Current Topics in Membranes and Transport, Volume 08

Current Topics in Membranes and Transport Volume 8

Advisory Board

Robert W . Berliner I . S. Edelman I . M. Glynn Franpis Morel Shmuel Razin Aser Rothstein H . J . Schatzmann Stanley G. Schultz Philip Siekevitz Daniel C. Tosteson

Contributors

William J . Adelman, Jr. Robert J . French R. P . Garay P. J . Garrahan R. Kinne M . A. Moscarello Rivka Panet D. Rao Sanadi

Current Topics in Membranes and Transport

VOLUME 8

Edited by Felix Bronner Department of Oral Biology llniversity of Connecticut Health Center Farmington, Connecticut and Arnort Kleinzeller Department of Physiology [Jniversity of Pennsylvania School of Medicine Philadelphia, Pennsylvania

1976

Acodernic Press

New York

San Froncisco

London

A Subsidiary of Harcourl Brace Jovanovich, Publishers

COPYRIGHT 0 1976, 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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LIBRARY OF CONGRESS CATALOG CARD NUMBER:70- 11709 1 ISBN 0- 12-153308-5 PRINTED IN THE UNITED STATES O F AMERICA

List of Contributors, vii Contents of Previous Volumes, ix

Chemical and Physical Properties of Myelin Proteins M. A. MOSCARELIJO

I. Introdurtion, 1 11. Ihcephalitogenic Protein of Myelin, 3 111. IV. V. VI. VII.

Physical Structure of Myelin Basic Protein, 7 l’roteolipid Protein Fraction of Myelin, 10 Other Protein Fractions Prepared from Whole Brain, White fut,ter, or Myelin, I Conclusions on the Nature of Myelin l’roteins, 18 Localization of Proteins i n Myelin, 21 Referencaes, 24

The Distinction between Sequential and Simultaneous Models for Sodium and Potassium Transporf 1’. J. GARRAHAN AND R. P. GARAY

I. Introduction, 29 11. Cation-Binding Sites of the Xu Pump, 31 111. Experimental Evidence for Simultaneous Existence of Inner and Outer CationBinding Sites, 40 IV. Intermediate Stages in Hydrolysis of ATP by the Na Pump, 68 V. Mechanism of Ion Transport, 77 References, 91 Soluble and Membrane ATPases of Mitochondria, Chloroplasts, and Bacteria: Molecular Structure, Enzymatic Properties, and Functions RIVKA PANET AND TI. RAO SANADI

I. Introduction, 99 11. Mitochondria1 Coupling Factor, 100 111. Oligomycin-Sensitive ATPase, 118 IV. Chloroplast Coupling Factor, 126 V

CONTENTS

Vi

V. Bacterial ATPase, 141 VI. General Conclusions and Perspective, 149 References, 150 Competition, Saturation, and Inhibition-Ionic Interactions Shown by Membrane Ionic Currents in Nerve, Muscle, and Bilayer Systems ROBERT J. FRENCH AND WILLIAM J. ADELMAN, JR.

I. 11. 111. IV. V.

Perspectives, 161 Saturation Phenomena, 166 Blocking and Competition, 169 Models and Analyses, 189 Concluding Remarks, 198 References, 200

Properties of the Glucose Transport System in the Renal Brush Border Membrane R. KINNE

I. Introduction, 209 11. Isolation and Characterization of Plasma Membranes from Proximal Tubular Epithelium, 211 111. Interaction of D-Glucose with Isolated Renal Plasma Membranes, 218 IV. Interaction of Phlorizin with Isolated Renal Plasma Membranes, 233 V. Molecular Characteristics of the Sugar Transport System in the Brush Border Membrane, 245 VI. Conformational Response of the Glucose Transport System, 251 VII. Relation of Renal Glucose Transport System to Enzymes Interacting with Carbohydrates, 256 VIII. One or Several Glucose Transport Systems in the Brush Border Membrane?, 257 IX. Summary and Conclusions, 259 References, 259 Note Added in Proof, 267 Subject Index, 269

Numhers in parentheses indicate the pages on which the authors’ contributions begin. Lahoratory of Biophysics, IRP, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Education and Welfare, Marine Biological Laboratory, Woods Hole, Massachusetts (161)

William J. Adelman, Jr.,

Laboratory of Biophysics, IRP, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Department of Health, Education and Welfare, Marine Biological Laboratory, Woods Hole, Massachusetts (161)

Robert J. French,

Departamento de Quimica Bioldgica, Facultad de Farmacia y Bioquimica, Buenos Aires, Argentina (29)

R. P. Gamy,

Departamento de Quimica Bioldgica, Facultad de Farmacia y Bioquimica, Buenos Aires, Argentina (29)

P. J. Garrahan,

R. Kinne,

Max-Planck-Institut fur Biophysik, Frankfurt, Germany (209)

M. A. Morcarello,

Research Institute, The Hospital for Sick Children, Toronto,

Canada (1) Department of Cell Physiology, Boston Biomedical Research Institute, Boston, Massachusetts (99)

Rivka Panet,*

Department of Cell Physiology, Boston Biomedical Research Institute, Boston, Massachusetts (99)

D. Roo Sanadi,

* Present address: Department of Nuclear Medicine, Hadassah Haspital, Jerusalem, Israel. vii

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Contents of Previous Volumes Volume 1

Some Considerations about the Stnicture of Cellular Membranes MAYNARD M. DEWEYAND LLOYDBARR The Transport of Sugars across Isolated Bacterial Membranes H. R. KABACK Galactoside Permease of Escherichia coli ADAMKEPES Sulfhydryl Groups in Membrane Structure and Function ASER ROTHSTEIN Molecular Architecture of the Mitochondrion DAVID13. MACLENNAN Author Index-Subject Index Volume 2

The Molecular Basis of Simple Diffusion within Biological Membranes W. R. LIER AND W. D. STEIN The Transport of Water in Erythrocytes

ROBERTE. FORSTER Ion-Translocation in Energy-Conserving Membrane Systems B. CHANCE AND M. MONTAL Structure and Biosynthesis of the Membrane Adenosine Triphosphatase of Mitochondria ALEXANDERTZAGOLOFF Mitochondria1 Compartments: A Comparison of Two Models HENRYTEDESCHI Author Index-Subject Index ix

X

CONTENTS OF PREVIOUS VOLUMES

Volume 3

The Na+, K+-ATPase Membrane Transport System : Importance in Cellular Function ARNOLDSCHWARTZ, GEORGEE. LINDENMAYER, AND JULIUS C. 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. AND Y. PALTI Properties of the Isolated Nerve Endings GEORGINA RODR~GUEZ DE LORESARNAIZ AND EDUARDO DE ROBERTIS Transport and Discharge of Exportable Proteins in Pancreatic Exocrine Cells : I n Vitro Studies J. D. JAMIESON The Movement of Water across Vasopressin-Sensitive Epithelia RICHARD M. HAYS Active Transport of Potassium and Other Alkali Metals by the Isolated Midgut of the Silkworm WILLIAMR. HARVEY AND KARLZERAHN Author Index-Subject Index Volume 4

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 HOWARD E. MORGAN AND CAROLF. WHITFIELD Secretory Events in Gastric Mucosa RICHARDP. DURBIN Author Index-Subject Index Volume 5

Cation Transport in Bacteria: K+, Na+, and H+ FRANKLIN M. HAROLD AND KARLHEINZ ALTENDORF Pro and Contra Carrier Proteins; Sugar Transport via the Periplasmic Galactose-Binding Protein WINFRIEDBoos

CONTENTS OF PREVIOUS VOLUMES

xi

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 WILLIAni A. BRODSKY AND THEODORE P. SCHILB Sodium and Chloride Transport across Isolated Rabbit Ileum A N D PETER F. CURRAN STANLEY G. SCHULTZ A Macromolecular Approach t.3 Nerve Excitation ICHIJI TASAKI AND EMILIO CARBONE Subject Index Volume 6

Role of Cholesterol in Biomembranes and Related Systems MAHENDRA KUMAR JAIN Ionic Activities in Cells A. A. LEV AND W. McD. ARMSTRONG Active Calcium Transport and Ca2+-ActivatedATPase in Human Red Cells II. J. SCHATZMANN The Effect of Insulin on Glucose Transport in Muscle Cells TORBEN CLAUSEN Recognition Sites for Material Transport and Information Transfer HALVOR N. CHRISTENSEN Subject Index Volume 7

Ion Transport in Plant Cells E. A. C. MACROBBIE H+ Ion Transport and Energy Transduction in Chloroplasts A. DILLEYAND ROBERTT. GIAQUINTA RICHARD The Present State of the Carrier Hypothesis PAULG. LEFEVRE Ion Transport and Short-circuit Technique S. REHM WARREN Subject Index

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Chemical and Physical Properties of Myelin Proteins M . A . MOSCARELLO Research Institide The Hospital for Sick Children Toronto, Canada

Introduction . . . . . . . . . . . . . . . . . . 1 3 Encephalitogenic Protein of Myelin . . . . . . . . . . . . Physical Structure of Myelin Basic Protein . . . . . . . . . 7 Proteolipid Protein Fraction of Myelin . . . . . . . . . . . 10 Physical Studies . . . . . . . . . . . . . . . . . 13 V. Other Protein Fractions Prepared from Whole Brain, White Matter, or Myelin . . . . . . . . . . . . . . . . . . . . 17 VI. Conclusions on the Nature of Myelin Proteins. . . . . . . . . 18 VII. Localization of Proteins in Myelin . . . . . . . . . . . . 21 References . . . . . . . . . . . . . . . . . . . 24

I. 11. 111. IV.

1.

INTRODUCTION

The word protein is derived from the Greek word proteios, meaning primary, the fundamental material. Until recently, models of membranes have ignored this primary role of proteins, but emphasis on the lipid components has been widespread. The myelin membrane, which consists of approximately 70-75010 lipid and 25-30010 protein, has always been considered as a bilayer of lipid with protein interacting with the charged groups of the lipids on the cxtcrnal surfaces (Danielli and Davson, 193435). This is the Danielli-Davson model based on the original work of Gortrr and Grrndel (1925) with cbrythrocytcs. Although it was generally agreed that the membranc proteins must be important, they were simply placed on the outside surface of the bilaycr. Thc more recent model of Singer and Nicolson (1972) emphasizes the nature of membrane proteins, some of which are visualized as being partially embedded in the lipid matrix, while others pass through the lipid. 1

2

M. A. MOSCARELLO

A single lipid bilayer appears to be a relatively fixed structure, impermeable to most water-soluble substances. On the other hand, large protein molecules, which can undergo many conformational transitions, might provide the needed flexibility required of a fluid mosaic membrane. It has become of central importance to the understanding of membrane structure and function to isolate and characterize membrane proteins. In the past, most membrane models were derived from data obtained from electron microscopy and X-ray diffraction studies. The lack of information concerning the number, nature, and characteristics of the various proteins in the membrane resulted in a simplistic model in which the protein was somehow stuck to the charged groups of the bilayer. An excellent critique of the methods used and interpretations made was published by Korn (1966). The possibility of artifacts arising from fixation techniques was carefully documented. In recent years, the primary structure of the encephalitogenic protein of myelin has been elucidated (Westall et al., 1971; Carnegie, 1971; Eylar, 1972). A “proteolipid” fraction consisting of several proteins and lipids soluble in chloroform-methanol has been known since 1951 (Folch Pi and Lees, 1951). A highly hydrophobic protein has recently been isolated from the myelin fraction soluble in acidified chloroform-methanol, purified (Gagnon et al., 1971), and some of its properties studied (Moscarello et al., 1973). Thus, on the one hand, a highly basic protein (the encephalitogenic or A1 protein) well suited for interactions with negatively charged phosphoryl groups was isolated from myelin. On the other hand, a totally different protein, the hydrophobic protein, ideally suited for interacting with the hydrocarbon chains of lipids and other apolar molecules was isolated from the same membrane. Both these very different proteins coexist in the myelin membrane along with other proteins still to be isolated. It is unlikely that these proteins coexist in a single 25-A layer of protein outside the bimolecular lipid leaflet as concluded by Finean (1953) and others from X-ray diffraction studies. The proteins must form an integral part of the membrane structure. The role they play must be determined by the properties of the proteins themselves. A highly charged protein, such as the basic protein of myelin, would be expected to take part in ionic interactions with the polar groups of lipids; the hydrophobic protein, on the other hand, would be expected to interact with fatty acid chains. Such interactions with different domains in the membrane must in some way reflect the functional role different proteins play in the membrane. The lipids, however, are usually associated with each other in the form of a bilayer structure. It is for this reason that the protein must be responsible for providing the “structural steel” of which membranes are made. It is proposed to discuss what is currently known

CHEMICAL A N D PHYSICAL PROPERTIES OF MYELIN PROTEINS

3

about myelin proteins with a view to showing that different proteins interact with the lipid in different ways, largely determined by the chemical and physical characteristics of the proteins themselves. Evidence from such studies will be used to show how these proteins may be responsible for the structural steel of the myelin membrane.

II. ENCEPHALITOGENIC PROTEIN OF MYELIN

Known as the basic protein of myelin (Kies, 1965) , the encephalitogenic protein, or the A, protein (Eylar et al., 1971), it comprises about 30% of the myelin protein. It is readily extracted from whole brain under a variety of conditions including buffers a t pH values from 3 to 4 and from isolated myelin by dilute mineral acid (Lowden et al., 1966). Because of these two features, i.e. , availability in rather large amounts and ease of extraction into aqueous media, it was possible to study it by conventional methods of protein chemistry (Eylar el al., 1969). The isolated material was shown to have potent biological activity. It induced experimental allergic encephalomyelitis (EAE) in the guinea pig a t concentrations of less than 5 pg per animal (Eylar, 1972). With the isolation of this protein, the earlier observations on EAE by RobozEinstein el al. (1962) and Kies et al. (1965) , who described EAE in a crude homogenate of brain, were extended significantly. It soon became obvious that a protein with such potent biological activity should be subjected to extensive physical and chemical studies in a number of laboratories, with a view to elucidating its complete structure and possibly defining its mechanism of action. The basic protein has been isolated in several laboratories (Carnegie, 1971; Lowden et al., 1966; Eylar et al., 1969; Itoboz-Einstein et al., 1962; Tomasi and Kornguth, 1967). The isolation from spinal cord (RobozEinstein et al., 1962) involved preliminary extraction in acetone-petroleumether followed by isolation of the protein with 5 1 0 % KCI. Other procedures have employed defatting the tissue with chloroform-methanol (Kies et al., 1965; Tomasi and Kornguth, 1967; Nakao et al., 1966; Wolfgram, 1965; Carnegie et al., 1967; Kibler and Shapka, 1968) prior to extraction of the protein. Basic protein has also been isolated from human central nervous system myelin by dilute mineral acid (Lowden et al., 1966). The isolation and purification of this protein was followed by structural studies that culminated in the elucidation of the complete amino acid sequence. The first significant chemical finding was the report (Lowden et al., 1966) that cystine was absent from the primary structure. The molecule was, therefore, not held together by disulfide bonds and, conse-

4

M. A. MOSCARELLO

quently, consisted of a single polypeptide chain. Eylar et al. (1971) isolated 27 tryptic peptides and 16 peptic peptides to establish the complete sequence of the 170 amino acid residues of the bovine spinal cord protein. No general periodicity of the basic residues was observed; the distribution appeared to be random. Several unusual features are noteworthy. The arginine residue at position 107 is present in both monomethylated and dimethylated derivatives. Not far from the methylated arginine residues is a proline-rich sequence, Pro-Arg-Thr-Pro-Pro-Pro, which may induce a sharp bend in the molecule, although other conformations are possible when the models of this region are constructed. If a sharp bend were present at this site near the center of the molecule, it may help to explain the axial ratio of 1O:l found by viscosity studies (Eylar et al., 1969). The Pro-ProPro sequence is not commonly found in proteins. It has been reported in rabbit immunoglobulin IgG (Smyth and Utsumi, 1967) in which it forms the hinge region. The threonine residue of this sequence has been reported by Hagopian and Eylar (1969) to be the site of glycosylation with N-acetylgalactosamine and an enzyme from submaxillary glands forming a GalNAc-0-Thr linkage. Only the deglycosylated natural acceptor of the enzyme served as an acceptor. Eylar et al. (1971) proposed that the A1protein (basic protein) may be glycosylated during myelin synthesis and deglycosylated when it becomes a constituent of the completed myelin membrane. If this were so, the glycosylated protein should be present in the Schwann cell. However, it has not as yet been isolated from this source. The structural determinants required for encephalitogenic activity vary from species to species. In the guinea pig, the sequence around the single tryptophan residue appears to be necessary for activity. The sequence of (Arg) . this region was shown to be Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys The C-terminal lysine could be replaced by arginine. An extensive study of this region was made by Westall et al. (1971). Using the solid-phase peptide synthesis technique (Merrifield, 1963), several peptides were prepared, each differing from the natural peptide by a single residue. Each peptide was tested for disease-producing activity in the guinea pig. As a result of these studies, it was concluded that the Trp - - -Gln-Lys (Arg) residues were essential. Activity was lost if the terminal Lys was removed. Replacement of the terminal Lys by Ile also resulted in loss of activity. Replacement of Gln by Ile and of Trp by Phe or Val produced inactive peptides. The evidence in favor of the view that the Trp region represents the major encephalitogenic site in the guinea pig can be summarized as follows: (i) the synthetic peptides have approximately the same activity on a mole/mole basis as the complete protein; (ii) modification of the Trp

CHEMICAL AND PHYSICAL PROPERTIES OF MYELIN PROTEINS

5

region with 2-hydroxy-5-nitrobenzyl bromide (Eylar et al., 1970), which attacks Trp exclusively, yields an inactive peptide; (iii) proteins isolated from several species including man, monkey, dog, rabbit, guinea pig, rat, mouse, and horse, all of which are equally encephalitogenic in the guinea pig, have preserved the Trp- - -Gln-Lys (Arg) sequence. This region is altered in the chicken and turtle proteins, but these are nonencephalitogenic. I n the chicken, the essential Gln residue is replaced by His (Eylar et al., 1974). Although the requirements for encephalitic activity in the guinea pig appear to be well defined, other requirements may be necessary for activity in the rabbit. An encephalitogenic peptide (Kibler et al., 1969) of 43 residues derived from another region of the molecule has been shown to be encephalitogenic in the rabbit but not in the guinea pig (Carnegie, 1969). Neither of the above two peptides, i.e., the one active in guinea pigs and the one active in rabbits, is active in the monkey (Eylar et al., 1972). This prptidc drrived from residues 117-170 (Iiibler et al., 1969) was produced by thc BNPS-skat ole [2- (2-nitrophenylsulfcnyl) -3-mcthyl-3’-bromo-indoleninc] reaction that cleaves the peptide chain a t a tryptophanyl residue. Thrrrforr, the Trp region, csscntial for activity in the guinra pig, is not essrntial for activity in the monkey. The main disease-inducing site cffrctive in the monkey resides in the carboxy terminal of the molecule (Eylar et nl., 1974). Since the region responsiblc for EAE activity varies from species to species, it appears unlikely that genrralizations of structural requirements for encephalitogenic activity can be made from the above studies, and it is reasonable to conclude that a large family of sequences may possess disease-inducing properties. I n discussing the possible viral etiology of multiple sclerosis, which may be similar to EAE, Westall (1974) speculates that the virus may bring about an appropriate change in a nucleic acid base which could result in an altered sequence. This sequence, in turn, would confer encephalitogenic activity on the protein. Other antigenic sites have been localized to different parts of the molecule (Borgstrand and Kallen, 1973). The regions that induce transformation of lymph node cells from immunized rabbits are located in regions 1 4 3 , 44-116, and 117-170. The Trp region was not active in these experiments. The complete amino acid sequence of the encephalitogenic, basic or A1 protein, is shown in Table I. No marked differences between the human myelin basic protein and those of other species have been observed. As found for the bovine protein, the N-terminal residue is blocked with an acetyl group (Carnegie, 1971). A deletion of His-Gly at residues 10 and 11 has been reported. The tryptophan nanopeptide responsible for EAE activity in the guinea pig

