonal Effects on Malpighian Tubules . . . . B. Autonomous Regulation . . . . . . . . . C . Inducible Transport Systems in Malpighian Tubules . V. Malpighian Tubule Action in the Absorption of Water Vapor VI. Summarizing Remarks . . . . . . . . . . . References . . . . . . . . . . . . . . .

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438 442 445 445 453 . 456 . 457 . 459 460

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Insect Malpighian tubules form an important part of the insect excretory system. They are responsible for the formation of the primary excretory fluid and some of its subsequent modification. They make excellent material for the study of many aspects of epithelial transport as they continue to function normally in isolated in v i m preparations. Isolated tubules can survive for prolonged periods; under suitable conditions they will often continue to secrete fluid for 2-3 days. When dissected from the insect they are long, easily unraveled tubules of diameter 50-200 /.m whose walls are of a single cell layer surrounded only by a thin acellular basement membrane which can relatively easily be penetrated by microelectrodes. It is simple to record the transmural potential difference simultaneously with measurements of the rate of fluid secretion. They can readily be cannulated so that their lumina can be perfused with saline solutions of varying composition. Most insects have two to six such 427

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tubules but in some t h e number is much higher (up to 200 occur in a few cases). In very small insects the tubules are less than 1 mm long but, in most cases, their length is in the range 20-100 mm. The range of transport functions they perform is very wide. They carry out fluid transport at rates that can be altered very considerably-as much as 1 0 0 0 ~in some cases, by hormones and by changes in ion levels. In extreme cases the rate of fluid secretion can reach 80 pl sec-' per gram of tissue, which is the highest yet recorded for any tissue. In addition they can actively transport such inorganic ions as sodium, potassium, chloride, magnesium, sulfate, and ammonium at high rates and for some of these ions the transport systems are inducible. Organic substances which are transported by Malpighian tubules include sugars, alkaloids (such as nicotine, morphine, and atropine), organic anions (such as sulfonates, acylamides, and acidic dyes), and even glycosides such as ouabain. Again in many cases the rate of transport of these substances depends on the extent and level of exposure to which the tubules have been subjected. In a few aberrant cases Malpighian tubules are known to be able to secrete silk or, even in the glowworm, Arachrzocampa (Green, 1979) emit light. In this article four aspects of Malpighian tubule function are dealt with. The first topic concerns the route and mechanism of water transport across the epithelium. Second, the permeability properties of Malpighian tubules to a wide variety of solutes are described and the implications of these for the functioning of the tubules are discussed. In the next section the question of how well structure and function are correlated is examined; the supposition that all cells of an epithelium that look alike function alike is criticized. Finally, several regulatory properties of Malpighian tubules-their responses to hormones, how they autonomously regulate the level of some blood solutes, the induction of specific transport systems, and their reabsorptive abilities.-are covered.

I. THE ROUTE OF WATER TRANSPORT

An important feature of much recent work on transepithelial fluid transport has been a concern with the route taken by water as it moves in response to solute transport. In such vertebrate fluid transporting epithelia as the gallbladder and proximal kidney tubules, it is now believed that a large fraction of the transepithelial water flow passes between the cells by the so-called paracellular route (Sackin and Boulpaep, 1975; Hill, 1977; Gupta et al., 1978). Passage between the cells obviates the need to suppose that the cell membranes of the epithelial

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cells have the extremely high permeabilities they would need if they were to achieve fluid transport by osmotic coupling (Hill, 1975). While this may be so, explaining how fluid can be transported through intercellular junctions which occupy only a tiny fraction of the area of the epithelium also presents great difficulties. At least, however, it is no longer necessary to suppose that the cells come into osmotic equilibrium with the transported fluid. A crucial element in this has been the discovery (Hill, 1977) that the gallbladder of Necturus can transport, out of the lumen, fluid whose osmotic concentration is only of the order of 2-3 mosmole liter-' when it is filled with solution of concentration about 1 mosmole liter1. Insect Malpighian tubules are passively permeable to organic solutes such as xylose, sucrose, and inulin. It is scarcely conceivable that all these substances cross the epithelial wall by other than paracellular routes. One might therefore expect a correlation between the rate of trans-wall movement of these markers and the rate of any water flow crossing the epithelium by the paracellular route. The extent of any such correlation would, of course, be dependent on the relative rates of diffusion of solute and the bulk flow rate of water. Fortunately many insect Malpighian tubules can be stimulated to secrete fluid at high rates, while electron microscopy shows that the area of their intercellular clefts is not large. It follows from this that if much of the transported fluid crosses via the intercellular clefts it must travel at a rate that is large in comparison to the speed of diffusion. For example, Malpighian tubule from the blood-sucking insect Rhodnius can each secrete fluid in vitro at rates above 120 nl min-'. The intercellular clefts are about 17 nm in width and occupy about 0.034% of the frontal area of the tubule. It follows from this that if all the fluid moved through the intercellular clefts it would pass through at a speed of 660 p m sec-'. Since each cleft is some 15 wm long the fluid would take only 22 msec to travel through it. Even small ions and solutes such as urea would take two to three times as long as this to diffuse 15 pm. In this case, therefore, one would perhaps expect a strong correlation between the rate of fluid secretion and passive transepithelial movements of extracellular markers. Preliminary experiments have been done by the author in which the rate of fluid secretion by the Malpighian tubules of Rhodnius has been increased from less than 1 nl min-l to more than 70 nl min-l by treatment with 5-hydroxytryptamine (which mimics the insect's own diuretic hormone in stimulating greatly accelerated fluid transport by the tubules). None of the extracellular markers used (xylose, sucrose, and inulin) were found to cross the walls of the tubules any faster during fast fluid secretion. This is powerful evidence that at least in this insect water flow does

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not occur primarily through the paracellular clefts. Confirmatory experiments have been made in which passive lumen to bath movements of the same markers have been measured during changes in bath to lumen fluid transport. Again no changes in flux were found. On the basis of these preliminary findings, it looks as if we have to conclude that water movement must largely be routed through the cells themselves. With the benefit of these findings one can now make several observations about the different route followed by water in the Malpighian tubules of at least this one insect. First, if molecules as large as inulin can penetrate the intercellular junctions it is difficult to see how an osmotic gradient of sufficient size could be created across the junction to draw water through at the observed rates. Therefore, it should not surprise us that water flow is not achieved in this way. In contrast the gallbladder epithelium must presumably be sufficiently “tight” not to allow passive transepithelial movements of substances of the size of the bile salts whose concentration in the lumen it is the function of the epithelium to achieve. Second, if Rhodnius Malpighian tubules could channel the water they transport through the 17-nm slits between the cells, calculation shows that the pressure head required, even assuming laminar Poiseuille flow through unimpeded parallel-sided apertures, would be of the order of 10 atmospheres. In fact, the intercellular boundaries are characterized by the presence of smooth septate junctions along nearly their entire lengths. As calculated by Filshie and Flower (1977), the presence of complex arrangements of septa, even if they are relatively short with frequent blind endings, dramatically reduces the cross-sectional area available for intercellular permeation and increases the transepithelial path length. These considerations make it very difficult to suppose that water follows an intercellular route in fast fluid secretion by Rhodnius Malpighian tubules. Third, if one compares the overall anatomy of the epithelium of the vertebrate gallbladder with that of an insect Malpighian tubule, the differences are pronounced. Furthermore, the differences can be reconciled with the different routes that water is thought to follow in the two cases. The gallbladder epithelium (Fig. la) is characterized by columnar cells with relatively tight intercellular junctions which, however, occupy only a tiny fraction of the epithelial thickness. The intercellular spaces over much of their length are wide, offering little resistance to longitudinal fluid flow. The apical cell membranes facing the side from which fluid is taken are not greatly folded or decorated with mitochondria. In contrast Malpighian tubule cells (Fig. Ib) are squamous in type being much wider than they are thick. Their intercellular clefts therefore

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( a ) Gallbladder

(b)

Malpighian tubu

FIG. 1. Diagrammatic representation of the structure of the epithelial walls of (a) vertebrate gallbladder and (b) insect malpighian tubule. In both cases, fluid secretion is directed toward t h e bottom of the diagram.

are not long, but the neighboring cells are held over their whole depth relatively much closer together than in the gallbladder. This is achieved by the extensive septate junctions. These junctions, although long, are much more permeable (in that they allow the passage of large molecules such as inulin) than those of the gallbladder. Both apical and basal faces of Malpighian tubule cells show extensive folding of the plasma membranes. Basally there are deep infolds which may reach close to the nearest parts of the apical membranes. Apically Malpighian tubule cells have close packed microvilli which greatly increase the luminal surface area. Mitochondria occur in large numbers in association with both surfaces of the cell. In fast secreting tubules, in particular, the apical microvilli are long and most have mitochondria in them. The evidence thus suggests that water moves through the cells of Malpighian tubules. This idea goes a long way to explain the structural and functional differences between the epithelia of Malpighian tubules and the gallbladder. However the very differences raise an obvious and

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important question. If paracellular water transport overcomes some of the many difficulties involved in explaining water transport via a cellular route, how is one to explain water transport in Malpighian tubules'? Before attempting to answer this, one further set of observations on insect Malpighian tubules should be described. We have seen how the finding that gallbladder epithelium can achieve almost isoosmotic transport of fluids as dilute as I mosmole liter-' (Hill, 1977) was finally responsible for the abandonment of mechanisms which proposed osmotic or electroosmotic coupling at two cell membranes in series. How do Malpighian tubules fare when presented with fluids of similarly low 0smotic concentrations'? The results of some recent experiments to test this are shown in Fig. 2 . The solutions were made by diluting standard saline with distilled water. The results show that while Rhodnius Malpighian tubules can continue to transport fluid when immersed in fluid of osmotic concentrations as low as 50 mosmole liter1, they clearly cannot function in the much lower concentrations that the gallbladder can. This is in spite of the fact that insects, as small animals with relatively high surface areas, are likely to have evolved tissues capable of functioning under a range of osmotic concentrations. The fact that the gallbladder can operate in fluids of much lower osmotic concentrations than can Malpighian tubules thus strongly suggests that the mechanisms of fluid secretion are fundamentally different. As seems likely from the other evidence discussed, water flow in Malpighian tubules may well occur through the cells. How then do Malpighian tubules secrete fluid'? Previous attempts to answer this (Maddrell, 1971b, 1977a) have pointed out that fluid transport depends on ion transport but have not been able to suggest any really satisfactory way of explaining how coupling between ion movements and water flow might be achieved. Some recent preliminary studies (Maddrell and Gardiner, unpublished results) have again raised the possibility that the coupling might involve osmosis. The experiments which have led to this suggestion are as follows. When the solution bathing a Malpighian tubule of Rhodnius was exchanged for one of lower osmotic concentration, there were very obvious rapid movements of water into the lumen. This suggested, of course, that the tubule wall had a very high osmotic permeability. This has now been investigated in a more systematic way by perfusing at a known rate fluid of known concentration through the lumen of a short length of tubule. The free length of tubule was run through a bathing drop of known lower osmotic concentration. From the rate at which fluid emerged from the open end of the tubule, the osmotic influx of water could be measured. From such measurements it is already clear that the effective osmotic

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permeability of the wall of the Rhodtzius Malpighian tubule is higher than cm sec-' osmole-' liter and it may be as high as 2 x cm sec-* osmole-' liter. With the very real possibility that the layers of luminal fluid nearest the tubule wall may not be effectively stirred by the perfusing fluid, the actual osmotic permeability may well be higher than indicated by these figures. These experiments suggested that it was worth reexamining the possibility that simple osmosis might explain water movements in Malpighian tubules. From first principles' it can readily be shown that the osmotic concentration, C,, of fluid produced in response to solute transport across a plane membrane from a fluid of osmotic concentration C,,, is given by the formula

c,.= C,,+ [ Cf +2 4 ( N / P ) ] 1 ' 2 It can be seen that the crucial parameter in determining C, is the rario between N , the rate of solute transport per unit area of membrane, and P, the osmotic permeability of the membrane. If ion transport across a membrane is to give rise, by simple osmosis, to a fluid of concentration not much above that, C,,, of the fluid on the other side of the membrane, then solute transport must be slow and spread over a large area of highly permeable membrane. In the case of Rhodnius Malpighian tubule, one can make estimates of the value of N from the known rate of solute transport and from electron micrographs showing the disposition of the membrane across which transport is thought to occur. The determinations of the overall osmotic permeThe flux of water, J , , , across a plane membrane separating two fluids of osmotic concentration C , and C , is P ( C , - C , , ) , where P is the osmotic permeability of the membrane. If N is the rate of solute transport across the membrane then C , = N / J , ; substituting for J , , we have

or, rearranging PC: - PC,C, - N

=0

from which

c, or

=

PC,, + ( P 2 C %+ 4NP)'" 2P

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Osmotic concentration of bathing fluid

(rnoirnole liter I I

FIG.2. The dependence of the rates of fluid secretion by isolated, stimulated Malpighian tubules of Rhodnius on the osmotic concentration of the bathing fluid. Each point represents the mean value at a particular osmotic concentration and the attached vertical lines indicate 2SE of the mean ( n = 6).

ability of the wall described above make it not unreasonable to suppose that the cell membranes are highly osmotically permeable. It is instructive to calculate the osmotic concentration of the fluids which would be transported by the individual cell membranes of the Rhodnius Malpighian tubule supposing for the moment that they were unfolded and plane. Let us take the case of a tubule secreting fluid at a rate of 100 nl min-I, a high rate but one often achieved by tubules under in virro conditions. In a bathing fluid of osmotic concentration 340 mosmole liter-l the rate of solute transport is close to 0.6 nosmole sec-' or 6.6 nosmole sec-' across each cm2 of tubule. To take first the apical cell membrane of the tubule, electron micrographs show that the cell membrane here is enormously elaborated. Not only is the cell surface increased by microvillar extensions but the extensions join together at intervals along their length increasing still further the membrane area in a unit volume of the cell border. Preliminary analysis suggests that the area of this side of the cell might be increased by 150 times or more. This gives a value for N of about 45 posmole sec-' c n r 2 of membrane.

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On the basal side of the cell, the membrane is less extensively folded, giving an increase in area of about 40 times. Solute transport across this side of the cell (thought to be driven by a chloride pump; see Maddrell, 1977b) would thus be at a density of 160 posmole sec-* cm-2. To gauge the effectiveness of these transport rates in promoting osmotic flow, one needs to know the actual osmotic permeability of the cell membranes. The experiments previously described provide figures only for the overall osmotic permeability of the epithelial wall. However, if one assumes that the fluids actually in contact with the cell membranes in these experiments were at the same osmotic concentrations as those in the bathing fluid and lumen (i.e., ignoring the unstirred layers in the basement membrane, in the basal infoldings, and in between the apical microvilli as well as any unstirred layers in the free bathing solution and luminal solution), and, in addition, supposing that apical and basal cell membranes have equal osmotic permeabilities, then one can estimate the osmotic permeability of the cell membrane. This is done using the formula 1 - 1 P , aP,

+-b P1 ,

(Hill, 1975), where P , is the overall effective osmotic permeability of the wall, P , is the actual osmotic permeability of the cell membranes, and a and b are the amplifications in area achieved by membrane folding on cm the two sides of the cell. Such a calculation gives a figure of 6 x sec-' osmole-' liter for the osmotic permeability of the cell membranes of the Rhodnius Malpighian tubule. This is a minimum figure as any unstirred layers have been ignored. Even using this figure, however, it follows from the formula given earlier that the fluid leaving the apical membrane of the tubule, if it were plane, would be more concentrated than the intracellular fluid by only about 2.2 mosmole liter-l. On the basal side, the fluid transported into the cell would be more concentrated than the bathing fluid by about 7.7 mosmole liter-l. From this it is not easy to determine the osmotic concentration of the fluid produced by both membranes acting together in series. One might argue along the following lines. Under steady-state conditions, fluids of the same osmotic concentrations must, of course, cross both cell membranes at the same rates. So if the fluid entering the cell were more concentrated than the bathing medium by 7.7 mosmole liter1, then this must also be the concentration of the fluid leaving the cell across the apical surface. It would then presumably be the case that the intracellular concentration would be lower than this by 2.2 mosmole liter-l. If this simple analysis is reasonable, then it follows that the fluid secreted by the tubule would be

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expected to be isoosmotic to the normal bathing fluid to within 2.2% which is similar to the value actually observed (Maddrell, 1969). Of course these calculations are unreal in that the cell membranes are anything but plane. The analysis of the osmotic flow to be expected from solute transport across folded cell membranes is complex and is rather sensitive to the assumptions and boundary conditions used. The analysis of Diamond and Bossert (1967) was confined to the situation of a single membrane-lined channel. They found that the fluid transported by osmosis in response to solute transport across the membrane of such a channel is markedly hyperosmotic unless the channel is very long and narrow. In Malpighian tubules, the channels are narrow but not long (usually less than 5 pm). However, as Fig. 1 shows, both apical and basal cell surfaces of Malpighian tubules are folded so as to produce an array of neighboring channels alternately opening to the cytoplasm and to the extracellular fluid. As pointed o u t before (Maddrell, 1971a), such an arrangement might act to improve osmotic coupling. The difficult analysis of the osmotic flow to be expected in response to solute transport across such an array is now being done and I am most grateful to Dr. D. L. S. McElwain for allowing me to quote from his unpublished work. The analysis is not yet complete, but at this stage it seems certain that osmotic coupling of solute and water in an array of channels is significantly better than in the single channel situation; i.e., the transported fluid has a lower osmotic concentration. Perhaps of greater importance, it turns out that the transported fluid has a lower osmotic concentration than would be expected from simple osmosis across a plane membrane. Since the calculations described suggest that Rhodnius Malpighian tubules might achieve nearly isoosmotic flow even if the cell membranes were plane, it does now seem likely that osmosis could play a significant part in explaining fluid transport in these tubules. If osmosis does play a significant part in explaining fast fluid secretion by Rhodnius Malpighian tubules, then the same explanation could well extend to the operation of other Malpighian tubules. They secrete fluid more slowly so that their rates of solute transport are lower. With cell membranes similar in osmotic permeability to those of Rhodnius, they need not have such extensive areas of membrane, and indeed the published micrographs show, for example, that their apical microvilli are shortest in the tubules that secrete fluid at the slowest rates, as has been pointed out before (Maddrell, 1971a). One can envisage that cell membranes of Malpighian tubules might have evolved with a suitable density of solute transport per unit area of osmotically permeable membrane. Tubules of different fluid secretion rates could then simply be produced by the inclusion of more or less membrane in unit length of tubule. Such

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an arrangement would have the advantage that the relative importance of trans-wall diffusion through the intercellular clefts and fluid secretion through the cells could be altered, with the benefits discussed in Sections I1 and IV. The conclusion we are left with is that it is likely that insect Malpighian tubules transport ions across cell membranes whose area, osmotic permeability, and folding are such as to allow osmotic movements of water sufficiently rapid that the fluid secreted is close to isoosmotic to the fluid bathing the tubules. None of this is to deny the possibility that solute transport and water movements might be coupled in other ways. However, the rapidity of osmotic flow observed across the tubule walls is so impressive as to give one a strong prejudice in favor of a mechanism involving osmosis. For example, in the experiments described on pp. 432-433, it was found that when a fluid hyperosmotic to the bathing fluid by 170 mosmole liter-' is rapidly perfused through the lumen of a Rhodrzius Malpighian tubule, nearly complete osmotic equilibration occurs in less than 5 seconds.

Transepithelial Potential Differences in Malpighian Tubules

Measurements of transepithelial electrical potential differences have emphasized the differences between Malpighian tubules on the one hand and epithelia such as gallbladder and proximal kidney tubule on the other. The cell-cell junctions of Malpighian tubules are permeable to substances as large as inulin, yet transepithelial potential differences as large as 100150 mV can often be measured (Maddrell, 1971a, 1977a). In contrast the trans-wall potentials of mammalian gallbladder and proximal kidney tubules are in the order of only a few millivolts (Giebisch, 1973; Diamond, 1962), though their cell-cell junctions are less permeable than those of Malpighian tubules. The most reasonable interpretation of this difference is that the electrical resistance of the intercellular clefts of Malpighian tubules is higher relative to that of the cells than is the case in gallbladder and kidney tubule. This presumably results from the low frontal area presented by the intercellular clefts of Malpighian tubules together with their narrowness over their whole length and the very large areas of plasma membrane of the apical and basal sides of the cells across which, presumably, the large potentials observed are developed. The differences in transepithelial potentials eloquently parallel the differences in importance that now seems to attach to the paracellular route in water transport by the two sets of epithelia. Mammalian gallbladder and kidney proximal tubule are absorptive epithelia in which fluid is transported into the

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blood. Malpighian tubules transport fluid in the opposite direction. The two types of epithelia differ in the relative positions of their closest cellcell junctions. In gallbladder and proximal tubule, sometimes known as “forward-facing” epithelia, the cells form their tightest junctions on the side nearer that from which they adsorb fluid (Fig. la). In “backwardfacing” epithelia, such as Malpighian tubules, the closest cell contact is on the side toward which fluid secretion is directed (Fig. Ib). A consequence of this is that the intercellular spaces face the direction of transport in epithelia such as gallbladder; this of course is why such epithelia are said to be forward facing. Because of this arrangement it is perhaps not surprising that at least some forward-facing epithelia have become adapted to transport fluid into the intercellular space; they then may be able to make use of the potentially very permeable cell-cell junctions. Such a strategy is not available to backward-facing epithelia.

II. THE PASSIVE EPITHELIAL PERMEABILITY OF MALPIGHIAN TUBULES As we have just seen, the intercellular clefts of Malpighian tubules are limited in area and must have a relatively high electrical resistance relative to the route across the cells. This suggests that even if the clefts allow large molecules to cross the tubule walls the overall permeability of the tubule to such substances would be low. How is it possible to square this with the idea that the tubules represent the essential permeable part of the excretory system of the insect? It may well be worth reiterating why it is believed that it is essential for excretory systems to be permeable to substances occurring in the extracellular fluid. As Ramsay (1958) has pointed out, such permeability allows animals automatically to excrete any novel toxic substances they might encounter. Such a substance would cross into the primary excretory fluid through the permeable areas of the system and then, because it would not selectively be reabsorbed, it would be eliminated. Evolution is bound to favor such a fail-safe mechanism, and all animals operate their excretory systems at least partly on the basis of the production of a primary excretory fluid, to which substances in the extracellular fluid have relatively free passive access, followed by selective active recovery of those substances the animal wishes to retain. Against this background it rings uncomfortably to emphasize the low overall permeability of insect Malpighian tubules. The following account attempts first to set out the relevant facts, then to reconcile the paradox just described, and finally to emphasize the advantages that have accrued

EPITHELIAL TRANSPORT IN INSECT MALPIGHIAN TUBULES

439

and the adaptations that have occurred during the evolution of the existing system. The first question to be decided is how permeable are Malpighian tubules or, more pertinently, how permeable are their intercellular junctions?. Compared with, say, the glomeruli of kidney tubules it would, at first sight, appear that they are much less permeable. For example, the fluid secreted by the tubules of the fly, Calliphora, contains D-glucose at concentrations less than 25% as high as in the bathing medium (Knowles, 1975). This is partly due to reabsorption, but even L-glucose appears in the secreted fluid at only half the concentration of that in the bathing medium. This contrasts very strongly of course with the situation in the human kidney where glomerular fluid/plasma ratios not significantly different from I are found with compounds as large as inulin (MW c5000) o r dextran (MW 17,000) (for references, see Smith, 1951). However, one has to remember that the intercellular junctions form only a small part of the area of Malpighian tubules and that fluid secretion almost certainly occurs through the cells. The only real way to examine the permeability of the junctions is to compare the rates at which substances of widely differing molecular size passively cross the tubule wall. Unfortunately such a survey has been attempted in very few cases. Ramsay (1958) examined the passive permeability of the Malpighian tubules of the stick insect, Carausius, to such substances as amino acids, sugars, and later inulin (Ramsay and Riegel, 1961). In broad terms what was found was that inulin crossed the tubule wall about three to five times more slowly than did small molecules such as amino acids and sugars. The evidence was that all these substances crossed the wall passively. That amino acids, for example, cross the tubule wall faster than inulin does not necessarily mean that the wall is fundamentally more permeable to them in the sense that the wall might slow passive trans-tubular movements of inulin more than amino acids. Large molecules diffuse more slowly than do small ones and so would be expected to penetrate more slowly. Also the possibility that amino acids and sugars cross the tubule wall at least partly through the cells cannot be excluded. Bearing this and the difference in diffusivity in mind, it seems very likely that, in Carausius, the intercellular junctions slow the diffusion of inulin little more than they do diffusion of smaller molecules. Using Malpighian tubules of Rhodnius the permeability to a wider range of substances has been examined (Maddrell and Gardiner, 1974, 1980). What has been found is that the tubules have a surprisingly low passive permeability to all substances, even amino acids as small as glycine. However, larger compounds such as inulin cross the tubule walls at measurable rates. The permeabilities of the wall to glycine and inulin