6

M. A. MOSCARELLO

TABLE I AMINOACIDSEQUENCEOF BOVINE A 1 PROTEIN' ~

~~~

~~

N-Ac-Ala-Ser-Ala-Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-~u Ala-Ser-Ala-Ser-Thr-Met-Asp-His-Ala-Arg-His-Gly-Phe-Leu-Pro-Arg-His Arg-Asp-Thr-Gly-Ile-Leu-Asp-Ser-Leu-Gly-Arg-Ph~Phe-Gly-Ser-Asp-Arg-Gly-AlaPro-Lys-Arg-Gly-Ser-Gly-Lys-Asp-Gly-His-His-Ala-Ala-Arg-Thr-Thr-His-Tyr-GlySer-Leu-Pro-Gln-Lys-Ala-Gln-Gly-His-Arg Pro-Gln-Asp-Glu-Asn-Pro-Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val Thr-Pro-Arg-Thr-Pro-Pro-Pro-Ser-Gln-Gly-Lys-Gly-Arg-Gly Leu-Ser-Leu-Ser-Arg-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys-Pro-Gly Phe-Gly-Tyr-Gly-Gly-Arg-Ala-Ser-Asp-Tyr-Lys-Ser-Ala-His-Lys-Gly-Leu-Lys-GlyHis-Asp-Ala-Gln-Gly-Thr-Leu-Ser-Lys Ile-Phe-Lys-Leu-Gly-Gly-Arg-Asp-Ser-Arg-Ser-Gly-Ser-Pro-Met

Ala-Arg-Arg-COOH From Eylar (1972).

is identical to the bovine sequence except for the replacement of the Cterminal Lys in the bovine protein for an Arg in the human. Other encephalitogenic determinants (residues 1-21 and 45-88) have been found, but these are 500-1000 times less active than the principal encephalitogen about the tryptophan region. Central nervous system myelin of rodents, of the suborders Myomorpha and Sciuromorpha, such as the rat, contain two basic proteins (Martenson et al., 1970). One is similar to that of other species in amino acid composition and size (18,400 daltons) ; the other is smaller by 4000 daltons. The smaller rat basic protein differs from the larger in having a 40 amino acid piece missing in the C-terminal half of the molecule. The complete sequence has been reported recently by Dunkley and Carnegie (1974). Carnegie (1971) has put forward an interesting proposal concerning a possible neuroreceptor role for the basic protein. It is based on the postulated binding site for 5-hydroxytryptamine (Smythies et al., 1970), formed by two molecules such as tryptophan, located one above the other, so that 5-hydroxytryptamine is held in position by the bonding energy from two a-cloud interactions. This allows it to intercalate between the two molecules. Carnegie suggests that the encephalitogenic determinant can accommodate the 5-hydroxytryptamine molecule, with the indole ring sandwiched between Phe 113 and Trp 115, held by a hydrogen bond to the peptide chain and an electrostatic interaction with Glu-118. Experimental evidence for such a binding site has as yet not been obtained. Although the major features of the primary structure of myelin basic protein have been elucidated, several problems remain unresolved. The

CHEMICAL A N D PHYSICAL PROPERTIES OF MYELIN PROTEINS

7

protein has been shown to be a suitable substrate for phosphorylation by B protein kinase (Carnegie et al., 1973; Miyamoto and Kakiuchi, 1974). The latt,er authors showed that myelin basic protein was phosphorylated by a cyclic AMP-dependent protein kinase from the brain. The phosphorylated amino acids were shown to be serine and threonine. Phosphorylation was found to take place both in vitro and in vivo. Miyamoto and Kakiuch postulated that the phosphorylation was in a dynamic state with dephosphorylation. Steck and Ape1 (1974) have shown that 14-day-old rat myelin was a better substrate for protein phosphorylation than adult myelin. They concluded that the basic protein was more accessible to the enzyme in young animals than in mature myelin. No biological role was postulated for the phosphorylated derivative.

111.

PHYSICAL STRUCTURE OF MYELIN BASIC PROTEIN

The elucidation of the complete amino acid sequence of basic protein from several species is a necessary prerequisite for understanding its secondary structure and its role in the structure of the myelin membrane. Unfortunately, studies of the secondary structure are not as advanced as the chemical studies. Some physical measurements (viscosity, optical rotary dispersion, and circular dichroism) were made early. These have led to the interpretation that myelin basic protein is not structured but has an open conformation. It has been argued that the open conformation, with a large number of basic residues (lysine, histitline, and arginine constitute 25% of all residues), is ideally suited for interacting with the phosphoryl groups of the lipid bilayer. That this conclusion may be premature will become evident from the data presented below. Some of the physicochemical properties of this protein were studied by Eylar and Thompson (1969). On the basis of viscosity studies, they determined an axial ratio of 1 : l O and concluded that the shape of the molecule was that of a prolate ellipsoid. Choa and Einstein (1970) found an axial ratio of 1:17. Optical rotary dispersion studies by Eylar and Thompson (1969), Choa and Einstein (1970), and Palmer and Dawson (1969) established that little helical structure was present in the molecule. However, both the high intrinsic viscosity and the optical rotary data are consistent with a highly ordered protein than has a specific tertiary structure and is relatively devoid of a-helical or P structures. In a recent study, Epand et al. (1974) showed that the protein had a nonrandom structure in solution. Intrinsic viscosity studies confirmed the axial ratio of 1 : l O found by Eylar and

8

M. A. MOSCARELLO

Thompson (1969). Circular dichroism studies in 0.2 M acetic acid and 0.1 M sodium hydroxide were very similar but different from the spectra in 6 M guanidine hydrochloride, a denaturing solvent. Moreover, no drastic temperaturedependent changes were observed in the spectra over 0-80°C. The radius of gyration, calculated from low-angle X-ray scattering measurements, was found to be 39 + 2 d. This corresponds to a prolate ellipsoid with an axial ratio of 10: 1. Sedimentation velocity data correspond to a prolate ellipsoid of axial ratio 12, corroborating the viscosity data. Therefore, X-ray scattering, viscosity, and sedimentation velocity are in good agreement with a prolate ellipsoid model for the basic protein of myelin with an axial ratio 1O:l and of dimensions 150 X 15 d. These studies indicate that the molecule is highly structured. Other recent data support the view that the basic protein of myelin is a nonrandom highly structured molecule. Circular dichroism spectra were run from 250-190 nm at pH values of 1.68, 11.3, and 12.1. At pH 1.68 and at 25"C, very low ellipticity values were observed at 220 nm. This indicates little or no helical structure. Below this wavelength, a large decrease in ellipticity occurred, with a minimum at 200 nm. When the molecule was heated to 85"C, its ellipticity at 220 nm decreased. This indicates an increase in helical content. Also, the ellipticity at 200 nm was less at 85°C than at 25"C, indicating a less random structure. The spectra a t pH 11.3 and 25°C showed little evidence of structure throughout the scan, with low ellipticity values. Heating to 85°C decreased the ellipticity, which was especially marked at 200 nm. At pH 12.1 and 25"C, some helical structure was evident at 220 nm. The helical content increased on heating to 85"C, similar to the observations at pH 1.68. The ellipticity at 200 nm was large at 25°C and decreased considerably on heating to 85°C. Support for the circular dichroism data has been obtained from surface tension measurements. The application of this technique to the study of the conformation of proteins has been described recently (Neumann et al., 1973). The technique was successfully applied to the study of the basic protein of myelin (Moscarello et al., 1974). The surface tension ( yLv) was studied as a function of temperature at different pH values from 2.2 to 12.5. The significant features are as follows: (i) the surface tension at 20°C decreased with increasing pH value-at pH 2.23, the surface tension was 46.1 ergs/cm2, whereas it was 41.6 erg/cm2 at pH 12.5; (ii) there was little evidence of phase transitions at intermediate pH values (3.7, 6.9, and 9.9) ; (iii) phase transitions occurred at extremes of pH, i.e., pH 2.2, 11.4, and 12.5; (iv) the low-temperature transition at 40°C was observed at low pH (pH 2.2), whereas the high-temperature transition at 80"-85"C was observed at high pH values.

CHEMICAL A N D PHYSICAL PROPERTIES OF MYELIN PROTEINS

9

The decrease in surface tension a t 20°C from 46.1 ergs/cm2 a t low pH to 41.G a t high pH is probably the result of the decrease in charge of the protein as the pK is approached. The decrease in charge is reflected in an increase in hydrophobicity so that more protein goes into the surface. The lack of phase transitions a t intermediate pH values is consistent with a highly ordered, rigid molecule. The conclusion from the optical, viscosity, X-ray, and surface tension data must be that the molecule is highly structured and nonrandom. This conclusion can be emphasized because the literature is full of references to the “open,” random conformation of this protein. I n fact, a whole theory of myelin breakdown has been proposed on the basis of an open conformation (Roboz-Einstein, 1972). It is argued that the basic nature of the protein, its open conformation and its external position in the myelin membrane, would render this molecule susceptible to attack by proteinases. Only the basic nature of this protein has been confirmed to date. The structure appears to be rigid and its position in the membrane has not been determined. Data from this laboratory, which support the nonexposed position of this prot,ein, are discussed in Section VII. The above observations may be pertinent to an observation first reported by Martenson and Gaitonde (1969) in their studies with basic protein from bovine white matter. When basic protein was subjected to polyacrylamide gel electrophoresis at acid pH values, a single component, was observed. However, when electrophoresis was done a t high p H values (pH lO.G), multiple components were detected. Isolation of these various components revealed that they had the same encephalitogenic activity and identical amino acid compositions (Martenson el al., 1970). A partial explanation for this heterogeneity was suggested by Baldwin and Carnegie (1971) as due to variable methylation of the arginine residues. However, Deibler and Martenson (1973) have shown that all components contain monomethylarginine and symmetrical dimethylarginine in approximately 4: 1 ratio. From our surface tension studies with basic protein, it was shown that phase transitions did not occur below pH 10, but do occur above this pH. A possible explanation for the heterogeneity observed a t high p H may he the presence of different conformational forms of the protein. The different conformations may migrate more slowly on acrylamide gels a t low than a t high pH values. The use of the myelin membrane for the studies of membrane structure has many advantages over the use of other membranes. At this stage in our understanding of membrane components and their assembly, the relatively simple structure of myelin represents almost a model system.

10

M. A. MOSCARELLO

With the elucidation of the structure of the basic protein, attention was focused on the interaction of this protein with lipids. Basic protein was shown to bind avidly to certain lipids resulting in large changes in the circular dichroism spectrum (Anthony and Moscarello, 1971s). More recently, basic protein was shown to interact with acidic phospholipids and sulfatides yielding an interesting lamellar phase. This phase contained two lipid bilayers in its unit cell: one contained mainly the phospholipids with the hydrocarbon chains in liquidlike conformation, and the other contained mainly the sulfatides, with at least one fraction of the chains stiff and hexogonally packed. The segregation of the lipids was brought about as a result of the interaction with basic protein. The double bilayer in the unit cell closely resembled myelin (Mateu et al., 1973). This interesting preliminary result suggests that the protein has “organized” the lipid so that the unit cell contained two bilayers with similar spacing to that of myelin. The data support the view expressed here concerning the central and possible primary role of proteins in the organization of this biological membrane.

IV.

PROTEOLIPID PROTEIN FRACTION OF MYELIN

The term proteolipid was proposed by Folch Pi and Lees (1951) to describe the unusual properties of proteinaceous material from the brain, soluble in mixtures of chloroform-methanol. The soluble material was shown to consist largely of lipids but also contained a significant amount of protein. Lipoproteins had been known for some time to be composed of protein with a small amount of lipid and thus to be water-soluble. The term proteolipid was used to distinguish chloroform-methanol-soluble material from water-soluble material. The term, therefore, has historical significance. A complete review of the historical background can be found in Folch Pi and Stoffyn (1972). The material soluble in chloroform-methanol has been shown to contain several proteins (Gagnon et al., 1971; Miyamoto and Kakiuchi, 1974; Chan and Lees, 1974); therefore, proteolipid as originally isolated is not a homogeneous protein. In fact, lyophilized myelin can be dissolved in chloroform-methanol. It is important to recognize this fact because the literature of the last 10 years is full of references to “proteolipid protein” and “proteolipid apoprotein,” creating the misleading impression that the term refers to a homogeneous protein. In the absence of evidence for homogeneity, it is necessary to use caution in interpreting data on amino acid composition, analytical ultracentrifugation, end-group analyses, or

11

CHEMICAL A N D PHYSICAL PROPERTIES OF MYELIN PROTEINS

optical measurements. For this reason, I have referred to proteolipid as the proteolipid protein fraction of myelin. The various methods available for the preparation of the proteolipid protein fraction include dialysis in organic solvents and Sephadex LH-20 chromatography. The methods reviewed by Folch Pi and Stoffyn (1972) have not yielded a single material. Therefore, the proteolipid of different workers may vary in composition, depending on the method of preparation. Some of the properties of this protein fraction have been reported by Folch Pi and his collaborators. The amino acid composition of their material is shown in Table I1 and compared to the purified proteins of Gagnon et al. (1971) and Nussbaum et al. (1974). A glance at the amino acid analyses indicates several similarities between the proteolipid proteins isolated from bovine brain by Folch Pi's group and the protein isolated from human myelin by Gagnon et al. (1971) and Nussbaum et al. (1974) from the rat brain. Differences in overall composition are noted in aspartic TABLE I1 AMINOACIDCOMPO5ITION OF RR.4IN WHITE MATTER,PROTEOLIPID FRACTION, AND PROTEINS PURIFIED FROM PROTEOLIPID PROTEIN FRACTION OF MYELIN"

Amino acid

Bovine proteolipid apoproteinb

Human myelin protein (N-2)C

Rat brain P7d

Aspartic Tlireonine Serine Glutamic Proline Glycine Alanine Valine Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine

4.2 8.5 8.5 6.0 2.8 10.3 12.5 6.9 4.0 1.9 4.9 11.1 4.6 7.9 4.3 1.8 2.6

5.4 8.2 5.9 6.4 3.1 10.4 10.6 6.8 2.9 1.6 4.9 11.2 4.2 8.3 4.7 2.6 2.9

5.5 8.5 6.0 6.5 3.0 11.0 12.0 8.5 4.0 1.5 6.0 12.0 5.0 8.5 5.0 2.5 3.0

a

Residue per 100.

* Folch Pi and Stoffyn (1972). Gagnon et al. (1971). Nussbaum et al. (1974).

12

M. A. MOSCARELLO

acid, serine, alanine, cystine, and histidine. Some of the differences may be attributed to species variation. A definite statement can be made only when the preparation of Folch Pi is obtained in higher purity. Gagnon et al. (1971) used several methods to monitor the purity of their preparation. In addition to sedimentation velocity, they used equilibrium ultracentrifugation. A single component was demonstrated on polyacrylamide gels using both the system of Takayama et al. (1964) and that of Weber and Osborn (1969) at different pH values. In a recent study of the Folch Pi apoprotein, Nicot et al. (1973) were able to isolate two major components, one of molecular weight 25,100 and the other 20,700. The two components were isolated by preparative electrophoresis. Amino acid analysis showed some differences in composition; notably threonine, glutamic, serine, and leucine. Minor differences were found in several other amino acids. Because the preparation was obtained by extracting bovine white matter and not purified myelin, the authors were not able to rule out contamination with a nonmyelin proteolipid. Chan and Lees (1974) have studied bovine white matter and myelin preparations in polyacrylamide-sodium dodecyl sulfate (SDS) gels with and without urea. Multiple bands were observed for white matter proteolipid. On indirect evidence, they concluded that the multiple bands represented different states of aggregation of a monomeric unit of molecular weight 5000. A homogeneous proteolipid apoprotein P7 has been reported by Nussbaum et al. (1974). Myelin was prepared from rat brain and extracted with chloroform-methanol. On preparative electrophoresis, they obtained three proteolipid proteins; P7 accounted for about 60%. The amino acid analysis reported by them is shown on Table 11. Differences in amino acid composition with that of Gagnon et al. (1971) were found in alanine, valine, cystine, and isoleucine. They reported a total of 219 residues for P7, whereas Gagnon et al. (1971) reported 223 residues for N-2. These differences in composition and total number of residues may reflect a species difference. By using an automatic Edman degradation technique, Nussbaum et aZ. ( 1974) have reported an N-terminal sequence after performic oxidation of the protein. The sequence as reported was Gly-Leu-Leu-Glu-CysSOs-

CysSO~-Ala-Arg-CysSO~-Leu-Val-Gly-Ala-Pro-Phe-Ala-X-Leu-Val-Ala - -.

This sequence must be interpreted with caution since the yields reported by them for each turn of the sequencer are very low, often less than 10%. An interesting structural feature of this group of proteins is the presence of about 2% fatty acid, apparently covalently linked to the protein. The first report of the presence of fatty acids was made by Wood et al. (1971)

CHEMICAL A N D PHYSICAL PROPERTIES OF MYELIN PROTEINS

13

who stated that glycerol was not found in either acid or alkaline hydrolyzates of the protein. Neither phosphorus nor sphingosine was detected by careful analysis. Attempts to remove the fatty acids, shown to be C16and C l ~ : lby several methods, were unsuccessful. These included Soxhlet extraction procedures and the charcoal method of Chen (1967) for the removal of fatty acids from serum albumin (Gagnon et al., 1971). It was concluded that the fatty acid was probably covalently bound to the peptide chain. About 3 months after the initial report of Wood et al. (1971), a similar study was published by Stoffyn and Folch Pi (1971). They showed that 2.2-3.0% fatty acids, which could not he removed by extensive dialysis with chloroform-methanol, remained associated with their proteolipid apoprotein. Choline, sphingosine, inositol, and hexosamine were not detected, and glycerol was less than 0.03%. The fatty acids were converted to their methyl esters after alkaline hydrolysis, and reaction with diazomethane and sodium borohydride reduction converted them to their corresponding alcohols. They concluded that the fatty acids were esterified to the peptide chain, probably with hydroxy amino acids. The role of fatty acids covalently bound to protein is not understood. They would certainly contribute to the hydrophobicity of the protein, important in interactions with lipid hydrocarbon chains of the bilayer. A similar, highly hydrophobic protein has been isolated from endoplasmic reticulum by MacLennan et al. (1972) as part of the ATPase complex. It has been demonstrated to contain 2% fatty acid also. They concluded that the fatty acid was responsible for the hydrophobic nature of their protein.

Physical Studies

Sherman and Folch Pi (1971) studied the rotary dispersion and circular dichroism of brain proteolipid protein. Their water-soluble preparation showed a helical content that varied between 16 and 40%, depending on the preparation. Using optical rotary dispersion (ORD) , they were unable to detect a random coil (characterized by a trough a t 205 nm). By circular dichroism (CD) , the protein was shown to be highly a-helical in chloroform-methanol but of low helicity in water. The N-2 protein isolated from normal human myelin by Gagnon el al. (1971) was studied by a number of physical techniques. The conformation of their protein was found to be very flexible. Anthony and Moscarello (1971b) prepared a water-soluble form of the protein by dissolving it in phenol-acetic acid-water (3: 1 :1) containing 2 M urea, followed by

14

M. A. MOSCARELLO

dialysis against decreasing concentrations of acetic acid. If the dialysis was begun with 50% acetic acid, followed by 25, 10, 5% and then water, an a-helical circular dichroism spectrum was obtained with minima at 210 and 220 nm. If the dialysis from phenol-acetic acid-water solution of the protein was started with 50% acetic acid containing 2 M urea in the first step, followed by 15, 10, 5% acetic acid and water, the circular dichroism spectrum was that of a /3 conformation with a minimum at 216 to 217 nm. The conformation obtained in either dialysis procedure was not affected by performic acid oxidation or by the presence of mercaptoethanol during dialysis. The conformation was not affected by varying the pH from 1.5 to 6.0. More detailed studies on the conformational flexibility of this protein (N-2) were reported by Moscarello et al. (1973). The ORD parameter b P 2 was calculated from the spectra run between 420 and 300 nm. The data for the two conformations are shown in Table 111. In the formation of the /3 form, the first step in the dialysis procedure following the removal of the phenol results in a conformation with low bO2l2,However, if phenol and urea were removed together with 25% acetic acid, the bO2l2value was -330, corresponding to about 50% helix. The subsequent stages in dialysis do not affect the bO2l2values. The a-helical and /3 conformations were confirmed by infrared analysis. The a-helical form showed a major infrared peak at 1650 cm-' and a smaller peak at 1630 cm-' representative of a-helical and /3 conformations, respectively. In the case of the /3 form, the major peak was at 1630 cm-l, with a smaller peak at 1650 em-'. The shoulder at 1690 cm-l in the /3 form TABLE I11 MOFFITT PARAMETER bozle OF N-2

DURING

VARIOUSSTAGES OF DIALYSIS

bf" or-Helical f o m

Dialysis stage ~

+ 2 M urea

50% Acetic acid

-440 -

25% Acetic acid

-330

10% Acetic acid

-320 -320 -320

Phenol-acetic acid-water (3: 1 :1) 2 M Urea in 50% acetic

5% Acetic acid

Water

9,

Form

~

-440 -110

-200 -200 - 160 - 180 - 185

15

CHEMICAL AND PHYSICAL PROPERTIES OF MYELIN PROTEINS

is attributed to the antiparallel /3 form (Miyazawa and Blout, 1961), The infrared spectrum of the protein in phenol-acetic acid (2 :l) showed a sharp peak at 1650 cm-l, representing n large amount of helix. In summary, the protein is highly helical in the presence of phenol. Removal of both phenol and urea simultaneously results in a preparation with about 50% helix. Equilibrium ultracentrifugation was carried out in water, 98% formic acid, and in 0.01 M sodium phosphate buffer containing 0.5% SDS. The data are shown in Table IV. In formic acid and SDS, the molecular weights obtained were 28,000 and 24,000, respectively. These values probably represent the molecular weights of the monomer. The a-helical form with a molecular weight of 86,000 consists of three subunits, whereas the /3 form, with a molecular weight of about 500,000, consists of many. The a-helical form was readily converted to the 0 form by heating. At 25"C, the protein was largely a-helical with minima in the CD a t about 220 and 210 nm. When heated to 9O"C, the spectrum changed to a /3 type with a single minimum at 217 nm. A small recovery was obtained on cooling to 25°C. The conversion to the /3 form was accomplished in the heating phase and not in the cooling one. The change in [ O ] 2 2 2 with temperature was studied. No change was observed until about 40"C, after which a steady decrease in [0]222 was recorded with increasing temperature. The broadness of the transitions in the temperature range from 40" to 90°C suggested that it was not highly cooperative or that the enthalpy of transition was small. Recently, the two conformations have been studied by electron microscopy after negative staining. The photographs are shown in Fig. 1. The a-helical form is much smaller than the ,8 form and corresponds to a molecular weight of less than 100,000. TABLE IV EQUILIBRIUM ULTRACEN.TRIFUGATION OF THE PROTEIN I N W A T E R , FORMIC ACID, AND SODIUM DODECYL SULFATE Sample a

Form (water)

@ Form (water)

Protein in 98a/, formic acid Protein in 0.01 M sodium phosphate buffer (pH 7.2) containing 0.5% SDS

Molecular weight 86,000 -500,000 28,000 24,000

FIQ.1. Electron micrographs of N-2 stained with 1% uranyl acetate. (A) The a-helical form; (B) the 0 form. X160,OOO; bar = 500 = 1 cm.

CHEMICAL AND PHYSICAL PROPERTIES OF MYELIN PROTEINS

V.

17

OTHER PROTEIN FRACTIONS PREPARED FROM WHOLE BRAIN, WHITE MATTER, OR MYELIN

A number of other workers have used a variety of methods to prepare chloroform-methanol-soluble proteins from whole brain or white matter. The relationship of any of these preparations to that of Folch Pi and Lees (1951) has not been determined. Wolfgram (1966) extracted a protein into acidified chloroform-methanol that differed from that of Folch Pi and Lees (1951) in being trypsindigestible. Recently, Wiggins el al. (1974) have resolved this proteolipid fraction into at least three different proteins, one of which, denoted WI, may be homogeneous. Braun and Radin (1969) prepared a water-soluble bovine brain myelin protein by modification of the method of Tenenbaum and Folch Pi (1966) and studied the interaction with lipids. They found that anionic lipids formed insoluble complexes that were stabilized by divalent cations. Nonionic lipids formed nonprecipitating complexes. Soto et al. (1969) fractionated proteolipids from cat gray and white matter by chromatography on an organophilic dextran gel (Sephadex LH-20) , similar to the method of Mokrasch (1967). Barrantes et al. (1972) prepared proteolipid proteins from bovine cerebral cortex using chromatography on Sephadex LH-20 and elution with NIN-dimethylformamide. Pasquini and Soto ( 1972) extracted proteolipids from bovine brain in n-butanol-water systems. Uda and Nakazawa (1973) prepared proteolipid from bovine brain with chloroform-methanol and then treated this material with ethanol-ther to extract sphingomyelin. The residue was dialyzed against chloroform-methanol. Nussbaum and Mandel ( 1973) extracted total brain proteolipids from whole brain of mutant mice in chloroform-methanol and then partitioned the protein between phases. Proteolipids have also been extracted from isolated myelin by the method of Gonzalez-Sastre ( 1970). The chloroform-methanol-soluble material was treated with 0.1 M KC1 to precipitate the basic protein. Eng et al. (1968) used several extraction procedures, including neutral salt and Triton X-100 and neutral salt extraction to obtain protein fractions from myelin. The soluble material was further fractionated with organic solvents. Several groups of workers (Mehl and Halaris, 1970; Eng et al., 1971) have isolated water-insoluble proteins by electrophoresis in acrylamide gel blocks with the aid of phenol-acetic acid-water solvents. Finch and Moscarello (1972) have reported the isolation of a protein fraction from normal human myelin with mercaptoethanol. The fraction is not homogeneous and appears to resemble the fraction of Wolfgram (1966) and the one recently reported by Wedege (1973), which has been obtained in homogeneous form. An interesting structural feature of the material of Finch et al. (1971) was the presence of the amino acid citrulline in covalent

18

M. A. MOSCARELLO

bond, which could be released by proteolytic digestion. It would be interesting to know if the homogeneous protein reported by Wedege (1973) contains citrulline. Agrawal et al. (1972) demonstrated the presence of a minor protein component on polyacrylsmide gels migrating between myelin basic protein and proteolipid in extracts of rat brain and white matter from bovine, dog, and rabbit brains. They called this material DM-20 and consider it to be a unique myelin component. This latter conclusion remains to be substantiated.

VI.

CONCLUSIONS ON THE NATURE OF MYELIN PROTEINS

In this article, I have reviewed what is known about the chemical and physical properties of myelin proteins. The basic protein of myelin is best understood, since the complete amino acid sequence of this encephalitogenic protein from the myelin of a number of animal species has been elucidated. A protein isolated from the chloroform-methanol-soluble fraction has also been purified in at least two laboratories. In our own laboratory, we have called it N-2 for convenience because it was the second Azsopeak of four, isolated from Sephadex LH-20 when a chloroform-methanol extract of normal human myelin was applied. Nussbaum et al. (1974) have reported the isolation of a similar protein from rat brain, which they called P7. Obviously at some point these trivial names must be replaced by descriptive names. Folch Pi and his collaborators have been in favor of using the name “proteolipid” protein for the chloroform-methanol-soluble material. As I have tried to point out in the foregoing, this fraction is not homogeneous. In fact, the basic protein of myelin will dissolve in chloroform-methanol in the presence of large amounts of myelin lipids (Gonzalez-Sastre, 1970). The main objection to the use of the term proteolipid is that it does not refer to a defined chemical entity. As pointed out above, different workers have isolated proteolipid protein by a variety of methods with no evidence that they all have isolated the same material. Although the name is useful, it must be clearly understood that it does not refer to a specific protein of defined chemical composition in contrast to the basic protein of myelin. It is my feeling that each of the proteins, isolated from the chloroformmethanol soluble material, should be given appropriate names that reflect the chemical composition. This point has been emphasized for myelin basic protein. It has been suggested that our protein N-2 be called lipophilin to describe its tendency to associate closely with lipids (D. Papahadjopolous, private communication).

CHEMICAL AND PHYSICAL PROPERTIES OF MYELIN PROTEINS

19

The two proteins that we have discussed, the myelin basic protein and the hydrophobic protein, must play very different roles in the maintenance of the integrity of the membrane. On the one hand, the basic protein with its rigid structure may have some function in maintaining a fixed structure since it would be less susceptible to denaturing conditions. On the other hand, the role of the hydrophobic protein (N-2) must be different. Because it is more flexible conformationally, it may be associated with functional aspects of myelin. An interesting view of the biological function of proteins has been put forward recently by Nickerson (1973). He proposes a concept of multistability. A protein is defined as multistable if it can be shown to have more than one most stable conformation. Multistability would permit a protein to function in different conformations depending on environmental circumstances, e.g., an enzyme that carries out a certain function when it is in a particular conformation may carry out the same reaction in a different conformation by altering its K , value. Such changes in K , values have been observed for pyruvate kinase of crab muscle and lactic dehydrogenase of rainbow trout as a mechanism of temperature compensation. Boos and Gordon (1971) have shown that the galactose-binding protein from Escherichia coli, responsible both for binding galactose and for its transport through the membrane, can exist in two conformational states-one with a high affinity for galactose and the other with a low affinity. They speculate that when galactose is bound a t the surface, the protein conformation is changed so that the resulting difference in surface charge allows the protein to move through the membrane. The second conformation has a lower affinity for galactose than the first so that the sugar is released on the inside of the membrane. The proteins of the sarcoplasmic reticulum have been shown to include ATPase of molecular weight 102,000 as the major protein; two other proteins of molecular weight 55,000 and 44,000 that are the high Ca2+binding protein and calsequestrin, respectively; several minor proteins; and a proteolipid (MacLennan, 197.5). The function of this proteolipid, which exists in a 1 :1 ratio with ATPase, is not known. It may have a structural role, forming a nucleus around which other proteins and lipids may bind. It may act as ionophore, transferring Cn2+ across the membrane. This latter role is especially attractive since the conformational flexibility described earlier for X-2 may be a characteristic of this protein as well, although this has not yet been established. Rhodopsin, an integral component of the disc membranes of the vertebrate rod outer segment of the eye, is a highly hydrophobic protein and similar in many respects to N-2. It has 235 residues and a molecular weight of 30,000, compared to 223 residues and a molecular weight of 25,000-

20

M. A. MOSCARELLO

28,000 for N-2. It contains 126 neutral residues compared to 124 in N-2 (calculated from the data of Heller, 1968). Although the role in visual excitation is not defined, it may store transmitter substance that can be released by photons of light, This role would be mediated by the conformational state of the protein. Conformational flexibility would appear to be important in this protein also (Chen and Hubbell, 1973). By freeze fracture studies (Chen and Hubbell, 1973; Vail et al., 1974), it has been shown to partition in lipid bilayers in a manner similar to N-2. Cytochrome b6 is a membrane protein of the endoplasmic reticulum of mammalian cells. In liver microsomes, it forms an important part of the system for the conversion stearoyl-CoA to oleoyl-CoA. It has been shown to exist in two domains-a hydrophilic and a hydrophobic one (Robinson and Tanford, 1975). The hydrophobic domain can produce a hydrophobic environment acting as a nucleus for the formation of micelles. Part of the molecule has an important enzymatic function, whereas the other part may function in organizing the membrane. Semlicki forest virus contains spikes protruding from the surface. These spikes are part of a membrane glycoprotein and can be cleaved by proteolytic digestion. When the spikes are gone, the virus is noninfectious; the remaining protein in the membrane is highly hydrophobic (Uterman and Simons, 1974). As in the case of cytochrome b6, the hydrophobic core appears to be important in anchoring the protein in the membrane. Although the above-mentioned hydrophobic proteins have been shown to possess a variety of functions related to enzymatic activities (ATPase and cytochrome b 6 ) , visual excitation (rhodopsin) , and viral infectivity (Semlicki forest virus), no functional role has been assigned to N-2. A possible role in energy transduction is mentioned in the following. Conformational changes in the protein components of the membrane may be important during some phase of the passage of an impulse. During the passage of an impulse, there is a net heat production in rabbit nerve of the order of 2 pcal/gm/impulse (Ritchie, 1973). Some mechanism must be available for the removal of this heat. If myelin is insulating the nerve, then the heat must be transmitted through the axonal membrane and through the myelin sheath in some way. A possible mechanism for the dissipation of heat may be a conformational change in a protein. The protein must be able to exist in more than one conformational state at any one time and these states must be interconvertible. The basic protein of myelin is too rigid a structure, since it has been shown to undergo conformational changes only at extremes of pH. However, the hydrophobic protein has been shown to be conformationally flexible. The absorption of heat by the molecule may result in a shift of equilibrium from one state to another that may help to dissipate the heat.

CHEMICAL A N D PHYSICAL PROPERTIES OF MYELIN PROTEINS

VII.

21

LOCALIZATION OF PROTEINS IN MYELIN

I n the lipid bilayer model of the myelin membrane developed from electron microscopic and X-ray diffraction data, the protein is considered to occupy an external position on either side of the lipid bilayer. A number of observations have forced us to reexamine this model to give to the protein components more defined and important roles. Napolitano et al. (1967) have reported electron micrographs of the myelin membrane after extraction of 98% of the lipids in chloroformmethanol and acidified chloroform-methanol. The micrographs showed the same multilamellar structure with 170-A spacings found in unextracted myelin. The authors concluded that the protein was responsible for maintaining the three-dimensional structure of the membrane. An earlier study by Shanthaveerappa and Bourne ( 1962) showed electron micrographs of optic nerve myelin in which a radial component was visualized. These “lines” were considered bridges between the layers. Akers and Parsons ( 1970) have discussed these observations and they considered the interesting possibility that protein bridges may be stabilizing the membrane structure. The above-mentioned observations support the view that the proteins of myelin may be inserted into the bilayer, in whole or in part, as has been proposed for rhodopsin in the retina. Studies in our laboratory have shown that the hydrophobic protein of myelin (N-2) can be inserted into lipid vesicles. I n a recent study, Vail el al. (1974) have shown that freeze fractures of liposomes membranes showed smooth fracture surfaces, whereas liposomes into which the hydrophobic protein (N-2) from human myelin had been incorporated showed particulate fracture surfaces. It was concluded that this protein was partially exposed and partially buried in the liposome membrane. These freeze fracture micrographs are shown in Fig. 2. A detailed study of this system by Papahadjopoulos et al. (1975) has shown that N-2 binds strongly to phospholipids irrespective of surface charge, the presence of cholesterol, or of double bonds on the fatty acyl chains. The buoyant density of the resulting lipoprotein membranes was intermediate between that of the pure lipids and proteins. The presence of N-2 in the membranes increased the permeability to sodium by 2-3 orders of magnitude. We concluded that N-2 was incorporated into the lipid bilayer and was partially exposed and partially buried. The myelin basic protein has been considered to be ideally suited for interacting with the phosphoryl groups of the lipid a t the surface of the myelin membrane. However, little evidence is available concerning the localization of this protein in myelin. Herndon et al. (1973) were unable to visualize the basic protein of myelin using immunoelectron microscopic

FIG.2. (1)Freeze fracture of a liposome. Note that the fracture surfaces are smooth without particles. (2) Freeze fracture of liposomes after the incorporation of a hydrophobic protein extracted from human myelin. Note the particles are present on the fracture surfaces. Total magnification, X75,OOO; bar = 0.1 pm.

CHEMICAL A N D PHYSICAL PROPERTIES

OF

MYELIN PROTEINS

23