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S. H. P. MADDRELL

are 0.08 and 0.02 nl min-l mm-2, respectively (0.133 and 0.033 x cm sec-l. A reasonable explanation of this might be that the tubules have regions which are highly permeable but which are limited in area. This, of course, fits well with what is known of the structure of the epithelium where the intercellular junctions are only 17 nm wide (Section I) and occupy only a small fraction of the overall surface area of the tubule. Urea penetrates the walls of the Malpighian tubules of Rhodriiirs some hundreds of times faster than most other substances. Since urea is known to be able to penetrate cell membranes with relative ease in other systems (Wright and Pietras, 1974), this is easily understandable, especially in view of the extensive area of the apical and basal cell membranes in Rhodriiris tubules. The overall conclusion to be drawn from these studies is that insect Malpighian tubules, although they are the sites of filtration of the extracellular fluid, have much lower passive permeabilities than do the analogous filtering parts of other excretory systems, for example, the glomerulus of the vertebrate kidney. This is due less to a restriction at the sites of permeability than to the limited area of these sites relative to the epithelium in which they occur. In this connection a fascinating contrast exists between the Malpighian tubules of insects and those of Myriapoda (millipedes), a group of animals thought to be closely related to insects. The tubules of the pill millipede. Glomeais, are very freely permeable to a wide range of solutes (Farquharson, 1974). N o potential difference can be recorded across the tubule walls and the secreted fluid is virtually identical in composition to the bathing fluid, even when this contains solutes of high molecular weight. If this tubule has sites of permeability similar to those in insect Malpighian tubules, they must occupy very much larger areas. This is exactly what is found; electron micrographs of the wall show that the tubules have very profuse lateral interdigitations so that any section of the wall shows many intercellular junctions (Farquharson, 1974). The structure is very reminiscent of the podocytes of the kidney glomeruli of vertebrates (see, e.g., Kummel, 1973) and is in sharp contrast to that of insect Malpighian tubules. We are now in a position to return to the question posed at the beginning of this section. How can insect Malpighian tubules with only very restricted areas of high permeability operate satisfactorily as excretory organs'? The explanation may be along the following lines. Insects, as small animals, have high surface area to volume ratios. Their body fluids are thus liable to be much affected by changes in their environment. In spite of this, the most striking feature about the distribution of insects is

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that they are virtually only to be found in environments which are not stable in terms of osmotic and ionic concentration. Thus they occur on land and in natural bodies of water of all sorts with the marked exception of the sea, which significantly is a very stable environment. It is hard to avoid the conclusion that the tissues of insects must have evolved a tolerance of changes in the hemolymph. Indeed the ability of most insect tissues to survive well in vitro is an indicator of this. The one tissue that is sensitive to its ionic environment is the central nervous system. It has been recognized for some years that the internal environment of the insect central nervous system is protected and controlled by a special epithelium, the perineurium, which completely envelops it (Treherne, 1974). That insects lack a blood-borne respiratory pigment (oxygen reaches the tissues directly via air-filled tubes, the tracheae) is presumably another important element in freeing insects from the need to have a more constant hemolymph. So, if insects can tolerate changes in their internal milieu, one can argue that the excretory system need not work as swiftly as it otherwise would have to to control the composition of the hemolymph. Perhaps insect tissues can tolerate potentially deleterious changes in their internal environment for long enough to allow the excretory system to correct the situation at a relatively leisurely rate. It may not matter as much then how quickly toxic materials are removed as long as they are eventually removed. A system which allows slow passive exit of most substances, even large substances such as inulin, would, therefore, suit insects well and, as we have seen, that is exactly what they have. The advantages of such a relatively impermeable system are many. Useful substances that the insect needs to retain appear only slowly in the primary excretory fluid so that less energy needs to be expended in their reabsorption. As an extension of this it becomes feasible for insects to maintain high levels of low-molecular-weight materials in the hemolymph. Many insects have 100-200 m M amino acids in the hemolymph. Flying insects have very high levels of the fuel used by their flight muscles in the hemolymph; in Locusta, there may initially be 100 m M disaccharide trehalose and, later, as much as 60 m M diglyceride lipid (Weis-Fogh, 1967). As the tubule wall is not very permeable, it is possible to transport substances actively into or from the lumen without the resulting gradient being immediately cancelled out by diffusion. Insects take advantage of this (see Section IV, C) by actively eliminating through the tubules ions such as phosphate, magnesium, and sulfate and organic materials such as sulfonates, acylamides, alkaloids, and glycosides. In addition active

442

S . H. P. MADDRELL

recovery of sugars from the lumen of the tubules occurs in at least one insect. None of these processes would be profitable in a tubule as permeable as that of Glomeris. Finally, it is worth mentioning that it is also a consequence of the low permeability of the tubules that their rate of fluid secretion can be varied, under hormonal control, and this affects the rate of passive removal of substances from the hemolymph. During very fast fluid excretion, however, this effect diminishes and little extra work needs to be done in the reabsorption of useful compounds from the primary fluid. This point is taken further in Section IV, A.

Ill. CORRELATION OF STRUCTURE WITH FUNCTION

It is often assumed that in an epithelium made up of cells of uniform structural appearance, each cell functions identically. Some recent work, again on the Malpighian tubule of the bloodsucking insect Rhodnius, has shown that such an assumption can be totally wrong. During diuresis after a blood meal Rhodnius excretes a sodium-rich potassium-poor hypoosmotic urine. The first step in this is the secretion by the upper Malpighian tubules of a slightly hyperosmotic fluid containing both sodium and potassium chloride. The fluid then passes through the lower parts of the tubules where a hyperosmotic solution of potassium chloride is reabsorbed leaving the final urine to be eliminated (Maddrell and Phillips, 1975a). The upper tubules have been examined both by light and electron microscopy (Wigglesworth, 1931 ; Wigglesworth and Salpeter, 1962) and the cells all appear similar to each other. No differences have been found in the function of different parts of this upper tubule (Maddrell, 1969). All the more reason to suppose, then, that the lower tubule, whose cells also appear similar to each other (Wigglesworth, 1931; Wigglesworth and Salpeter, 1962), would function uniformly along its length. What has actually been found (Maddrell, 1978) is that the absorption of KCl is confined to the lowermost 30% of the length of the lower tubule which is effectively almost osmotically impermeable. By contrast, the upper part of the tubule is osmotically very permeable and reabsorbs no KCI. Two experiments which point up these differences are shown in Figs. 3 and 4. In other respects the lower tubule is uniform in properties along its length. Its passive permeability to solutes such as urea, chloride, ions, and sulfate ions is not different between its upper and lower regions.

443

EPITHELIAL TRANSPORT IN INSECT MALPIGHIAN TUBULES 0 rthcdrom ic pe rfusion

% \

Emerging fluid i s hypoosmotic : 151 m O s M ; 81 nl min-l

-Fluid

f

,

\

Hyperosmotic perfusion fluid: 138 mM K, 15 mM Na; 340 m O s M ; 75 nl min-'

I n lumen 1s now isoosmotic

Bathing fluid: 5 mM K, 135 mM Na; M 5-HT 280 mOsM : 5 x

\ Fluid in lumen i s now hypoosmotic

FIG. 3. The different results which follow from perfusing hyperosmotic K-rich fluid through a lower Malpighian tubule of Rhodnius either in the normal direction (above) or from the opposite end (below). (From Maddrell, 1978.)

Similarly all parts of the lower tubule carry out transport of nicotine into the lumen. The upper part of the lower tubule is osmotically permeable as previously mentioned. In light of the importance of the osmotic permeability of the upper tubule in explaining its function, it is of interest to enquire how permeable the upper part of the lower tubule is. The results of some experiments measuring the net transepithelial water flux in response to a known osmotic concentration difference (Maddrell, 1978) provide suitable figures for a calculation of osmotic permeability. In one experiment it was found that full osmotic equilibration occurred when fluid lower in

444

S. H. P. MADDRELL

93 "I

"I,"-'

280mOshl

ul n

-

upper

Lower

Bathing fluid

Perfused f l u i d 138 mLI K; 340 mOsM; flow rate 75 "I

5mMK; M 5-HT; 280 m 0 s M

73 "I rn1n-l95 mos M

Lawer

rnln-1

upper

FIG.4. The different results which follow the positioning of a K-poor, 5-HT containing drop of saline on the upper o r lower part of the lower Malpighian tubule of Rhodnius while K-rich saline is perfused through the lumen. (From Maddrell, 1978.)

osmotic concentration than the bathing fluid by 130 mosmole-' was perfused through a 2.2 mm length of the tubule. This resulted in a net water flow of about 40 nl min-'. Even assuming the average osmotic concentration difference along the tubule was 65 mosmole liter-' (it would be less if, as seems probable, the concentration in the lumen asymptotically approached that of the bathing fluid) the osmotic permeability of the wall must be about 1.8 x lop3cm sec-' osmole-l liter. It is easy to believe that the osmotic permeability of the wall of the upper tubule is at least as high as this, since the plasma membranes of the lower tubule are not as extensively folded as those of the upper tubule (Wigglesworth and Salpeter, 1962). Because KCl reabsorption in the lower tubule is confined to the lowermost part, its rate has to be very high. In life, fluid passes through the reabsorptive region in less than 10 seconds. Yet in this time its potassium content falls from 80 m M to less than 5 m M and its osmotic concentration falls from 370 to 250 mosmole liter-'. This means that the net flux of potassium chloride is about 0.80 pmole cmP2min-', which is apparently higher than the rates of solute absorption achieved by any vertebrate epithelia. This discovery of regionalization of function in an apparently uniform tubule has the important consequence that one can no longer assume that all the cells even in an apparently uniform epithelium are functionally equivalent. In fact, many Malpighian tubules are not uniform in appearance (see, e.g., Irvine, 1969). Their structure may change steadily or discontinuously with length; in other tubules there is a minority of cells of unusual structure known as stellate cells and these are distributed along the length of the tubule among cells of more typical appearance (Maddrell, 1971b). Very little is known of the significance and function of the different regions and cells in these cases.

445

EPITHELIAL TRANSPORT IN INSECT MALPIGHIAN TUBULES

IV.

REGULATORY PROPERTIES OF MALPIGHIAN TUBULES

Malpighian tubules as important parts of the insect excretory system have, as their main function, regulation and control of the hernolymph composition and volume. In carrying out this function they respond in appropriate ways to changes in hernolymph composition either autonomously or as directed by circulating hormones. A. Hormonal Effects on Malpighian Tubules

From what is known of the effects of hormones on kidney function in vertebrates, one might expect hormonal effects both on fluid secretion and on ion regulation by Malpighian tubules. In fact, in most insects, hormones are as yet known only to affect rates of fluid secretion. However, in one insect there is evidence of hormonal control of ion levels and this may turn out to be of wide occurrence. During the last twenty years or so, a variety of insects have been shown to be able to regulate with hormones the rates at which their Malpighian tubules secrete fluid. These hormones are usually referred to as diuretic hormones. In some cases, though, because water reabsorption is also accelerated there is no diuresis in the sense of rapid fluid elimination. A list of those insects known to use hormones to regulate fluid transport by their Malpighian tubules is shown in Table I . An example

INSECTS Order

Hymenoptera Diptera

Lepidoptera Coleoptera Hemipterd

Orthoptera

TABLE I WHICH THEREIs EVIDENCE THATTHE RATEOF FLUIDTRANSPORT BY THEIRMALPIGHIAN TUBULES Is REGULATEDBY HORMONES

FOR

Species Apis me//rjka Calliphora vomitoria Glossitla morsitaris Aedes taerriorhyrichus Ariopheles freeborrri Pieris brassicae Ariisotarsus cupripenrris Rhodriius prolixus, Triatoma itrfestatrs, Triatoma phyllosoma, Dipetalogaster maximu Dysdercus fasciatus Periplatieta americana Locusta migratoria Schistocerca gregaria Carausius morosus

Reference Altmann (1956) Knowles (1976) Gee (1975) Maddrell and Phillips (1978) Nijhout and Carrow (1978) Nicolson (1976) Nuiiez (1956) Maddrell (1%2) Maddrell (unpublished observations) Berridge (1966) Mills (1967) Cazal and Girardie (1968) Mordue (1969) Pilcher (1970)

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S.H. P. MADDRELL

of the effect of one diuretic hormone on fluid secretion rate is shown in Fig. 5. How, in detail, the acceleration of fluid secretion is achieved is, in most cases, not known. In several insects, bathing the tubules in solutions containing 3’ ,5’-cyclic adenosinemonophosphate (cyclic AMP) will stimulate rapid fluid secretion. The tubules appear to be more sensitive to extrinsically applied cyclic AMP than many other tissues; for example Rhodnius tubules are half maximally stimulated by 8 X M cyclic AMP. This sensitivity may well be due not to greater intrinsic permeability of the cell membranes but to the relatively larger area of membrane that Malpighian tubule cells expose to their bathing media. It seems likely from this, of course, that one element in the response of insect Malpighian tubule cells to circulating hormones is a change in the intracellular level of cyclic AMP. In Rhodnius this level has been measured and it does indeed increase following treatment with the insect’s diuretic hormone (Aston, 1975) (Fig. 6). Transport across an epithelium, especially rapid transport, is very likely to involve changes at both apical and basal cell membranes and the use of intracellular second messengers such as cyclic

T

Brain extract added

I

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d

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FIG.5 . The effect on the rate of fluid excretion by isolated Malpighian tubules of Aedes faeniorhynchus of adding an extract of the brain to the bathing medium. Each point indicates the mean value at a particular time and the attached vertical lines show ?SE of the mean. (From Maddrell and Phillips, 1978.)