techniques. They concluded that the antigenic determinants of the basic protein are occluded in vivo. A different approach to the localization of myelin proteins was reported recently (Wood and Moscarello, 1975) using a nonpenetrating reagent, 4,4’-diisothiocyano-2,2’-ditritiostilbenesulfonate ( DID5L3H). This reagent has been used by Cabantchik and Rothstein (1972) to label membrane proteins of the red cell. It is presumed to react with exposed amino groups of proteins, forming a covalent linkage. The myelin membrane was labeled with DIDS-”, and the basic protein and the hydrophobic protein (N-2) were isolated. After a %minute exposure to the reagent, the specific activity of N-2 was 30 times greater than that of basic protein. Water shock or sonication treatments of myelin did not affect the specific activities of the isolated proteins. When the isolated proteins were reacted with DIDS-3H the specific activity of basic protein was 10 times greater than that of K-2, the reverse of the situation found when the membrane was labeled. Therefore, we concluded that the basic protein was not accessible to the reagent when it formed a part of the myelin membrane. The basic protein could either be buried inside the bilayer or else it could be near the surface but occluded from the hydrophilic environment as a result of interactions with other membrane components such as phospholipids. Recently, Poduslo and Braun (1975) have shown that basic protein was not labeled by lactoperoxidase1251reaction when applied to the intact myelinated nerve bundle. Cyanogen bromide fragments of N-2 were prepared and resolved into a t least four Azso peaks on hydroxylopatite. The specific activity of one fragment was G times greater than the others implying that this fragment was more exposed to the hydrophilic surface than other parts of the protein. A similar study was done with myelin isolated from cases of multiple sclerosis (Wood et al., 1975). The specific activities of the basic protein and the hydrophobic protein isolated from chronic multiple sclerosis myelin were similar to the normals. By contrast, the specific activity of the basic protein isolated from acute multiple sclerosis myelin was about 400% higher than that of the basic protein from either normal or chronic multiple sclerosis material. The specific ttctivity of K-2 from the acute case was only 50% of that of N-2 isolated from chronic multiple sclerosis or from normal myelin. It was concluded that the arrangements of proteins in isolated chronic multiple sclerosis myelin was not markedly altered in comparison to that in isolated normal myelin. However, the arrangement of proteins in acute multiple sclerosis myelin appeared to be considerably different from that of the other two myelins. The experimental work on the arrmgement of proteins in myelin is just beginning. It is clear from this early work that the proteins are interacting

24

M. A. MOSCARELLO

in a complex manner with other membrane components and are not merely stuck on the outside of a lipid bilayer. This finding lends support to the fluid mosaic model of biological membranes (Singer and Nicolson, 1972). REFERENCES Agrawal, H. C., Burton, R. M., Fishman, M. A,, Mitchell, R. F., and Prensky, A. L. (1972). Partial characterization of a new myelin protein component. J . Neurochem. 19, 2083-2089. Akers, C. K., and Parsons, D. F. (1970). X-ray diffraction of myelin membrane. 11. Determination of the phase angles of the frog sciatic nerve by heavy atom labeling and calculation of the electron density distribution of the membrane. Biophys. J . 10, 116-136. Anthony, J., and Moscarello, M. A. (1971a). A conformation change induced in the basic encephalitogen by lipids. Biochim. Biophys. Acta 243, 429433. Anthony, J., and Moscarello, M. A. (1971b). Conformat,ional transition of a myelin protein. FEBS (Fed. EUT.Biochem. Sac.) Lett. 15, 335-339. Baldwin, G. S., and Carnegie, P. R. (1971). Isolation and partial characterization of methylated arginines from the encephalitogenic basic protein of myelin. Biochem. J . 123, 69-74. Barrantes, F.J., La Torre, J. L., Llorente de Carlin, M. C., and De Robertis, E. (1972). Studiea on proteolipid proteins from cerebral cortex. I. Preparation and some properties. Bwchim. Biophys. Acta 263, 368-381. Boos, W.,and Gordon, A. (1971). Transport properties of the galactose-binding protein of Escherichia coli. J . Bwl. Chem. 246, 621-628. Borgstrand, H., and Kallen, B. (1973). Antigenic determinants on bovine encephalitogenic protein. Localization of regions that induce transformation of lymph node cells from immunized rabbits. Eur. J . Immunol. 3, 287-292. Braun, P. E., and Radin, N. S. (1969). Interactions of lipids with a membrane structural protein. Biochemistry 8, 43104318. Cabantchik, Z. I., and Rothstein, A. (1972). The nature of the membrane sites controlling anion permeability of human red blood cells as determined with disulfonic stilbene derivatives. J . Membr. B i d . 10, 311-330. Carnegie, P. R. (1969). Digestion of an arg-pro bond by trypsin in the encephalitogenic basic protein of human myelin. Nature (London) 223, 958-9. Carnegie, P. R. (1971). Properties, structure and possible neuroreceptor role of the encephalitogenic protein of human brain. Nature (London) 229, 25-28. Carnegie, P. R., Bencina, B., and Lamoureux, G. (1967). Experimental allergic encephalomyelitis isolation of basic proteins and polypeptides from central nervous tissue. Biochem. J . 105, 559-568. Carnegie, P. R., Kemp, B. E., Dunkley, P. R., and Murray, A. W. (1973). Phosphorylation of myelin basic protein by a adenosine 3', 5'-cyclic monophosphate-dependent protein kinase. Biochem. J . 135, 569-572. Chan, D.S., and Lees, M. B. (1974). Gel electrophoresis studies of bovine brain white matter proteolipid and myelin proteins. Biochemistry 13, 2704-12. Chao, L. P., and Einstein, E. R. (1970). Physical properties of the bovine encephalitogenic protein; molecular weight and conformation. J . Neurochem. 17, 1121-1132. Chen, R. F. (1967). Removal of fatty acids from serum albumin by charcoal treatment. J . B i d . Chem. 242, 173-181. Chen, Y. S., and Hubbell, W. L. (1973). Temperature- and light-dependent structural changea in rhodopsin-lipid membranes. Exp. Eye Res. 17, 517-532.

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Danielli, J. F., and Davson, H. (1934-35). A contribution of the theory of permeability of thin films. J . Cell. Comp. Physaol. 5 , 495408. Deibler, G. E., and Martenson, 11. E. (1973). Chromatographic fractionation of myelin basic protein. Partial characterization and methylarginine contents of the multiple forms. J . Biol. Chem. 248, 2392-6. Dunkley, P. R., and Carnegie, P. R. (1974). Amino acid sequence of the smaller basic protein from rat brain myelin. Biochern. J . 141, 243-261. Eng, L. F., Chao, F. C., Gerstl, B., Pratt, I)., and Tavastsjevna, M. G. (1968). The maturation of human white matter myelin. Fractionation of the myelin membrane proteins. Baochemistry 7, 4455-4465. Eng, L. F., Bond, P., and Gerstl, B. (1971). Isolation of myelin proteins from disc acrylamide gels electrophoresed in phenol-formic acid-water. Neurobiology 1, 58-63. Epand, R. M., Moscarello, M. A., Zirenberg, B., and Vail, W. J. (1974). The folded conformation of the encephalitogenic protein of the human brain. Biochemistry 13, 1264-1267.

Eylar, E. H. (1972). The structure and immunologic properties of basic proteins of myelin. Ann. N . Y . Acad. Sci. 195, 481-91. Eylar, E. H., and Thompson, M. (1969). Allergic encephalomyelitis: The physicochemical properties of the basic protein encephalitogen from bovine spinal cord. Arch. Biochem. Biophys. 129, 46&479. Eylar, E. H., Salk, J., Beveridge, G., and Brown, L. (1969). Experimental allergic encephalontekutus : An encephalitogenic basic protein from bovine myelin. Arch. Biochern. Biophys. 132, 3 H 8 . Eylar, E. H., Caccam, J., Jackson, J., Westall, F., and Robinson, A. B. (1970). Experimental allergic encephalomyelitis : Synthesis of disease-inducing site of the basic protein. Snence 168, 1220-3. Eylar, E. I%.,Brostoff, S., Hashim, G., Caccam, J., and Burnet, P. (1971). Basic A1 protein of the myelin membrane. The complete amino acid sequence. J . Bzol. ChenL. 246, 5770-5784.

Eylar, E. H., Brostoff, S., Jackson, J . J., and Carter, H. (1972). Allergic encephalomyelitis in monkeys induced by a peptide from the A1 protein. Proc. Natl. Acad. S’ci. U . S . A . 69, 617-9. Eylar, E. H., Jackson, J. J., Bennett, C. D., Kniskern, P. J., and Brostoff, S. W. (1974). The chicken A1 protein. Phylogenetic variation in the amino acid sequence of the encephalitogenic site. J . Biol. Chem. 249, 3710-6. Finch, P. R., and Moscarello, M. A. (1972). A myelin protein fraction extracted with thioethanol. Brain Res. 42, 177-187. Finch, P. R., Wood, D. D., and Moscarello, M. A. (1971). The presence of citrulline in a myelin protein fraction. FEBS (Fed. Evr. Biochcin. SOC.)Lett. 15, 145-148. Finean, J. B. (1953). Further observations on the structure of myelin. Exp. Cell Res. 5, 202-215. Folch Pi, J., and Lees, M. B. (1951). Proteolipides, a new type of tissue lipoproteins, their isolation from the brain. J . Biol. Chenz. 191, 807-817. Folch Pi, J., and Stoffyn, P. J. (1972). Proteolipids from membrane systems. Ann. N . Y . Acad. Sci. 195, 86-107. Gagnon, J., Finch, P. It., Wood, D. D., and Moscarello, M. A. (1971). Isolation of a highly purified myelin protein. Biochemistry 10, 4756-4763. Gonzalez-Srtstre, F. (1970). The protein composition of isolated myelin. J . Neurochem. 17, 1049-56.

Gorter, E., and Grendel, F. (1925). Bimolecular layers of lipoids on chromocytes of blood. J . Exp. Med. 41, 439443.

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Hagopian, A., and Eylar, E. H. (1969). Glycoprotein biosynthesis: The purification and characterization of a polypeptide: N-acetylgalactosaminyltransferase from bovine submaxillary glands. Arch. Biochem. Biophys. 129, 515-524. Heller, J. (1968). Structure of visual pigments. I. Purification, molecular weight, and composition of bovine visual pigment. Biochemistry 7, 2906-2913. Herndon, R. M., Rauch, H. C., and Einstein, E. R. (1973). Immuno-electron microscopic localization of the encephalitogenic basic protein in myelin. Zmmunol. Commun. 2, 163-172. Kibler, R. F., and Shapira, R. (1968). Isolation and properties of an encephalitogenic protein from bovine, rabbit and human central nervous system tissue. J. Biol. Chem. 243, 281-286. Kibler, R. F., Shapira, R., McKneally, S., Jenkins, J., Selden, P., and Chou, F. (1969). Encephalitogenic protein : Structure. Science la, 577-580. Kies, M. (1965). Chemical studies on an encephalitogenic protein from guinea pig brain. Ann. N . Y . Acad. Sci. 122, 161-70. Kies, M., Thompson, B. E., and Alvord, E. C. (1965). The relationship of myelin proteins to experimental allergic encephalomyelitis. Ann. N . Y . Acud. Sci. 122, 148-60. Korn, E. D. (1966). Structure of biological membranes. The unit membrane theory is reevaluated in light of the data now available. Science 153, 1491-1498. Lowden, J. A., Moscarello, M. A., and Morecki, R. (1966). The isolation and characterization of an acid-soluble protein from myelin. Can. J.Biochem. Physiol. 44,567-,577. Martenson, R. E., and Gaitonde, M. K. (1969). Comparative studies of highly basic proteins of ox brain and rat brain. Microheterogeneity of basic encephalitogenic (myelin) protein. J. Neurochem. 16, 889-898. Martenson, R. E., Deibler, G. E., and Kies, M. W. (1970). Rat myelin basic proteins: Relationship between size differences and microheterogeneity. J. Neurochem. 17, 1329-1330. Mateu, L., Luzzati, V., London, Y., Gould, R. M., Vossberg, F. G. A., and Olive, J. (1973). X-ray diffraction and electron microscope study of the interacJions of myelin components. The structure of a lamellar phase with a 150 to 180 A repeat distance containing basic proteins and acidic lipids. J. Mol. Biol. 7 5 , 697-709. MacLennan, D. H. (1975). Resolution of the calcium transport system of sarcoplasmic reticulum. Can. J. Biochem. 53, 251-261. MacLennan, D. H., Yip, C. C., Iles, G. H., and Seeman, P. (1972). Isolation of sarcoplasmic reticulum proteins. Cold Spring Harbour Symp. Quant. Biol. 37, 469-477. Mehl, E., and Halaris, A. (1970). Stoichiometric relation of protein components in cerebral myelin from different species. J. Neurochem. 17, 659-668. Merrifield, R. B. (1963). Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. SOC.85, 2149-2154. Miyamoto, E., and Kakiuchi, I. (1974). I n vitro and in vivo phosphorylation of myelin basic protein by exogenous and endogenous adenosine 3', 5'-monophosphatedependent protein kinases in brain. J. Biol. Chem. 249, 2769-2777. Miyazawa, T., and Blout, E. R. (1961). The infrared spectra of polypeptides in various conformations: Amide I and I1 bands. J. Am. Chem. SOC.83, 712-719. Mokrasch, L. C. (1967). A rapid purification of proteolipid protein adaptable to large quantities. Lije Sn'. 6, 1905-1909. Moscarello, M. A,, Gagnon, J., Wood, D. D., Anthony, J., and Epand, R. M. (1973). Conformational flexibility of a myelin protein. Biochemistry 12, 3402-3406. Moscarello, M. A., Katona, E., Neumann, W., and Epand, R. M. (1974). The ordered

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27

structure of the encephalitogenic protein from normal human myelin. Biophys. Chem. 2, 290-295. Nakao, A., Davis, W. J., and Einstein, E. R. (1966). I. Isolation and Characterization. Basic proteins from the acidic extract of bovine spinal cord. Biochim. Biophys. Acta 130, 163-170. Napolitano, L., Le Baron, F., and Scaletti, J. (1967). Preservation of myelin lamellar structure in the absence of lipid. A correlated chemical and morphological study. J . Cell Biol. 34, 817-826. Neumann, A. W., Moscarello, M. A., and Epand, R. M. (1973). The application of surface tension measurements to the study of conformational transitions in aqueous solutions of poly-blysine. Biopolymers 12, 1945-1957. Nickecson, K. W. (1973). Biological functions of multistable proteins. J . Theor. Biol. 40, 507-515. Nicot, C., Le Neuyen, T., Le Pretre, M., and Alfsen, A. (1973). Study of Folch-Pi apoprotein. I. Isolation of two components, aggregation during delipidation. Biochim. Biophys. Acta 322, 109-123. Nussbaum, J. L., and Mandel, P. (1973). Brain proteolipids in neurological mutant mice. Brain Res. 61, 295-310. Nussbaum, J. L., Rouayreng, J. F., Mandel, P., Jolles, J., and Jolles, P. (1974). Isolation of terminal sequence determination of the major rat brain myelin proteolipid P7 apoprotein. Biochem. Biophys. Res. Cotnmirn. 57, 1240-1247. Palmer, F. B., and Dawson, K.hf. C. (1969).The isolation and properties of experimental allergic encephalitogenic protein. Biochern. J . 111, G29-636. I’apahadjopoulos, D., Vail, IV.J., and Moscnrello, 3f. A . (1975). Interaction of a purified hydrophobic protein from niyelin with phospholipid membranes: Studies on ultrast,ructure, phase transitions and permeability. J . Mernbr. Biol. 22, 143-164. Pasquini, J. M., and Soto, E. F. (1972). Extraction of proteolipids from nervous tissue with n-BUTANOL-WATER. Life Sci. 11, Part 11, 433443. Poduslo, J. F., and Braun, P. (1975). Topographical arrangement, of membrane proteins in the intact myelin sheath. J . Biol. Chew. 250, 1099-1105. Ritchie, J. M. (1973). Energetic aspects of nerve conduction. The relationship between heat production, electrical activity and metabolism in progress. Biophys. Mol. Biol. 26, 147-187. Robinson, N. C., and Tanford, C. (1975). The binding of deoxycholate, trit,on X-100, sodium dodecyl sulfate, and phosphatidycholine vesicles to cytochrome b5. Biochemistry 14, 369-377. Roboz-Einstein, 13. (1972). Basic protein of myelin and its role i n experimental allergic encephalomyelitis and multiple sclerosis. Handh. Nwrochent. 7, 107-122. Roboz-Einstein, E., Robertson, D., Dicaprio, J., and Moore, W. (1962). The isolation from bovine spinal cord of a homogeneous protein with encephalitogenic activit>y. J . Neurochem. 9, 353-61. Shanthaveerappa, T. R., and Bourne, G. 1%. (1962). Radial bands in the optic nerve myelin sheat,h. Nature (London) 196, 1215-17. Sherman, G., and Folch-Pi, J, (1971). On the type of linkage binding fatty acids present in brain white matter proteolipid apoprotein. Biochem. Biophys. Res. Commun. 441, 157-161. Singer, S. J., and Nicolson, G. L. (1972). The fluid mosaic model of the structure of cell membranes. Science 175, 720-731. Smyth, D., and Utsumi, S. (1967). Structure a t the hinge region in rabbit immunoglobulin-G. Nature (London) 216, 332-335.

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The Distinction between Seauential and Simultaneous Models for Sodium and Potassium Transport P. J . GARRAHAN A N D R. P. GARAY Departamento de Quimica Bioldgica Facultad de Farmacia y Bioqimicu Buenos Aires, Argentina

I. Introduction. . . . . . . . . . . . , . . . . . 11. Cation-Binding Sites of the Na Pump . . . . . . . . . . A. General Properties , . . . . . . . . . . . . . B. Distinction between Affinity of Cation-Binding Sites and Reactivity of Cationsite Complexes . . . . . . . . . . . . C. Interactions among Cation-Binding Sites of the Na Pump . . . D. Are Cation-Pump Complexes in Equilibrium with Free Cations? . E. Inner and Outer Cation-Binding Sites . . . . . . . . . F. Relation between Inner and Outer Cation-Binding Sites. . . . 111. Experimental Evidence for Simultaneous Existence of Inner and Outer Cation-Binding Sites . . . . . . . . . . . . . . A. Biochemical Evidence. . . . . . . . . . . . . . B. Kinetics of Cation Fluxes . . . . . . . . . . . . IV. Intermediate Stages in Hydrolysis of ATP by the Na Pump . . . . A. Phosphorylation and Dephosphorylation of the Pump . . . . B. Apparent Uncoupling between Partial Reactions and Cationic Fluxes C. Meaning of Apparent Uncoupling between Partial Reactions and Cationic Fluxes . . , . . . . . . . . . . . . V. Mechanism of Ion Transport . . . . . . . . . . . . . A. Na Pump as a V System. . . . . . . . . . . , . B. Are Changes in Affinity of the Cation-Binding Sites Necessary for Active Transport?. . . . . . . . . . . . . . C. Other Transport Systems . . . . . . . . . . . References . . . . . . . . . . . . . , , .

.

1.

. . .

29 31 31

. . . . .

33 34 33 37 38

. .

40 40 44

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. . .

68 69 72

. . .

76 77 77

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84 90 91

.

INTRODUCTION

The active transport of Na and I< driven by the Na pump is one of the most important energy-transducing functions of cell membranes. Although 29

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P. GARAY

a large amount of experimental data about this system is now available, no coherent scheme has yet been evolved to integrate these data into a plausible molecular mechanism for the coupling of the hydrolysis of ATP to the active movement of cations. We do not intend in this article to propose a detailed hypothesis to account for this phenomenon. Rather we will try to analyze the available experimental evidence to see if it can be used to place restrictions on the possible molecular mechanisms of active transport. It is hoped that these restrictions, by circumscribing the possible modes of operation of the Na pump, will help to reach the goal of elucidating its mechanism. To do this we will concentrate on one particular problem within the gencral question of the mechanism of active transport, namely, the relation between the inward-facing and the outward-facing, cation-binding sites of the Na pump. One of the salient features of the Na pump is that all the cation movements it is able to drive have strict cation requirements at both the inner and the outer surfaces of the cell membrane. This almost certainly means that cation movements only take place if inward-facing and outward-facing sites of the pump have a definite state of occupation by cations. It is obvious, therefore, that the nature of the linkage between inner and outer sites is a problem that has to be considered by any hypothesis accounting for tho mechanism of active transport. In the second section of this paper, we intend to define with precision the concept of cation-binding sites and to characterize the interactions between sites and with cations. The section ends with a statement of the two alternative explanations that have been proposed to account for the linkage between inner and outer sites: (i) these sites are alternative states of the same set of sites (sequential models) or (ii) they are two physically distinct and independent sets of sites that coexist in each transport unit (simultaneous models). We think that available experimental evidence rather strongly favors the idea of the simultaneous existence of inner and outer cation-binding sites. A critical analysis of this evidence is undertaken in the third section. Although structural and biochemical data are taken into account, the main weight of our reasoning is placed on the results of experiments on the kinetics of cation fluxes. In this respect we are convinced of two facts, which we would like to state clearly before detailing, in the corresponding section, the reasons of our belief. 1. If, as it seems likely, (a) free cations are in equilibrium with the cation-pump complexes and (b) the rate equation for cation fluxes can be expressed as a function of external cations only, times a function of internal cations only, then, not only the simultaneous existence of inner and outer sites may be considered as having been demonstrated, but also only a

31

SEQUENTIAL AND SIMULTANEOUS MODELS FOR Na AND K TRANSPORT

particular class of the set of simultaneous models is able to account for the behavior of the Na pump. 2. Since i t can be demonstrated that only a particular class of simultaneous models gives rate equations with separation of intra- and extracellular variables, lack of separation of variables is not a n argument against simultaneous, or in favor of sequential models, but only against a particular class of simultaneous models. The fourth section of this papcr deals with the biochemical aspects of the Na pump, namely, with the intermediate steps and the partial reactions of the hydrolysis of ATP catalyzed by this system. Most experiments performed on this subject have been interpreted within the conceptual framework of sequential schemes of transport. Our aim in this section is to show that the present knowledge of the mechanism of the hydrolysis of ATP by the Na pump is not incompatible with thc postulates of simultaneous models for act.ive transport. The last section is the most speculative one of this paper. In it we take for granted the simultaneous schcmc devcloped from the equilibrium treatment of the kinetics of cation fluxes, and, in order to further circumscribe the possible mechanisms of active transport, confront this scheme with tho available information on the interaction between cation-binding and metabolite-binding sites and on the degrccs of freedom of mcmbranebound proteins. The picturs that emerges from this confrontation seems to indicate that, in the operation of the Na pump, intcractions in reactivity predominat,e over interactions in affinity and that cation translocation probably takes place by some sort of “internal transfer” mechanism. This article does not intend to present a comprehensive survey of the copious literature on active transport of Na and I<. For this the reader is referred to the excellent and up-to-date rcview of Glynn and Karlish (1975) (for a more partial view, see also Skou, 1975). A good survey of the prcsent trends of research in the field can be found in the proceedings of the International Conference on the Properties and Functions of Naf Kf ATPase (Aska,ri, 1974) sponsored by thc New York Academy of Sciences.

+

II.

CATION-BINDING SITES OF THE Na PUMP

A. General Properties

Although the molecular mechanism of the Na pump is almost completely unknown, few workers in the field would disagree with the assertion that an active transport cycle begins when cations become bound to the pump

32

P. J. GARRAHAN AND

R. P. GARAY

and ends after the bound cations are released into the solutions bathing the cell membrane. The process of binding and release of cations implies that the Na pump possesses a discrete number of sites capable of forming specific complexes with the cations that are transported. The evidence for the existence of such sites is wholly kinetic in nature, and rests on the usually accepted criteria of specificity, saturation, and competition of the activation curves of cation fluxes, or of the coupled metabolic events. No successful direct study of cation binding to the Na pump has yet been performed, mainly due to the practical difficulties involved in the application of conventional binding techniques to relatively low-affinity sites as those of the Na pump. 1. BOUND CATIONS AND TRANSPORTED CATIONS

Most kinetic schemes of the Na pump assume that all the cations that have to be bound to the pump to induce cation translocation are in themselves transported. Although there is a rather large body of circumstantial evidence in favor of this view (see for instance Glynn, 1968; Garay and Garrahan, 1973), it must be borne in mind that in many cases kinetic studies cannot distinguish between binding that leads to the transport of the bound cation and binding that is necessary to activate a transport event in which the bound ion does not participate directly. The only conclusive way to distinguish between these alternatives is to compare the stoichiometry of transport with the stoichiometry of cation binding. No study of this kind has yet been performed with the Na pump. Ligand binding has been measured in the Ca pump of sarcoplasmic reticulum. In this case, it seems that the identity of bound with transported ions is true, since this system is able to bind 2 calcium ions per molecule of ATP bound, and it transports 2 calcium ions per molecule of ATP that is split (Meissner, 1973). 2. ENTERING AND LEAVING SITES

An important point in the development of kinetic schemes for the Na pump concerns the relationship between sites to which ions are bound to initiate a transport cycle (enterine sites) and the sites from which ions are released at the end of a transport cycle (leaving sites). Most models that have been proposed to account for active transport assume that there is no essential distinction between entering and leaving sites. More specifically, it is usually considered that entering sites for cations coming from one of the surfaces of the membrane are the leaving sites for cations coming from the opposite surface of the membrane (see Caldwell, 1970). Since this assumption has not yet led to any inconsistency in the kinetic treatment of experimental data, we will take it for granted in the rest of this article.

SEQUENTIAL AND SIMULTANEOUS MODELS FOR Na AND K TRANSPORT

33

B. Distinction between Affinity of Cation-Binding Sites and Reactivity of Cation-Site Complexes

The ability of a given site to form a complex with the caiton can be measured by the value of the apparent affinity of the site for the cation, obtained from the cation-activation curve. Apparent affinities may or may not be equal to the value of the thermodynamic equilibrium constant for the formation of the cation-site complex. The ration-binding sites of the Na pump are able to bind all alkali cations, and in some cases also cations that do not belong to this class such as NH4+ and TI+ (see, for instance, Post and Jolly, 1957; Maizels, 1968; Skulskii et al., 1973). Cation-binding sites show selectivzty, that is, they bind some cations more strongly than others. The mechanism of cationic selectivity in biological systems has been extensively studied (for a review, see Diamond and Wright, 1969). As a consequence of the formation of cation-site complexes, observable transitions may be induced. These transitions may involve the bound cation, which may, for instance, undergo translocation, or some other process for which the bound cation is acting as an activator, as, for instance, the elicitation of an enzymatic activity. It is important to stress a t this point that the ability of a cation-site complex to undergo or induce observable transitions, which we may loosely call its “reactivity,” is independent of the solectivity of the site and cannot, therefore, be predicted from the properties of the process of equilibration of cations with the site. In consequence the analysis of the interactions between cations and the Na pump involves not only measuring the affinity of each site but also assessing the reactivity of each of the possible cationsite complexes. A good example of the distinction between affinity and reactivity is provided by the properties of the inner sites of the Na pump. It is almost certain that these sites are able to bind, although with different affinities, all alkali cations (Maizels, 1968). In spite of this it is now evident that only some of the possible complexes between inner sites and Na ions confer on the pump the ability to induce active transport (see Garay and Garrahan, 1973). Although in general cations that form reactive complexes have higher affinities for the pump sites than those that do not, this is not always the case. A clear example of this assertion is provided by the inner sites of the Na pump of low-potassium goat red cells. These sites bind K more strongly than Na. I n spite of this, only the Na-site complexes are reactive, as is shown by the fact that only internal Na can induce active transport and (Na I<)-dependent ATPase activity (Glynn and Ellory, 1972). The distinction between the affinity of a site and the reactivity that

+

34

P. J. GARRAHAN AND R. P. GARAY

complexes of the site with cations may confer on the pump h a s two important consequences that must be kept in mind when interpreting the kinetic and biochemical properties of the Na pump. (i) The fact that a cation is required for a given elementary step of ion transport docs not necessarily mean that the species involved in this step are the only ones that possess sites or have selective affinity for the activating cation, and (ii) the fact that a cation is not required for a given elementary step, does not necessarily mean that the species involved in this step do not possess binding sites for this cation. C. Interadions between Cation-Binding Sites of the N a Pump

1. INTERACTIONS AMONG SITES BELONGING TO DIFFERENT TRANSPORT

UNITS These kind of interactions will exist when the affinity and/or the rcactivity of sites in a transport unit depend on the state of occupation of sites in neighboring units. Interactions between units in a membrane have been analyzed in general by Changeux et al. (1967), who showed that they give highly cooperative responses, which may even be of thc “all” or “nonc” kind. Since it is now accepted that interaction between sites in macromolecules are mediated by eonformational transitions caused by changes in the number and/or strength of noncovalent bonds (see Whitehead, 1970), interactions among units will only be possible when these units are arranged in close contact with each other. Available experimental evidence (see Baker and Willis, 1972) from a wide variety of tissues indicates that the number of pumps per unit membrane area is small, which almost certainly means that pump units are sufficiently far away from each other so as t o exclude the possibility of intcractions among them. The lack of interaction among pump units leads to a considerable simplification in the mathematical treatment of the kinetics of active transport. It allows us t o calculate the state of occupation by cations of a pump unit independently of the state of occupation of the rest of the units, and it makes the rate of the transitions of the different cation-pump complexes first-order or pseudo-first-order in the concentration of the cation-pump complex. 2. INTERACTIONS AMONG SITESWITHIN

A

PUMPUNIT

a. Interactions between Aginity Constants. K Systems. There is interaction among affinity constants when the affinity of a site varies with the state of occupation of other sites in the same unit. Following Monod et al. (1965),

SEQUENTIAL AND SIMULTANEOUS MODELS FOR Na AND K TRANSPORT

35

W P distinguish hetnc.cn intcractions among sitcs for tlir samr ligand (Iiomotropic intcmctions) and intrractions lwtivccm sitcs for differtint ligands (hcterotropic interactions). T h c interactions bctwccn affinity const:tnts are always linked, in the sense that if occupation of sitc A modifies the affinity of sitc B, thc occupation of sitr B will also modify the affinity of sitc &4.Linkage is a conscqucnce of t h P srcond law of thcrmodynamics and has hten rxtrnsivdy studied by Wyman (1964). Monod et al. (1965) have callcd K s!jstpms those systems whose operation is rrgulated by ligand-induccd changrs in affinity. h. Interactions among Rate Constants. V Systems. Thcrc is interaction among rate coilstants when thc existrnco and/or th(. nature of the transitions that a given cation-pump complm may undergo arc governed ljy the stat(’ of occupation of other sites in tho sanw pump unit. These effccts are callvd interactions between rate constants brcausc they can he cxprcssed formally by assigning to thr rate constants of the transitions of a ligand bound to a sitc, valurs that, d(yxnd on thc1 stat(’ of occupation of thr ot1it.r sites. As in th(1 case of intcractions in :tffinity, intwactions among rate c3onst:tnts may bc either homo- or hctrrotropic but, i n contrast with the effocts on affinity, intcractions h t w w n rat(>constants a r t not necessarily linkcd. Systtms whose operation is govcv-nrd by lig:tnd-inducrd changrs in reactivity, and riot hy changrs in affinity, have hem c a l l d V systems by Moriod et 02. (1965).

D. Are Cation-Pump Complexes in Equilibrium with Free Cations?

Cations in thc solutions bathing tho mmmbranc will be in equilibrium with those bound to the pump if, during a transport cycle and for cach of the stagrs of the cycle, thc lifctimc of the binding sites is long compared with thc half-time of cxchangc between bound and free cations. If cquilibrium holds, the state of occupation of tho pump sitrs will be indcpcridcnt of the rate of transition of the pump betwcrri thc various stagrs of a transport cycle. Thc assumption that equilibrium holds for cation binding seems to be rcasonablc on the gcnrral ground that it is likcly that cation binding will br ;t faster phenomenon than a proccss that, like ion translocation, is limited by thc rate of onc or more conformational transitions of a macromol(wdar complex. In addition, sevcral lines of experimental evidence lend morci direct support to the view that cation binding to the pump is an cquilibrium proccss.

36

P. J. GARRAHAN AND R. P. GARAY

1. BINDINGEXPERIMENTS The conclusive test for equilibrium binding during active transport would be the demonstration that affinities detected by kinetic experiments are identical wit,h those obtained by the direct determination of cation binding under zero-turnover conditions. As we have already mentioned, no such study has been performed in the Na pump. Cation binding has been measured in the Ca pump from sarcoplasmic reticulum and, in this case, kinetic and binding data yield almost identical affinities for Ca ions (Meissner, 1973). The fact that equilibrium holds for the binding of Ca to the Ca pump is suggestive and may be relevant in the analysis of the mechanism of the Na pump, since it is known that the Ca pump and the Na pump share a sufficiently large number of structural and functional properties to make it likely that both systems have evolved from a common ancestor (Bastide et al., 1973). 2. IRREVERSIBLE INHIBITORS

An indirect method for measuring cation affinities of the Na pump under zero-turnover conditions is to study the effect of cations on the rate of onset of inhibition of the Na pump by irreversible inhibitors. Cations will modify the rate of inhibition if the reactivity of the pump toward the inhibitor depends on whether the pump has cation-binding sites empty or occupied by cations. If it can be demonstrated that these sites are the same sites that are involved in active transport, the measurement of the rate of inactivation at different cation concentrations will give a measure of the af€inity of these sites. Studies along these lines have been performed using N-ethylmaleimide (NEM) (Skou, 1974a,b), dicyclohexylcarbodiimide (DCCD), Be2+,and F- (Robinson, 1973, 1974a,b). In all cases, the affinities detected by these procedures are very close to those obtained from kinetic measurements (compare, for instance, the left and right curves in Fig. 2, Section 111, A, 2).

3. EFFECTOF TURNOVER ON APPARENT AFFINITIES

If during cation pumping, cations were unable to reach equilibrium with their binding sites, their apparent affinity would depend on the rate of turnover and would approach the value of the real affinity as the rate of turnover was reduced. In contrast with this prediction, all the available experimental evidence shows that the apparent affinities of the Na pump for both Na and K, when the turnover of the pump is drastically reduced by cooling, are not very different from those observed under physiological conditions (see, for instance, Post et al., 1965; Kanaeawa et al., 1970).

SEQUENTIAL A N D SIMULTANEOUS MODELS FOR Na A N D

K TRANSPORT

37

In conclusion, experimental evidence, in some cases obtained by several independent approaches, suggests that during the operation of the Na pump, cations bound to the system approach equilibrium with those in the bathing solutions to a degree sufficient to make it almost certain that if deviations from equilibrium exist, they are not very large. [The reader may be reminded for instance of the 400-fold difference between real and apparent affinities detected by Chance (1943) in his classic studies on peroxidase.]

E.

Inner and Outer Cation-Binding Sites

It is now clear that the Na pump can be made to operate in five different modes. Four of them represent exchanges of internal for external cations: (i) the exchange of internal Na for external K coupled to the hydrolysis of ATP which is the physiological mode of operation of the pump (Nu:K exchange) (Glynn, 1956; Post and Jolly, 1957; see also Glynn, 1968) ; (ii) the reversed mode, that is, the exchange of internal K for external Na coupled to the net synthesis of ATP, which represents the reversal of Na: K exchange that takes place when the thermodynamic conditions-i.e., the cationic gradients and the ATP/ADP X Pi ratio-are unfavorable for the forward running of the pump (Garrahan and Glpnn, 1967e; Glynn and Lew, 1970; Lant el al., 1970; Lew et al., 1970) ; (iii) the exchange of internal for external Na ( N a : N a exchange) observed when cells are suspended in high-Na, K-free media (Garrahan and Glynn, 1967a,c; Baker et al., 1969; Sachs, 1970) ; and (iv) the exchange of internal for external K ( K : K exchange) observed in low-Na high-Pi cells (Glynn et al., 1970; Simons, 1974). The fifth mode of operation of the Na pump is the efflux of Na not accompanied by the uptake of Na or K (uncoupled N u e f l u x ) , which is observed when red cells are suspended in Na K-free media (Garrahan and Glynn, 1967a,b; Lew et al., 1973; Karlish and Glynn, 1974). For each of the different modes of operation of the Na pump to be manifest, definite cationic requirements have to be simultaneously fulfilled at both sides of the cell membrane. In the case of the four exchange fluxes, the requirements are for the cations that are transported. The uncoupled Na efflux, on the other hand, requires the presence of intracellular Na, plus the absence of extracellular Na or K, since addition of low concentrations of any of these cations leads to inhibition of this mode of behavior of the Na pump (Garrahan and Glynn, 1967a,b). It is clear, therefore, that for all the known modes of behavior of the pump its cation-binding sites show “sideness,” that is, some can be occupied

+

38

P. J. GARRAHAN AND R. P. GARAY

only by intracellular cations (inner sites), whrrras others can be occupied only by extracellular cations (outer sites). In the case of thc exchange fluxes, both inner and outer sites have to bc occupied by the cations that will be exchanged. The uncoupled Na efflux, on the other hand, will takc place only in those pump units whose inner sitrs arc occupied by Na and whose outer sites are free of both Na or I<. To bc completr, thereforr, any study on the interaction of a cation with the Na pump must include experiments to ascertain on which kind of sites the observed effect is exerted. Thc definite demonstration of the sideness of an effect requires experiments with membrane preparations in which the macroscopic distinction between intra- and extracellular phases is preserved. Howrver, sufficient knowledge on the properties of the inner and outer sites of the pump has accumulated so as to allow their identification in many casrs cven in broken-membrane preparations. In this respect, inner sites may bc identified as those sites a t which Na has to combine to induce phenomena associated with the forward running of the pump. These sites usually show more affinity for Na than for the other alkali cations (but, cf. Glynn and Ellory, 1972). Conversely, outer sites may be identified with those whose occupation by K or its congeners (Li, Rb, Cs, N&, and TI) is necessary for the K-dependrnt events associated with the forward running of the pump to takc place. These sites usually show higher affinity for I< than for Na. F. Relation between Inner and Outer Cation-Binding Sites