EPITHELIAL TRANSPORT IN INSECT MALPIGHIAN TUBULES

447

Time after Stimulation (min)

FIG.6 . The time course of changes in cyclic A M P concentration in cells of the Malpighian tubules of Rl?odriiiis in response t o stimulation with the insects diuretic hormone. (Redrawn from Aston, 1975.)

AMP would seem to be imperative if the changes are to be coordinated in an effective manner. In Rhodnius at least some of the intracellular events accompanying hormonal stimulation are known (Gupta et al., 1976; Maddrell, 1977b). Figure 7 shows how the composition of the fluid secreted by Rhodnius Malpighian tubules differs according to whether or not fast fluid secretion has been stimulated. Under stimulation the tubules produce a fluid much richer in sodium than when they secrete fluid slowly. This behavior is appropriate, as the main function of accelerated fluid secretion is to eliminate most of the sodium-rich plasma from the blood meal. After diuresis, as the blood meal is slowly digested, potassium from the blood cells needs to be excreted and now the tubules slowly secrete a fluid suitably rich in potassium. The question arises as to how the stimulation of fluid secretion affects the function of Malpighian tubules in removing substances from the hemolymph. One consequence of faster fluid secretion is that substances,

440

S. H. P. MADDRELL l a ) Slow secretion

(b)

Fast (stimulated) s e c r e t i o n

18Or

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K I

Na 1 5 0

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(mM)

FIG.7. A comparison of the cationic composition of the fluid secreted by (a) unstimulated and (b) stimulated Malpighian tubules of Rhodnius. The points shown in (a) represent the mean values and the attached vertical lines indicate ?SE ( n = 6); (b) is redrawn from Maddrell (1969).

other than the ions and water of the secreted fluid, will be diluted in the tubule lumen. This will increase the concentration gradient for those substances entering passively and will reduce the tendency for substances actively concentrated in the lumen to leak out. In both cases it would seem that removal of the substances from the hemolymph would be accelerated. Figures 8 and 9 display the results of calculations done for the Malpighian tubules of Caffiphora to show how the active and passive removal of substances from the bathing medium depends on the rate of fluid secretion in the range 0-16 nl min-’. Unstimulated tubules of Cafliphora secrete fluid at about 5 nl min-’ (Berridge, 1968). The calculations have been done for substances to which the tubule walls are relatively permeable (Fig. 8) and for substances to which the walls are less permeable (Fig. 9). The bases for these calculations are given in Maddrell et a f . (1974) and Maddrell and Gardiner (1974). The calculations show that: 1 . Changes in the rate of fluid secretion in the range 5-16 nl min-l (i.e., stimulated rates) have relatively greater effects on the removal of permeable substances than they have on substances to which the tubules are relatively impermeable (compare “extra removed” in Figs. 8 and 9).

EPITHELIAL TRANSPORT IN INSECT MALPIGHIAN TUBULES

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2 . Active and passive entry of substances to which the tubule wall is equally permeable are affected in a similar way by changes in fluid transport (compare the upper and lower line in Fig. 8). However, the extra amounts of material removed from the hemolymph on stimulation is higher in the case of a substance subject to active transport, particularly where the tubule is not very permeable to the substance (compare the "extra removed" shown by the upper lines in Figs. 8 and 9 with the "extra removed" shown by the lower lines).

Figure 10 shows that actual measurements made on dye transport by Calliphora tubules are comptabile with the theoretical predictions given above. What the calculations reveal is that changes in fluid secretion have their greatest effects on substances which the tubule cells actively transport into the lumen. Since these substances are, of course, the ones which the insect wishes most to eliminate, this makes very good sense.

FIG.8. The expected dependence on rate of fluid secretion of the rate of removal of a substance from the medium by a Malpighian tubule of Calliphora. For the calculation it was assumed that the substance was present in the medium at I mM, that the wall has an area of 4 mm2, and a passive permeability to the substance of 5 nl min-l mm-*. The upper line is for the case where the substance is transported into the lumen at a rate of 75 pmole min-I. The lower line shows net entry for the case where the substance enters only by diffusion. The extra amounts of solute removed by an increase in rate of fluid secretion from 5 to 16 nl min-l are shown.

450

S.H. P. MADDRELL

Actively transported 101"te

..........................................................

.........................................

Solute entering by diffusion .

0

2

4

6

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I2

14

. -3 16

Rate of fluid secretion in1 min 1

FIG.9. As in Fig. 8 but for a substance to which the tubule wall is less permeable, the passive permeability being 0.25 nl min-l mm-*. The dotted line is a replot of the lowest line, but with the ordinate expanded in scale by 40 times.

Further it provides a reason for the very existence of a so-called diuretic hormone in insects where no rapid elimination of water follows the appearance of the hormone in circulation. A case in point concerns the diuretic hormone of the locust. It appears that the Malpighian tubules of the locust are stimulated to secrete fluid faster during flight, but faster fluid reabsorption in the hindgut means that no fluid is lost. It has been suggested that this would result in faster elimination of waste products appearing in the hemolymph as a result of the intense metabolic activity during flight. From the analysis given above it can now be seen that acceleration of fluid secretion by the tubules does not affect passive excretion very much but does greatly increase elimination of actively transported materials. Since evolution is bound to have favored the production of active transport mechanisms for the more toxic materials that appear in the hemolymph, it follows that increased circulation of fluid through the locust excretory system will, appropriately, increase most the excretion of potentially damaging substances. On the other hand the rate of appearance of useful substances in the primary excretory fluid will be less affected and so energy is saved in not having to provide for their more intense reabsorption.

EPITHELIAL TRANSPORT

IN

451

INSECT MALPIGHIAN TUBULES

Although these conclusions provide a satisfying rationale for the way in which the excretory system operates during changes in its rate of operation, it must be said that the experimental evidence supporting the main points is still thin. The few available facts support the interpretation given but much more work needs to be done really to put these ideas on a firmer foundation. Before leaving this area there are some remarks to be made about diuresis in Rhodnius. This insect takes enormous meals of blood (up to 12 times it own body weight) and then excretes the excess fluid at an extraordinarily high rate. The rate of excretion can be as high as 1.2 p1 min-l which is equivalent to excreting a volume of fluid equivalent to its whole hemolymph volume in I5 minutes. This goes on until a weight of fluid equal to 40-45% of the meal has been excreted. Why is fluid excreted so fast? Apart from the advantages of rapidly recovering its shape and increasing its maneuverability, one can suggest that very rapid fluid excretion drains less useful solutes from the hemolymph. For example, Rhodnius hemolymph contains more than 20 m M amino acids (Maddrell

/---

expected Y ~ I U ~ E

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-

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12

I 14

I 16

’

Rate of fluid ~ecretionin1 mln I

FIG.10. The dependence of the net rate of secretion of indigo carmine into the lumen of a Malpighian tubule of Calliphora on the rate of fluid secretion (observed values from Maddrell et al., 1974), compared with that expected if the tubule wall has a permeability to indigo carmine of 0.77 nl min-l mrn+ and actively transports the dye into the lumen at a rate of 81 pmole min-I.

452

S. H. P. MADDRELL

and Gardiner, 1980). To these substances, the tubules have a permeability of about 0.15 nl min-I mm+ (Maddrell and Gardiner, 1980). One can calculate the fraction of the hemolymph content of amino acids that would be passively lost at, say, an excretory rate of 1.2 pl min-l (that actually observed) and compare it with the loss that would occur at 80 nl min-' (equivalent to the stimulated rate of fluid secretion by the tubules of other insects). If the insect took a blood meal of 330 p1 and excreted 140 p1 of fluid, calculation shows that at 1.2 p1 min-' only 3% of the hemolymph amino acids would be lost (actual measurements agree with this figure); at 80 nl min-I, more than 30% of the amino acids would be lost. There is an evident advantage to the insect in excreting fluid at as high a rate as possible. This may help explain why the tubules of blood sucking insects secrete fluid at the highest rates known for any tissue. It can be seen from Fig. 1 1 that very fast fluid secretion offers no advantage to insects using accelerated fluid secretion as a means of hastening elimination of actively transported substances. Once faster fluid secretion has lowered the luminal concentration of transported ma-

L

0

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1W

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2w

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Rate of fluid secretion In1 min 1

FIG. I I. The calculated effect on indigo carmine excretion by a Malpighian tubule of Calliphora of raising the rate of fluid secretion to the very high levels found in bloodsucking insects. As in Fig. 10, it was assumed that the tubule has a permeability to indigo carmine of 0.77 nl min-I rnmP and transports the dye into the lumen at a rate of 81 pmole min-I.

453

EPITHELIAL TRANSPORT IN INSECT MALPIGHIAN TUBULES

terial down to a level not much above that of the bathing medium, further increases in the rate of fluid secretion increase the elimination of actively transported substances relatively little and would of course require the more rapid expenditure of energy. The differences in the rates of fluid secretion by the tubules of blood sucking insects and of other insects are thus seen to be appropriate. B. Autonomous Regulation

Regulation of a tissue by a hormone involves a chain of neurons which monitor some parameter and stimulate suitable hormone release. A more economical system might be one in which the tissue itself directly responded in an appropriate way to a change in the composition of its environment. Just such behavior is seen in some insect Malpighian tubules. Many insects are phytophagous and in consequence face the problem of an excess of potassium in the diet. Most insects seem to absorb the constituents of their diet into the hemolymph in a surprisingly unselective way. Potassium levels in the hemolymph are known to be strongly affected by the nature of the diet (Pichon and Boistel, 1963). The Malpighian tubules of many insects respond appropriately to increasing levels of potassium in the hemolymph. Figure 12 shows how the composition and rate of secretion of the fluid produced by isolated insect Malpighian tubules change with the potassium concentration in their environment. Over the physiological range, below 20 m M , it is clear that potassium excretion increases rapidly with

Nalto 120 100

80

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Ned0

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FIG. 12. The cationic composition and rate of secretion of fluid produced by isolated Malpighian tubules of Culliphoru as affected by the composition of the bathing medium. (Redrawn from Berridge, 1968.)

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S. H. P. MADDRELL

increasing bathing concentrations and that this does not rely on hormonal regulation. In recently fed bloodsucking insects the problem is a different one. We have seen how it is to their advantage to eliminate fluid at high rates. However, the ingested plasma contains only 5 m M potassium. If the tubules were to secrete potassium-rich fluid at a rate dependent on the potassium level, as do other insects, they would soon not only exhaust the hemolymph potassium but would be forced to secrete fluid at very low rates. Appropriately the rate of fluid secretion in the tubules of those bloodsucking insects so far examined is not dependent on the potassium concentration. Furthermore, in the tsetse fly, Glossina, the tubules secrete fluid rich in sodium and containing only low levels of potassium (Fig. 13). Rhodnius’ tubules, though the rate of fluid secretion is unaffected by the potassium concentration, perversely enough secrete fluid about as rich in potassium as it is in sodium; it contains 10-20 times as much potassium as the hemolymph (Fig. 7). The situation is rescued by the activity of a further part of the Malpighian tubule, the lower region (Section 111), where potassium chloride is rapidly returned to the hemolymph leaving a suitably hypoosmotic, potassium-poor sodium-rich fluid to be excreted. What is of concern here is that the avidity with which the lower tubule recovers potassium from the lumen is directly affected by the potassium concentration of the bathing fluid (Fig. 14). Since potassium transport into the lumen by the upper tubule is similarly sensitive, the performance of the whole tubule acts to regulate the hemolymph 180r

*

K 150

1

120

90

60

30

C o n c e n t r a t i o n of i o n s i n b a t h i n g f l u i d

0

(mM)

FIG.13. The cationic composition of fluid secreted by isolated Malpighian tubules of Glossina in different bathing media. The secreted fluid is very rich in sodium; compare

with Figs. 7 and 12. (Redrawn from Gee, 1976.)

455

EPITHELIAL TRANSPORT IN INSECT MALPIGHIAN TUBULES

I0

1

10

I

20

I

30

I

40

I

50

I

60

I

70

Concentration of potassium in bathing fluid (mM)

FIG. 14. The effect of altering the potassium concentration of the fluid bathing stimulated lower Malpighian tubules of Rhodnius on the osmotic concentration of initially K-rich fluid passing through the tubule lumen. (Redrawn from Maddrell and Phillips, 1976.)

potassium level. If the hemolymph level falls, the upper tubule produces fluid containing less potassium (Fig. 7) and the lower tubule recovers more potassium from the fluid passed to it. Opposite changes follow rises in potassium level. All these changes occur autonomously without changes in hormone stimulation. The ability of Rhodnius tubules to secrete at a constant rate a fluid containing amounts of sodium and potassium which vary in a complementary way raises the question of how this is achieved. Most vertebrate epithelia selectively transport one of these ions and are not able to substitute the other one. This is not the place to examine the evidence in detail, but it seems possible that insect Malpighian tubules achieve transepithelial transport of sodium and potassium ions with a cation pump, situated on the apical cell membrane, able to transport either ion but having a higher affinity for sodium (see Maddrell, 1977a). The secretion of potassium-rich fluid by most insect Malpighian tubules probably

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S. H. P. MADDRELL

depends on the potassium-selective basal membrane; since little sodium reaches the cation pump it is largely potassium ions that are transported into the lumen.

C. Inducible Transport Systems in Malpighian Tubules

We have seen in the preceding two sections how Malpighian tubules respond to excretory needs of the insect either under hormonal control or by making direct, and virtually immediate changes in their functioning. Other changes in the hemolymph composition are known to lead more slowly, within hours or days, to counteracting changes in Malpighian tubule function. It is not yet clear whether these changes are mediated via some hormonal action or are direct responses of the tubules. First, we shall consider the excretion of organic anions. Insect Malpighian tubules have for years been known rapidly to accumulate acidic dyes such as atnaranth and phenol red when these are injected into the hemolymph (Lison, 1937; Palm, 1952). Until recently the significance of this was not at all clear. However, it now appears that these dyes are transported by two noncompeting systems, one of which handles acylamides and the other sulfonates (Maddrell e f al., 1974). In vertebrate kidneys a single transporting system is known to be responsible for the elimination of these two classes of substances (Smith, 1951). Recent research has shown that the rate of transport of these organic anions in insect Malpighian tubules depends very much on the nutritional state of the insect. As Fig. 15 shows, transport of paminohippuric acid (PAH) is slow in tubules from unfed Rhodnius, but, after the insect has fed, the tubules soon gain the ability to excrete PAH at a much higher rate. The ability to transport PAH is evidently inducible and the induction appears to depend not simply on the act of feeding but on the meal containing substantial amounts of protein. PAH transport by vertebrate kidneys is similarly affected by changes in the protein content of the diet (Calcagno and Lowe, 1963). Many insect Malpighian tubules can excrete toxic organic cations such as alkaloids (Maddrell and Gardiner, 1976). In this case the ability is not inducible but is independent of the presence of alkaloid in the diet. Presumably these compounds are so toxic that insects do not run the risk of encountering them unprepared. Larvae of saline-water mosquitoes can thrive in waters of very varied composition. Larvae of Aedes taeniorhynchus can be found in water very rich in magnesium and sulfate ions (Scudder, 1969). They ingest the water and absorb it and its content of ions. The Malpighian tubules of these insects can actively excrete both magnesium and sulfate ions (Maddrell

457

EPITHELIAL TRANSPORT IN INSECT MALPIGHIAN TUBULES

0

5

10

15

20

2s

Time (davs)

FIG.15. Changes in rate of transport of PAH by Malpighian tubules isolated from adult Rhodnius. From day 0 to day 9, the insects were kept unfed; on day 9 they were given a blood meal. (From Maddrell and Gardiner, 1975.)

and Phillips, 1975b; Phillips and Maddrell, 1974). It has now transpired that at least in the case of sulfate ions, transport is inducible (Fig. 16) (Maddrell and Phillips, 1978). Malpighian tubules from larvae reared in sulfate-free water cannot carry out active transport of sulfate ions. However within 4 hours of transferring the larvae to sulfate-rich water, the tubules develop an ability to transport these ions. After 18 hours the tubules can transport sulfate at least half as fast as those from larvae reared from birth in the water. The evidence, as far as it goes, does not support the idea that the tubules respond directly to the appearance of sulfate ions in their environment, as isolated tubules kept in sulfate-rich saline for 7 hours do not develop any ability to transport sulfate. This evidence does not, however, exclude the possibility that the saline was, in some respect, sufficiently dissimilar to the hemolymph to prevent an induction of transport. It remains to be seen just what the mechanism of induction is. It is also not yet known whether magnesium transport in these larvae is inducible or not. Finally, ouabain transport by Malpighian tubules has recently been shown to be inducible (Rafaeli-Bernstein and Mordue, 1978). V.

MALPIGHIAN TUBULE ACTION IN THE ABSORPTION OF WATER VAPOR FROM THE AIR

As a fitting tailpiece to this article, it is worth describing an extreme ability of Malpighian tubules to secrete a solution of such high osmotic

458

S. H. P. MADDRELL

T

100 c

80

-

-._ x

E

*

I

Su Ifate-free

0

5

10

15

20

25

30

Concentration of SO4 in bathing medium (mMI

FIG. 16. Characteristics of sulfate transport by Malpighian tubules isolated from larvae of Aedes faeniorhynchus reared in seawater enriched with sulfate (upper line), normal seawater (middle line), or sulfate-free seawater (lower line). (From Maddrell & Phillips, 1978.)

concentration that water vapor can be drawn from air of humidity less than 90% RH. This most unusual ability is found in larvae of the meal worm, Tenebrio, which lives in drying environments such as stored flour. The upper parts of the Malpighian tubules of this insect run into a virtually impermeable chamber round the rectum. Here they make intermittent contacts with the external walls of the chamber and at these points the wall is missing so that the tubules are in contact with the hemolymph. At these sites, the tubule wall has specialized cells called leptophragma cells. They are very much reduced in thickness (down to about 0.25 pm) and have a plane membrane facing the hemolymph (Grimstone et al., 1968). An extremely concentrated solution of potassium chloride is transported from the hemolymph into the lumen through these cells. The concentration of this solution was measured as at least 2 M

EPITHELIAL TRANSPORT IN INSECT MALPIGHIAN TUBULES

459

potassium chloride (Ramsay, 1964), but recent melting point determinations suggest that it may be considerably more concentrated than this, to perhaps 7 osmole liter-' (Machin, 1978). It is thought that this concentrated solution then abstracts water from the fluid in the chamber round the rectum, raising its osmotic concentration to similarly high values. In turn water is drawn through the rectal wall anteriorly from fluid passing down the gut, but posteriorly from air entering the rectum from the air surrounding the animal. In this way the insect can continuously absorb water from air at 88% RH. An ability to take up water vapor from the air like this is unknown in vertebrates, but helps explain how some insects can survive in exceptionally dry environments despite their small size.

VI.

SUMMARIZING REMARKS

The picture of insect Malpighian tubules which has become apparent in the course of the preceding is as follows. They are epithelia capable of fluid transport at a great variety of rates. Where it has been measured, the movement of water across their walls in response to an applied osmotic gradient is so rapid as to revive interest in the possibility that fluid transport might here involve osmotic coupling of ion and water movements. However, there is yet no satisfactory suggestion as to how, in detail, this might be achieved. The route between the cells is restricted in area, but is very permeable. Hemolymph solutes diffuse through this paracellular pathway unaffected by the cellular fluid transport, except insofar as this alters the transepithelial concentration gradient. This independence means that the insect can greatly accelerate fluid secretion without passively losing large amounts of useful substances occurring in the hernolymph. The overall permeability of the tubule wall is low, so that active secretion of excretory materials into the tubule lumen and active reabsorption of useful substances from the lumen can go on without diffusion rapidly degrading the resulting concentration gradients. Different regions of the tubule often have cells of different structural appearances. In some cases these can be correlated with a difference in function. The apparent correlation of function with appearance is not however a sure guide, since, in at least one case, an apparently uniform part of a tubule has very different properties in different regions. Many insects can vary the rate of tubule fluid secretion under hormonal control. Many other transporting systems of the tubules alter in response to a change in the concentration of the transported substances. This

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change may be rapid and autonomous or it may depend on relatively slow induction, whose mechanism is not yet understood. The range of transport abilities possessed by Malpighian tubules of insects is very wide. They are known to possess active transport mechanisms for all the following: potassium, sodium, chloride, ammonium, magnesium, phosphate, and sulfate ions; acylamides, sulfonates, alkaloids, and glycosides; and amino acids and sugars. In most cases very little is known about the detailed mechanisms involved. Malpighian tubules are so easily studied in isolated preparations that it should be easy to make rapid advances in understanding these transport mechanisms. REFERENCES Altmann, G. (1956).Die regulation des Wasserhaushaltes der Honigbiene. Itisectes SOC.3,

33-40. Aston, R. J. (1975).The role of adenosine 3‘:5’-cyclic monophosphate in relation to the diuretic hormone of Rhodriius prolixus. J. Insect. Physiol. 21, 1873-1877. Berridge, M. J. (1%6). The physiology of excretion in the cotton stainer, Dysdercus fasciarus Signoret. IV. Hormonal control of excretion. J. Exp. Biol. 44, 553-566. Berridge, M. J. (1%8). Urine formation by the Malpighian tubules of Calliphora erythrocephala. I. Cations. J . Exp. Biol. 48, 159-174. Calcagno, P. L.,and Lowe, C. V. (1963).Substrate induced renal tubular maturation. J. Pediatr. 63, 851. Cazal, M., and Girardie, A. (1968). Controle humoral de I’equilibre hydrique chez Locusta migratoria migratorioiodes. J . Insect Physiol. 14, 655-668. Diamond, J. M. (1962).The mechanism of solute transport by the gallbladder. J. Physiol. (London) 161, 474-502. Diamond, J. M., and Bossert, W. H. (1%7). Standing-gradient osmotic flow. A mechanism for coupling of water and solute transport in epithelia. J. Geti. Physiol. 50, 2061-2083. Farquharson, P. A. (1974).A study of the Malpighian tubules of the pill millipede, Glomeris rnarginata (Villers), 111. The permeability characteristics of the tubule. J. Exp. Biol.

60,41-51. Filshie, B. K., and Flower, N . E. (1977).Junctional structures in Hydra. J . Cell Sci. 23,

151-172. Gee, J. D. (1975).The control of diuresis in the tsetse fly Glossiria austeni, a preliminary investigation of the diuretic hormone. J. Exp. Biol. 63, 391-401. Gee, J. D. (1976).Active transport of sodium by the Malpighian tubules of the tsetse fly Glossiria morsitans. J. Exp. Biol. 64, 357-368. Giebisch, G. (1973). Some transport properties of amphibian and mammalian nephrons. 1ti “Comparative Physiology: Locomotion, Respiration, Transport and Blood” (L. Bolis, K. Schmidt-Nielsen, and S. H. P. Maddrell, eds.), pp. 241-291. North-Holland Publ., Amsterdam. Green, L. B. F. (1979).The fine structure of the light organ of the New Zealand glowworm Arachriocampa lumiriosa (Diptera : Mycetophilidae). Tissue Cell 11, 457-465. Grimstone, A. V., Mullinger, A. M., and Ramsay, J. A. (I%@. Further studies on the rectal complex of the mealworm Tetiebrio rnolitor, L. (Coleoptera, Tenebrionidae). Philos. Trans. R . SOC. Lotidoti, Ser. B 253, 342-382.

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Gupta, B. L., Hall, T. A., Maddrell, S. H. P., and Moreton, R. B. (1976). Distribution of ions in a fluid-transporting epithelium determined by electron-probe x-ray microanalysis. Nature (London) 264, 284-287. Gupta, B. L., Hall, T. A., and Naftalin, R. J. (1978). Microprobe measurements of Na, K and C1 concentration profiles in epithelial cells and intercellular spaces of rabbit ileum. Nature (London) 272, 70-73. Hill, A. E. (1975). Solute coupling in epithelia: a critical examination of the standing gradient osmotic flow theory. Proc. R . SOC. London, Ser. B 190, 99-114. Hill, A. E. (1977). Fluid transport across Necturus gallbladder epithelium. III “Comparative Physiology: Water, Ions atid Fluid Mechanics” (K. Schmidt-Nielsen, L. Bolis, and S. H. P. Maddrell, eds.), pp. 41-42. Cambridge Univ. Press, London and New York. Irvine, H. B. (1%9). Sodium and potassium secretion by isolated insect Malpighian tubules. Am. J. Physiol. 217, 1520-1527. Knowles, G. (1975). The reduced glucose permeability of the isolated Malpighian tubules of the blowfly Calliphora vomitoria. J. Exp. Biol. 62, 327-340. Knowles, G. (1976). The action of excretory apparatus of Calliphora vomiroriu in handling injected sugar solution. J. Exp. Biol. 64, 131-140. Kummel, G. (1973). Filtration structure in excretory systems-A comparison. I I I “Comparative Physiology: Locomotiorr, Respiration, Transport arid Blood” (L. Bolis, K. Schmidt-Nielsen, and S. H. P. Maddrell, eds.), pp. 221-240. North-Holland Publ., Amsterdam. Lison, L. (1937). Etudes histophysiologiques sur les tubes de Malpighi des Insectes. I. Elimination des colorants acides chez les Orthoptkres. Arch. Biol. 48, 321-360. Machin, J . (1978). Water vapour uptake by Tenebrio: a new approach to studying the phenomenon. I n “Comparative Physiology: Water, Ions and Fluid Mechanics” (K. Schmidt-Nielsen, L. Bolis, and S. H. P. Maddrell, eds.), pp. 67-77. Cambridge Univ. Press, London and New York. Maddrell, S. H. P. (1%2). A diuretic hormone in Rhodniusprolixus Stahl. Nature (London) 194, 605-606. Maddrell, S. H. P. (1%9). Secretion by the Malpighian tubules ofRhodnius. The movements of ions and water. J. Exp. Biol. 51, 71-97. Maddrell, S. H. P. (1971a). Fluid secretion by the Malpighian tubules of insects. Philos. Trans. R . SOC. London Ser. B 262, 197-207. Maddrell, S. H. P. (1971b). The mechanisms of insect excretory systems. Adv. Insect Physiol. 8, 199-331. Maddrell, S. H. P. (1977a). Hormonal action in the control of fluid and salt transporting epithelia. In “Water Relations in Membrane Transport in Plants and Animals” (A. M. Jungreis, ed.), pp. 303-313. Academic Press, New York. Maddrell, S. H. P. (1977b). Insect Malpighian tubules. Itr “Transport of Ions and Water in Animal Tissues’’ (B. L. Gupta, R. B. Moreton, J. L. Oschman, and B. J. Wall, eds.), pp. 541-569. Academic Press, New York. Maddrell, S. H. P. (1978). Physiological discontinuity in an epithelium with an apparently uniform structure. J. Exp. Biol. 75, 133-145. Maddrell, S. H . P., and Gardiner, B. 0. C. (1974). The passive permeability of insect Malpighian tubules to organic solutes. J. Exp. Biol. 60, 641-652. Maddrell, S. H. P., and Gardiner, B. 0. C. (1975). Induction of transport of organic anions in Malpighian tubules of Rhodnius. J. Exp. Biol. 63, 755-761. Maddrell, S. H. P., and Gardiner, B. 0. C. (1976). Excretion of alkaloids by Malpighian tubules of insects. J. Exp. Biol. 64, 267-281.