We have seen in Section 11, E that each of the known modes of operation of the Na pump has definite cationic requirements both at the inner and at the outer cation-binding sites. This fact indicates that for each mod0 of behavior there is a close interplay betwccn the state of occupation of inner and outer sites. Therefore the relation between the inner and outer sites of the Na pump unavoidably bccomcs one of the main questions that any detailed investigation into the mechanism of active Na and I< transport must try t o solve. There are a t present two alternative, and mutually exclusive, ways of looking at the relation betwcen inner and outer cation-binding sites. These ways allow us to separate models of the Na pump into two general classes, i.e., sequential models and simultaneous models (Baker and Stone, 1966; Hoffman and Tosteson, 1971; Skou, 1971) (Fig. 1 ) . 1. SEQUENTIAL MODELS

It is proposed that in sequential models, each transport unit has a single class of cation-binding sites that, during a transport cycle, are alternately

SEQUENTIAL AND SIMULTANEOUS MODELS FOR Na AND K TRANSPORT

A

EXTRACELLULAR

39

B

1

t

3iZL

f INTRACELLULAR

FIG.1. The two alternative explanations for the relation between t he inward-facing and the outward-facing cation-binding sites of the Na pump. The accessibility of sites for cation exchange are represented by the vertical arrows. In sequential schemes (A), inner and outer bites represent two alternative states of the same set of sites present in a t least two different conformational states of the pump. In these models, inner and outer sites cannot exist a t the same time on the same pump unit. In sznatrltuwocrs (B) models, inner and outer sites are two physically distinct set of sites that exist at the same time on the same pump unit.

~xposcdto t h c b intra- and c.xtracdlular solutions (Fig. 1 A ) . Thv main feature of t h t w models is therefore that intwr and ( J U t W cation-binding sitcs cannot exist a t the same time on thti s a m pump ~ unit sincc they rc.prcs m t successivcb stagcls of a singlc sc.t of sitos going through a transport cyclc. These schemes cxplairi the linkage twt n the intra- and c.xtraccllular cationic requirements on the assumption that the transitions bct w o n the outnwd-facing and the. inv\ard-fnc~ings t a t r s of thv pump t:tkc place only in thosc, pump units whost. sitvs arc. suitably occupied by cations. Srqumtial tnodcls for the N a pump are nxtinlv dwivcd from the classic circulating carrier hypothrsis of Shau (cittd by Glynn, 19.56), originally proposc3d to account for the coupling 1)c~twcwiNa efflux and I< influx ( s w Glynti, 19.56). At that time conccpts of nwnihranc. structurv ww h s c d on the Davson and Daniclli modcl ( s w , for itistanw, Ccreijido and 110tunno, 1070) which did riot allow for the. t.xistc1ncc of proteins spanning the mcmbrancb so as to c~xposc~ sitw to hoth thc intra- and the extracrllular solutions. During tho 1960s sccluential n i o d ~ l ssecimcd to gain furthcr support from biochemical st1idic.s showing that th(3 hydrolysis of ATP by tho Na pump could bc separated into a Na-chycmdrnt step, followd by a Ii-dcpend(mt s t q (see Scction IV, A \ , and tnorc rc4ned schcmcls inrorporating new findings on membrane. structure. and function w(w proposed st^ Chldwell, 1970) , The kinetic treatment of scqucnti:d transport is indrpcndrnt of the

40

P. J. GARRAHAN AND R. P. GARAY

molecular assumptions in particular models that have been made to account for the alternative exposure of sites to the inner and outer surface of the cell membrane. In all models of this kind, the kinetic entities that intervene in the rate equation are the cation-site complexes, and it is, therefore, irrelevant from the point of view of kinetics what kind of molecular mechanism moves the cation-site complexes from one surface of the membrane to the other. 2. SIMULTANEOUS MODELS In sharp contrast with sequential models, in simultaneous models, it is proposed that inner and outer cation-binding sites are physically distinct and independent entities that exist at the same time on the same pump unit (Fig. 1B). In these models, coupling between intra- and extracellular cationic requirements will take place if at least one of the elementary steps of an active transport cycle occurs only in those pump units having both their inner and their outer cation-binding sites adequately occupied by the relevant cations. Simultaneous models require that the Na pump be arranged within the membrane structure in such a way as to allow it to have both regions exposed to the intracellular medium and regions exposed to the extracellular medium. The existence of such structures is compatible with the present knowledge of the chemical organization of cell membranes [for an up-to-date review of membrane structure, see Bretscher (1973)l.

111.

EXPERIMENTAL EVIDENCE FOR SIMULTANEOUS EXISTENCE OF INNER AND OUTER CATION-BINDING SITES

A. Biochemical Evidence

1. STRUCTURE OF THE Na PUMP

We have already mentioned that the existence in a pump unit of sites exposed to the extracellular medium together with sites exposed to the intracellular medium necessarily requires that the arrangement within the membrane structure of the system possessing these sites be such as to allow it to bridge the membrane from side to side. Available experimental evidence strongly suggests that this is so. The molecular weight of the pump has been estimated to be about 2 X loKdaltons, which corresponds to that of a sphere about 80 A in diameter,

SEQUENTIAL A N D SIMULTANEOUS MODELS FOR Na A N D K TRANSPORT

41

and is comparable to the width of the membrane ( I h p n r r and Maccy, 1968). The main protein component of purifird (Na K)-ATPasc preparations is a polypeptide chain of a molvcular weight of about lo5 daltons (Kytc, 1971; Uesugi et nl., 1971; Hokin et nl., 1973; Hokin, 1974; Jorgcnsen, 1974). It has been convincingly shown that, in red cell membranes, polypeptidc chains, whose molecular wright is similar to that of the ATPase, are attacked by specific reagents from both thc inner and thc outer surface of the membrane. This provides conclusivc evidcnce that these chains span the membrane from the inner to the outrr surface (Bretscher, 1971). Analogous experimental evidence indicates that the protein component of the Ca pump of sarcoplasmic reticuluni is also cxposrd to both surfaces of the cell membrane (Hasselbach, 1973). Thc availability of purified ATPasc preparations has made it possible to obtain specific antibodies against the Na pump. Kyte (1974) has shown that some of these antibodies do not inhibit the Na pump when bound to thc inward-facing part of the (Na I<)-ATPasc. Although his results are compatible with some of the proposed sequential models, they seem to rule out those mechanisms for active transport that require large movrments of the system within the membrane, such as those based on the translational or rotational diffusion of carrirr molecules across the plane of the membrane. It is difficult to see how thew transitions could takr place without apparrnt hindrances when a large hydrophilic molecule, such as 7globulin, is strongly bound to the transport systcm.

+

+

2.

ItEQUIREMENT FOR SIMULTANEOUS OCCUPATION SITESUNDER ZERO-TURNOVER CONDITIONS

OF I N N E R AVD

OUTER

N-Ethylmaleimide is an irreversible inhibitor of the Na pump (for references, see Glynn and Karlish, 1975). Adrnosine triphosphatasc protects the pump against inhibition, and K partially abolishes this protection. In the presence of ATP, replacement of K by Na, keeping the sum of the concmtrations of Na and I< constant, progressively eliminates the effect of I<. As the concentration of Na is increased, the rractivity of the pump toward NEM reaches a minimum and then increascs again when all the K is replaced by Na (Fig. 2) (Skou, 1974s). The parallelism betwecn the effects of Na and K on the susceptibility to Ii)-ATPasc, activity (cf. left and right NEM inhibition and on (Na curves in Fig. 2) a t different ATP concentrations, strongly suggests that thc effect of cations on inhibition is mediated by their combination at sites a t which they combine to inducc active transport (Skou, 1974a,b). The most straightforward explanation for the combined effect of Na and K on the reactivity of the Na pump toward NEM is to suppose that inner

+

42

P.

J. GARRAHAN AND R. P. GARAY

I W

z

loot

o;

-

90 I20 1o; 150 120 90 60 30 0 K+ Cations in preincubation medium (mM 1

“0

$0

150

N a+ 120 90 60 30 0 K+ Cations in test medium cmM)

+

FIQ.2. A comparison of the effects of Na and K on the reactivity of (Na K)ATPase toward N-ethylmaleimide (NEM) and on the rate of (Na K)-dependent ATP hydrolysis. In the experiment on the left, the enzyme preparation was preincubated at 37”C, for 30 minutes, in media containing 1 m M NEM, 3 mM ATP, 5 mM EDTA, and the concentrations of Na and K shown on the abscissa. After this treatment, (Na K)ATPase was assayed in a medium containing 3 mM ATP, 3 mM MgC12, 120 m M NaCl, and 30 m M KCl. I n the experiment on the right, (Na K)-ATPase was estimated in untreated enzyme in media containing 3 m M ATP, 3 mM MgCl,, and the concentrations of Na and K shown on the abscissa. Note that the concentration of Na for half-maximal effect on the ascending part of both curvea is 37 mM, indicating that the apparent affinityfor Na under zero-turnover conditions is practically identical with the apparent affinity for Na under normal-turnover conditions. (Redrawn from Skou, 1974a, by permission of the publishers.)

+

+

+

and outer cation-binding sites exist a t the same time on thc same pump unit. If this assumption is taken for granted, when Na is increased at the expense of K, then Na will first displace K from the inner sites of the pump because their affinity for Na is higher than for K. I n addition, as all the K is replaced by Na, both inner and outer sites will become occupied by Na. The protective effect of Na in the presence of K would, therefore, be owing to the fact that when the pump has its inner sites occupied by Na and its outer sites occupied by K, its reactivity toward NEM is less than that observed when both inner and outer sites are occupied by either N a or K. It is difficult to find a simple explanation for Skou’s findings, if inner and outer cation-binding sites are taken as existing in alternative conformations of the pump. I n his experiments, Skou pretreated the enzyme with NEM in the presence of EDTA. Although it has been argued that under these conditions the pump may still become phosphorylated (Glynn

SEQUENTIAL AND SIMULTANEOUS MODELS FOR Na AND K TRANSPORT

43

and Rarlish, 1975), it is almost certain that EDTA will drastically reduce the net turnover of the pump. If sequential schemes are assumed, in tho abscncc of turnover, conformations of the pump possessing outer sites that prefer Ii: ovcr Na will be in equilibrium with conformations of the pump possessing inncr sites that prefer Na ovcr I<.Under these conditions, addition of cations to the incubation media will result not only in their binding to the pump sites but also in the displacement of the equilibrium toward the conformation having the highest affinity for the added cation. Hcnce, in a high-I<, Na-free medium, the pump will be in the conformation possessing cxternal sites, whercas in the high-Na, I<-free medium, the pump will mainly be in the conformation posscssing internal sites. In Skou’s experiments the ratio of Na to I< concentration giving optimal protection against NEM is identical to that giving optimal activation of ATPase activity (cf. left and right curves in Fig. 2 ) . Within this range of cation concentration, in sequential schemes this would mean that the pump exists mainly as a mixturc of conformations having outer sitras occupied by I< and inner sites occupied by NR,that is, a mixture of the states that predominate in thr all-I< mrdia and of the states that predominatc in thc all-Na media. It is difficult to see how thc reactivity toward NEM of a mixturr of thesc specirs can be less than the individual rcactivity of each of them.

3.

PERSISTENCE OF I N N E R .4ND

OUTER SITES I N DIFFERENT LIGANDSTATES

OF THE P U M P

I n contrast with the postulates of simultaneous models, sequential models require that the conformations of the pump that carry inner and outer sitcs represent mutually exclusivP statcs of the system. Therefore, if scqurntial models gave an adequated description of the process of active transport, one ought to be able to detect conformations having only inner or only outer cation-binding sites. This dctrction is, in fact, possible since, by adequate manipulations of the ligands present in the incubation media, the pump may be “frozen” in each of the various stages it travcrscs during cation transport, and the prcscnce or absrncc of a given class of sites in each state may be detected by the already mmtioned procedure (sre Scction 11, D) of studying the action of cations on thc rate of onset of the effect of irreversible inhibitors. This approach has bern applicd by Robinson (1973, 1974a,b) t o detect the pres(’ncc and the affinity of both inncr and outer sites during the different ligand statas of the pump. a. Inner Sites. Dicyclohexylcarbodiimide is an irrrversiblc inhibitor of (Na I<)-ATPase. The rate of inhibition of the pump by DCCD is decreased by Na. The apparent affinity for the protectivc effect of Na is very

+

44

P. J. GARRAHAN AND R. P. GARAY

close to the apparent affinity of the inner sites of the Na pump, and both are similarly increased by phlorizin. It seems, therefore, almost certain that t8heprotective effect of Na is due to its combination with the inner sites of the Na pump (Robinson, 1974a,b). Robinson (1974a,b) has taken advantage of the interactions of Na and DCCD with the Na pump to show that sites for Na with nearly constant affinity are detectable in the ATP-containing, in the ATP-free, and in the phosphorylated states of the pump. This result strongly suggest that, in contrast with the predictions of sequential models, inner sites exist in all the various states of a pump cycle. b. Outer Sites. The existence and the affinity of the outer sites of the Na pump in different ligand states has been investigated taking advantage of the increase by K of the rate of onset of the irreversible inhibition of the (Na K)-ATPase by beryllium or fluoride ions. There is strong experimental evidence that the effects of K are mediated by its combination with the external sites of the Na pump (Robinson, 1973, 1974b). The results obtained with this procedure agree with those obtained for the inner sites, in the sense that external K-binding sites are detectable in all the ligand states representing the various steps of a transport cycle. I n contrast with the invariance of the affinity of the inner sites, the results also show that, as a consequence of phosphorylation, external sites go from a state of moderate affinity for K t o a state of high affinity for K. Comparison of the increase in the affinity of the external sites in the phosphorylated pump with the lack of effect of phosphorylation on the affinity of the inner sites provides strong additional evidence in favor of the distinct and independent existence of inner and outer sites in the Na pump.

+

B.

Kinetics of Cation Fluxes

1. EXPERIMENTAL RESULTS

The strongest experimental evidence in favor of the simultaneous existence of inner and outer cation-binding sites comes from the study of the kinetic properties of the cationic fluxes driven by the Na pump. The study of the kinetics of ion fluxes can be approached in two different ways. The first one is to look at the shape of the flux vs. cation concentration curve when cation concentration is varied at one of the surfaces of the cell membrane and is kept constant at the opposite surface of the cell membrane. Although the shapes of these curves provide useful information about the number of binding sites, their affinities, their interactions, and the like, they do not in themselves indicate or exclude any particular kinetic mechanism.

SEQUENTIAL AND SIMULTANEOUS MODELS FOR Na AND K TRANSPORT

45

The second approach, originally suggested by Baker and Stone (1966), is to see how, for each kind of flux, changes in cation composition a t one of the surfaces of the cell membrane alter the shape of the curves relating cation flux with cation composition a t the opposite surface of the cell membrane. This approach, not only allows one to distinguish among different classes of models but also has thc considerable practical advantage that most of the information that can be obtained from it is independent of any dctailed knowledge of the actual shape of the cation activation curve. The experimental study of the effects of trans cations on the shape of the flux curves has become feasible since the development of techniques for changing independently the cation composition of the solutions bathing both surfaces of the cell membrane (Garrahan and Rega, 1967). Studies performed mainly in red cells (Hoffman and Tosteson, 1971; Garay and Garrahan, 1973; Garrahan and Garay, 1974) have shown that under all the conditions testcd, curves relating cation flux with cation concentration a t a given surface of the cell mcmbranr change only by a constant factor when the cation composition a t the opposite surface of the cell mombrane is altered. The following is an account of the experimental evidence supporting this statement. 1. The relation between internal Na concentration and the efflux of Na in exchange for external K differs only by a constant factor from the relation between internal Na concentration and the efflux of Na in exchange for external Na (Fig. 3) (Garay and Garrahan, 1973). 2. Changes in the external Na concentration only alter by a constant factor the activating effect of internal Na on the rate of Na:Na exchange (Garay and Garrahan, 1973). 3. Intracellular K is a competitive inhibitor of the effect of internal Na on Na efflux. The inhibitory effect of I< is identical, regardless of whether the efflux of Na is taking place in exchange for external Na or for external K (Garay and Garrahan, 1973). 4. Tho curve relating the rate of Na:Na exchange to the external concentration of Na changes only by a constant factor when the internal concentration of Na or I< is altered (Garay and Garrahan, 1973; Garrahan and Garay, 1974). 5. The relation between K influx and the external concentration of K is altered only by a constant factor by changes in the intracellular concentration of Na or K (Baker et al., 1969; Hoffman and Tosteson, 1971; Garay and Garrahan, 1973; but see Chipperfield and Whittam, 1974). 6. The curve relating external Na concentration to the influx of Na in exchange for internal Na (Na: Na exchange) is similar to the curve relating external Na concentration to the influx of Na in exchange for internal K (the reversed mode of operation) (Glynn et al., 1970).

46

P. J. GARRAHAN AND R. P. GARAY

5.0

0 Internal No concentration (mM)

Rate of No: No exchange

FIG.3. Left: effect of internal Na concentration on the ouabain-sensitive efflux of Na into media containing nonlimiting concentrations of K ( 0 )(Na:K exchange) and into high-Na media without K ( 0 )(Na:Na exchange). Right: the rate of Na:K exchange plotted against the rate of Na:Na exchange for each of the internal N s concentrations tested. This plot yields a straight line of zero intercept, indicating unequivocally that replacement of K by Na a t the external sites of the Na pump affects only the value of the efflux at nonlimiting internal Na concentrations and has no effect on the affinity, number, and interactions of the inner sites of the Na pump [see Eqs. (2) to (4)]. (Redrawn from Garrahan and Garay, 1974, by permission of the publishers.)

7. The activation by external K of K: K exchange follows a curve similar t o that of the activation by exkrnal K of Na:K exchange (Glynn et al., 1970). 2. OVERALL RATEEQUATION FOR CATION FLUXES

The shape of a curve relating cation flux to cation concentration will depend on the number and properties of the cation-binding sites and on the reactivity of each of the possible cation-site complexes. Since none of the different cation fluxes driven by the Na pump shows evidence of inhibition by excess activating cation, any flux equation will be expressible as

where J is the flux, J,, is the flux at nonlimiting concentrations of the activating cations, and f (C+) is a function of cation concentration that goes from m to 1 as (C+) goes from 0 to m . Inspection of Eq. (1) makes it clear that, if the only effect of changes in

SEQUENTIAL A N D SIMULTANEOUS MODELS FOR No A N D K TRANSPORT

47

cation composition a t thc oppositc surface of the cell mcmbranc is to altcr by a constant factor the shape of the flux curve, the effect of trans cations must necessarily be caxcrtcd only on the value of J,,,, that is, on the turnover of the system, and not on function f(C+), which includes all the other variables that affect the flux. Wc have mentioned before that all the known modes of opwation of the pump have strict rcquiremcnts concerning cation composition a t the surface of the membrane that occupies a trans position with respect to thc surface of the mcmbrane from which the flux is taking place. This togrther with the already mentioned fact that the requirement is cxpressed only in the value of the maximum flux ncccssarily implies that the overall rate equation for flux as a function of both inner (C;) and outer (C,+,,) cation concc~ntrationmust be expressible as a product of a function of inner cations only times a function of outer cations only, that is,

Equation (2) implies that J will tend to 0 when eit,hcr X or Y tend to Q) . This fact expresses the tight linkagc botwcm intra- and cxtracellular cation requiremcnts, which is a salicnt feature of all the modes of operation of thc Na pump. In thc case of thc four exchange fluxes, X and Y go from to I as t>hcconcentrat’ion of the activating cation goes from 0 to =. The applicability of Eq. (2) to the uncoupl(Ad nxtrusion of Na has not yet been tested. If it were applicable, X would havc the samc limits as for the cxchange fluxes, whereas Y would go from 1 to 00 as external Na or I< goes from 0 to 0 0 . The validity of Eq. (2) is indcpondcnt of any detailed knowledge on the actual shapc of functions X and Y . This is an cxtremely convenient property since, as we will see later, most of the information on thc mechanism of cation transport provided by equations such as Eq. (2) comes from the fact that the terms containing inner and outer cations are separable into a product and not from the shape of functions X and Y . Mornover, the cxperimental tcst of thc validity of Eq. (2) requires us to know neither the functions X and Y nor the value of Jmax.I n fact, if we call J I and J z the values of thc fluxes a t two constant extcrnal cation concentration, it is evident from Eq. (2) that Q)

JdJ2

=

Y ( C L )/ Y ( G u t 1 )

(3)

or

Obviously, a similar cxpression will hold when internal cation conccntration is kept constant.

40

P. J. GARRAHAN AND

R. P. GARAY

If an equation such as Eq. (2) expresses the kinetics of the process, when a cation flux measured at different cation concentrations at the cis surface of the cell membrane is plotted against the flux measured at the same cis cation concentration but at a different trans cation concentration, a straight line of zero intercept and positive slope will be obtained and this will be true however complicated or unknown functions X and Y may be. In Fig. 3 is shown an example of this kind of plot. 3. EQUILIBRIUM TREATMENT OF THE KINETICS OF CATION FLUXES We have shown in Section II,D that experimental evidence makes it quite likely that during active transport, cations in the solutions bathing the membrane are in equilibrium with those bound to the pump sites. We will try to demonstrate now that if equilibrium actually held, the kinetic properties of the cation fluxes driven by the pump, summarized in Eq. (2), would not only demand the simultaneous existence of inner and outer cation-binding sites, but also place a sufficient number of additional restrictions on the possible modes of operation of the Na pump, to indicate that only a particular class of simultaneous model, can account for the kinetic properties of the Na pump. Under equilibrium conditions, separation of variables as in Eq. (2) is a sufficient, but by no means a necessary, condition for demonstrating the simultaneous existence of inner and outer cation-binding sites. Lack of separation of variables, therefore, must not be used as an argument against the simultaneous models, or in favor of sequential schemes but only as evidence against the particular class of models which we will develop in the following sections. In fact a simultaneous model of the Na pump not involving separation of variables has been proposed by Chipperfield and Whittam (1974). a. Equilibration of Two Diferent Ligands with a System Possessing Two Diferent Sets of Binding Sites. The simultaneous models for active transport assume that inner and outer cation-binding sites exist at the same time on the same transport unit. The analysis of the equilibrium kinetics of this class of system must, therefore, start with the development of a general equation for the process of addition of cations to such a system. Let us, therefore, define a system El having q sites for cation x and r sites for cation y. There is no loss in the generality of our reasoning if, for the sake of simplicity, we do not consider competitive interactions between x and y. If sites for x and y are taken as the inner and outer cation-binding sites of the Na pump, the fact that they face different solutions makes this assumption a physically feasible one. We will, furthermore, assume that, within each set of sites, the probability of occupying a site in the empty system is the same for each site.

SEQUENTIAL A N D SIMULTANEOUS MODELS FOR Na A N D K TRANSPORT

49

The fraction of the sitesfor x occupied by x will be

a=

j-0

i-1

(5)

The addition of ligands can be considered to be the consequence of 1) equilibria

q(T

+

governed by q ( r

+

+ +

E ix j y Exiy, 1) macroscopic association constants,

(6)

The concentration of each of the species Exiy, can, therefore, be expressed as a function of the concentration of the empty system ( E ) , the free ligands, and the respective association constant, i.e.,

Insertion of this expression into Eq. (5) yields

x=

i-q

j=r

i-1

j-0

C C iKij (2)

(Y)j

(9) q

i-q

j-r

i-0

j-0

C C Kii(zIi(yI’

where KW always = 1. A similar expression will hold for the fraction of sites occupied by the ligand y (P). The fraction of molecules of system E having its sites for 2 with a state of saturation and its sites for y with a state of saturation I’ will be, for a given concentration of x and y,

F

=

a.9

(10)

From the preceding analysis, it is obvious that

F

=

a@,Y)

P(X1

Y)

(11)

Hence, in general, the function governing the simultaneous occupation of

50

P. J. GARRAHAN AND R. P. GARAY

a system by two ligands cannot b e separated into a product of functions each of them containing only one of the ligands. Only when

K 20. -K. - K c2. 21

-

=

Kij =

...

=

Kir

=

Ki

(12)

for each value of index i docs Eq. (9) become s=q

j=r

i= q

i-0

i- 1

C i K i ( ~C) ~yi i1

Z=

C~ K < ( x ) ~

-

-

=

X(x)

(13)

The equalities of constants in Eq. (12) signify that the affinity of the sites for x are independent of the state of occupation of the sites for y, that is, there are no interactions betwcen the affinities of each set of sites. We have already mentioned that interactions in affinity are linked, so that if the equalities of Eq. (12) hold, the affinities of the sites for y must also be independent of the state of occupation of the sites for x (see Section II,C,2). Hence if Eq. (13) is valid, it necessarily follows that

Y

=

P(g)

(14)

Under these conditions, Eq. (10) can be writtcn

F

=

X(x)P(y)

(15)

Equation (15) , which can be extendcd to any number of ligands, demonstrates that, if the probability of finding a system with a degree of occupation 8 by ligand x and a degree of occupation Y by ligand y is expressible as a product of functions, each containing only one of the ligands, there must be no interactions between the affinities of the sites for each of the different ligands. Thus, there must be no heterotropic (see Section II,C,2) interactions in affinity. Equation (15) , on the other hand, has been developed without postulating any restriction to the shape of each of its constitutive functions, so that it allows the existence of any kind of interaction in affinity within each set of sites. Homotropic interactions (see Section II,C,2) may, therefore, exist without affecting the separation of variables of Eq. (15). The lack of heterotropic interactions between affinities necessarily demands the independent and simultaneous existence of each set of sites in each of the molecules to which ligands are bound. If this postulate is applied t o the case of the cation-binding sites of the Na pump, it is evident that equilibrium binding functions for inner and outer cations having the

SEQUENTIAL A N D SIMULTANEOUS MODELS FOR N o A N D K TRANSPORT

51

shape of Eq. (15) ar(1 not compatible with stqucntisl mod& for activc. transport, in which inner and outer chation-binding sites reprcwnt two mutually cxclusive statcs of the same sot of sites. This assertion can bc il1ustratc.d in a moro quantitative way by working out thc expression for the saturation by a ligand x, in thc prcwncc of anothcr ligand ( ! I ) of a systen1 ( E ) that exists in state El having sittls only for 2 and is in rquilibrium with anothrr state, E2, which has sitvs only for y. Using csscntially similar reasoning to that employed to drvclop Eq. (9), it is vasy to show that in this casc the fraction of sitcls owupied hy x will lw

q

(2K , \,=a

( 2 )i

+ rL g Kj (y) i) j=0

1

where q is the total number of sites i n El; r is the total number of sitvs in E z ; L is the equilibrium constant brtwcvn thc two cmpty states L = E 2 / E , ;K , = ( E P x l ) (/ E l )(x),; and K , = (E2y,)/ ( E z )(y) 1. Inspcction of Eq. (16) makvs it char that under no conditions can X he madc indeprndcnt of y. I n s e q u c d a l schcmc~s,thcroforc, the fraction of molecules simultaneously occupied by scvwal ligands will never bc expressiblc as a product of functions of cacah of the ligands. b. Transjormation of Saturation us. Ligaiad Coricenlration Function into Velocity us. Laganrl Concentratzon Functzons. When the rate of transitions are slow compared with the rate of quilibration with ligands, it is easy to transform the fractional equilihration with ligand x [lCq. (5)], into an PXpression for thc fractional rate of the transition that the systeni induces in thc same ligand, i.c.,

whcrc. k,, is tho rate constant per site for the transition, e.g., the translocattion, of ligand x in the species Exly,, and k,, has the same meaning for the fully saturated species EX,^,; J and J,,, have the same meaning as in Eq. (1). It is clear that an analogous expression will govern the rate of transitions of ligand y. We have shown in Section III,B,2 that the kinetic properties of the

52

P. J. GARRAHAN AND R. P. G A M Y

cation fluxes driven by the Na pump demanded that the terms containing intra- and extra-cellular &ion concentration should be separable into a product in the rate equation [see Eq. (2)]. If Eq. (17) is taken as a flux equation in which x and y represent intra- and extracellular cations, it is clear that in general the terms containing x and y will not be separable into a product. For this t o be possible the following set of restrictions necessarily hold. 1. There must be no interactions between the ajinities of the sites for x and the sites for y. As we have shown in Eq. (13) only if this is true the terms containing x and y in the denominator of Eq. (17)become separable into a product. Lack of interactions in affinity necessarily demand the independent existence of sites for x and sites for y in each molecule of E [see comments about Eq. (16) 1. Under equilibrium conditions, therefore, separation of variables in the rate equation for cation flux [Eq. (2)] is a suflcient condition for demonstrating the simultaneous existence of inner and outer cationAinding sites. 2. There must be interactions among rate constants, that is, the rate constant for the transition of ligand x when bound to a site must depend on the state of occupation of the sites for ligand y . This postulate can be clearly demonstrated by analyzing the case of no interactions among rate constants. In fact, if we assume that, for all values of index i, kio =

kil

=

kiz =

... =

kij

E

k ar. - ki

then we may define, in the absence of interactions in affinity, (q new constants, Ki' = kiKi

(18)

-

1)

(19)

When introduced into the numerator of Eq. (17), these constants allow the terms containing y to be factored out from the terms containing x [see Eq. (13)], yielding an expression for y that cancels out with the same expression for y appearing in the denominator, as a consequence of the absence of interactions in affinity. This demonstrates the intuitively obvious fact that, in the absence of interactions among affinities and among rate constants, the flux of ligand x will be independent of the concentration of ligand y. In the case of the Na pump, the tight linkage between intra- and extracellular cationic requirements demands that some of the l c i j be zero. In the case of the four exchange fluxes that the pump is capable of, it is evident that this requirement must be fulfilled at least by all the kio (and by symmetry by all the k,o of the equation governing the flux of ligand y), that is, the rate of transition of all the ion-pump complexes having sites a t one of the surfaces of the cell membrane either unoccupied or occupied by the wrong cation

SEQUENTIAL AND SIMULTANEOUS MODELS FOR Na AND

K TRANSPORT

53

must be zero. I n the case of the uncoupled Na efflux, the krOmust be diffcrent from zero and a t least the k,, must bc zero. 3. Only some kind of interactions among rate constants will yield separation of variables in the ratr equation. The mraning of this statement can be seen more clearly if we notice that the double summation in the numerator of Eq. (17) is expressible as a sum of terms of the form

i= I

where lack of interactions in affinities is implicit in tho fact that K i is indcpendent of the value of index j . It is evident that, for the terms containing y and the terms containing x to be separable into a product, the sum over i must be either zero or equal for all thc values of index j . As the only term undcr the summation sign that depends on i is k i j , this restriction applies to the possible values of the rate constant,s. The nature of the restrictions is seen morc clearly by writing down the matrix of rate constants:

Inspection of the matrix makrs it clear that separation into a product of the tcrms containing z and y in th r numerator of Eq. (17) will only be possible if the following restrictions on thc intrractions between rate constants are satisfied: (i) in each column of the matrix, all the elements that are different from zero have th r same value; (ii) if an element in a column is equal to zero, all the elements of th r row to which this elcmcnt belongs must also be equal to zero; and (iii) only one of the clrments of the matrix is different from zero. When these restrictions on th r possiblr intclractions between rate constants are applied to the interactions bctwem inner and outer sitrs of the Na pump some interesting points a r k . Thc first of thrsc is of very general

54

P. J. GARRAHAN AND R. P. GARAY

nature and concerns separation of variables in the rate equation [see Eq. (2)]. From the preceding paragraphs, it is evident that coexisting sites that do not interact in their affinity and, therefore, give saturation functions with the shape as expressed by Eq. (15) may not yield separation of variables in the rate equation. The second point refers to the stoichiometry of cation exchanges through the Na pump. The idea that the stoichiometry of the pump is fixed and independent of cationic concentrations comes mainly from experiments performed in red cells (Glynn, 1962; Post and Jolly, 1957; Sen and Post, 1964; Whittam and Ager, 1965; Garrahan and Glynn, 1967d). On the other hand, data from dialyzed squid axons (Mullins and Brinley, 1969) and muscle (Adrian and Slayman, 1966) seem to favor the idea of variable coupling between Na extrusion and K uptake during Na: K exchange. If the number of ions that are transported is equal to the number that are bound to the system, inspection of the restrictions we imposed to the rate equation [Eq. (17)] makes it evident that scparation of variables is consistent with variable stoichiometry for cation exchange. Even more, if fixed stoichiometry is taken for granted, some additional restrictions to the interactions betwcen rate constants must be taken into account. In fact, the general case of fixed stoichiometry of cation exchange would be that in which only k i j , in which i/j = constant, is different from zero. Under these conditions there would be no separation of variables. Fixed stoichiometry will give separation of variables in the rate equation only in the particular case in which only one of the rate constants is different from zero. This would mean that the stoichiometry is not only fixed with regard to the ratio of cation exchanges but also with reference t o the number of cations transported during each transport cycle. The experimental behavior of transport systems, such as those of red cells, which show both separation of variables in the rate equation and fixed stoichiometry, seems to agree with this requirement, since in red cells not only the ratio between Na extrusion and K uptake but also the ratio of cation transport to ATP hydrolysis is constant (Sen and Post, 1964; Whittam and Ager, 1965; Garrahan and Glynn, 1967d). The third point concerns an experimental restriction on the interactions among rate constants. In fact, when tested in intact cell preparations, the active transport system shows no evidence of inhibition by excess cations (see, for instance, Sachs, 1970; Garay and Garrahan, 1973). This places a restriction on the possible values of the rate constants in Eq. (17) which is purely experimental: if there is no inhibition by excess cation, the rate constant of the fully occupied states cannot be zero. Therefore, in the case that only one of the rate constants is different from zero, it is unavoidable to conclude that it must be the one governing the transitions of the fully

SEQUENTIAL AND SIMULTANEOUS MODELS FOR Na AND K TRANSPORT

55

occupied species [i.e., k,, in Eq. (17) and k,, for the equation govrrning the transition of ligand y]. Multiple sites for morr than one ligmd, on the othrr hand, do not allow us, evrn whrn equilibrium kinetics holds, to cquate fractional vrlocity with fractional occupation of sites. Thus, the (quality,

JIJ,,..

=

fraction of transport units in conditions to induce transport

will only be valid in the case in which all thr rato constants that are diffcrent from zero have the same valur. Only if this is true will apparent affinities mrasurrd on the flux curvrs bc q u a 1 to the true affinities for the formation of the cation-pump complexes. c. Rate Equation for More Than One Conformation of Cation-Rznding System. The kinetic analysis w h a w prrformed up to now was based on the assumption that thr systrm to which ligands are bound exists in a singlc state. This assumption rcprcscwts a gross oversimplification when applird to the operation of thci Na pump, since it is now quite char that during a transport cyclr, the N a pump travcmrs a srries of conformational states ( s c ~Scction IV). We will, therrfore, extend our kinetic analysis to a system that, in order to operate, has to undergo a cycle of conformational transitions and drterminr to what rxtcnt, brrausr of this new complication, additional restrictions have to hti postulated in order to krcp the rsscntial properties of the kinrtics of cation fluxrs defincd by Eq. (2). To perform our analysis, we start from thc assumption that there is a tight coupling betwern the ratr of transition bctwc~mconformations and the rate of cation translocation. i.e., thr “conformational flux” diffrrs only by a constant factor from the cationic fluxes. With this assumption, wc may distinguish t w o clearly distinct casrs among the diff (.rent modes of operation of the Na pump. (i) T h r Na:K exchange and the uncoupled Na efflux arc, under physiological conditions, essentially irreversible phcnomena in which unidirrctional fluxrs can bc equated with net fluxes through the pump. This ovrrall irrev&bility means that a t lrast one transition of the ion pump complcx is irrcversible. Using our initial assumption, it is obvious that the rat(. of this transition will be proportional to the overall ratr of pumping. (ii) The Na: Na and I<:I< exchanges are 1:1 exchanges of internal for cxtrrnal cations, net fluxes arr zero, and unidircctional fluxrs have the same magnitude in both directions. I n these cases the value of the flux will b r proportional to thc value of the unidirectional ratr of the slowrst transition. Therefor(), for both casrs (i) and (ii), if n is the number of states the pump has to traverse in order to induce fluxes, wo can order them in a way that makes the unidirectional rate of transition of the n-th species proportional to thr value of the flux. For instance, for the case of a pump fully