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Maddrell, S. H. P., and Gardiner, B. 0. C. (1980). Retention of amino acids by the excretory system of Rhodnius prolixus. J. Exp. Biol. In press. Maddrell, S. H. P., and Phillips, J. E . (1975a). Active transport of sulphate ions by the Malpighian tubules of larvae of the mosquito, Aedes campestris. J. Exp. Biol. 62, 367378. Maddrell, S. H. P., and Phillips, J. E. (1975b). Secretion of hypo-osmotic fluid by the Malpighian tubules of Rhodtiius prolixus. J. Exp. Biol. 62, 671-683. Maddrell, S. H. P., and Phillips, J. E. (1976). Regulation of absorption in insect excretory systems. Iti “Perspectives in Experimental Biology. Vol. 1: Zoology” (P. SpencerDavies, ed.), pp. 179-185. Pergamon, Oxford. Maddrell, S. H. P., and Phillips, J. E. (1978). Induction of sulphate transport and hormonal control of fluid secretion by Malpighian tubules of larvae of the mosquito Aedes taeniorhynchus. J. Exp. Biol. 15, 133-145. Maddrell, S. H. P., Gardiner, B. 0. C., Pilcher, D. E. M., and Reynolds, S. E. (1974). Active transport by insect Malpighian tubules of acidic dyes and acylamides. J. Exp. Biol. 61, 357-377. Mills, R. R. (1967). Hormonal control of excretion in the American cockroach. I. Release of a diuretic hormone from the terminal abdominal ganglion. J . Exp. Biol. 46, 35-41. Mordue, W. (1969). Hormonal control of Malpighian tubules and rectal function in the desert locust Schistocerca gregaria. J. Insect Physiol. 15, 273-285. Nicolson, S. W. (1976). The hormonal control of diuresis in the Cabbage white butterfly, Pieris brassicae. J. Exp. Biol. 65, 565-575. Nijhout, H. F., and Carrow, G. M. (1978). Diuresis after a bloodmeal in female Anopheles freeborni. J. Iiisect Physiol. 24, 293-298. NuAez, J . A. (1956). Untersuchungen uber die Regelung des Wasserhaushaltes bei Anisotarsus cupripeririis Germ. Z . Vgl. Physiol. 38, 341-354. Palm, N.-B. (1952). Storage and excretion of vital dyes in insects. Ark. Zool. 3, 195-272. Phillips, J . E., and Maddrell, S. H. P. (1974). Active transport of magnesium by the Malpighian tubules of the larvae of the mosquitoe, Aedes campestris. J. Exp. Biol. 61, 761-77 1 . Pichon, Y., and Boistel, J. (1%3). Modifications of the ionic content of the haemolymph and of the activity of Periplaiieta americanu in relation t o diet. J. Insect Physiol. 9, 887-891. Pilcher, D. E. M. (1970). Hormonal control of the Malpighian tubules of the stick insect, Carausius morosus. J. Exp. Biol. 52, 653-665. Rafaeli-Bernstein, A., and Mordue, W. (1978). The transport of the cardiac glycoside ouabain by the Malpighian tubules of Zonocerus variegatus. Physiol. Enr. 3, 59-63. Ramsay, J. A. (1958). Excretion by the Malpighian tubules of the stick insect Dixippus morosus (Orthoptera, Phasmidae): amino acids, sugars and urea. J. Exp. Biol. 35, 871691. Ramsay, J. A. (1964). The rectal complex of the mealworm, Teriebrio molitor, L. (Coleoptera, Tenebrionidae). Philos. Trans. R . Soc. London Ser. B 248, 279-314. Ramsay, J . A., and Riegel, J. A. (1961). Excretion of Inulin by Malpighian tubules. Nature (London) 191, 1 1 IS. Sackin, H., and Boulpaep, E. L. (1975). Models for coupling of salt and water transport: proximal tubular reabsorption in Necturus kidney. J . Geri. Physiol. 66, 671-733. Scudder, G. G. E. (1969). The fauna of saline lakes of the Fraser Plateau in British Columbia. Verh. I n t . Vereiii. Theor. Angew. Limriol. 17, 430-439. Smith, H. W. (1951). “The Kidney.” Oxford Univ. Press, London and New York. Treherne, J . E. (1974). The environment and function of insect nerve cells. I t i “Insect

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Neurophysiology” (J. E. Treherne, ed.), pp. 187-244. North-Holland Publ., Amsterdam. Weis-Fogh, T. (1967). Metabolism and weight economy in migrating animals, particularly birds and insects. I n “Insects and Physiology” (J. W. L. Beament and J . E . Treherne, eds.), pp. 143-159. Oliver & Boyd, Edinburgh and London. Wigglesworth, V. B. (1931). The physiology of excretion in a blood-sucking insect, Rhodriius prolixus (Hemiptera, Reduviidae). 11. Anatomy and histology of the excretory system. J . Exp. B i d . 8, 428-442. Wigglesworth, V. B., and Salpeter, M. M. (1962). Histology of the Malpighian tubules in Rhodnius prolixus Stal. (Hemiptera). J . Insecr Physiol. 8 , 299-307. Wright, E. M., and Pietras, R. J . (1974). Routes of non-elecrolyte permeation across epithelial membranes. J . M e m b r . B i d . 17, 293-312.

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Subject Index A Acetylcholine receptor, reconstitution of, 93-99 Acetylcholinesterase, at aidwater interface, 20-22 Acronycin, 289 Adenine transport kinetics, 242, 254 Adenosine transport kinetics, 242, 254, 258, 269 Adsorption potential dipole component of, 7-8 influence of solution composition on, 910 at interface of two immiscible liquids, 67 Affinities, asymmetric derived from inhibitor studies, 173- 177 membrane transfer kinetics for, 181-202 two site model and, 182-187 Aflatoxin, 289 Airiwater interface acetylcholinesterase at, 20-22 adsorption of enzymes of, 16-19 enzymatic reactions at, 16-25 equipment for study of, 19-20 galactosyltransferase at, 22-24 Volta potential of complex system at, 2425 Amino acid entry into blood, 364-365 intestinal sites of maximal absorption, 353-355 muscosal uptake, 346 in developing animals, 356-357 relative absorption rates, 349-35 I Amino acid auxotrophs, in peptide transport studies, 372-373, 391 Amino acid transport in bacteria, 375-376 in barley scutellum, 401-402 in yeast, 391

p-Aminohippuric acid, transport in Malpighian tubules, 456-457 Anion transport protein, reconstitution, 106- I08 Asymmetric carrier kinetics, applications, 198-200 ATPase, oligomycin-sensitive mitochondrial, reconstitution, 99- 101 Autoradiography, in reconstitution experiments, 80-81 8-Azaguanine transport, 269

B Bacteria, nature of cell surface, 369-370 Bacteriochlorophyll, reconstitution, 104 Bacteriorhodopsin at interface, 43-45 reconstitution, 103- 104 Band 3, reconstitution, 106- 108 Barley scutellum, peptide transport in, 398403 Blood, entry of amino acids and peptides into, 364-365 Brain, peptide transport in, 365 Bromohexadecanoic acid, 8

C Calcium pump, reconstitution of, 86-90, 106, 110 Calorimetry, for reconstitution systems, 76 Ca2+ + Mg2+-ATPase reconstitution of, 86-90, I10 from sarcoplasmic reticulum, 128- 159 activity and lipids, 137-144, 148-150 lipid annulus, 150-154, 157-158 purification, 131 - 133 reconstitution in sealed vesicles, 144148 Capacitance, membrane, measurement of, 71-72 465

466 Carrier model, see also Mobile carrier hypothesis applicability to nucleoside and base transport, 235-253 Cell surface, microbial, nature of, 369-371 Charge transfer, at interface, oil phase dielectric constant and, 45-46 Chemiosmotic hypothesis, 46-47 p-Chloromercuribenzene sulfonate, 173 Chlorophyll electron transfer by, 33-34 at interface, 30-42 oxidation-reduction transformations of, 30-33 photooxidation of water and, 35-40 proton phototransfer, 34-35, 40-42 Colchicine, 289, 304 Compensation potential, in water/oil chain, 13-16 Computer enhancement of image, in reconstitution experiments, 81 Conductance, measurement of, 69-71 Coproporphyrin derivative, photooxidation of water and, 35-40 Critical micelle concentration, 132 Current fluctuations, measurement of in reconstitution systems, 74-76 Cytidine transport kinetics, 242, 254, 274 Cytochalasin B asymmetric affinity, 177- 179 effect on nucleoside and base transport, 289 Cytochrome oxidase, 43 reconstitution, 101- 102 Cytosine arabinoside transport kinetics, 242 Cytosine transport, 269

SUBJECT INDEX

5,5‘-Dimethyl-2,4-oxazolidinedione transport, 269 Dimethyl sulfoxide, effect on nucleoside uptake, 291 2,4-Dinitrophenol as proton acceptor, 36-42 role in proton transfer, 40-42 Dipeptide transport in bacteria, 375-376 in yeast, 391-392 Dipyridamole, 234, 288-291, 305 Distribution potential, at interfxe of two immiscible liquids, 5-6, 7, 9 DMO, see 5,5’-Dimethyl-2,4-oxazolidinedione DNP, see 2,4-Dinitrophenol

E

Electrical measurements, in reconstitution experiments, 68-76 Electrical potential difference, intestinal transport of peptides and, 339 Electron microscopy, 76-82 conventional sample preparation, 76-78 freeze-fracture techniques, 78-82 Electron paramagnetic resonance, reconstitution experiments and, 82-85 Electron transfer, by chlorophyll, 33-34 Enzymatic reaction at interface, experimental approaches to, 16-25 Enzymatic reaction rate, determination, equipment for, 19-20 Enzyme adsorption, at interface, 16-19 hydrolytic, effect on nucleoside and base transport, 295 Enzyme-membrane system, coupling of, D 46-48 DCDS, see 3,3’-Di-2-chloroallyIdiethylstil- Equilibrium exchange experiments bestrol with glucose, 171 Deoxyadenosine transport kinetics, 254 with nucleosides and bases, 238, 240-244 Deoxycytidine transport kinetics, 242, 254, Erythrocyte membrane 274 asymmetry of, 202-206 Deoxyuridine transport kinetics, 274 environmental asymmetry of, 206-209 Detergent, for solubilization of SR, 133 hexose transfer system of, 166-215 Diaphragm, peptide transport in, 366 peptide transport across, 366 3,3’-Di-2-chloroallyIdiethylstilbestrol, 173 Ethanol, effect on nucleoside uptake, 291 Dielectric constant, oil phase, charge trans- Ethylidene glucose, 173, 174, 175, 197 fer and, 45-46 N-Ethylmaleimide, 173, 288

467

SUBJECT INDEX

F Facilitated diffusion, of nucleosides and bases, 235-253 FDNB, see I-Fluoro-2,4-dinitrobenzene 1-Fluoro-2,4-dinitrobenzene, 167 Freeze-fracture techniques, 78-82

G Galactosyltransferase, at aidwater interface, 22-24 Gallbladder, epithelial structure, 430-431 Galvani potential, 10 Glucose, see also Hexose transfer system half-saturation constant determination, 168-170, 171 Glucose transport, model, 176 L-Glucose transport, 269 Goldman equation, 72-73 Guanine transport, 274 Guanosine transport kinetics, 254