56

P. J. GARRAHAN A N D R. P. GARAY

saturated with internal Na and external K, lENa,K,

e WNa,K,

*ENa,K, e

. . . + IENa,K,

. . . $ nENa,K,

--t

(21)

Superscripts 1, 2, . . . , n refer to different states of the pump, and q and T are the maximum number of internal and external sites, respectively, in each conformation. In Eq. (21), and the following, Na and K are taken to represent inner and outer cations, respectively, and can therefore be replaced by the adequate cations when fluxes other than Na:K exchange are considered. If equilibrium held for the addition of cations to each species and steady state for the transitions between the cation-pump complexes, the ratio of the concentrations of the fully saturated species would be constant and independent of the cationic concentration. We can, therefore, define (n - 1) constants

which allows us to express the concentration of each of the fully saturated species as a function of the n-th species, i.e., "Na,K,

= KinENa,K,

(23)

The Ki are combinations of rate constants some of which will be of the form

k'

=

k(X)

(24)

where X is a substrate or a product of the metabolic reaction coupled to the ionic fluxes. Therefore, in general, K i will be constant only at a constant concentration of metabolites. The total concentration of pump will be

If 'F(Na, K)-l is a function of internal and external cation concentraE in the state "Na,K, (in the tion expressing the probability of finding C case of the uncoupled Na efflux the proper function would be that giving the probability of finding iE in the state "Na,Ko), then {Etotal = 'ENa,K,('F(Na,

K))

(26)

Equation (23) can now be written {Etotnl = K i nENa,K, ('F(Na, K) )

(27)

SEQUENTIAL A N D SIMULTANEOUS MODELS FOR Na A N D K TRANSPORT

57

Inspection of Eq. (28) makes it clcar that "ENa,I<, is exprcssiblc as a product of a function of internal Na times a function of extcrnal I( only if (i) for each state of the pump, iniicr and outer sitw coexist and do not interact in their affinity, i.c., for each value of index i

%F(Na,K )

=

tf(Na)"(I<)

(29)

and (ii) the summation of the denominator of Eq. (28) is expressible as a function of inner cations times a function of outer cations only. For this to be possible not only Eq. (29) must hold, but the saturation function Zf(Na) and 'y(I<) and the values of K , must be such as to make th e fractional distribution of the pump aniong its different affinity states independent of thc cationic concentration. This statement extends to the whole system the restraint, which Eq. (29) places on each of its states, namely, a lack in hcterotropic interactions in affinity. The simplest, but not the only, way of allowing separation of variables is to assume that a t least one of the saturation function is the same in all the states of the pump, that is, the number, affinity, and interactions of either inncr or outer sites rcmain constant during a transport cycle. For instance, if

*f(Na) = 2j(Na) = 3f(Na) =

---

=

"f(Na)

= f(Na)

(301

In this equation the terms containing innw and outer cations are separated into a product as is demanded by the experimental results.' Although Eq. (31) was derived for the case that the flux is proportional to an irreversible transition of "ENa,K,, the general restrictions will also be valid if the transition were reversible, that is, if the rate of pumping were proportional to the net rate ( k , *ENa,K, - k-I (n+l)ENa,K,). I n this case the concentration of (n+l)ENa,K, would be given by an expression differing only in the values of Ki from those in Eq. (31). Hence, the net rate would be expressible as a function of inner, times a function of outer, cations if the restrictions used to develop Eq. (31) are applied.

58

P. J. GARRAHAN AND R. P. GARAY

Equation (31) may be generalized to any number of different sets of sites for different ligands. In this case, separation of variables will be possible when all the saturation functions but one are the same for all the various states of the system. On the other hand, Eq. (31) provides a clear illustration of the impossibility of separating into a product the terms containing intra- and extracellular cations in sequential models of active transport. I n this sense, it completes the demonstration of this impossibility as stated in Eq. (16). In fact, if the sequence of steps in Eq. (21) represented sequential transport, by definition each of the species involved would be accessible either to the internal or to the external cations, but never to both, and, therefore, ;F(Na, K )

=

F(Nain, Kin)

or F(Nao,t, K o d

(32)

This clearly forbids one to express "ENa,I<, as f(Na) y (K) . The value of K i [see Eq. (22)] of one or more of the species may be small enough so as to make the term containing this constant negligibly small within the experimentally accessible range of cation concentrations. In this case, the terms containing these constants may be dropped from the overall rate equation. The overall kinetic properties, therefore, cannot rule out the possibility that during some stage or stages of a pump cycle the restrictions stated in Eqs. (29) and (30) are not fulfilled; but they demand that these stages occupy only a very small fraction of the total duration of a pump cycle. Equation (31) is valid as a flux equation only if the fully saturated species "ENa,K, are able to undergo transition, as seems to be the case in red cells (see Section III,B,3). In systems in which the stoichiometry is not fixed, species with a lesser degree of saturation should also intervene in the turnover of the pump. To analyze this case, let us postulate dissociation equilibria of the form: "ENa,K,

+ iNa + j K

nENa(q-i)K(r-j)

(33)

Governed by dissociation constants, Kij

=

(Na) "K) i(nENa(q-i)K+j)) "ENa,K,

(34)

and hence

If, regardless of its state of occupation, the transitions of "E were limiting,

SEQUENTIAL AND SIMULTANEOUS MODELS FOR No AND

K TRANSPORT

59

the expression for, e.g., the flux of Na would be given by (36) i=o

i=o

Then, using Eqs. (35) and (31) ,

where Y (K) represents the summation over the different functions of K in the denominator of Eq. (31). Inspection of Eq. (37) makes it clear that separation of variables in this case needs to take into consideration, in addition to the restrictions used to develop Eq. (31), the lack of interactions among affinities and interactions among rate constants, as already stated in the development of Eq. (17) and in the discussion that follows Eq. (17). Analysis of Eqs. (31) and (37) shows that, when a transport system has to undergo a series of conformational changes in order to induce cation movements, the requirement of simultaneous occupation of inner and outer cation-binding sites, which is mandatory to explain in terms of equilibrium kinetics thc experimental properties of the flux curves [see Eq. (2)], refer only to the species whose ratc of transition is proportional to the flux, i.c., to the rate-limiting step. Spccies other than this may not require simultaneous occupation of inner and outer sites t o undergo their transitions, but they require the simultaneous existence of inner and outer sites, with the restrictions stated in the discussion of Eq. (31). If this were not so, the stationary distribution of the pump among its diff ercnt states, and, hence, the concentration of the n-th species [Eq. (21)] would depend on inner and outer cation concentration in such a way as not to allow separation of variables in the rate equation. The fact that the rate of transition of the n-th specks is proportional to the flux does not necessarily mean that this transition is physically responsible for ion translocation. Wit,h simultaneous models, it seems difficult to decide on kinetic grounds alone, which step of a pump cycle is associated with the movement of cations. I n this respect, simultaneous schemes for active transport differ from sequential modcls, in which the transitions producing cat'ion translocation must necessarily be those that transform inner into outer sitcs and vice versa. Expttrimental evidence seems to indicate that in agreement with the as-

60

P. J. GARRAHAN A N D

R. P. GARAY

sumption we made to devclop Eq. (31) , the properties of the inner sites of the Na pump remain constant in the states of the pump that predominate during a pump cycle. We have already mentioned the invariance of the apparent affinity of the inner sites of the Na pump revealed by the protectivc effect of Na on the DCCD inactivation of the pump (see Section 111, A,3 and Robinson, 1974a,b). Invariance in the properties of the inner sites is also suggested by the fact that the equilibrium constant for the binding of ATP to its catalytic site in the Na pump, is unaffected by Na (Hegyvary and Post, 1971). Further evidence in favor of the invariance of the properties of the inner sites of the pump comes from the comparison of the kinetics of the Na:K and the Na: Na exchanges catalyzed by the Na pump. During Na:Na exchange the rate of hydrolysis of ATP by the pump is very low, and presumably most of the pump is in a phosphorylated state (Garrahan and Glynn, 1967d). On the other hand, during Na: K exchange the rate of ATP hydrolysis is fast, and the distribution of the pump is likely to be heavily in favor of its dephosphorylated forms. In spite of this the apparent affinity for both Na and K of the inner sites of the Na pump is independent of the kind of exchange the pump is driving (see Fig. 2) (Garay and Garrahan, 1973; Garrahan and Garay, 1974). This strongly suggests that phosphorylation has no effect on the properties of the inner sites. Additional evidence in favor of the invariance in the affinity of the inner sites of the Na pump is provided by the fact that the shape of the Na efflux curves is not affected by changes in the intracellular levels of ATP or of Pi (Garay and Garrahan, 1975). A biochemical finding, which may at first seem to be in contradiction to the proposed constancy in the properties of the inner sites, is the finding by Skou (1974a,b) that, in the presence of enough K to saturat,e the external sites of the pump, the ratio of Na to K necessary to get half-maximal occupation by Na of the inner sites of the pump goes from 0.4: 1, in the absence of ATP, to 3 :1, in the presence of excess ATP. It is almost certain that this contradiction is only apparent and that it arises from the different conditions used during flux experiments, as compared to those employed by Skou in his experiments. In fact, the effect of ATP on the selectivity of the inner sites is 80% complete at 0.1 m M ATP (see Fig. 12 in Skou, 1974a). This concentration is well below the range of concentrations, 0.3-1.0 mM (Garay and Garrahan, 1975), used to demonstrate both the validity of separation of variables in the flux equations [Eq. (2)] and of the independence from the concentration of ATP of the properties of the inner sites of the Na pump. This consideration highlights the different but complementary roles that flux and biochemical studies play in the analysis of the mechanism of the

SEQUENTIAL AND SIMULTANEOUS MODELS FOR No AND

K

TRANSPORT

61

Na pump. In a biochrmical expcrimcnt , t he syst em may, in many casvs, be frozon in a particular stat(’, whosc propertics can t h m be studird. On the othrr hand, thv proprrtics measured in a flux c.xprrimcnt unavoidably rvflrct a statistical average over all the states thv pump has to traverse in ordcr to induce thc flux under study. Kinetic and biochemical data arc complcmentary in thc sens(b that kinctics can define thc relativc abundance of a statr, which has bcrn charactrrizrd in a biochrmical rxperimrnt during the actual working of the pump. I n this srnsc thc findings of kinetic studies may be reconciled with Skou’s findings by assuming that, in thc prcsrncc. of high ATP concentrations, thc ATP-frw states of the pump occupy a very small fraction of thc total duration of a pump cyclr. If this wrrc the casc thr cation affinities of the ATP-frrr states of thtt pump would not havc an appreciable weight in thr overall ratr (quation [see discussion about Eq. (31)i. d. Apparent Afinitg of the Set of Sites of Variable Affinity. Wc havc demonstrated in Eqs. (31) and (37) that scparation of variablrs in thr flux equation [Eq. ( a ) ] holds w e n if the propertics of oiic sct of sitrs change during t he various steps of a transport c y l e . Available exprrimrntal cvidenee strongly suggests that, in contrast with the invariance of the affinity of the internal sitrs, the affinity of the external sites of the pump deprnds on thc ligand state of thr pump. More specifically, it scems that, as a consequencr of phosphorylation, thrsc sites go from a statr of modrratc. affinity to I< to a statr of high affinity to I<.Wc havc already discussed (see Section III,A,3) thr studies with irrevrrsiblr inhibitors supporting this view. Indrpendent evidence for an effcct of phosphorylation on the affinity for I< of the external sites of the pump comrs from studies of the I<-activated phosphatase activity of the Na pump (for a revirw and references on this activity, see Glynn and Karlish, 197,5; Rega and Garrahan, 1976). Phosphatase activity requires thc occupation by I< or its congcncr of the external sites of the Na pump (Itega et nl., 1970). The concentration of I< necessary for half-maximal activation of thc phosphatase is reduced by about twenty-fold when ATP and Na are prcsent in the incubation media. This effcct is almost certainly due to the phosphorylation of the pump (Garrahan et al., 1970; Robinson, 1970; Koyal et al., 1971 ; Skou, 1 9 7 4 ~ ) . In view of these data, it is of interest to know what information can be obtained from the shape of thc flux vs. cation concentration curves, using cations that, like K, occupy sites whosr affinity changrs during the diffrrent stages of a transport cyclr. I n thcb absencc of extcrnal Na, thc I< influx curvc is reasonably wcll drscribed by a Michaelis-likc quation (Glynn, 1956; Garrahan and Glynn,

62

P. J. GARRAHAN AND R. P. GARAY

1967b; Sachs, 1967). We may, therefore, assume that the 'y(K) in Eq. (31) are of the form 'y(K) = (1

+ iK~/(K))

(38)

where iKK is an apparent dissociation constant for K from the i-th species of the pump. In the presence of nonlimiting concentrations of internal Na, Eq. (31) can now be written kEtota1

J =

(39)

In this case the half-maximal rate will be reached when the concentration of K is

i-1

= i-n

C Ki

i-1

since [see Eq. (22)] Ki

--

-

"Na,K,

i-n

CK; i-1

<ENa,K, i-1

It is clear that the weight the affinity of each of the states of the pump will have on the overall apparent affinity will depend on the relative abundance of each state. In this sense, experimental evidence is conclusive in indicating that the apparent affinity for K influx is much nearer to that corresponding to the high-affinity state than to that corresponding t o the low-affinity state of the pump (see Garay and Garrahan, 1975). Confrontation of this fact with Eqs. (40) and (41) strongly suggests that the high-affinity states of the pump occupy a much larger fraction of the total duration of a pump cycle than the low-affinity states of the Na pump. This again illustrates the already mentioned complementary nature of biochemical and flux studies. The conclusions we have reached in this section may, in fact, be generalized and allow us to state that, provided equilibrium holds, if the affinity of one of the stages of the pump is very different from the apparent affinity measured from the flux curves, the lifetime of this species will be very short.

63

SEQUENTIAL A N D SIMULTANEOUS MODELS FOR Na A N D K TRANSPORT

e. Occluded Forms of the Pump. In order to translocate a cation, the Na pump must first pick it up from thc solution bathing one surface of the membrane, then move it across the mcmbrane, and finally release it into the solution bathing the opposite surface of the membrane. It seems reasonable to think that, during a part of its journey across the membrane, the ion-pump complex will be unable to exchange with cations in the bathing solutions. Hence, on a time average, a fraction of the pumps may have its cation-binding sites in an occluded state. Since there seems to be no a priori reason for discarding the possibility that occluded conformations occupy a large fraction of the total duration of a pump cycle, it seems pertinent to analyze in what way the existrnce of a significant amount of occluded conformations will affect the overall kinetic behavior of the Na pump. To do this we will assume that the n-th stage of the pump [see Eq. (21)] passes through a state in which it is unable to exchange bound cations with the bathing solutions and that cation translocation is proportional to the concentration of this occluded statr, ic., ki

"ENa,K,

k-I

("ENa,K,),,~

ka +

(42)

In the steady state,

(nENaqli,)/(nENaqli,).,,I = ( k , + k-l)/lcl

=

Kt

(43)

If ("ENa,I(,) is expressed as in Eq. (31), tho flux is

The additive constant in the denominator of Eq. (43) makes it impossiblc t o express it as a function of inner cations times a function of outer cations. This means that, in thc presenco of a significant amount of occluded conformations, the apparent affinities for cations a t one of the surfaces of the membrane will depend on the cationic concentration a t the opposite surface of the cell membrane, even if the other criteria for separation of variables are fulfilled. Equation (44) will approach a form that fits with the demands of the experimental results only when K , tends to infinity. Therefore, in order to preserve separation of variables in the flux equation [Eq. (2)], occluded conformations should either not exist or, if they do, they must occupy a very small fraction of the total duration of a pump cycle. It is easy to show that the restrictions concerning occluded conformations are valid also if occlusion occurred a t a step different from the one we assumed in developing Eq. (44),or if inner and outer sites underwent occlusion at different stages of the transport cycle.

64

P. 1.

GARRAHAN A N D R. P. GARAY

Partial occlusion. The only strong biochemical evidence of occlusion of cations comes from the studies of Post et al. (1972). These authors showed that, at least at low ATP concentrations, the overall rate of turnover of the pump is limited by its rate of reconversion, after dephosphorylation, into an ATP-reactive form. The authors provided strong indirect evidence that this process is slow because K or its congeners remain bound to the pump in an occluded state after the release of phosphate. It is easy to show that occlusion of only one set of sites can be made to be consistent with the experimental kinetics of cation fluxes. For this purpose, let us assume that the transition of Eq. (42) implies only the occlusion of the external sites. Using a procedure similar to that employed to derive Eq. (44), it can be shown that in this case

where f'(Na) is the function governing thc saturation of the inner sites [see Eq. (26)] of the state having its outer sites in the occluded conformation andthe remaining symbols have the same meaning as in Eqs. (43) and (44). When f'(Na) = f ( N a ) ,

Clearly, the occlusion of one set of sites is compatible with the experimental demands provided that the properties of the other set of sites remains constant during occlusion. Partial occlusion and apparent afinity. In the absence of hcterotropic interactions in affinity, the fraction of sites in a system that are occupied f(x)]-', wheref(x) is a by the ligand x will always be expressible as [l continuous and monotonically decreasing function of x whose limits are infinity when x tends to zero and zero when x tends to infinity. Equation (46) can, therefore, be written

+

Now y (K) has the same meaning as f(x) just defined. The apparent affinity for K of the system described by Eq. (47) can be estimated from the concentration of K a t which the flux is half-maximal (Ko.6).In the absence of occlusion ( K , = ~3 ), KO.b will be the value of K for which y ( K ) = 1. In the presence of occlusion, on the other hand, Ko.6 will be that value of K for which y ( K )K,/ (1 K,) = 1. Since K t is al-

+

SEQUENTIAL A N D SIMULTANEOUS MODELS FOR N o A N D K TRANSPORT

65

ways positive, it is evident that IG.5 nonoccl

> IG.5 ocol

(48)

Thus, when equilibrium kinetics holds, the apparent affinity of the sites that undergo occlusion is higher than the real affinity of these sites. 4. STEADY-STATE TREATMENT OF THE KINETICS OF CATIONFLUXES

The most general way of dealing with the kinetic properties of the Na pump would be to see under which conditions steady-state kinctics yield equations that are compatible with the known biochcmical features of the Na pump as well as with the experimental properties of the cationic fluxes driven by this system. Active transport is a phenomenon ,involving not only the addition and release of differmt, ligands from multiple sites, but also transitions within the system itself. Therefore, a general steady-state treatment of this process would givc! immensely complicated equations of little practical value. No complete steady-state treatment of active transport has yet been developed, and it will not be attempted here mainly bccause, as we have already mentioned, it seems likcly that equilibrium kinetics is a valid tool in the quant,itativeanalysis of the rate equations for active transport. I n this section we will, therefore, analyze only some simplificd steadystate schemes and show that they impose restrictions on the possible modes of operation of the pump that, in general, confirm those that wc have developed from the equilibrium treatment. a. The Classical Sequential Scheme.. By using t'he steady-state approach, Hoffman and Tosteson (1971) devclopcd the rate equation for the circulating carrier hypothesis of Shaw (cited by Glynn, 1956). Assuming that the pump has a single cation-binding site and that the external Na and internal K concentrations are constant, t.hey derived the following equation for the rate of Na: I< exchange as a function of internal Na and external I< Concentrations:

Here A is a product, of rate constants, and B, C , D, and E are combinations of thc rate constants. Hoffman and Tosteson demonstrated that Eq. (49) is expressible as a product of a function of inner cations times a function of outer cations only if physically unlikely, fortuitous, relationships exist between rate constants arid the concentrations of external Na and internal I<. Moreover, even if these relationships held, the authors showed that separation of

66

P. J. GARRAHAN A N D R. P. G A M Y

variables would only be possible when the external Na concentration is not zero, which is not compatible with the experimental results since at least for the case of sheep red cells, independence between inner and outer sites holds even in the absence of external Na. The circulating carrier model of Shaw is kinetically indistinguishable from most sequential models. The latter differ from Shaw’s (see, for instance, Jardetsky, 1966; Caldwell, 1970) in the molecular process proposed as the mechanism of the change of orientation of the sites, so that the aforementioned conclusions seem to be of general validity. We have demonstrated before [see comments about Eqs. (16), (17), and (31)] that when equilibrium kinetics holds, sequential schemes are not compatible with the kinetic properties of cation fluxes. The steady-state treatment of Hoffman and Tosteson (1971) strongly suggests that this is also true even when the restrictions concerning equilibrium binding of cations are lifted. In view of this, it seems safe to conclude that classic sequential models have to be abandoned as a plausible explanation of the mechanism of active cation transport. b. The TheoreZZ-Chance Kinetics. Dr. R. L. Post (personal communication) suggested a kinetic scheme for active transport. His modcl is neither sequential nor simultaneous and may yield, if certain assumptions are made, rate equations in which the terms containing inner and outer cations are separated into a product. The scheme involves two states of the pump: (i) a state having sites facing the outer surface of the membrane, which, during Na: K exchange, act as leaving sites for internal Na and as entering sites for external K; and (ii) an inward-facing state of the pump in which coexist separate sites for Na and K. The release of K into the intracellular medium necessarily requires the previous occupation by Na of its intracellular site, with the formation of a ternary complex, pump- (Na, K) , a fact also implying that Na can only get on or off the inward-facing state of the pump if K is bound to it. The ternary complex has, therefore, an obligatory participation in the functioning of the pump. This mechanism of transport has been called “overlapping transport” by Post and, for the case of single cation-binding sites, may be schematized as follows:

If it is assumed that (i) the rate constants for the dissociation of the ternary

SEQUENTIAL A N D SIMULTANEOUS MODELS FOR N o A N D K TRANSPORT

67

complcx are cqual and much largcr than the rcst, i.e., k7 = k9 >>> k l , . . . , k6, (ii) k2 = ks = k8, and (G) the reaction is irrrversiblr, thcn thc following equation will describe the rate of Na:K exchange its n function of inner and outer Na and I< concentrations: C’ J =

(501

Equation (50) fits with thc c.xpt.rimcnta1 rcsults of thc kinrtics of Na: K exchange, a t lcast to the cxtcnt that thc tcrms containing inner and outer cations arc separatcd into a product and that there arc competitive cffccts between Na and I< a t each surface of the cell mcmbrane. Howcver, before accepting this ltinetic scheme as a plausible basis for a n active transport mechanism, scvcral additional propcrtics of this mcchanism, which arc rather difficult to fit with the experimrntal behavior of thc Na pump, have to bc taken into account. 1. I n Eq. (50) thc apparrnt affinitieb for rxtcrnal Na and Ii are equal to the real affinitirs. On the other hand, the apparcnt affinity for internal Na (k8/k3) must be very diffrrent from thr real affinity for internal Na (ke/ko), since to get Eq. (50) it is necessary that lcs >> k,. This secms to contradict the cxpcrimental evidence rather strongly suggesting that apparent and real affinities of the inner sites arc not very different (see Section II,D). 2. In the scheme we are considrring, inner sitrs have only a very transient cxistcncc. This also seems to contradict the biochemical evidence showing that innrr sites are detectablr during the whole duration of a pump cyclc (see Section III,A,3). 3. The obligatory nature of the trrnary complex E-(Na, I<) makcs all phrnomena mediatcd by the pump dcpcndcnt on the presence of both Na and K. More specifically, in this scheme Na:Na exchange will have a n absolutc requirement for internal I( and K : I< exchange a n absolute requircment for internal Na. The Na:Na cxchange is, in fact, activated by internal K (Garay and Garrahan, 1973), a phenomenon that may be related to thc activation by K of the ATP:ADP exchange catalyzed by the pump (Banjerec and Wong, 1972a,b). Howcver, in contrast with the predictions of the model we arc discussing, the expcrimental requirements for internal I< of Na:Na cxchange do not seem to be absolute and are exerted with a n apparent affinity that is considerably lowcr than the apparent affinity for the competitive effect of I< on Na efflux. An evcn morc srrious contradiction between this scheme and thc experimental results is that it predicts a strict depcndence on internal Na for K: K exchange, since experimental evidence

60

P. J. GARRAHAN AND R. P. GARAY

clearly indicates that internal Na is, in fact, a powerful inhibitor of K:K exchange (Simons, 1974). It is also difficult to explain with the scheme we are analyzing the biochemical events that require only the occupation by Na of the inner sites of the Na pump, as, for instance, the Na-dependent phosphorylation of the pump by ATP. c. Nonequilibrium Simultaneous Models. Suppose that cation transport required the simultaneous occupation of inner and outer cation-binding sites but that the transition induced by the occupation is fast enough to impede the equilibration of the cation-pump complexes with free cations. In this case, since the transition that impedes equilibration will only take place when trans sites are occupied by cations yielding reactive complexes, the occupation by cations of sites at one of the surfaces of the cell membrane will depend on the state of occupation of sites at the opposite surface of the cell membrane. Therefore, steady-state simultaneous models will not give, even in the absence of interactions in affinity, rate equations in which the terms containing inner and outer cations are separable into a product (for a quantitative treatment of this case, see Cleland, 1963). Thus, from our brief analysis of some simplified steady-state treatments of the kinetics of cation transport by the Na pump, it would seem that this approach provides additional and, perhaps, conclusive evidence against the classic sequential models. What does not yet seem to be certain is whether steady-state models provide a reasonable alternative to the simultaneous equilibrium models, particularly if the kinetic evidence is taken together with the biochemical data on the coexistence of inner and outer sites. Evidence favoring the idea that cation-pump complexes are in equilibrium with free cations has also to be considered.

IV.

INTERMEDIATE STAGES IN HYDROLYSIS OF ATP BY THE Na PUMP

One of the main problems yet to be solved to understand better the molecular mechanism of the Na pump is the relation between the intermediate steps of ATP hydrolysis and the various stages of an active transport cycle. It is beyond the scope of this work to present either a comprehensive survey of the copious literature on the subject (for an excellent review, see Glynn and Karlish, 1975) or to argue for or against the main two alternative hypotheses that have been proposed to account for the intermediate stages of ATP hydrolysis by the Na pump (Fahn et al., 1966a,b; Siege1 and Albers, 1967; Post et al., 1969; Kanazawa et al., 1970; Fukushima

SEQUENTIAL AND SIMULTANEOUS MODELS FOR N o AND K TRANSPORT

69

and Tonomura, 1973; Post el al., 1973; Tonomura and Fukushima, 1974). Rather, we will try to show in this section that what is known on this subject may be interpreted in such a way so as to be compatible with the properties we have attributed up to this point to the mechanism of the Na pump. This task is not made easier by the fact that, with very few exceptions (see, for instance, Skou, 1975), experiments on the intermediate steps of the hydrolysis of ATP by the Na pump have been performed within the conceptual framework of sequential schemes for active transport. Sequential schemes have been used not only to interpret results of biochomical experiments but also as working hypotheses to design experiments. This way of thinking obviously leads to “sequential experiments,” that is, to experiments in which activating cations are added one after the other and which, therefore, unavoidably give “sequential” r e s u lk A. Phosphorylation and Dephorphorylation of the Pump

It is now accepted that in thc presence of Na ions, the Na pump is able to catalyze the transfer of the terminal phosphate of ATP to an aspartyl (Post and Kume, 1973; Nishigaki et al., 1974) residue of a membrane protein with the formation of an acylphosphate bond. The apparent affinity of this effect of Na strongly suggests that it is the consequence of thc combination of Na with the inner sites of the Na pump (Post et al., 1965; Kanazawa et al., 1970; Fahn et al., 1968). In the prcsence of Na alone the phosphate in the protein is slowly transferred to water. Potassium (Post et al., 1965; Kanazawa et al., 1970) or its congeners (Post et al., 1972) markedly increase the hydrolysis of the acylphosphate bond. As a consequence, the steady-state level of thc phosphoprotcin in the presence of concentrations of Na and K optimal for Na: K exchange is very low. There is rather strong experimental evidence suggesting that Na-dependent phosphorylation by ATP and I<-dependent hydrolysis of the phosphorylated intermediate take place in two different conformational states of the pump, which probably represent two successive steps of the various stages of a transport cycle (see Post et al., 1973). The two conformational states of the phosphorylated intermediate seem to differ in the value of the free energy of hydrolysis of the acylphosphate bond, which, in a state that transfers phosphate to water, is sufficiently low to allow the incorporation of phosphate from inorganic phosphate into the pump protein (Post et al., l974b) and a rapid I<-stimulated exchange of ‘*O between water and Pi (Dahms and Boyer, 1973). It is obviously tempting to suppose that tho occurrence of a sequence of Na-dependent phosphorylation, followed by a I<-stimulated dephosphorylation of the pump is the expression of the sequential binding and release

70

P. J. GARRAHAN AND R. P. GARAY

of these cations from pump sites and that the chemical changes catalyzed by each kind of cation somehow results in the sequential transfer of cations of that kind across the membrane (see Post et al., 1969, 1973). This interpretation implicitly makes use of the different cationic requirements of phosphorylation and dephosphorylation as evidence that sites for Na and sites for K exist in different states of the Na pump (see Post et al., 1969). Even if we disregard the fact that this hypothesis runs into serious contradictions with the kinetic and biochemical facts we have discussed up to now, it is clear that the use of phosphorylation and dephosphorylation experiments for supporting sequential schemes for cation binding and release from the pump has the intrinsic weakness of confusing the affinity of a cation-binding site with the reactivity of the resulting cation-site complex (see Section II,B) . On their face value, phosphorylation experiments only indicate that it suffices for Na to occupy the inner sites of the pump to confer on the pump the ability to catalyzc a transphosphorylation reaction between ATP and an aspartyl residue of a pump protein; on the other hand, it suffices for K or its congeners to occupy the outer sites of the pump to make the phosphoprotein much more reactive toward water than when these sites are either empty or occupied by Na. We have already pointed out (Section II,B) that because a cation is not required in a given elementary step of transport does not imply that sites for this cation do not exist in the chemical entities involved in this step. Moreover we have shown [see discussion about Eq. (37)] that simultaneous systems may undergo a sequence of transitions, some of which may require cations only a t one of the surfaces of the cell membrane and still yield kinetic equations that are compatible with the experimental properties of the pump. This may occur provided that (i) inner and outer sites coexist and (ii) transport is proportional to a state of the pump having both its inner and outer sites occupied by the relevant cations. It would seem, therefore, that there is no serious contradiction between phosphorylation experiments and the simultaneous models for active transport. The simplest, but not necessarily the correct, way of interpreting these experiments from the point of view of simultaneous models would be to assume that the states, p u m p (Nain) , p u m p (Nain, NaOut), p u m p (Nai,, KO“,),are able to become phosphorylated by ATP but that the reactivity toward water of the phosphorylated state pump(Nain, KOut)is much larger than that of the other states. If we look at things in this way, it is clear that the usual statement that Na is needed for phosphorylation and K is required for dephosphorylationwhich has clear “sequential” connotation-can be replaced by the assertion that, although the presence of Na suffices for phosphorylation t o proceed, only those pumps occupied by Na and K are able t o yield a phosphorylated

SEQUENTIAL A N D SIMULTANEOUS MODELS FOR Na A N D K TRANSPORT

71

intermediate whose reactivity toward water is high enough to allow rapid turnover. This statement is in agrcemcnt not only with thc kinetics of phosphorylation and dephosphorylation but also with the fact that sites wit,h high affinity for K cocxist with sites with high affinity for Na in the phosphorylated pump (see Section III,A,3). The scqucntial requirements for Na and K would be, thareforc, a consequence more of the sequential nature of the phosphorylatiori and dephosphorylation reactions than of the sequential appearance of sites for Na and K. The distinction between phosphorylation in tho presence of Na and phosphorylation in the presence of Na and K has been taken a step further by Skou (1975) who has raised the question as to whether or not the phosphoprotein formed in the presence of Na is a part of the reaction when both Na and I< are present. Skou suggested that the phosphate bond formed under these conditions may be very transient and have properties intermediate between an electrostatic and a covalent bond (Skou, 1975). Quite different experimental evidence for the participation of I< and, hence, for thc existence of sites for I< during or even before phosphorylation has been provided by the experiments of Tonomura and his co-workers (Kanasawa et al., 1970; Fukushima and Tonomura, 1973). These workers showed that (i) when I< is prescmt, the ratc constant for the hydrolysis of phosphate from the phosphoprotein, measured from the ratio of ATPase activity to the steady-state concentration of phosphoprotcin, exceeds significantly the value of the rate constant describing the fall of the concentration of phosphoprotein when rephosphorylation is stopped; and (ii) in the presence of I< the amount of Pi released from the phosphoprotein is larger than the amount of phosphoprotein lost when phosphorylation is stopped. These results can be explained by supposing that t,here is an equilibrium between a form of the pump with tightly bound ATP and the phosphoprotein (EP) and that this equilibrium, which is in favor of EP in the absence of I<, is displaced to the left by K. This interpretation gains further support by the fact that dephosphorylation in the presence of K results in a rapid decay of EP, followed by a slower exponential loss. The rapid initial fall of EP is associated with thc synthesis of an equivalent amount of ATP (Fukushima and Tonomura, 1973). If K displaces the equilibrium between EATP and EP toward EATP, either EATP or a preceding state of the transport cycle must have sites for Ii whose affinity is even greater than those of EP. These sites, which, because of their high affinity for K, are likely to be extracellular ones, would therefore exist in a state of the pump that, according to sequential interpretations of phosphorylation kinetics (see, for instance, Post, et al., 1969), would correspond to a state of the pump in which sites face the intracellular medium.

72

P. J. GARRAHAN AND R. P. GARAY

B. Apparent Uncoupling between Parlial Reactions and Cationic Fluxes

+

The cationic requirements of the (Na K)-dependent hydrolysis of ATP by the Na pump are, under all the conditions tested, identical with the cationic requirements of the Na: K exchange (see Glynn, 1968). The identity of cationic requirements is almost certainly the expression of the fact that (Na K)-ATPase activity is always coupled to cation movements. To the author’s knowledge, no instance of ATPase activity uncoupled to cation movements has been reported in preparations in which ATP hydrolysis and cation movements are measurable; this is true even for highly purified ATPase preparations (Hokin, 1974; Hilden and Hokin, 1975; Goldin, cited by Kyte, 1974). In contrast to this strict coupling, the cationic requirements of the partial K)-ATPase may be different from those of the reactions of the (Na fluxes that these partial reactions may eventually drive. This would seem to indicate that a givcn partial reaction will be coupled to a cationic flux only if the cationic requirements for both the reaction and the flux are simultaneously fulfilled. If this is not so, the partial reaction will proceed without driving, that is, uncoupled, from the flux. This assertion, which may be relevant in the interpretation of the intermediate stages of the hydrolysis of ATP from the point of view of simultaneous models for active transport, can be illustrated comparing the reK)quirements of the Na-dependent partial reactions of the (Na ATPase with those of the Na-dependent fluxes driven by the Na pump.

+

+

+

1. TRANSPHOSPHORYLATION AND Na :Na EXCHANGE

We have already mentioned that, in the presence of Na, the Na pump catalyzes the transference of the terminal phosphate from ATP t o a membrane protein. After phosphorylation it seems that the phosphoprotein undergoes a transition from a state in which it is able to donate its phosphate moiety to ADP (EIP) to a state in which it is able to donate its phosphate to water (E2P).This transition can be inhibited by pretreating the systcm with NEM (Fahn et al., 1966a), by reducing the concentration of Mg (Fahn et al., 1966b), by an equimolar mixture of BAL and arsenite (Siegel and Albers, 1967), or by olygomycin (Fahn et al., 1966b). The concentration of Na necessary for half-maximal initial rate of phosphorylation (Kanazawa et al., 1970), half-maximal steady-state level of phosphorylation (Post et al., 1965; Fahn et al., 1968), or half-maximal rate of ADP-ATP exchange (Fahn et al., 1966b; Siegel and Albers, 1967) is low (less than 8 mM) , and none of these phenomena show inhibition by excess Na (Fahn et al., 1968; Siegel and Albers, 1967). These results strongly

SEQUENTIAL AND SIMULTANEOUS MODELS FOR Na AND K TRANSPORT

73

suggest that Na occupying only the internal sites of the pump enables the pump to catalyze a t an optimal rat(. all the Na-dependent transphosphorylation reactions of the (Na I<)-ATPase. In the presence of Na alone, the Na pump is able to drive a 1 :1 exchange of Na across the cell membrane. This exchange requires ATP but is not associated with an appreciable hydrolysis of the nucleotidc. (see Section I1,E) (Garrahan and Glynn, 19G7d). The concentration of ATP nwded for optimal Na:Na exchange is considerably lower than that needed for optimal Na:K exchange (Glynn and Hoffman, 1971), a fact that agrees well with the much lower value of the K , for ATI’ for phosphorylation (Post et al., 1965; Iianazawa et al., 1970) as compawd to the K , for ATP K-ATPast. (Robinson, 1967; Neufcld and Levy, 1969). of the (Na Furthermore, Na: Na exchange is blocked by oligomycin (Garrahan and Glynn, 1967c) which blocks the EJ’ to E2Ptransition. In view of this it is difficult to avoid the conclusion that the Na-dependent phosphorylation of the pump and associated phrnomma are the biochemical events underlying Na: Na exchange. Moreover, the fact that the rate of Na: Na cxchange is a linearly increasing function of the internal concentration of ADP (Glynn and Hoffman, 1971) strongly suggests that Na:Na exchange rcquires not only the forward running of thf>phosphorylation reaction but also the reversal of this reaction, with the rephosphorylation of ADP by the phosphorylated pump protein. If Na :Na exchange and the Na-drpendent transphosphorylation rcactions of the Na pump showed the same dcgrcc of tight linkage as that shown by Na:I< exchange to the overall (Na I<)-dependent ATPase activity, one would expect that the requircmcnts for Na for transphosphorylation and for Na:Na exchange to be the same. Experimental evidence, however, does not uphold this view. In fact, the rate of Na:Na exchange can be made proportional to thc number of pump units having both their inner and outer sites simultaneously occupird by Na (Garay and Garrahan, 1973). The affinity of the external sites for Na is low, as shown by the fact that Na:Na exchange is half-maximal a t about 120 mM external Na (Garay and Garrahan, 1973). Hence, if the Na-dependent partial reactions of the pump were strictly linked to Na:Na exchange, their rate would be limited by the state of occupation of the external sites of the pump. In contrast with this, we have shown that for thesc reactions to proceed it suffices to occupy the inner sites of the pump. These rcsults can be interpreted by supposing that, whereas Na-depcndent transphosphorylation will take place in all pump units that have their inner sites occupied by Na, they will becomcl coupled to Na:Na exchangc only in those pump units in which outer sites are also filled with Na ; that is, external Na is not necessary for the catalysis of the transphosphoryla-

+

+

+

74

P. J. GARRAHAN AND R. P. GARAY

tion reactions but is required to couple transphosphorylation to cation exchange. A clue to the possible mechanism of the coupling effect of external Na is suggested by the fact that, although high-Na concentrations have no effect on the main features of the transphosphorylation reactions, they appear to shift the equilibrium between the two states of the phosphorylated pump toward the ADP-reactive state. This assertion is based on the following evidence. 1. In rat brain preparations (but not in guinea pig kidney or in “electrophorus” electric organ) , when the concentration of Na is increased from 12 to 120 mM, the fraction of the phosphoprotein that is sensitive to ADP increases from about 10 to almost 100% of the total (Tobin et al., 1973). 2. Inorganic phosphate inhibits the phosphorylation of the (Na I<)ATPase by ATP-32Pbecause inorganic phosphate is itself able to phosphorylate the protein. The inhibitory effect of inorganic phosphate is halved when the concentration of Na is raised from 16 to 160 mM. This effect has been interpreted as caused by the displacement by Na of the phosphorylated intermediate away from a water-reactive state and toward an ADP-reactive state (Post et al., 1974b). 3. Phosphoenzyme formed from inorganic phosphate can donate its phosphate to ADP with net synthesis of ATP, if Na in high concentrations (up to 1.5 M ) is added after phosphorylation (Post et al., 1974; Taniguchi and Post, 1975). In sequential schemes, these effects of Na are explained on the basis of E2P representing a state of the pump possessing outward-facing sites, which, for this reason, have a much lower affinity for Na than E1P or a state preceding EIP whose sites face the inner surface of the cell membrane. If this were true, then, as Na is increased, the equilibrium would be displaced toward the ADP-reactive state EIP (Post et al., 1974, 1975; Taniguchi and Post, 1975). A hint of a difference in affinity for Na between E1P and E2P is provided by the fact that, when the transition between both states is prevented by BALarsenite, the concentration of Na for half-maximal steady-state phosphorylation drops from 4 to 0.8 mM (Siege1 and Albers, 1967). The interpretation of this effect as a change in affinity is risky. It should take arsenite on the into account the possibility of a direct effect of BAL sites for Na and the fact that, in general, the effect of a ligand on a steadystate level depends not only on the affinity but also on the rate constants of the transitions that intervene in the generation of the steady state. Even if the aforementioned half-maximal values were taken as indicatory of the affinities of all the sites for Na of EIP and E2P,it is clear that they are

+

+

SEQUENTIAL A N D SIMULTANEOUS MODELS FOR N o A N D K TRANSPORT

75

considrrably lower than the concentration of Na necessary for half-maximal occupation of the external sites of the pump (120 mM). Moreover, the effects of Na mentioned in preceding paragraphs 1 arid 2 are exerted in a 12-160 m M Na range, and tJhe effects mentioned in paragraph 3 a t an even higher concentration of Na. In this range of Na concentrations, sites having values of 0.8 to 4 m M will be almost fully occupied and, hence, only negligible displacement of equilibria can be expected by changes in the concentration of Na in thc rangrs mentioned in paragraphs 1-3. On the other hand, the sequential interpretation of the effects of Na on the distribution of EP between its two possible states is incompatible both with the kinetics of Na:Na exchange [which follows an equation such as Eq. (2)] and with the fact that studies with inhibitors indicate that, when most of the pump is in its E2Pform, sites for Na whose affinity is high and not very different from that of those present in the dephosphorylated states of the pump coexist with high-affinity sites for I< (see Section 111, A,3) * A plausible interpretation of the eff ects of high-Na concentrations, which may also account for the requirements for external Na for the coupling between transphosphorylation reactions and Na: Na exchange, is to suppose that internal sites do not significantly change in affinity in going from Ell' to E2Pbut that the affinity for Na of the external sites, whose oecupation is not necessary to catalyze transphosphorylation, is higher in the EIP than in the E2Pstate. This assumption would explain the apparent ability of high-Na concentrations to drive the phosphoenzyme toward an ADPreactive state, without running into contradictions with other experimental findings, since it preserves the requirement for both the distinct existence of inner and outer sites and the constancy of the affinity a t one of the surfaces of the cell membrane. Both conditions are necessary t o account for the kinetics of Na: Na exchange. The fact that thc apparent affinity for external Na during Na:Na exchange is higher than the apparent affinity for the inhibitory effect of external Na on the influx of K (Garay and Garrahan, unpublished results) is consistent with the idea that the affinity for external Na of E1P is higher than that of E2P. 2. Na-DEPENDENT ATPASEAND UNCOUPLED Na EFFLUX We have already mentioned that when red cells are suspended in solutions lacking both Na and K, an ouabain-sensitive efffux of Na, not associated with the entry of Na or K can be detccted (see Section 11,E). This flux is coupled to the hydrolysis of ATP (Glynn and Karlish, 1975), suggesting that it may be related to the ATPase activity detected in brokcn

76

P. J. GARRAHAN A N D R.

P. GARAY

cell preparations when Na is present without K (Czerwinski et al., 1967; Blostein, 1968; Neufeld and Levy, 1969; Post et al., 1972). This activity presumably is the expression of the slow hydrolysis of the phosphorylated intermediate when K is absent. However, the cationic requirements of the uncoupled Na efflux and of the Na-ATPase differ in a way that again suggests that coupling between these two phenomena only takes place in those pump units in which there is a definite state of occupation of the external sites. In fact, the uncoupled Na efflux is inhibited more or less completely by external Na concentrations as low as 5 mM (Garrahan and Glynn, 1967b), yet Na-ATPase is observed with broken cell preparations when the concentration of Na must be high a t both faces of the cell membrane. This result may be taken as indicating that, as we have already suggested in the case of the transphosphorylation reactions, the coupling beK)-ATPase to Na tween Na-dependent partial reactions of the (Na fluxes is governed by the state of occupation of the external sites of the pump. In the case we are analyzing, when the sites are empty the spontaneous dephosphorylation of the pump gives rise to the uncoupled Na efflux. There is no reason to think that occupation of external sites will inhibit dephosphorylation and the resulting Na-ATPase activity. What seems to happen is that, when external sites are filled, Na-ATPase is no longer able to drive the uncoupled Na efflux; under these conditions, the efflux becomes an Na: Na exchange that seems to be associated not with the hydrolysis of the phosphoenzyme but with the reversible transphosphorylation between the pump and ADP.

+

C. Meaning of Apparent Uncoupling between Partial Reactions and Cationic Fluxes

The usual procedure for dealing with the connection between partial K)-ATPase and cationic fluxes is to order the reactions of the (Na partial reactions in an unbranched sequence and then to relate each step of the sequence to cation transport on the basis of the cationic requirements of each partial reaction. We have tried to show in the preceding paragraphs that this procedure is not always acceptable since, in some cases, cationic requirements, in addition to those necessary for the catalysis of the partial reaction, are necessary for the biochemical event to be coupled to observable transport phenomena. In the two cases we have analyzed, coupling between the reaction and cation movements only takes place when sites at the opposite surface of the cell membrane are either empty (uncoupled Na efflux) or occupied by Na (Na:Na exchange), whereas

+

SEQUENTIAL A N D SIMULTANEOUS MODELS FOR Na A N D K TRANSPORT

77

catalysis requires only t'he occupation of the inner sites. We think that this fact may prove to be of general relevance in the interpretation of the linkage between cation movements and biochemical transit,ions. Some apparent discrepancies between the sequence of reactions describing the biochemical behavior of the Na pump and the requirement of simultaneous existence and occupation of inner and outer cation-binding sites, which seems to be demanded by the kinetics of cation movements, may also be explained by this approach. V.

MECHANISM OF ION TRANSPORT

We have already mentioned in the Introduction that much more detailed knowledge is still required before any molecular model for activr transport can represent something more concrete than a simple working hypothesis. The kinetic properties of the Na pump allow us, however, to place a set of rather stringent restrictions on the possible modes of operation of the Na pump. These restrictions have been analyzed in dctail in the preceding sections, and may be summarized as follows: 1. Inner and outer cation-binding sites coexist, and their affinities are independent. 2. At least one of the steps of a transport cycle requires the pump to have its inner and outer sites adequately occupied by cations. 3. The apparent affinity of at least one set of cation-binding sites probably remains invariant during a transport cyclr. 4. Occluded states of the pump either do riot exist or represent a very transient stage of the pump. I n this section we further circumscribe the possible modes of behavior of the pump, combining the preceding restrictions with the available experimental evidence on the mechanisms of the intwaction between cation- and metabolite-binding sites, and with present knowledge of the structure and of the degrees of freedom of membrane-bound proteins. A. Na Pump as a V System

A question that is very relevant to understanding the mechanism of active transport and which can be, a t least tentatively, answered by the present experimental evidence concerns which kind of interactions predominate among sites (see Section II,C,2) in governing the operation of the Na pump. Available experimental evidencc rather strongly suggests that interac-

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P. J.

GARRAHAN A N D R.

P. GARAY

tions among rate constants predominate over interactions among affinities and, hence, that the Na pump is mainly a V system. 1. INTERACTIONS AMONG CATION-BINDING SITES

We have already shown (see Section III,B,3) that, provided equilibrium kinetics holds, the tight linkage between intra- and extracellular cationic requirements, exhibited by all the modes of behavior of the Na pump, is fully and unambiguously accounted for by (a) assigning to the rate constants of the transitions induced by the binding of cations a t one of the surfaces of the cell membrane values that are strictly dependent on the state of occupation of sites a t the opposite surface of the membrane and by (b) keeping the affinity of a given set of sites independent of the state of occupation of the other set of sites. It would seem, therefore, that, a t least with regard to the interactions between inner and outer sites, the Na pump behaves as a pure V system. The fact that inner and outer sites only show interactions among rate constants raises the question on whether a similar kind of interaction also governs the linkage of cation-binding sites a t one of the surfaces of the membrane. This possibility has been submitted to detailed experimental testing for the case of the inner sites of the Na pump of red cells (Garay and Garrahan, 1973). At high internal K concentration, the efflux of Na from red cells increases with internal Na following an S-shaped curve. As internal K is reduced, both the S-shaped region and the value of internal Na for which Na efflux is half-maximal are shifted progressively toward zero. The effects of internal Na and K on the shape of the Na efflux curve can be quantitatively accounted for if it is assumed that only those pump units having three sites occupied by internal Na are able to induce translocation and that these sites are identical and noninteracting so far as their affinities are concerned. The interactions between the inner sites of the Na pump can, therefore, be regarded as interactions solely between rate constants, given that the rate constant for the transition of a Na-site complex will be zero when the remaining sites are either empty or occupied by K and that it will be different from zero when the sites are occupied by Na. In this respect, therefore, the Na pump also seems to behave as a V system. Although the outer sites have not been studied in such detail as the inner ones, it seems that most but perhaps not all (see Section IV,B,l and Cavieres and Ellory, 1975) the effects of extracellular cations on fluxes can also be adjusted by schemes that postulate interactions between rate constants and no interactions among the affinities of the outward-facing sites of the Na pump (Sachs and Welt, 1967; Sachs, 1967; Garay and Garrahan, 1973).

SEQUENTIAL A N D SIMULTANEOUS MODELS FOR Na A N D K TRANSPORT

79

It may be worth stressing at this point that the definition of interactions on the basis of cation-activation curves has considerably less weight than that based on the studies of the effects of trans cations on the shape of the flux curves. We have demonstrated in Section III,B,3 that, provided equilibrium holds, the V-like behavior of the pump necessarily follows from the shape of the overall rate equation [Eq. (2)]. On the other hand, even when equilibrium holds, the validity of the preceding interpretation for the mechanism of activation by Na of the efflux of Na rests solely on the fact that it yields equations that fit the experimental results and provides a simple explanation for the fact that three Na ions are transported per pump cycle by the Na pump in red cells (Glynn, 1962; Whittam and Ager, 1965; Garrahan and Glynn, 1967d; Sen and Post, 1964). The only way of demonstrating unambiguously which kind of interactions are operative in a cation-activation curve is to compare this curve with the curve relating cation binding to cation concentration. We have already mentioned that no data of this kind are available for the Na pump but they do exist for the case of the Ca pump of sarcoplasmic reticulum. Binding of Ca to this system fits linear Scatchard plots, indicating that thew is a single class of noninteracting Ca-binding sites. The number of these sites is about twice the number of ATP-binding sites, strongly suggesting that each Ca pump possesses two Ca-binding sites (Meissner, 1973). The shape of the Ca-binding curve strongly contrasts with the curve of the initial rate of Ca-dependent phosphorylation of the Ca pump by ATP, whose sigmoid shape can be accounted for on the assumption that only those pump units having their two sites occupied by Ca are reactive (Kanazawa et d.,1971). This supposition agrws well with the finding that two Ca ions are transported per molecule of ATP that is split by the Ca pump (see Hassclbach and Makinose, 1972). In the case of the Ca pump, therefore, data on cation activation and cation binding are conclusive in the sense that intrractions among Cabinding sites are interactions among rate constants and not interactions in affinity. There are striking analogies betwccn the interactions demonstrated in the Ca pump and those we havr proposed to account for the shape of the Na activation curve of the Na pump. These analogirs may, perhaps, be used as evidence for a mechanism in common, in view of the alrrady mentioned structural, biochemical, and perhaps evolutionary similarities between the Ca and the Na pump. 2.

INTERACTION BETWEEN

CATION- A N D hfETABOLITE-BINDING SITES

The Na pump behaves as a V system with regard to the interactions among sites that are responsible for the Na-dependent phosphoryla-

80

P. J. GARRAHAN A N D R. P. GARAY

tion of the pump by ATP. Kanazawa el al. (1970) have shown that the initial rate of this phenomenon varies with the concentration of ATP following a Michaelis-like curve. Sodium ions modify this curve acting on the VmaX,which tends to zero as Na tends to zero, but has no effect on the K , for ATP. The absolute dependence on Na for the phosphorylation of the Na pump by ATP can, therefore, be fully explained by assuming that only those pump units whose sites arc occupied by Na, can catalyze the transphosphorylation reaction. It is suggestive in this respect, that a similar lack of interaction among affinities has been demonstrated for the Cadependent phosphorylation of the Ca pump of sarcoplasmic reticulum (Kanazawa el al., 1971). The interactions between metabolite- and cation-binding sites have also been analyzed by studying the effects of changes in the intracellular level of ATP and inorganic phosphate on the kinetics of Na: K exchange in red cells (Garay and Garrahan, 1975). The results of these studies may be summarized as follows: 1. A reduction from 1.0 to 0.3 mM in the intracellular level of ATP halvcs the rate of Na :K exchange but alters only by a constant factor the curves relating Na efflux to internal Na concentration and K influx to external K concentration (Fig. 4 ) . 2. An increase in the intracellular concentration of inorganic phosphate reduces the rate of Na:K exchange, but, in this case too, the Na efflux and K influx curves are altered only by a constant factor (Fig. 4 ) . 3. The effects of ATP and Pi are mutually independent in the sense that the fractional inhibition of Na:K exchange by Pi is independent of the concentration of ATP and vice versa, These results imply that, within the concentration range tested, the ovcrall rate equation for Na: li exchange as a function of internal cations, ATP and Pi, is expressible (see Section 111,B12)as a product of functions each of them containing only one ligand, i.e., J

= Jmax/W(Nain,Kin)X(Koutl N

a d Y(ATP)Z(Pi)

(51)

where W ,X, and Y go from 0 to 1, and Z goes from 1 to m as the respective variables go from 0 to 00. A t its face value, Eq. (51) would seem to mean that the rate of Na:K exchange is proportional to a state of the pump having its inner sites occupied by Na, its outer sites occupied by K, an ATP-binding site occupied by ATP, and a Pi-binding site unoccupied by Pi. If this were the case, Eq. (51) would describe a typical V system, since none of the ligands affect the affinity of the pump for the other ligands. However, a given state of occupation of the pump sites by each ligand is necessary to give to the resulting complex adequate reactivity to induce cation translocation.

SEQUENTIAL A N D SIMULTANEOUS MODELS FOR N o A N D

Externol K concentration (mM)

K

TRANSPORT

81

Ouabain-sensitive K-influx into low ATP (a) or into high P i ( d cells (mmole/liter cells hr)

FIG.4. Effect of external K concentration on the ouabain-sensitive K influx into control cells (0)into cells whose ATP content has been reduced from 1.0 to 0.3 mM ( 0 )by incubation in glucose-free media, and into cells whose concentration of Pi had been increased from 1 to 25 mM (A)by the citrate procedure (Glynn e t al., 1970). The cells were suspended in Nafree isotonic choline media. The plot at the right shows that changes in the concentration of ATP and Pi alter the value of the flux at nonlimiting external K concentration but have no effect on the number, affinity, or interactions of the outer sites of the Na pump [see Eqs. (2) to (4) and (51)) (Redrawn from Garay and Garrahan, 1975, by permission of the publisher.)

That the rate of Na: K exchange, as is assumed by Eq. (51), is proportional to the concentration of a dephosphorylated state of the pump agrees well with the biochemical evidence suggesting that a step subsequent to dephosphorylation is rate-limiting in the rate of hydrolysis of ATP by the Na pump [Post et d.,1972; see also Glynn and Karlish's (1975) comments on Mardh and Zetterqvist, 19721, As already mentioned, the lack of effect of ATP and of Pa on the properties of the inner sites of the pump is consistent with the idea that the properties of these sites remain invariant during a transport cycle [see Eq. (31)]. The inhibitory effect of P, is almost certainly due to the reduction in the net rate of dephosphorylation of the pump. I n the formalism we used to develop Eq. (31), this would mean that Paalters the value of some of the K i [see Eqs. (22) and (24)] in such a way as to decrease the concentration of that state of the pump whose transition is ratc-limiting in the overall turnover of the system. I n Section III,B,3 [see comments about Eq. (41)], we concluded, from the comparison of the affinity for I< obtained from the K influx curve with the affinity for I< of the various states of the pump, that those states endowed

P. 1. GARRAHAN A N D R. P. GARAY

82

with high affinity for K predominated during Na: K exchange. The lack of effect of Pi on the apparent affinity for K strongly suggests that the predominating states have the same high affinity for K, in both their phosphoand their dephosphorylated states (Garay and Garrahan, 1975). To the authors’ knowledge no direct study of the interactions between K and Pi during the dephosphorylation of the pump is available. It is, however, suggestive that the apparent affinity for exteroal K during K: K exchange, which requires internal Pi (Glynn et al., 1970; Simons, 1974), is identical with the apparent affinity for external K during Na:K exchange, which is maximal when internal Pi is zero [Eq. (51)]. Since dephosphorylation requires external K, these findings are compatible with the view that K is necessary to confer to the pump the necessary reactivity so as to catalyze the transference of phosphate from the protein to water, but it has no effect on the affinity for Pi of the system. If the only action of ATP were to phosphorylate the pump, then it is difficult to see how the effects of ATP and Pi could be exerted independently, as is demanded by the experimental results summarized in Eq. (51). Any simple kinetic scheme involving phosphorylation and dephosphorylation of the pump will predict interactions between ATP and Pi if, as it is usually done, the ATP-containing and the phosphorylated forms are taken as mutually exclusive states of the pump. The affinity for Pi becomes independent of ATP if it is assumed that, under the experimental conditions tested, the catalytic site of the pump is fully saturated by ATP but that the rate of pumping is controlled by the state of occupation with ATP of a second, nonphosphorylating site whose affinity for the nucleotide is much lower and whose occupation has no effect on the affinity for the rest of the ligands. The proposed effect of ATP at a low-affinitysite is an effect on reactivity and, if this is taken for granted, it would represent another example of the V-like behavior of the pump. There are several lines of evidence that, independently of the demands of Eq. (51), suggest that a low-affinity site for ATP exists in the Na pump K)-dependent ATP hyand that its occupation is necessary for (Na drolysis. This evidence may be summarized as follows: a. In the presence of K, the number of moles of ATP that are bound to the (Na I()-ATPase is twice the amount of phosphate that can be incorporated from ATP (Hegyvary and Post, 1971). b. The curvc relating ATP hydrolysis to ATP concentration shows a biphasic response with an apparent K , of 0.001 m M as well as a Km near 0.5 mM (Neufeld and Levy, 1969). A similar biphasic response has also been observed by Kanazawa et al. (1970). In both cases, VmaXassociated with the high-affinity site is quite small.

+

+

SEQUENTIAL AND SIMULTANEOUS MODELS FOR No AND K TRANSPORT

83

c. A low-affinity site for ATP has been implicated (Post et al., 1972) in the increase in the rate of reconversion of E2 into El. d. The requirement for the occupation of a low-affinity site for ATP may be responsible for the fact that K:I< exchange does not occur in the absence of ATP (Glynn et al., 1971; Simons, 1974), in spite of the fact that K:K exchange is not associated with hydrolysis of ATP (Simons, 1974) or with phosphorylation of the pump from ATP, as is shown by the fact that nonphosphorylating analogs of ATP are satisfactory substitutes for ATP during K: K exchange (Simons, 1975). If Ii :I< exchange is, as seems likely, associated with the reversible transfer of phosphate from the phosphoenzyme to water and from P, to the dephosphoenzymc, the requirement for ATP during K:I< exchange is almost certainly an effect on reactivity and not on affinity. This follows from the observation that the relation between the concentration of K and the ratc of K-dependent dephosphorylation of the pump is independent of the concentration of ATP (see Fig. 5 in Kanazawa et al., 1970). e. If under physiological conditions the K , of the phosphorylating site were similar to that detectable by ATP binding or phosphorylation studies (0.1-30 p M ) (see, for instance, Hegyvary and Post, 1971), its valuc would be considerably lower than the concentration of ATP attained in the experiments that lead to Eq. (51). Such evidence would support our assumption that under these conditions tht. phosphorylating site is fully saturated by ATP.

3. CHANGES CYCLE

IN

AFFINITYVS. CHANGES

IN

REACTIVITY

DURING A P U M P

I n the course of this work we have mentioned a series of ligand-induced changes in the affinity of the pump sites. In contrast with the interactions in rate constants, which are necessary to account for the kinetic properties of the cation fluxes driven by the Na pump, ligand-induced changes in affinity are not manifest and, hcnce, are not required to develop kinetic equations to describe the operation of the pump. This is probably because ligand-induced changes in affinity either take place at ligand concentrations far from physiological levcls, as, for instance, the effect of I< on the affinity for ATP (Hcgyvary and Post, 1971), the effect of ATP on the Na/K selectivity (Skou, 1974a,b), and the effect of Na on the synthesis of ATP from ADP and EP (Taniguchi and Post, 1975), or result in a distribution of the pump heavily favoring a particular affinity state, as, for instance, the effect of phosphorylation on the affinity for I< (see Section II11B,3,d). It would seem, therefore, that affinity states significantly different from

04

P.

J. GARRAHAN AND

R. P. GARAY

the average represent, at most, very short-lived stages of the pump cycle when ligands are present a t physiological concentrations. It is tempting to conclude that, in contrast to what happens in soluble regulatory enzymes, in the operation of the Na pump, ligand-induced changes in reactivity are more important than ligand-induced changes in affinity. A plausible explanation for this difference may reside in the different role that conformational changes induced or stabilized by ligands have in soluble regulatory proteins as compared to the role of these transitions in transport systems (Lieb and Stein, 1970). In transport systems it is likely that transport is caused by a ligandinduced conformational change, that is, in these systems the rate of catalysis is probably limited by the rate of transition between different conformational states. This contrasts with the behavior of soluble regulatory enzymes, in which the rate of conformational transition is probably much less important than the ligand-dependent equilibrium proportion of the different affinity states. 8. Are Changes in Affinity of the Cation-Binding Sites Necessary for Active Transport?

It is customary to read in descriptions of the mechanisni of active transport that the existence of cyclic changes in the affinity of the cation-binding sites is a mandatory requirement for an active transport scheme. This assertion is usually justified on the basis that the pump sites pick up cations from media in which their concentration is low and releases them into media in which their concentration is high. It may be important to point out that this statement is only generally valid for the case of sequential schemes for active transport, since in these schemes it is the same set of sites that binds cations at one of the surfaces of the membrane and releases them at the opposite surface of the membrane. In these models, therefore, cyclic changes in affinity are not only necessary to explain the binding and release of cations but are also required to account for the difference in the affinity of inner and outer cation-binding sites. For similar reasons, cyclic changes in affinity would also be mandatory if, in simultaneous models, transport took place as a consequence of the rotation of the pump through the membrane with a change in the orientation of the cation-binding sites. A rotation of this kind would have to be accompanied by a change in the affinity of the binding sites, so that the outward-facing sites always prefer K and the inward-facing sites always prefer Na. Although a model of this kind would account for the kinetics of cation fluxes, the mechanism on which it is based does not seem to be physically feasible, since several lines of evidence strongly suggest that proteins in

SEQUENTIAL A N D SIMULTANEOUS MODELS FOR No A N D K TRANSPORT

85

the cell membrane are unable to rotate in the way envisaged by this mechanism. The following evidence argues against this model: a. For a protein to be exposed simultaneously to both surfaces of the cell membrane, it must have two polar ends joined by a long hydrophobic region. It is very difficult, on energetic grounds, to see how such structures would be capable of rapid rotation across the membrane. A good example of the energetic hindrance to this kind of movement is provided by the very slow rate of exchange of phospholipids from one side of the bilayer to the other (Bretscher, 1973). b. One of the major protein component of the rcd cell membrane (component a ) is exposed to both surfaces of the membrane. The molecular weight of this protein (about 105,000) is similar to that of the major polypeptide of the Na pump (Usegui et al., 1971; Hokin et al., 1973; Jorgensen, 1974; Kyte, 1971). Peptide patterns of component a labeled with nonpermeable reagents, either in intact cells (only from the outside) or ghosts (from both sides), are different showing that component a does not rotate across the membrane (Bretscher, 1971). c. We have already mentioned that specific binding of a hydrophilic protein of a molecular weight of the same order of magnitude as the (Na K)-ATPase, e.g., a y-globulin, has no effect on active transport (Kyte, 1974). This seems conclusively to rule out rotation across the membrane as a physically plausible mechanism for cation transfer.

+

INTERNAL TRANSFER If the Na pump is unable to rotate, cation-binding sites will always face the same surface of the cell membrane. The obvious choice for a mechanism of cation transport in a system in which inner and outer sites not only coexist but also always face the same surface of the membrane are the internal transfer (Lieb and Stein, 1970) mechanisms. I n this type of mechanism, transport results from the migration of cations within the structure of the transport system, from entering sites facing one of the faces of the membrane and toward leaving sites facing the opposite surface of the membrane. In this case, changes in affinity, a t least in the exposed sites, do not need to be postulated to account for the cyclic binding and release of cations. In these models and during Na: K exchange, the process of internal transfer in itself will move t,he cations from high-affinity entering sites to lowaffinity leaving sites a t the opposite surface of the cell membrane. Nothing is known about the mechanism that brings about the process of internal transfer. However, it seems possible to postulate at least two different classes of mechanism to account for this phenomenon. a. Internal Transfer through Occlusion of Exposed Sites. Figure 5 shows an example of this class of mechanism. The scheme does not intend t o give a detailed account of all the properties of the active transport system but

86

P. J. GARRAHAN AND R. P. G A M Y

FIG.5. Schematic representation of a simultaneous model for active transport that drives cation translocation through a n internal transfer mechanism involving the occlusion of the exposed sites. The A, B, and C are different conformational states of the pump. Vertical arrows indicate accessibility of cation-binding sites for cation exchange; horizontal arrows indicate the possible transitions between conformations. These transitions depend on the state of occupation of the cation-binding sites, and their net direction is given by the free energy gradient. For more details see text.

rather to illustrate in a very general way the main features of what appears to be a plausible mechanism for the Na pump. In the scheme of Fig. 5, a transport unit may exist in three different conformations (A, B, and C). In conformation B, sites cannot exchange cations~withthe solution bathing the membrane but are able to exchange cations between themselves. I n conformations A and C, sites can only exchange with the cations of the media to which they are exposed. Only the transitions A 2 B, B S C, and C A take place a t a measurable rate. Let us now assume that the transitions among conformations only take place when the state of occupation of cation-binding sites shown in Table I is fulfilled. Although the eationic requirements are necessary for the transitions to take place at a measurable rate, their net direction will be given by the sum of the free energy change of the transition and the free energy of the metabolic event coupled to the transition. Even though it does not appear in our scheme (Fig. 5 ) , the coupling of each transition with an elementary step of the hydrolysis of ATP catalyzed by the pump is an experimental requirement. If there were no coupling, the system would be unable to drive active transport, and if the coupling were loose in the absence of ATP, the system would allow passive movements of cations. We are now ready to analyze under which conditions the system of Fig. 5 will drive cationic fluxes. These conditions may be elaborated as follows: 1. Inner sites occupied by N a and outer sites occupied by K . The allowed transition is A B, which in turn drives C $ A. If in state B, the Na and

SEQUENTIAL AND SIMULTANEOUS MODELS FOR N o AND K TRANSPORT

87

TABLE I TRANSITIONS AMONG CONFORMATIONS A N D OCCUPATION ~~EQL-IREMENTS O F C.4'rION-I3rNI>INt: SITES

Transition

Itcquirement at thc inner sitesa

Requirement at the outer sitesu

Nu

Na or K Na or K

A=B B=C C e A

Ii

K also represents its congeners, Li, Rb, Cs, TI, and NH,.

Ii change places, then transition B