H Heat shock, effect on nucleoside and base transport, 295 Heterologous exchanges, 170 Hexose transfer system, 166-215 affinity constants for, 168- 170 asymmetric proteolytic responses, 179I 80 asymmetry of, 166-215 equilibrium exchanges, 186- 187 flux measurements, 172- 173 implications of asymmetry, 21 1-215 inhibitor studies, 173-177, 192-198 initial transfer rates, 170- 172 kinetic asymmetry, 166-202 basis of, 181-182 definition, 166 effects of pH and temperature, 200-202 history, 166-167 membrane transfers with asymmetric affinities, 181-202 model, 176 morphological asymmetry, 202-21 1 physical asymmetry of, 209-21 1 Sen Widdas exits, 168-171, 185 Hormones, regulatory role in Malpighian tubule transport, 445-453

p-Hydroxymercuribenzene sulfonate, 287288 p-Hydroxymercuribenzoate, 287-288, 305 2-Hydroxylnitrobenzylthioguanosine,234 Hypoxanthine transport kinetics, 242, 254, 256, 257, 262, 263, 269, 275

I Incorporation, definition, 227 Infinite-cis procedure, 238, 242, 245-248 Infinite-trans procedure, 245-246 Inosine transport kinetics, 242, 256, 258 Interface, see also Airiwater interface enzyme adsorption at, 16-19 of two immiscible liquids, 2-49 electron transfer at, 33-34 oil phase dielectric constant and charge transfer through, 45-46 potential jumps at, 4- I I , 25-30 proton transfer at, 34-35, 40-42 Iodoacetate, 230 Ion currents, measurement of, 72-74 Ion transport systems, reconstitution, 57Ill experimental systems, 86- 108 future of, 108- I I I measurements, 64-86 preparation of components, 60-64 Isopropylidene glucose, 175, 197

K Kidney, peptide transport in, 365, 366

L Lipid annulus composition, 150- 154 definition, 149-150 transbilayer disposition, 157- 158 asymmetry, 154- 155 Ca-ATPase activity and, 137- 144, 148I 50 distribution across SR membrane, 155I57 headgroup, and Ca-ATPase activity, 141144 phase transition temperatures, and CaATPase activity, 137- 140

468 pools, equilibration of, 133- 137 role in functioning of ATPase, 127- 159 Lipid substitution technique, 134- 135 Lipid titration procedure, 135- 137 Lipopolysaccharide, in mixed monolayer. 22-23

SUBJECT INDEX

Noise analysis, in reconstitution experiments, 74-76 Nonmediated permeation, role in nucleoside and purine uptake, 267-271 Nuclear magnetic resonance, reconstitution experiments and, 82-85 Nucleic acid base transport M in animal cells, 226-313 effect of hydrolytic enzymes, 295 Magnetic resonance, reconstitution experiof pH, 281-282 ments and, 82-85 of sulfhydryl reagents, 287-288 Malpighian tubule, insect of temperature, 279-281 absorption of water vapor from air, 457equilibrium exchange studies, 240-244 459 facilitated diffusion, 235-253 autonomous regulation, 453-456 heat shock and, 295 epithelial structure, 430-43 I infinite-cis procedure, 245-248 epithelial transport in, 428-460 infinite-trans procedure, 245 hormonal effects on, 445-453 in nonmammalian organisms, 284-287 inducible transport systems, 456-457 nonmediated permeation and, 267-271 passive epithelial permeability, 438-442 presumptive cell clones defective in, 282regulatory properties, 445-457 284 structure and function, 442-444 regulation, 295-303 transepithelial potential differences in, specificity for natural substrate, 271-277 437-438 substrate analog transport, 277-279 water transport route, 428-438 i n tandem operation with metabolism, MANQ,see 2-N-Methylamino- 1,4,-naphtho255-261 quinone in vesicles, 264-267 Mechanochemical hypothesis, 47 zero-trans kinetics, 237-240, 260-264 Membrane-enzyme system, coupling of, Nucleic acid base uptake, 253-271 46-48 general characteristics, 227-235 2-Mercapto- I -p-4-pyridethylbenzimidazole, regulation, 295-303 289 Mercuric chloride-sodium iodide solution, Nucleoside transport, 226-3 13 effect of hydrolytic enzymes, 295 234 of pH, 281-282 2-N-Methylamino- I ,4-naphthoquinone, 43 of p-nitrobenzylthiopurine nucleosides, Micelle formation, I32 293-295 Microsomal redox chain, reconstitution, of sulfhydryl reagents, 287-288 102-103 of temperature, 279-281 Mitochondria1 respiratory chain, membrane equilibrium exchange studies, 240-244 enzyme systems of, 42-43 facilitated diffusion, 235-253 Mobile carrier hypothesis, 187- 192, see also heat shock and, 295 Carrier model infinite-cis procedure, 245-248 infinite-trans procedure, 245 N inhibitors of, 287-295 NADH dehydrogenase, 42 in nonmammalian organisms, 284-287 Na+ + K+-ATPase, reconsitution of, 90-93 nonmediated permeation and, 267-271 NEM, see N-Ethylmaleimide presumptive cell clones defective in, 282Nitrobenzylthioinosine, 234 284 p-Nitrobenzylthiopurine nucleosides, effect regulation, 295-303 on nucleoside transport, 293-295 specificity for natural substrates, 27 1-277

469

SUBJECT INDEX

substrate analog transport, 277-279 in tandem operation with metabolism, 255-261 in vesicles, 264-267 zero-trans kinetics, 237-240, 260-264 Nucleoside uptake, 253-271 general characteristics of, 227-235 regulation, 295-303 Nucleotides, permeation in animal cells, 303-3 10

0 Octane/water interface, photooxidation of water at, 35-40 OiUwater interface, see Interface, of two immiscible liquids Oligopeptide transport in bacteria, 375-376 in yeast, 391-392 Open bilayer membrane area determination, 65 definition, 59 experimental advantages of, 59 preparation, 63-64 Optical measurements, in reconstitution experiments, 65-68 Optical probes, in reconstitution experiments, 66 Oxidation reaction, coupling to phosphorylation reactions, mechanisms of, 46-48 Oxidation-reduction potential, determination in bilayers, 74 Oxidation-reduction transformations, of chlorophyll and porphyrins, 30-33 Oxygen photogeneration, 37-39

P PAH, see p-Aminohippuric acid Papaverine, 289, 304-305 Partition coefficient, of sugar and base substrates, 269 PCMBS, see p-Chloromercuribenzene sulfonate Peptides, labeled, in transport studies, 373375 Peptides, nonprotein, transport of, 396-397 Peptides, small active uptake by absorptive cells, 335-336

effect on electrical potential difference, 339 entry into blood, 364-365 foliar absorption by SarraceniaJlava, 398 intracellular hydrolysis of, 357-358 intralumen and brush border hydrolysis of, 334-335 kinetics of intestinal absorption, 352-353 nutritional utilization in microorganisms, 367-369 relative absorption rates, 349-35 I Peptide permeases genetics of, 376-378 membrane location, 369-371 substrate specifities, 378-383 Peptide transport, 331-407 absorption mechanisms, 358-361 in algae, 395 amino acid side-chains, 342, 382-383 amino-terminal group and, 339-340, 379 in animal nonintestinal tissues, 365-367 in animal small intestine, 333-365, 406 in bacteria, 375-390, 406 of biologically active peptides, 365 carboxy-terminal group and, 340, 379 defining characteristics of, 371 effects of dietary alterations and disease on, 355-356 energy coupling to, 383-386, 393 in higher plants, 397-403 a-hydrogen atom and, 380 interrelations between uptake, exodus, and metabolism, 389-390 intestinal sites of maximal absorption, 353-355 kinetics, 402-403 in lichens, 395 methods of study, 371-375 in microorganisms, 367-397, 406 molecular size and, 343-344, 383, 401 molecular structure and, 339-342 mucosal uptake, 344-346 competitive, 346 in developing animals, 356-357 independence of, 344-346 quantitative importance of, 362-364 multiple systems, 347-349 Naf replacement and, 336-338 in Neurospora crassa, 394-395 peptide bond and, 340-341, 379-380

470 physiological advantages of, 403-407 regulation, 387-389, 393-394 by scutellum of germinating barley, 398403, 406 stereochemical specificity, 341-342, 380382, 392-393, 400-401 in yeast, 390-394, 406 Persantin, see Dipyridamole Phenethyl alcohol, effect on nucleoside uptake, 291, 305 Phloretin, 289 Phlorizin, 289 Phosphatidylethanolamine, in mixed monolayer, 22-23 Phospholipase, D, in digestion of lipids surrounding Ca-ATPase, 157- 158 Phospholipid, see Lipid Phosphorylation reaction, coupling to oxidation reactions, mechanisms for, 4648 Photobleaching techniques, in reconstitution experiments, 66-68 Photosystem I , reconstitution, 104 Plateau-Gibbs border, 59 open bilayer membrane diameter, 65 Podophyllotoxin, 289 Porphyrin at interface, 30-42 oxidation-reduction transformations of, 30-33 Potassium cyanide, 230 Potassium transport, in Malpighian tubules, 453-456 Potential, see Adsorption potential; Compensation potential; Distribution potential; Transmembrane potential; Volta potential Potential jump at interface of two immiscible liquids, 4II mechanism of generation of, 25-30 Principle of uniformity of nature, 210 Probenecid, 304, 305 Propyl-P-D-glucopyranoside, 176 Prostaglandins, 289 Protein, intralumen and brush border hydrolysis of, 334-335 Proton-potassium pump, reconstitution, 104-106

SUBJECT INDEX

Proton transfer chlorophyll and, 34-35 role of water and DNP in, 40-42 Purine uptake, 253-271, see crlso Nucleic acid base transport inhibition, 287-293 nonmediated permeation and, 267-27 I in tandem operation with metabolism, 255-261 in vesicles, 264-267 zero-trans kinetics, 237-240, 260-264

R Reaggregation, definition, 58 Recombination, definition, 58-59 Reconstitution, 58-59, see also Ion transport systems, reconstitution of Reformation, definition, 58 Regen and Tarpley kinetics, applied to nontransportable inhibitors, 192- 198 Relaxation analysis, in reconstitution experiments, 74-76 Respiratory chain, mitochondrial, membrane enzyme systems of, 42-43 Rhodopsin, at interface, 43-45

S Sarcoplasmic reticulum isolation and enzymes of, 128- 13I reconstituted membrane of, effect of different lipids on, 145-148 solubilization, I33 Sarraceniu f l a v a , foliar absorption of peptides, 398 Scanning electron microscopy, in reconstitution experiments, 81 Sen-Widdas exits, 168-170, 171, 185 Small intestine disease of, and peptide absorption, 355356 maximal absorption sites, 353-355 multiple peptide uptake systems, 347-349 peptide transport in, 333-365 “Smugglin” concept, 396 Sodium ion, peptide uptake and, 336-338 Sodium-potassium pump, reconstitution, 90-93 Sterigmatocystin, 289

47 1

SUBJECT INDEX

Streptovaracin, 289 Substrate facilitated transfers, 167- 168 Succinate-cytochrome c reductase, 43 Sugar transport, model, 176 Sulfate transport, in Malpighian tubules, 457, 458 Sulfhydryl reagents, effects on nucleoside and base transport, 287-288 Surface pressure determination, 20 of mixed chlorophyll-cytochrome monolayer, 31 of mixed chlorophyll-ferredoxin monolayer, 33 reconstitution experiments and, 86 Symbols, list of, 49-50

T TF$F,, reconstitution, 99- 101 Theophylline, 289, 304, 305 Thymidine transport kinetics, 242, 254, 269 Transmembrane potential, in nitrobenzene/ water system, 14 Transport, definition, 227 Trimethyl glucoside, 197 Two complex model, 193 Two site model, 182- 187

U Uptake, definition, 227 Uracil transport kinetics, 242, 269, 274 Uridine transport kinetics, 238, 241, 242, 244, 246, 248, 254, 262, 269, 274, 275, 290, 295-303

V VDAC, see Voltage-dependent, anion-selective channel

Vesicle area determination, 65-66 definition, 59 experimental advantages of, 59-60 in nucleoside and purine uptake studies, 264267 preparation of, 61-63, 109 from sarcoplasmic reticulum, 129- 130 Vibrating plate method, 11-13 Vinblastin, 304 Voltage-dependent, anion-selective channel, reconstitution, 108 Volta potential, 9 in complex enzyme system, 24-25 determination experimental, 11-13 theoretical, 10- I I mixed chlorophyll-ferridoxin monolayer and, 33 in nitrobenzeneiwater system, 14 in octane/water system, 41 oil phase dielectric constant and, 46

W Water intestinal transport of peptides and, 338 photooxidation, 35-40 role in proton transfer, 40-42 Water transport, in Malpighian tubules, 428-438. 457-459

XYZ Yeast, peptide transport in, 390-394 Zero-trans experiments initial transfer rates, 170- 172 with nucleosides and bases, 242, 237-240, 260-264

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Contents of Previous Volumes Volume 1 Some Considerations about the Structure of Cellular Membranes MAYNARD M. DEWEYA N D LLOYDBARR The Transport of Sugars across Isolated Bacterial Membranes H. R. KABACK Galactoside Permease of Escherichiu coli ADAMKEPES Sulfhydryl Groups in Membrane Structure and Function ASER ROTHSTEIN Molecular Architecture of the Mitochondrion DAVIDH. MACLENNAN Author Index-Subject Index