C will takc place. I n this way we end up with C having its inner sites occupied by the initially clxtcrnal Ii, and its outer sites by the initially intcrnal Na. Thc low affinity of the sites for these cations favors their wIeas('. The releasc is complctcld because, to initiate a new transport cycle, C has to return to A and acquire a state of occupation by cations which is th r opposite to that it had a t the end of the preceding transport cyclr. It is clear that a similar reasoning will c>xpl:tinthe reversed operation of the pump if we start with C having its irincr sitrs occupied by I< and its outer sites occupied by Na. The diffrrence is that in this case the release of cations will be a consequcncc of the encrgctic situation that drives the systc.m in the dirrction C -+ B -+ A and of the fact that, in order to observe reversal, external I< and intrrnal Na conccntrations must be very low. 2. I n n e r and outer sates occupzed by Nu. The only possible transition is A B. The reversible oscillation of thc systcm betwetm these two states will drive the Na: Na exchange. 3. I n n e r and outer sites occupied by K . The allowcld transition will be B C. As the result of the oscillation of thc system between these two states, the I(:I< exchange will be observed. 4. Thr system in Fig. 5 would also account for the. uncoupled Na efflux if thc transition A -+ B took place also when the external sitw are empty, and thp transition B -+ C when the internal sitcs are occupied; either one or both transitions are much slower undm these conditions. The schcme of Fig. 5 would fit the main kinrtic properties of the Na pump provided the lifetimr of conformation B (the occluded statc.) is short. As pointed out by Lieb and Stclin (1975), intcmml transfer models based on the occlusion of exposed sitcs may he refined in a way that allows avoidance of the restriction concerning the short life-span of the occluded states. An example of this is provided by the tetramclric model for active transport proposcd by Stein et al. (1973). .--)

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P. J. GARRAHAN AND R. P. GARAY

In this model, each transport unit is envisioned as formed by the association of a pair of identical dimers, each having inner and outer facing subunits. Each dimer drives cation translocation in a way that is essentially similar to that proposed in the scheme of Fig. 5. A distinctive feature of the model is that, in each transport unit, the dimers are 180" out of phase, so that when sites in one dimer are occluded they are exposed in the other and vice versa. If transitions between exposed and occluded conformations are fast, the tetrameric transport unit will expose to the bathing solution the same number of sites during the whole pump cycle. Thus, in this model, there will be occlusion of cations but not occlusion a j sites. Occluded cations in a dimer may occupy any fraction of the total duration of the transport cycle, without affecting the separation of variables in the flux equation CEq. (2)l. b. Internal Transfer with Coexistence of Occluded and Exposed Sites. The class of internal transfer models analyzed on the basis of the scheme in Fig. 5 are V systems, in the sense that their operation does not require changes in affinity of the exposed sites and that their behavior can be explained by assigning particular reactivities to the various cation-pump complexes. In these models, however, the affinities of the sites in the occluded state (state B, in Fig. 5) may differ from those of the exposed states. It has in fact been proposed that the affinities of the sites in the occluded state are inverted, so that now outer sites prefer Na and inner sites prefer K (Stein et al., 1973; Garrahan and Garay, 1974; Skou, 1975). Although such a change would help the interchange between Na and K in the occluded state during the Na:K exchange, it would hinder the same phenomenon when the pump is operating in its reversed mode, since, by the principle of microscopic reversibility, the pump must traverse in opposite direction all the elementary steps of the forward reaction. On the other hand, changes in affinity in the occluded state are not necessary to account for either the Na: Na exchange or the K: K exchange. In view of this it may perhaps be simpler to suppose that in the occluded state inner and outer sites have the same affinity for Na and for K. A way out of the problems raised by the changes in affinity and by the duration of the occluded states would be to assume that the pump always possesses, in addition to sites that are exposed to the bathing solutions, occluded sites. These sites would be able to exchange cations either with exposed sites, at only one of the surfaces of the cell membrane, or between themselves. A scheme based on this hypothesis is shown in Fig. 6. Our pump can exist in five different states (A, A', B, C', and C) . All of them possess sites that exchange with cations in the solutions together with occluded sites.

89

SEQUENTIAL A N D SIMULTANEOUS MODELS FOR N o A N D K TRANSPORT

EXTRACELLULAR A

A'

B

C'

C

~~

INTRACE LLULAR

FIG.6. Schematic representation of a simultaneous model for active transport that drives cation translocation through an internal transfer mechanism involying the coexistence of exposed and occluded cation-binding sites. The A, A', B, C', C are different conformational states of the pump, Vertical arrows indicate accessibility of cation-binding sites for cation exchange; horizontal arrows indicate the possible transitions between conformations. For more details, see text.

In states A and C, occluded sites are unable to exchange their cations; in states A' and C' occluded sites exchange cations with their corresponding exposed sites but not directly with the media; and, in conformation B, occluded sites exchange cations between themselves but not with the exposed sites. It is clear that such a system will be capable of driving all the different modes of operation of the Na pump if we impose on the A B, B C, A transitions, cationic requirements similar to those we assigned and C to the transitions of the scheme in Fig. 5. Systems such as that in Fig. 6 may be pure V systems since their operation does not necessarily require changes in affinity, but only changes in the accessibility of the cation-binding sites. In these systems, on the other hand, exposed sites will exist throughout the pump cycle, regardless of the duration of tho occlusion of cations that are transported. c. Molecular Events during Internal Transfer. In the preceding paragraphs we concluded that internal transfer models appeared as the most adequate to accommodate the evidence for the coexistence of inner and outcr sites with the structural evidence that seems to prohibit the rotation of proteins across the membrane. It seems rather obvious that the molecular event underlying internal transfer is a change in the orientation of the cation-binding sites, induced by the combination of the pump with the adequate ligands. A general way of defining these changes would, perhaps, be to say that, as a consequence of a ligand-induced conformational change, the accessibility to the solvent

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of cation-binding sites is altered. Changes in the properties of side groups of proteins in solution resulting from conformational transitions are a welldocumented fact, and these changes can at least in part be attributed to changes in the degree of exposure of the group to the solvent. Such changes are probably the cause of the different degrees of accessibility to NEM of K)-ATPase sulfhydryl groups in the different ligand states of the (Na (Hart and Titus, 1973). The explanation of internal transfer in terms of changes in the accessibility of cation-binding sites has the advantage that it interprets this process as mediated by the adaptation to a definite function of a mechanism whose existence has been demonstrated and whose operation does not require large conformational changes in the transport system. The latter is important since it now seems clear that ligand-induced functional changes in proteins are generally mediated by quite small rearrangements within the protein structure (see, for instance, Peruta, 1970) ; this also seems to be true for the case of the Na pump, in view of the already mentioned lack of effect on active transport of the binding of some antibodies against the Na Pump. d. Does Simultaneous Binding Imply Simultaneous Transport?Sequential models for active transport necessarily imply sequential transport of cations, because, in these models, the transition of cation-binding sites from their inner- to their outer-facing states is necessarily associated with the transference of cations in the direction of the transition. In the case of simultaneous models, there is no such strict requirement, since, in general, sequential transport does not seem to contradict any of the restrictions on the possible modes of behavior of the Na pump that we have analyzed in this paper. In the schemes of Figs. 5 and 6, simultaneous binding is associated with simultaneous transport, but it appears possible that mechanisms similar to those proposed in these schemes could be adapted to transfer cations first in one direction and then in the opposite direction, provided that the ratelimiting step of the overall process is the transition of a species having its inner and outer sites occupied by the cations that have to be transported.

+

C. Other Transport Systems

In this paper we have developed a set of restrictions on the possible modes of behavior of the mechanism of the Na pump starting from the fact that all modes of operation of this system have strict cationic requirements at both surfaces of the cell membrane which requires the linkage between inner and outer cation-binding sites to be explained.

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91

It is intcrcsting to see to what extent the propertirs we have attributed to the Na pump can be extrapolatrd to propose a similar mechanism for transport systems that do not show ligand rcquiremrnts a t both surfaces of the mrmbrane. In this class we may includc both facilitated diffusion systems, such as that responsible for glucose transport in red cells, as well as activr transport systems, such as the Ca pump of sarcoplasmic reticulum. To fit this class of systcms within th r general scheme wc have developed for the Na pump, it is necessary to consider that, although neither glucose nor Ca transport have requirements at sites that occupy a trans position with rcspect to the surface of the mrmbrane from which the flux is taking plaer, trans sitcs must also exist in these systems. This follows from the facts that glucose transport takes place in both directions and that the entry of Ca ions into sarcoplasmic reticulum vesicles is a reversible process (Hasselbach and Makinosc, 1972). Glucose and Ca transport systems would operate using the same kind of mechanism that we have proposed for the Na pump if inward- and outward-facing sites in these systems were two different classes of sites that cwxist in thc same' transport unit ; the main difference between these systcms and thc Na pump would then be that simultaneous occupation of sites is not rcquircd for transport to take place. Similarily, sequential schemes, in this class of transport systems, would rrquire thc oscillation of a single set of sites from an inward- to an outward-facing conformation. If thc simultaneous view is accepted, it is clear that Ca or glucose transport may be the result of the internal transfrr of th r ligand from one set of sites to the other. A model for glucose transport in red cells, basrd on considerations similar to those we have made here, has been proposed by Lirb and Stein (1970) (see also Lieb and Stein, 1972). They showed that this model was able to account for propcrtics of this transport system that in tclrms of the classic, sequcntial, circulating carrier schemes would prcsent contradictions. A “simultancous” mechanism has also been proposed by Ho and Guidotti (1975) to account for the structural features of the anion transport system in red cells. ACKNOWLEDGMENTS The work reported in this paper was partially supported by grants from the Consejo Nacional de Investigaciones Cienttficas y TBcnicas (CONICET) and from the Univenidad de Buenos Aires, Argentina. One of the aut.hors (P, J. G.) is an Established Investigator from CONICET, Argentina. REFERENCES Adrian, It. H., and Slayman, C. I,. (1966). Membrane potential and conductance during transport of sodium, potassium and rubidium in frog muscle. J . Physwl. (London) 184,970-1014.

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Askari, A., ed. (1974). International conference on the properties and functions of the Na+ K+ ATPase. Ann. N.Y. A d . Sci. 242,6-741. Baker, P. F., and Stone, A. J. (1966). A kinetic method for investigating hypothetical models of the sodium pump. Bwchim. Biophys. Acta 126,321-329. Baker, P. F., and Willis, J. S. (1972). Binding of the cardiac glycoside ouabain to intact cells. J. Physiol. (London)224, 441462. Baker, P. F., Blaustein, M. P., Keynes, R. D., Manil, J., Sham, T. I., and Steinhart, R. A. (1969). The ouabain-sensitive fluxes of sodium and potassium in squid giant axons. J. Physiol. (LomGn) 200,459-496. Banerjee, S. P., and Wong, S. M. E. (1972a). Potassium ion stimulated and sodium ion-dependent adenosine diphosphate-adenosine triphosphate exchange activity in a kidney microsomal fraction. Biochem. J . 129, 775-779. Banerjee, S. P., and Wong, S. M. E. (1972b). Effect of potassium 0n sodiumdependent adenosine diphosphate-adenosine triphosphate exchange activity in kidney microsomes. J . Biol. Chem. 247, 5409-5413. Bastide, F., Meissner, G., Fleischer, S., and Post, R. L. (1973). Similarity of the active site of phosphorylation of the adenosine triphosphatase for transport of sodium and potassium ions in kidney to that for transport of calcium ions in the sarcoplasmatic reticulum of muscle. J . Bwl. Chem. 248, 8385-8391. Blostein, R. (1968). Relationship between erythrocyte membrane phosphorylation and adenosine triphosphate hydrolysis. J. Biol. Chem. 243, 1957-1965. Bretscher, M. S. (1971). A major protein which spans the erythrocyte membrane. J. Mol. BWl. 59,351-357. Bretscher, M. S. (1973). Membrane structure. Some general principles. Science 181,

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Czerwinski, A., Gitelman, H. J., and Welt, L. G. (1967). A new member of the ATPase family. Am. J . Physwl. 213, 786-792. Dahms, A. S., and Boyer, P. D. (1973). Occurrence and characteristics of ‘80 exchange reactions catalyzed by sodium- and potassium-dependent adenosine triphosphatase. J . Bwl. Chem. 248,3155-3162. Diamond, J., and Wright, E. M. (1969). Biological membranes: Physical basis of ion and non-electrolyte selectivity. Annu. Rev. Physsiol. 31, 581-589. Fahn, S., Hurley, M. R., Koval, G. J., and Albers, R. W. (1966s). Sodium-potassiumactivated adenosine triphosphatase of Electrophorus electric organ. 11. Effects of N-ethylmaleimide and other sulphydryl reagents. J . Biol. Chem. 241, 1890-1895.

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Jorgensen, P. L. (1974). Purification of Na+, K+-ATPase: Active site determination and criteria of purity. Ann. N . Y . Acad. Sei. 242, 36-52. Kanazawa, T., Saito, M., and Tonomura, Y. (1970). Formation and decomposition of a phosphorylated intermediate in the reaction of Na+-K+dependent ATPase. J . Biochem. (Tokyo) 67, 693-711. Kanazawa, T., Yamada, S., Yamamoto, T., and Tonomura, Y. (1971). Reaction mechanism of the Ca2+-dependentATPase of sarcoplaamic reticulum from skeletal muscle. V. Vectorial requirements for calcium and magnesium ions of three partial reactions of the ATPase: Formation and decomposition of a phosphorylated intermediate and ATP formation from ADP and the intermediate. J . Bwchem. (Tokyo) 70,95-123. Karlish, S. J. D., and Glynn, I. M. (1974). An uncoupled efflux of Na from human red cells probably wociated with Nadependent ATPase activity. Ann. N . Y . Acad. Sd. 242,461-470.

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Post, R. L., and Kume, S. (1973). Evidence for an aspartyl phosphate residue at the active site of sodium and potassium ion transport adenosine triphosphatase. J . Biol. Chem. 248,6993-7000. Post, It. L., Sen, A. K., and Rosenthal, A. S. (1965). A phosphorylated intermediate in adenosine triphosphate dependent-sodium and potassium transport across kidney membranes. J . B w l . Chem. 240, 1437-144.5. Post, 1%.L., Kume, S., Tobin, T., Orcutt, B., and Sen, A, K. (1969). Flexibility of an active center in sodium-plus-pot assium adenosine triphosphatase. J . Gen. Phyeiol. 54, 3069326s.

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Post, R. L., Hegyvary, C., and Kume, S. (1972). Activation by adenosine triphosphate in the phosphorylation kinetics of sodium and potassium ion transport adenosine triphosphatase. J . Bwl. Chem. 247,6530-6540. Post, R. L., Kume, S., and Rogers, F. N. (1973). Alternating paths for phosphorylation of the sodium and potassium ion pump in plasma membranes. Mech. Bioenerg. Proc. Znt. Conj., 197.2 pp. 203-218. Post, R. L., Taniguchi, K., and Toda, G. (1974). Synthesis of adenosine triphosphate by the Na+, K+-ATPase. Ann. N.Y. Acad. Sci. 242,80-91. Post, R. L., Toda, G., and Rogers, F. N. (1975). Phosphorylation by inorganic phosphate of the sodium plus potassium ion transport adenosine triphosphatase. Four reactive states. J . Biol. Chem. 250,691-701. Rega, A. F., and Garrahan, P. J. (1976). K+-activated phosphatase. In “MembraneBound Enzymes” (A. Martonosi, ed.), Plenum, New York. (In press.) Rega, A. F., Garrahan, P. J., and Pouchan, M. I. (1970). Potassium activated phosphatase from human red blood cells. The asymmetrical effects of K+, Na+, MgH and adenosine triphosphate. J . Membr. Biol. 3, 14-25. Robinson, J. D. (1967). Kinetic studies on a brain microsomal adenosine triphosphatase. Evidence suggesting conformational changes. Biochemistry 6, 3250-3258. Robinson, J. D. (1970). Phosphatase activity stimulated by Na+ plus K+: Implications for the (Na+plus K+)-dependent adenosine triphosphatase. Arch. Biochem. Biophys. 139, 164-171. Robinson, J. D. (1973). Variable affinity of the ( N d K+)-dependent adenosine triphosphatase for potassium. Studies using beryllium inactivation. Arch. Biochem. Biophys. 156, 232-243. Robinson, J. D. (1974a). Affinity of the (Na+ K+)-dependent ATPase for Na+ meavured by Na+-modified enzyme inhibition. FEBS Lett. 38, 325-328. Robinson, J. D. (1974b). Cation interactions of different functional states of the Na+, K+ ATPase. Ann. N . Y . Acad. Sci. 242, 185-202. Sachs, J. R. (1967). Competitive effect of some cations on active potmium transport in the human red cell. J . Clin. Znuest. 46, 1433-1441. Sachs, J. R. (1970). Sodium movements in the human red blood cells. J . Gen. Physiol. 56,322-341. Sachs, J. R., and Welt, L. G. (1967). The concentration dependence of active K transport in the human red cell. J . Clin. Invest. 46, 65-76. Sen, A. K., and Post, R. L. (1964). Stoichiometry and localization of adenosine triphosphate dependent sodium and potassium transport in the erythrocyte. J . Biol. Chem. 239,345-352. Siegel, G. J., and Albers, W. R. (1967). Sodium-potamium activated adenosine triphosphatase of Electrophorus electric organ. IV. Modifications of response to sodium and potassium by arsenite plus 2,3-dimercapto propanol. J . Biol. Chem. N 2 , 4972-4979. Simons, T. J. B. (1974). Potassium:potassium exchange catalyzed by the sodium pump in human red cells. J . Physiol. (London) 237, 123-155. Simons, T. J. B. (1975). The interaction of ATP-analogues possessing a blocked 7-phosphate group with the sodium pump in human red cells. J . Physiol. (London) 244, 731-739. Skou, J. C. (1971). Sequence of steps in the (Na+ K+)-activated enzyme systems in relation to sodium and potassium transport. Curr. Top. Bioenerg. 4, 357-398.

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Skou, J. C. (1974a). Effect of ATP on the intermediate steps of the reaction of the (Na+ K+)-activated enzyme system. I. Studied by the use of N-ethyl maleimide inhibition as a tool. Biochim. Biophys. Acta 339, 234-245. Skou, J. C. (1974b). Effect of ATP on the intermediate steps of the reaction of the (Na+ K+)-activated enzyme system. 11. Effect of a variation in the ATP/Mg* ratio. Biochim. Biophys. A c h 339, 246-257. Skou, J . C. (1974~).Effect of ATI' on the intermediate steps on the reaction of the (Na+ K+)-activated enzyme systems. 111. Effect on the p-nitrophenyl phosphatase activity of the system. Biochim. Biophys. Acta 339, 258-273. Skou, J. C. (1975). The (Na+ K+)-activated enzyme system and its relationship to transport of sodium and potassium. Q. Rev. Biophys. 7, 401434. Skulskii, I. A., hlanninen, V., and Jiirnfelt, J. (1973). Interaction of thallous ions with the cation transport system in erythrocytes. Riochim. Biophys. Acta 298, 702-709. Stein, W. D., Lieb, W. R., Karlish, S. J. D., and Eilam, Y. (1973). A model for active transport of sodium and potassium ions as mediated by a tetrameric enzyme. Proc. Natl. Acad. Sci. U.S.A. 70, 275-278. 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. Tobin, T., Akera, T., Baskine, S. T., and Brody, T. M. (1973). Calcium ion and sodium and potassium-dependent adenosine triphosphatase. Its mechanism of inhibition and identification of the El-P intermediate. Mol. Pharmacol. 9, 336-349. Tonomura, Y., and Fukushima, Y. (1974). Kinetic properties of phosphorylated intermediates in the reaction of the Na+, I<+-ATPase. Ann. N.Y. Acud. Sca. 242, 92-105. Uesugi, S., Dulak, C. N., Dixon, F. J., Hexum, D. T., Dahl, .J. I,., Perdue, J. F., and Hokin, L. E. (1971). Studies on the characterization of the sodium-potassium transport adenosine triphosphatase. VI. Large scale partial purification and properties of a Lubrol-solubilized bovine brain enzyme. J . Riol. Chem. 246,531-543. Whitehead, E. (1970). The regulation of enzyme activity and allosteric transitions. Prog. Biophys. Mol. Biol. 20, 321-397. Whittam, R., and Ager, M. E. (1965). Connexion between active cation transport and metabolism in erythrocytes. Riochem. 1.97, 214-227. Wyman, J. (1964). Linked functions and reciprocal effects in hemoglobin: A second look. Adv. Protein Chem. 19, 223-286.

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Soluble and Membrane ATPases of Mitochondria. Chloroplasts. and Bacteria: Molecular Structure. Enzymatic Properties. and Functions R I V K A PANET"AND D . RAO SANADI Deparlnient of Cell Physiology Boston Biomedical Research Institute Boston. Massachusetts

I . Introduction . . . . . . . . . . . . . . . . . . I1 . Rlitochondrial Coupling Factor 1 . . . . . . . . . . . . A . Role in Oxidative Phosphorylation . . . . . . . . . . B . Enzymatir Properties . . . . . . . . . . . . . . C . Molecular Properties . . . . . . . . . . . . . . 1) . Summary . . . . . . . . . . . . . . . . . I11. Oligomycin-Sensitive ATPase . . . . . . . . . . . . . A . Enzymatic Properties . . . . . . . . . . . . . . B . Molecular Properties . . . . . . . . . . . . . . C. . The Oligomycin and Dicyclohexylcarbodiimide Site of Action . . I) . Summary . . . . . . . . . . . . . . . . . I v . Chloroplast Coupling Factor 1 . . . . . . . . . . . . A . Reaction Catalyzed by Chloroplast Coupling Factor 1 . . . . B. Rnzymatic Properties . . . . . . . . . . . . . . C . Molecular Properties . . . . . . . . . . . . . . D . Effect of Light on Conformational Changes in Chloroplast Coupling Factor 1 . . . . . . . . . . . . . . . . . E . Summary . . . . . . . . . . . . . . . . . V. Bacterial ATPase . . . . . . . . . . . . . . . . A . Reactions Catalyzed by Bacterial ATPsse and the Use of Mutants B. Enzymatic Properties . . . . . . . . . . . . . . C . Molecular Properties . . . . . . . . . . . . . . D . Comparison of Bacterial ATPase w i t h F1 arid CF1 . . . . . E . Summary . . . . . . . . . . . . . . . . . VI . General Conclusions and Perspect.ive . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

.

1

99 . 100 . 101 . 104 . 114 . 117 . 118 . 118 . 120 . 12.5 . 126 . 126 . 126 . 129 . 135

. 139

. 140 . 141 . 141 . 143 . 147 . 148 . 148 . 149 . 150

INTRODUCTION

Mitochondria1 oxidative phosphorylation can be grouped into a number of discrete processes catalyzed by integrated enzyme complexes that are

* Present address: Department of Nuclear Medicine. Hadassah Hospital. Jerusalem. Israel.

99

100

RIVKA P A N B AND D. RAO SANADI

held together to form a membrane structure. The complexes of the electron transport system (Hatefi, 1966) are the NADH dehydrogenase (complex I), succinate dehydrogenase (complex 11) , cytochrome b-c (complex 111) , and cytochrome aag (complex IV) . Ubiquinone (coenzyme Q ) communicates among complexes I, 11, and 111, and cytochrome c links the electron transport between complexes I11 and IV (Fig. 1). There is good evidence that an energized intermediate (chemical hypothesis) or state [chemiosmotic hypothesis (Mitchell, 1966) and conformational coupling hypothesis (Boyer et al., 1973)] is generated during the oxidations. Its utilization is coupled to ATP formation by another enzyme complex, the oligomycinsensitive ATPase complex (OS-ATPase) (Tzagoloff et al., 1968a). The ATP generation process in chloroplasts and bacteria is quite analogous in regard to both the electron transport and the ADP phosphorylation reaction. The properties of the mitochondria1OS-ATPase complex and F1-ATPase have been reviewed recently (MacLennan, 1970; Racker, 1970; Tzagoloff, 1971a; Tzagoloff, 1973; Kagawa, 1972; Penefsky, 1974b; Senior, 1974). This review emphasizes the comparative enzymatic properties and inhibitor sensitivities of F1-ATPase, OS-ATPase, chloroplast coupling factor 1 (CF,), and the related bacterial ATPase. Molecular properties are discussed briefly for the sake of completeness.

II. MITOCHONDRIA1 COUPLING FACTOR 1

The approach to the study of oxidative phosphorylation utilizing the chemical resolution-reconstitution approach has led to the purification of several soluble proteins that restore phosphorylation activity in deficient submitochondrial particles. These soluble preparations are called coupling factors or energy transfer factors. Coupling factor 1 or F1was first purified from bovine heart mitochondria by Pullman (Pullman et al., 1960). It increased the phosphorylation activity and P,-ATP exchange activity of the Nossal particles and ammonia particles (Fessenden and Racker, 1966) which are deficient in this activity. A similar factor was found also in rat liver (Catterall and Pedersen, 1972; Lambeth and Lardy, 1971; Senior and Brooks, 1971), yeast (Schatz et al., 1967; Tzagoloff and Meagher, 1971), bacteria (see Harold, 1972; Abrams and Smith, 1974), and chloroplasts (Avron, 1963; Vambutas and Racker, 1965; McCarty and Racker, 1966; Lynn and Straub, 1969a; Karu and Moudrianakis, 1969; Farron, 1970).

101

MEMBRANE ATParcr AT P

Aurovertin bide

NADH

4

F

+

-(

A

COO 4 Cyt b

F1)

4

Cyt c

f

pD

,--

Cyt

c +Cyt

0 a3

4

O2

F

f ps Succina t e

FIG.1. Hypothetical scheme for the respiratory chain-linked energy transfer system and the proposed sites of action of oligomycin and aurovertin. DCCD, dicyclohexyl, dehydrogenase; Lee and Ernster (1968). F P ~ succinate , cerbodiimide; F P ~ NADH dehydrogenase.

A. Role in Oxidative Phorphorylation

The site of F1 action in the sequence of reactions leading to phosphorylation coupled to respiration is believed to be the last step in which the phosphorylation of ADP to ATP occurs. The evidence that supports this conclusion includes the following observations: ( I ) F1has ATPase activity that appears to be the reverse of the reaction catalyzed by F1 in oxidative phosphorylation; ( 2 ) F1 increases P/O ratio and P,-ATP exchange in Fldeficient membranes; ( 3 ) it increases ATPdriven reactions in submitochondrial particles (SMP) deficient in F1; and ( 4 ) its ATPase activity is resistant to oligomycin but is inhibited by aurovertin, indicating that F1 catalyzes only the last step in ATP synthesis and is not inhibited by oligomycin. Tightly coupled mitochondria have low ATPase activity. Uncoupling by aging or freezing and thawing, and trypsin digestion produce structural disorganization of the membrane, and a result activate the mitochondrial ATPase roughly ten-fold. The activation of ATPase by uncouplers may be a more complex process. 1. ATPase ACTIVITYOF F1 AND ITSRELATION TO COUPLING ACTIVITY

The ATPase activity of membrane-bound FI is quite low but is increased considerably when it is released into solution by exposure to sonic oscilla-

102

RIVKA PANET AND D. RAO SANADI

tion. Further increase is induced by heat, which is used as a purification step (Penefsky et al., 1960; Pullman et al., 1960; Andreoli et al., 1965). Coupling factor preparations with low (Andreoli et al., 1965; Warshaw et al., 1967) or no ATPase activity (Sone and Hagihara, 1966) have been obtained from beef heart mitochondria. Factor A, isolated in Sanadi’s laboratory, using as an assay (Andreoli et al., 1965; Warshaw et al., 1967) its activity in stimulating ATPdriven reversed electron flow in SMP depleted by treatment with low urea, was later shown to be functionally identical with F1. It has low ATPase activity since the isolation steps are mild and do not involve exposure to heat. It catalyzes the same coupling and energy-linked reactions as F1 does, and the two preparations are interchangeable in the assays involving stimulation of energy-linked reactions. Activation of the ATPase activity of Factor A by heat does not change its coupling activity (Warshaw et al., 1967). The coupling factor from beef heart mitochondria isolated by Sone and Hagihara (1966) has no ATPase activity, nor could it be induced by trypsin digestion. The ATPase activity of F1could be inhibited very strongly by complexing with a low molecular weight peptide isolated from mitochondria (Pullman and Monroy, 1963). Inhibition of the ATPase activity, however, does not affect the coupling activity of F1 (see Section 11, B, 4). Thus, it appears that the coupling activity of F1 is not affected by its level of ATPase activity. The ATPase activity, however, is a convenient handle for the purification of F1and relating its enzymatic and molecular properties. There has been confusion regarding the difference between F1,which has a high activity of cold-labile ATPase, and Factor A, which has a low activity of cold-stable ATPase. Recent work in this laboratory (Joshi and Sanadi, unpublished) may provide a reasonable explanation. Samples of F1 and Factor A prepared by a revised method were analyzed for their content of bound Pullman inhibitor by two methods. One was to release the inhibitor by heating at 75°C (Warshaw et al., 1967; Brooks and Senior, 1971) and analysis of the extract by its inhibitory activity. The second was sodium dodecyl sulfate (SDS) gel electrophoresis both of the heated extract and the coupling factor preparations. The F1 with a specific ATPase activity of 70 pmoles/min x mg yielded 0.05-0.11 mole inhibitor/mole of F1 by both methods. The inhibitor content of Factor A with an ATPase activity of 5-10 pmoles/min X mg was 0.025-0.045 mole/mole Factor A. Since both the high and low ATPase-active preparations had extremely low inhibitor content, it would appear that it is not valid to explain the latency of the ATPase in Factor A as due to bound inhibitor (Pullman and Schatz, 1967). On the other hand, there are abundant data that the inhibitor does inhibit ATPase activity, which means that one way of altering ATPase activity is by changing its inhibitor content. It has also been established

MEMBRANE ATPascs

103

that the ATPase activity of Factor A can be increased several-fold by mild heating (Andreoli et al., 1965; Warshaw et al., 1967). Since the inhibitor content is far less than stoichiometric and insufficient to account for the ATPase modulation, the explanation of Andreoli et al. (1965) that the activity change is related to a conformational change in the enzyme appears valid and may represent a second method of ATl’ase regulation. 2. ACTIVITIESOF F1 FOLLOWING CHEVICAL MODIFICATION

Chemical modification of FI has led to further understanding of the relationship between the hydrolytic and the coupling activity of FI and of its molecular properties. Chemical modifications could be achieved by iodination with Iz (Penefsky, 1967), reaction with dicychlohexylcarbodiimide (DCCD) (Penefsky, 1967), and with inhibitors that react specifically with tyrosine, e.g., tetranitromethane ( Penefsky and Warner, 1965; Senior, 1973) and 7-chloro-4-nitrobenzo-2-oxa-l , 3-diazole (NBD-Cl) (Ferguson et al., 1974). Penefsky (1967) showed that iodination of F1 inactivated the ATI’ase. The loss of ATPase activity was accompanied hy disappearance of -SH groups but subsequent treatment, with -SH compounds [such as dithiothreitol ( D TT) or mercaptoethanol] did not, restore the ATPase activity. From this it appears that the -SH group disappearance is probably a secondary effect, and, indeed, it is accepted now that there is no -SH a t the active site of F1 (see tzlso Section 11, C, 3 ) . When added in relatively high concentrations, DCCD inhibits also F1-ATPase activity (Penefsky, 1967). Tetranitromethane, a rcagent with considerable specificity for tyrosine, caused complete loss of ATPase activity even under mild conditions (Penefsky and Warner, 1963). In a recent paper, Fl-ATPase was found to be inactivated by treatment with NBD-Cl. From the spectral properties of the reaction product, the authors suggest that the modified group is tyrosine (Ferguson et aI., 1974). The inactivation of ATPase by treatment with 1 2 , DCCD, or tetranitromethane did not affect the coupling activity of F1. The chemically modified F1 stimulated oxidative phosphoryl:ttion, I>,-ATP exchange, and RTPdriven reactions such as NAD reduction by succinate in N particles that are not completely free of F1. (The N particles are obtained by disrupting of mitochondria in a Nossal shaker, which removes some FI, but not all of it.) It was found, however, that the chemically modified FI preparations failed to stimulate oxidative phosphorylation in SU particles which are even more depleted of F1. [The SU particles are submitochondrial particles that have been passed through a Sephadex column and subsequently treated with urea (Schatz et al., 1967) .] The effects of chemically modified F1 on oxidative phosphorylation are

104

RIVKA PANET AND D. RAO SANADI

very similar to those of low concentrations of oligomycin (Lee and Ernster, 1965) and of yeast F1 (Schatz et al., 1967). Low levels of oligomycin stimulate oxidative phosphorylation in SMP that contain some F1, but not in SMP better resolved with respect to F1 (Fessenden and Racker, 1966; Racker and Horstman, 1967). Similarly, yeast F1 stimulates coupled phosphorylation in N particles but not in SU particles prepared from beef heart mitochondria (Schatz et al., 1967). The stimulating effect of yeast FI in N particles was inhibited by an antiserum to beef F1, not by antiyeast FI. On the basis of these experiments, it was suggested that yeast FI acts in the same manner as chemically modified F1 or low oligomycin, by blocking an energy “leak.” Racker considers this evidence for a structural role for FI (Schatz et al., 1967). MacLennan et al. (1968) have suggested that this leak “plugging” involves reconstitution of F1 on the membrane, by insertion of the missing subunits of F1. Their suggestion is based on reconstitution experiments in which ATPase and ATP-energized reactions of depleted SMP were restored by adding a mixture of F1subunits which by themselves did not have ATPase activity. Futai et al. (1974) identified the subunit of the bacterial ATPase that is required for the binding of the enzyme to the membrane and as a result reconstitutes coupling activity (see Section V, C). His results are in agreement with MacLennan’s hypothesis. B. Enzymatic Properties 1. COLDLABILITY

The ATPase and coupling activities of FI are cold-labile when isolated from beef heart (Pullman et al., 1960; Penefsky and Warner, 1965; Sone and Hagihara, 1966), rat liver (Lambeth and Lardy, 1971; Catterall and Pedersen, 1971), and yeast (Schatz et al., 1967), in contrast to their cold stability on the mitochondria1 membrane. Both activities are stable at 25”-30”C particularly in the presence of ATP (Pullman et al., 1960). Inactivation at 0°C is accompanied by dissociation of the complex into subunits. The 11.9 S Fl is dissociated into a 9.1 S and a 3.5 S subunit; by prolonged incubation at OOC, most of the complex is dissociated to the 3.5 S subunit (Penefsky and Warner, 1965; Forrest and Edelstein, 1970). The depolymerized subunits could reassociate into an active complex if the incubation was limited to 2 hours. Over longer periods of exposure to cold, the 3.5 S subunits aggregated irreversibly into a large inactive polymer. Penefsky and Warner (1965) found that the rate of dissociation was strongly influenced by anions present in the medium; I-, NQa-, and Br- accelerated cold dissociation.

105

MEMBRANE ATPoses

When the ATPase activity is not activated by heat, as in Factor A, its intrinsic ATPase activity declines in the cold, but the coupling activity is maintained (Andreoli et al., 1965). Low ATPase preparations, when activated by heat, become irreversibly cold-labile (Sone and Hagihara, 1966; Sone et al., 1969; Warshaw et al., 1967). The cycling of activation by heat and inactivation by slow cooling could be carried out but to a limited extent (Andreoli et al., 1965; Warshaw et al., 1967). ATP, ADP, Mg2+,or combination of these substances stabilize the enzyme from cold inactivation (Penefsky and Warner, 1965). Phospholipids also protect F1 against cold inactivation (Racker, 1965) , a fact that may explain its cold stability on the mitochondria1 membrane. 2. RESISTANCE TO OLIGOMYCIN AND DCCD

The ATPase activity of F1is resistant to oligomycin, rutamycin, DCCD, and tri-n-butyltinchloride (Pullman et al., 1960). The resistance to oligomycin and related compounds is a characteristic of all similar factor preparations isolated from different sources. Beef heart F1 (Pullman et al., 1960), rat liver F1 (Lambeth and Lardy, 1971), and yeast F1 (Schatz et al., 1967; Tzagoloff, 1969b; Sone et al., 1969) are all resistant to oligomycin. The oligomycin and related compounds are believed to act at a site between the nonphosphorylated energized state (X I or [H+]) and the hypothetical phosphorylated intermediate (see Fig. 1 ) . The OS-ATPase is probably not catalyzed by F1alone; some membrane compounds (Fo) are also needed for this more complex reaction (see Section 111). In contrast to oligomycin, rutamycin, and tri-n-butyltin chloride, DCCD does inhibit F1-ATPase activity directly, except at concentrations higher than those necessary to inhibit the membrane activity (20 moles DCCD/moIe F1) (Penefsky, 1967). The F1 inhibition by DCCD is probably by a different mechanism and at a different site compared to the inhibition of the membrane-bound enzyme system.

-

3. ACTIVATION BY UNCOUPLERS AND ANIONS

The ATPase activity of F1can be induced by 2,4dinitrophenol (DNP) although to a lesser extent than the activation of the membranous enzyme. The activation, which is no higher than 50% (Pullman et al., 1960), is pH-dependent with maximal activation between pH 6.5 and 7.0. It seems that the degree of activation depends on the basal ATPase activity; the lower it is, the higher the activation, and the basal activity depends on whether or not heat activation was introduced during the isolation of FI. Factor A (Andreoli et al., 1965) with low ATPase activity and Hagihara’s coupling factor with no ATPase (Sone et al., 1969) could be activated by

106

RIVKA PANET AND D. RAO SANADI

uncouplers to a higher extent than F1.Yeast F1(Schatz et al., 1967; Sone et al., 1969) and rat liver F1(Sone et al., 1969; Lambeth and Lardy, 1971) were activated more than the beef heart enzyme. Two- to threefold activation by DNP was obtained with the yeast and the rat liver F1,but activation of the beef heart enzyme was only 30-50%. Besides uncouplers, different anions also activate the ATPase, probably by a similar mechanism (Sone et al., 1969; Lambeth and Lardy, 1971). A similar activation by anions is seen also with CF1 (Nelson et al., 1972a; see also Section IV, B) . Both groups found that at pH 8.0, which is optimal for the ATPase, the rat liver, beef heart, and yeast enzymes are activated by anions such as chromate, bicarbonate, maleate, sulfide, and by uncouplers such as DNP and pentachlorophenyl (PCP). The rat liver (Lambeth and Lardy, 1971) and the yeast enzymes (Sone et al., 1969) are activated strongly by the anions, and the beef heart F1 less so. Sone et al. (1969) have evidence that the uncouplers such as DNP and PCP activate F1probably by the same mechanism as the activation by carboxylic acids. They showed that Tris-maleate activates their yeast F1 more than Tris-acetate. The DNP and PCP stimulate the activity in acetate much more than in the presence of maleate, which itself is an activator. Very similar results were obtained by Lambeth and Lardy (1971) with beef heart and rat liver F1. When the enzyme is maximally activated by bicarbonate, DNP has no stimulatory effect; with lower levels of anions when F1is not fully activated, DNP does produce activation (Table I). TABLE I EFFECT OF ANIONSAND AUROVERTINON THE ACTIVITY OF RATLIVER AND BEEFHEART ATPasea ~~

~~

Rat liver ATPase activity in

Addition None 3 mM Chromate 2 mM Dinitrophenol 4 mM Sulfite 2 pM Aurovertin 0.1 mM Azide a