Volume 2 The Molecular Basis of Simple Diffusion within Biological Membranes W. R. LIEBA N D W. D. STEIN The Transport of Water in Erythrocytes ROBERTE. FORSTER Ion-Translocation in Energy-Conserving Membrane Systems B. CHANCE A N D M . MONTAL Structure and Biosynthesis of the Membrane Adenosine Triphosphatase of Mitochondria ALEXANDERTZAGOLOFF

Mitochondria1 Compartments: A Compari. son of Two Models HENRYTEDESCHI Author Index-Subject Index

Volume 3 The Na+, K+-ATPase Membrane Transport System: Importance in Cellular Function ARNOLDSCHWARTZ, GEORGEE. LINDENMAYER, AND JULIUSC. ALLEN Biochemical and Clinical Aspects of Sarcoplasmic Reticulum Function ANTHONY MARTONOSI The Role of Periaxonal and Perineuronal Spaces in Modifying Ionic Flow across Neural Membranes W.J. ADELMAN, JR., A N D Y. PALTI Properties of the Isolated Nerve Endings GEORGINA RODRfGUEZ DE LORES AND ARNAIZ EDUARDO DE ROBERTIS Transport and Discharge of Exportable Proteins in Pancreatic Exocrine Cells: In Vitro Studies J. D. JAMIESON The Movement of Water across Vasopressin-Sensitive Epithelia RICHARD M. HAYS

473

474 Active Transport of Potassium and Other Alkali Metals by the Isolated Midgut of the Silkworm WILLIAM R. HARVEYA N D KARLZERAHN

CONTENTS OF PREVIOUS VOLUMES

A Macromolecular Approach to Nerve Excitation AND ICHUITASAKI EMILIO CARBONE Subject Index

Author Index-Subject Index

Volume 6

The Genetic Control of Membrane Transport CAROLYN W. SLAYMAN Enzymic Hydrolysis of Various Components in Biomembranes and Related Systems MAHENDRA KUMARJAIN Regulation of Sugar Transport in Eukaryotic Cells HOWARDE. MORGAN AND CAROLF. WHITFIELD Secretory Events in Gastric Mucosa RICHARD P. DURBIN

Role of Cholesterol in Biomembranes and Related Systems MAHENDRA KUMARJAIN Ionic Activities in Cells A. A. LEV A N D W. McD. ARMSTRONG Active Calcium Transport and Ca2+-Activated ATPase in Human Red Cells H. J . SCHATZMANN The Effect of Insulin on Glucose Transport in Muscle Cells TORBENCLAUSEN Recognition Sites for Material Transport and Information Transfer HALVOR N. CHRISTENSEN

Author Index-Subject Index

Subject Index

Volume 4

Volume 5 Cation Transport in Bacteria: K+, Na+, and H + FRANKLIN M. HAROLDA N D KARLHEINZ ALTENDORF Pro and Contra Carrier Proteins: Sugar Transport via the Periplasmic GalactoseBinding Protein WINFRIEDBoos Coupling and Energy Transfer in Active Amino Acid Transport ERICHHEINZ The Means of Distinguishing between Hydrogen Secretion and Bicarbonate Reabsorption: Theory and Applications to the Reptilian Bladder and Mammalian Kidney WILLIAM A. BRODSKY AND THEODORE P. SCHILB Sodium and Chloride Transport across Isolated Rabbit Ileum STANLEY G. SCHULTZA N D PETERF. CURRAN

Volume 7 Ion Transport in Plant Cells E . A. C. MACROBBIE H+ Ion Transport and Energy Transduction in Chloroplasts RICHARD A. DILLEYA N D ROBERTT. GIAQUINTA The Present State of the Carrier Hypothesis PAULG. LEFEVRE Ion Transport and Short-circuit Technique WARRENS. REHM Subject Index

Volume 8 Chemical and Physical Properties of Myelin Proteins M. A. MOSCARELLO The Distinction between Sequential and Simultaneous Models for Sodium and Potassium Transport P. J . GARRAHAN A N D R. P. GARAY

475

CONTENTS OF PREVIOUS VOLUMES

Soluble and Membrane ATPases of Mitochondria, Chloroplasts, and Bacteria: Molecular Structure, Enzymatic Properties, and Functions RIVKAPANETA N D D. RAOSANADI Competition, Saturation, and InhibitionIonic Interactions Shown by Membrane Ionic Currents in Nerve, Muscle, and Bilayer Systems AND ROBERTJ . FRENCH WILLIAM J . ADELMAN, JR. Properties of the Glucose Transport System in the Renal Brush Border Membrane R. KINNE Subject Index

Volume 9 The State of Water and Alkali Cations within the Intracellular Fluids: The Contribution of NMR Spectroscopy SHPORER AND MORDECHAI MORTIMER M. CIVAN Electrostatic Potentials at Membrane-Solution Interfaces STUARTMCLAUGHLIN A Thermodynamic Treatment of Active Sodium Transport S. ROYCAPLANA N D ALVINESSIG Anaerobic Electron Transfer and Active Transport in Bacteria W I L N. KONINGS AND JOHANNES BOONSTRA Protein Kinases and Membrane Phosphorylation M. MARLENE HOSEYA N D MARIANO TAO Mechanism and Physiological Significance of Calcium Transport across Mammalian Mitochondria1 Membranes LEENAMELA Thyroidal Regulation of Active Sodium Transport F. ISMAIL-BEIGI Subject Index

Volume 10 Membrane Properties: Mechanical Aspects, Receptors, Energetics and Calcium-Dependence of Transport Mechanochemical Properties of Membranes AND E. A. EVANS R. M. HOCHMUTH Receptor-Mediated Protein Transport into Cells. Entry Mechanisms for Toxins, Hormones, Antibodies, Viruses, Lysosomal Hydrolases, Asialoglycoproteins, and Carrier Proteins DAVIDM. NEVILLE,JR., AND TA-MINCHANG The Regulation of Intracellular Calcium ERNESTO CARAFOLI A N D MARTINCROMPTON Calcium Transport and the Properties of a Calcium-Sensitive Potassium Channel in Red Cell Membranes VIRGILIO L. LEW A N D HUGOG. FERREIRA Proton-Dependent Solute Transport in Microorganisms A. A. EDDY Subject Index

Volume 11 Cell Surface Glycoproteins: Structure, Biosynthesls, and Biological Functions The Cell Membrane-A Short Historical Perspective ASERROTHSTEIN The Structure and Biosynthesis of Membrane Glycoproteins JENNIFER STURGESS, MARIOMOSCARELLO, AND HARRYSCHACHTER Techniques for the Analysis of Membrane Glycoproteins R. L. JULIANO Glycoprotein Membrane Enzymes AND JOHNR. RIORDAN GORDONG. FORSTNER

476 Membrane Glycoproteins of Enveloped Viruses W. COMPANS AND RICHARD MAURICE C . KEMP Erythrocyte Glycoproteins MICHAELJ. A. TANNER Biochemical Determinants of Cell Adhesion LLOYDA. C U L P Proteolytic Modification of Cell Surface Macromolecules: Mode of Action in Stimulating Cell Growth KENNETHD. NOONAN Glycoprotein Antigens of Murine Lymphocytes MICHELLELETARTE Subject Itidcx

Volume 12 Carriers and Membrane Transport Proteins Isolation of Integral Membrane Proteins and Criteria for Identifying Carrier Proteins MICHAELJ. A. T A N N E R The Carrier Mechanism S. B. HLADKY The Light-Driven Proton Pump of H d o bacterium hnlohiirtn: Mechanism and Function AND MICHAELEISENBACH S. ROY CAPLAN Erythrocyte Anion Exchange and the Band 3 Protein: Transport Kinetics and Molecular Structure P H I L I PA. KNAUF The Use of Fusion Methods for the Microinjection of Animal Cells R. G. KULKAA N D A. LOYTER Subject Index

Volume 13 Cellular Mechanisms of Renal Tubular Ion Transport PART I: ION ACTIVITY A N D ELEMENTAL COMPOSITION OF INTRAEPITHELIAL COMPARTMENTS

CONTENTS OF PREVIOUS VOLUMES

Intracellular p H Regulation WALTERF. BORON Reversal of the pH,-Regulating System in a Snail Neuron R. C. THOMAS How to Make and Use Double-Barreled Ion-Selective Microelectrodes THOMAS ZUETHEN The Direct Measurement of K , CI, Na, and H Ions in Bullfrog Tubule Cells MAMORUFUJIMOTO, K U N I H I K KOTERA, O AND Y UT A K A M ATSU M u RA Intracellular Potassium Activity Measurements in Single Proximal Tubules of Necturus Kidney TAKAHIRO KUBOTA,BRUCEBIAGI, A N D GERHARD GIEBISCH Intracellular Ion Activity Measurements in Kidney Tubules RAJA N. KHURI Intracellular Chemical Activity of Potassium in Toad Urinary Bladder JOEL DELONGA N D MORTIMER M. C I V A N Quantitative Determination of Electrolyte Concentrations in Epithelial Tissues by Electron Microprobe Analysis ROGERRICK,ADOLFDORGE, RICHARDBAUER,FRANZ BECK, J U N EMASON,CHRISTIANE ROLOFF, A N D KLAUSTHURAU PART 11: PROPERTIES OF INTRAEPIT H E L I A L MEMBRANE BARRIERS I N T H E KIDNEY Hormonal Modulation of Epithelial Structure JAMESB. WADE Changes in Cell Membrane Surfaces Associated with Alterations of Transepithelial Ion Movement MICHAELKASHGARIAN The Dimensions of Membrane Barriers in Transepithelial Flow Pathways LARRYw. WELLING A N D DANJ . WELLING Electrical Analysis of lntraepithelial Barriers E M I L EL. BOULPAEP A N D HENRYSACKIN

477

CONTENTS OF PREVIOUS VOLUMES

Membrane Selectivity and Ion Activities of Mammalian Tight Epithelia S I M O NA. LEWIS,NANCYK. WILLS, A N D DOUGLAS c. EATON Ion Conductances and Electrochemical Potential Differences across Membranes of Gallbladder Epithelium LUISREUSS A Kinetic Model for Ion Fluxes in the Isolated Perfused Tubule BRUCEBIAGI,ERNESTO GONZALEZ, A N D GERHARD GIEBISCH The Effects of Voltage Clamping on Ion Transport Pathways in Tight Epithelia ARTHURL. FINNA N D PAULARoGENES

Tubular Permeability to Buffer Components as a Determinant of Net H Ion Fluxes G. MALNIC,V. L. COSTASILVA, S. S. CAMPIGLIA, M. DE MELLO AIRESA N D G. GIEBISCH Ionic Conductance of the Cell Membranes and Shunts of Necrurus Proximal Tubule GENJIROKIMURAAND KENNETHR. SPRING Luminal Sodium Phosphate Cotransport as the Site of Regulation for Tubular Phosphate Reabsorption: Studies with Isolated Membrane Vesicles HEINIMURER,REINHARD STOLL, CARLAEVERS,ROLF KINNE, JEAN-PHILIPPE BONJOUR, A N D HERBERT FLEISCH The Mechanism of Coupling between Glucose Transport and Electrical Potential in the Proximal Tubule: A Study of PotentialDependent Phlorizin Binding to Isolated Renal Microvillus Membranes PETERS. ARONSON Electrogenic and Electroneutral Na Gradient-Dependent Transport Systems in the Renal Brush Border Membrane Vesicle BERTRAM SACKTOR

PART 111: INTRAMEMBRANE CARRIERS AND ENZYMES IN TRANSEPITHELIAL TRANSPORT Sodium Cotransport Systems in the Proximal Tubule: Current Developments R. KINNE,M. BARAC, A N D H. MURER ATPases and Salt Transport in the Kidney Tubule MARGARITA P~REZ-GONZALEZ DE LA MANNA,FULGENCIO PROVERBIO, AND GUILLERMO WHITTEMBURY Further Studies on the Potential Role of an Anion-Stimulated Mg-ATPase in Rat Proximal Tubule Proton Transport E. KINNE-SAFFRAN A N D R. KINNE Renal Na+-K+-ATPase: Localization and Quantitation by Means of Its K+-Dependent Phosphatase Activity REINIERBEEUWKES 111 SEYMOUR ROSEN

AND

Relationship between Localization of Na+K+-ATPase, Cellular Fine Structure, and Reabsorptive and Secretory Electrolyte Transport STEPHEN A. ERNST, CLARAV. RIDDLE,A N D KARLJ. KARNAKY, JR. Relevance of the Distribution of Na+ Pump Sites to Models of Fluid Transport across Epithelia JOHNW. MILLSA N D DONALDR. DIBONA Cyclic AMP in Regulation of Renal Transport: Some Basic Unsolved Questions THOMASP. DOUSA Distribution of Adenylate Cyclase Activity in the Nephron F. MOREL,D. CHABARD~S, A N D M. IMBERT-TEBOUL Subject Index

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