~~~~

~~

Beef heart ATPase activity in

20 mM Tris-sulfate

30 mM Tris-bicarbonate

20 mM Tris-sulfate

30 mM Tris-bicarbonate

(%I

(%)

(%)

(%)

100 235 200 360 40 15

310 250 270 365 65 65

100 135 130 195 25 15

200 180 155 220 35 70

Data from Lambeth and Lardy (1971).

MEMBRANE ATPases

107

The ATPase activated by the above anions is just as sensitive to aaide and aurovertin as the initial activity, and it is resistant to tri-n-butyltin chloride and oligomycin (Lambeth and Lardy, 1971). Thus, it appears from the above results that uncouplers and the different anions increase the maximal velocity of F1-ATPase activity, probably by improving the access of water to the hydrolytic site, i.e., producing a conformational change at the active site. This effect appears different from the uncoupling effect on phosphorylation coupled to respiration. 4. ATPase INHIBITORS

Azide, aurovertin, and the ATPase inhibitor block ATPase activity on the mitochondrial membrane as well as inhibit F1-ATPase from various sources. Dio 9 inhibits only the yeast F1 (Tzagoloff, 1969b). It is of significance that these inhibitors act at, the last step in ATP synthesis in mitochondria, whereas inhibitors such as oligomycin have no effect on F1-ATPase and act at the preceding step in ATI' synthesis (see Fig. 1 ) . a. Azide. Pullman et al. (1960) first demonstrated that azide inhibits beef heart F1-ATPase. Later the inhibition was shown also with yeast F1 (Sone et al., 1969) and rat liver F, (Lambeth and Lardy, 1971). Azide similarly inhibits the ATPase after its activation by anions and DNP (Lambeth and Lardy, 1971). b. Aurovertin. The antibiotic aurovertin was introduced by Lardy et al. (1964) as a tool for the study of Oxidative phosphorylation. They established that aurovertin inhibits the mitochondrial ATPase induced by some uncouplers and also Pi-ATP, H218@-P,,and H2"Q-ATP exchanges. Later it was shown that the forward reaction, i.e., ATP synthesis, is much more sensitive to aurovertin than the reverse energy-transfer reactions (Lenaa, 1965; Lee and Ernster, 1968). The ATPase activity of SMP was less sensitive to aurovertin than oxidative phosphorylation (Lee and Ernster, 1968). Lardy et al. (1964) have proposed a branched pathway of energytransfer reactions from the respiratory chain to the terminal transphosphorylation step and suggested that aurovertin acts on only one pathway, at a point between the respiratory chain and the oligomycin site. Lee and Ernster (1968) and Roberton et al. (1968) , on the other hand, proposed that only one pathway exists and aurovertin acts on the ATP side of the oligomycin site (see Fig. 1 ) . It was found that aurovertin inhibits also soluble F1 and forms a stoichiometric fluorescent complex with it (Roberton et al., 1967; Chang and Penefsky, 1973; Layton et al., 1973). Catterall and Pederson ( 1972) showed that aurovertin increased twenty-one-fold the Ki of rat liver F1 for ADP, indicating that in the presence of aurovertin ADP binds better to F1. By binding to mitochondrial membrane, the aurovertin

108

RIVKA PANET AND D.

RAO SANADI

.s maximal fluorescence is increased up to 100 fold. This fluore, during state 3 and is partially quenched on anaerobiosis L ;he presence of oligomycin or of uncouplers (Bertina el al., 1973). The careful studies of Chang and Penefsky (1973) might explain the unidirectional inhibition of aurovertin and its mechanism of action. They showed that ATP quenched the fluorescence of the F1-aurovertin complex; ADP, on the other hand, enhanced this fluorescence and also reversed the quenching by ATP. Similar effects of aurovertin were found with SMP (Layton et al., 1973; Van de Stadt el al., 1974). Chang and Penefsky measured the binding of aurovertin to soluble F1 and calculated from their data that there were two aurovertin binding sites in the presence of ATP and a single site in the presence of ADP. They proposed that only one out of the two aurovertin binding sites is inhibitory. The KD of F1-aurovertin calculated from the fluorescence experiments was 0.52 p M in the presence of ATP and 0.07 pM on adding ADP to the F1-aurovertin complex (Chang and Penefsky, 1973). The F1-aurovertin KD, measured recently by Bertina et al. (1973), is comparable (0.06 p M ) to the KD in the presence of ADP reported by Chang and Penefsky. The higher aflinity of F1 to aurovertin in the presence of ATP explains the greater inhibition of ADP phosphorylation by aurovertin than the reversed reactions utilizing ATP. They showed also that aurovertin inhibits F1 by decreasing its V,,, and increasing its K , for ATP. Very similar results were found in the inhibition of mitochondrial reactions by aurovertin (Layton et al., 1973; Van de Stadt and Van Dam, 1974a, b) . They observed the quenching of the fluorescence of aurovertin bound to mitochondria by ATP and its enhancement by ADP. They speculate from these results that ATP causes attachment of the ATPase inhibitor, whereas ADP promotes its release, but there is little evidence to support this conclusion. In a recent paper it was demonstrated that the fluorescence of aurovertin bound to mitochondrial particles that contain the Pullman ATPase inhibitor was lower than that of aurovertin bound to inhibitor-depleted particles (Van de Stadt and Van Dam, 1974a). They suggest that the ATPase inhibitor causes some conformational changes in F1 so that it binds aurovertin with a lower quantum yield, a decreased aflinity, and an increased binding capacity. This is of interest, since the Pullman inhibitor is believed to act unidirectionally, inhibiting F1 activity in the direction opposite to aurovertin action. The finding that the fluorescence of aurovertin in solution is increased by glycerol or by exposure to low temperature (Layton et al., 1973; Chang and Penefsky, 1973), and is not affected by polar solvents, might indicate that any change in the fluorescence of the bound aurovertin may be the result of a change in the viscosity of the probe environment rather than a change in the polarity of the site.

MEMBRANE ATPorcs

109

c. ATPase Inhibitor (Pullman Inhibitor). A low molecular weight protein isolated by Pullman and Monroy (1963) specifically inhibits the ATPase activity of F1. They purified this protein from beef heart mitochondria and showed that it formed a stoichiometric complex with F1. Most of the ATPase activity in mitochondria is believed to be masked by this inhibitor and could be unmasked by either trypsin treatment or Sephadex filtration which result in removal of the inhibitor (Racker and Horstman, 1967). Yeast mitochondria yield a relatively small amount of inhibitor, and attempts to purify it have not succeeded (Tzagoloff, 1971a). There is as yet no evidence for an inhibitor protein in rat liver F1. The ATPase inhibitor blocks yeast F1, OS-ATPase complex, and also the ATPase activity of SMP (Pullman and Monroy, 1963). The inhibitor is a basic protein (Horstman and Racker, 1970; Knowles and Penefsky, 1972b) with the molecular weight reported to be 15,OOO (Pullman and Monroy, 1963) and later 10,500 (Brooks and Senior, 1971) or 5700 (Knowles and Penefsky, 1972b). The molecular weight of the chloroplast ATPase inhibitor is nearly the same (9500) (Nelson et al., 1972a). The F1-ATPase inhibitor complex is cold-stable; the inhibitor blocks ATPase activity but not its coupling activity (Pullman and Monroy, 1963). Horstman and Racker found that Mg2+ is needed for the binding of the inhibitor to FI (1970). Senior and Brooks (1970) suggested that the inhibitor is a subunit of F1, which is easily separated. They showed that the inhibitor is present in different amounts in different preparations of F1, the amount also varied with the preparative procedure for F1. Knowles and Penefsky (1972a, b) found that subunit 5 of F1is the inhibitor and isolated it from preparations of F1.According to their data, the inhibitor is a mixture of a monomer of 5500 daltons and dimer (mol wt of 11,300), and - S H compounds convert the dimer to monomer. Senior and Brooks (1971) claimed that Pullman inhibitor was not present in several different preparations of F1and that the only preparation that contained it was that of Horstman and Racker (1970), Heat activation of F, removes the ATPase inhibitor from F1 (Horstman and Racker, 1970). High pH and trypsin digestion also dissociate the inhibitor (Horstman and Racker, 1970), and gel filtration through Sephadex G-50 accomplishes similar separation (Senior and Brooks, 1970). All of the above treatments produce activation of the ATPase. Asami et a2. (1970) showed that ATPase inhibitor inhibits ATP-driven NAD reduction by succinate and ATPdriven transhydrogenase in EDTA particles in the presence of low oligomycin. By contrast, the energy-linked reactions driven by respiration, e.g., NAD reduction by succinate and nicotinamide nucleotide transhydrogenase coupled to succinate oxidation were un-

110

RlVKA PANET AND D. RAO SANADI

affected. Thus, it is of interest that the ATPase inhibitor blocks energy transfer reactions unidirectionally, and in the direction opposite to the effect of aurovertin. Van de Stadt et al. (1973) showed that the kinetic constants of F1 and of SMP are not changed by the ATPase inhibitor; the K , for ATP and Ki for ADP are not affected by the inhibitor, only the VmaXis decreased. They found that when the ADP/ATP ratio is high, the inhibition is diminished, which is in agreement with the idea that the inhibitor controls oxidative phosphorylation by inhibiting the backward reactions. When the ADP/ ATP ratio is increased, the inhibition decreased. d. Quercetin ( 3,3' ,4' ,6,7-Pentahydr0xyJlavone),Quercetin inhibits the mitochondrial, as well as the Na+-K+ transport ATPase (Carpenedo et al., 1969). It also inhibits both soluble and particulate mitochondrial ATPase at low concentrations but has no effect on oxidative phosphorylation in SMP (Lang and Racker, 1974). The mechanism of action of quercetin appears to be similar to that of Pullman inhibitor (see above). Both compounds inhibit ATPase activity of F1 and of SMP as well as ATPdependent NAD reduction by succinate in SMP, and both have no effect on oxidative phosphorylation. Based on the above results, the direction of quercetin inhibition is proposed to be the reversal of oxidative phosphoryl% tion, which is the same unidirectional inhibition as with Pullman inhibitor. 5. SPECIFICITY TOWARD NUCLEOSIDE DI-

AND

TRIPHOSPHATE

Soluble F1 loses its specificity to nucleotide triphosphate once it is removed from the mitochondrial membrane. The F1 (Pullman et al., 1960; Schatz et al., 1967) and Factor A (Andreoli et al., 1965) split all nucleoside triphosphates with the exception of CTP. Thus, ATP, ITP, and UTP are all cleaved by F1 at different rates. The K m for ATP of the yeast F1 is 0.25 mM (Schatz et al., 1967) or 0.5 mM (Sone et al.,1969), the K , of the beef heart enzyme is in the same range, 0.21-0.29 mM (Hammes and Hilborne, 1971) or 1.25 mM (Van de Stadt et al., 1973). The K , for ATP of the membrane-bound enzyme is not significantly different (0.1-0.38 mM) (Hammes and Hilborn, 1971; Van de Stadt et al., 1973). ADP inhibits competitively with ATP and is the only diphosphate that inhibits FI (Pullman e l al., 1960). The K i for ADP is 30-100 p M for FI (Mitchell and Moyle, 1970; Hammes and Hilborn, 1971; Van de Stadt el al., 1973) and 80-170 pM for the membranous enzyme. Selwyn (1967) showed that the Mg-ATP complex is the substrate for F1; free ATP and free Mg2+were inhibitors. Penefsky (1974a) has recently studied the interaction of the ATP analog, adenylyl imidodiphosphate (AMP-PNP) , with the mitochondrial ATPase

MEMBRANE ATParer

111

and showed that it inhibits competitively both the soluble F1 and the membrane-bound enzyme. The affinity of the enzyme is much higher for the analog than for its substrate, as judged by the K ; of AMP-PNP (0.33 p M for F1) compared to the K , for ATP (see above). The author showed that AMP-PNP inhibits also ATP-dependent energy-linked reactions. However, it does not affect the forward reactions of oxidative phosphorylation, i.e., the synthesis of ATP from ADP and P, linked to oxidation of succinate. In this regard the AMP-PNP resembles the Pullman inhibitor, which also inhibits the backward reactions such as ATP-dependent reduction of NAD by succinate, although only in the presence of rutamycin or oligomycin (see Section 11, B, 4). It is proposed, based on these studies, that F1 has two catalytic sites that are specialized, respectively, for ATP synthesis and ATP utilization. A similar suggestion has been made based on the effects of aurovertin on mitochondria (see Section 11, B, 4). 6. NUCLEOSIDE DI- AND TRIPHOSPHATE BINDING TO F1

It was reported by Zalkin et al. (196.5) that soluble F1 has no ADP but binds added ADP and ATP. ADP could be bound to F1 either by adding it directly or following ATP hydrolysis (Zalkin et al., 1965). In the latter case, the ADP binding needs Mg2+. The maximum binding was 2 moles ADP/mole of F1, when the Mg:ATP ratio was 2. Binding of added ADP did not require Mg2+, and the ADP-F1 complex was relatively stable and could be precipitated with 2 M ammonium sulfate (Zalkin et al., 1965; Catteral and Pedersen, 1972). Factor A, as isolated following ammonium sulfate precipitation and DEAE-cellulose chromatography, had 1.3 moles of bound ADP/340,000 gm of protein (Sanadi et al., 1971; Warshaw el al., 1967). Hilborn and Hammes (1973) measured the binding of ADP to F1 by equilibrium binding, which is probably a better assay than ammonium sulfate precipitation. They found two binding sites for ADP with different binding constants, 0.28 and 47 p M . Each site binds one ADP (Table 11). Hilborn and Hammes showed that one site needs Mg2+for ADP binding; its Ki without Mgz+ (in the presence of EDTA) is 11 pM compared to 0.28 pM with Mg2+.The other site is not affected by Mg2+;its Ki is 43 pM without Mg2+ and 47 p M in the presence of Mg2+ (see Table 11). The results of Hilborn and Hammes might explain some of the discrepancies. First, they explain why Zalkin et al. (1965) did not notice a difference in ADP binding to F1 by adding Mg2+: by adding an excess of ADP in their binding assay they apparently saturated both sites. The second discrepancy, namely, the higher KD found by Sanadi et at. (1971) and by Catterall and Pedersen (1972) is probably due to differences in experimental conditions. In agreement with the specific inhibition of FI-ATPase by ADP (Pull-

TABLE I1 DISSOCIATION CONSTANTS AND STEADY-STATE KINETICCONSTANTS FOR SOLWILIZED ATPasea Compound

ADP (+ Mg2+> ADP (+ EDTA) eADPb (+ Mg'+) cADPb (+ Caz+) SHDP" IDP SHTPe ATP ADP

ADP (+ Mg*+) ADP ADP

Ki

bM) 0.28

11

0.5 0.5 42

> 10 >40

-

0.92d 2.04d 16.6' 4.Y

Technique Gel filtration Ultrafiltration Fluorescent titration Fluorescent titration Absorbance difference spectrum titration Competitive ultrafiltration Competitive ultrafiltration

-

Equilibrium dialysis Ammonium sulfate precipitation Ultrafiltration Ammonium sulfate precipitation

Kz

W)

47 30 43 100-200 -

Technique Gel filtration Steady-state kinetics Ultrafiltration Estimated from gel filtration

-

.-2oo(i

Estimated from steady-state kinetics

-2000

Estimated from steady-state kinetics Steady-etate kinetics Steady-state kinetics

-

650 220

-

-

-

-

-

-

The experimental conditions are described in Hilborn and Hammea (1973). rADP is 3-&~-ribofuranosylimidazo(2,l-i)purine-5'-diphosphate. SHDP and SHTP are 6-mercapto-9-p-~-ribofuranosylpurine-5'-d~phosphate and -5'-triphosphate, respectively. dFrom Hilborn and Hammea (1973). 'From Sanadi et al. (1971). From Zalkin et d.(1965). b

113

MEMBRANE ATPasss

man et al., 1960), the binding of nucleoside diphosphate is also very specific. ADP and 3-~-~-ribofuranosylimidazo ( 2 ,1 4 ) -purine-5’-diphosphate (EADP) are the only diphosphates that bind to F1 in nearly equivalent amounts, whereas IDP, UDP, and AMP bind very little (see Table 11). Different nucleoside diphosphates compete with ADP for the binding, but their dissociation constant is higher (Hilborn and Hammes, 1973). Thus, it appears that the tight binding site (site 1) measured by Hilborn and Hammes is very specific, whereas the weak site (site 2) is less specific toward nucleoside diphosphates (see Table 11). Harris el al. (1973) have claimed that F1 in both soluble and membrane-bound form contains 3 tightly bound molecules of ATP and 2 of ADP per molecule. The nucleotides are not removed from the enzyme by standard physical techniques such as repeated precipitation, treatment with activated charcoal, or filtration through Sephadex and anion exchange resins, but they are removed by perchloric acid denaturation or cold inactivation in the presence of nitrate. Warshaw et al. (1967) also noticed that the bound ADP in Factor A is released by heat inactivation. In a recent paper, Rosing et al. (1974) found that adenine nucleotides (AdN) are released from F1 in parallel with cold activation. They suggest that the first product of cold inactivation still contains bound nucleotides but in a form removable by ammonium sulfate precipitation. They have proposed the following scheme for cold inactivation:

irreversible

00

subunits - AdN

F1 - AdN 30’

subunits

+ AdN

Although their proposal is attractive, more supporting data are needed. 7. DIVALENT CATIONS

Membrane-bound as well as soluble F1 requires divalent cation for maximal activity. The purified F1-ATPase activity shows complete dependency on Mgz+,but, in contrast to the ATPase activity of the mitochondrial membrane, other divalent cations such as Mn2+, Go2+,and Fe2+ could replace Mgzf with varying degrees of effectiveness (Pullman et al., 1960; Schatz et al., 1967; Selwyn, 1967). Stimulation of ATPase by D N P was observed only in the presence of Mg2+ (Pullman et al., 1960; Selwyn, 1967). The lack of specificity toward cations is true also for the yeast F1 (Schatz et al., 1967; Sone et al., 1969). Adolfscn and Moudrianakis (1973) found that the optimal concentration for Mg2+ is not dependent on ATP concentration, but the ATP concentrations that they used were below the K,.

114

RIVKA PANET A N D D. RAO SANADI

C. Molecular Properties

1. MOLECULAR WEIGHTAND SEDIMENTATION COEFFICIENT

Factor F1 from beef heart is globular with S20,wof 11.9 S (Penefsky and Warner, 1965; Schatz et al., 1967; Forrest and Edelstein, 1970) ; rat liver F1 has a sedimentation coefficient of 12.1-12.9 S (Lambeth and Lardy, 1971; Catterall and Pedersen, 1971) ; and the yeast enzyme is 11.6-13.0 S (Schatz et al., 1967; Tzagoloff and Meagher, 1971; Sone et al., 1969). The molecular weight of beef heart F1was first estimated to be 284,000 (Penefsky and Warner, 1965; Kagawa, 1969; Forrest and Edelstein, 1970), but Lambeth et al. (1971) obtained a value of 360,000 by gel filtration and high-speed sedimentation. The revised molecular weight is very similar t o that of rat liver F1 (384,000) by gel filtration and sedimentation on sucrose gradient (Catterall and Pedersen, 1971), or it is 360,000 by sedimentation equilibrium (Lambeth and Lardy, 1971). The yeast F1 has also roughly the same molecular weight, namely, 340,000 (Tzagoloff and Meagher, 1971). It appears that all F1 preparations from different sources are of nearly the same molecular size. Electron micrographs of mitochondria1 ATPase from several sources reveal spherical particles approximately 90 A in diameter with five to six symmetrically arranged subunits (Kagawa and Racker, 1966a; Racker and Horstman, 1967; Schata et al., 1967). 2. SUBUNIT STRUCTURE OF F1

In contrast to previous reports that F1has ten to twelve (Penefsky and Warner, 1965) or six (Datta and Penefsky, 1970; Forrest and Edelstein, 1970) identical subunits, electrophoresis in the presence of phenol-urea (Tzagoloff et al., 1968a; Sanadi et al., 1971) or SDS showed that the subunit composition is considerably more complex. Senior and Brooks (1970,1971) showed that the phenol-urea gels were prone to artifacts. From the summary of the results from different laboratories using SDS gel electrophoresis (Table 111),it is seen that F1 preparations from different sources have a uniform subunit structure, consisting of five to six distinct subunits. The similarities in the subunit structure of F1and Factor A has also been demonstrated (Sanadi et al., 1971). Subunits 1 and 2 are in the highest concentration and nearly equal in amount. They are close in molecular weight and have only a slight difference in amino acid composition (Knowles and Penefsky, 1972; Brooks and Senior, 1972). Knowles and Penefsky separated F1 subunits in bulk and determined their molecular weights by four different methods: (1) SDS gel electrophoresis, (2) amino acid

TABLE I11 SUBUNITSTRUCTURE OF MITOCHONDRIAL ATPase

FROM

DIFFERENTSOURCES' Subunit:

Source

Method

1

2

3

1. SDS electrophoresis 2. Gel filtration in 6 M guanidine 3. Amino acid composition 4. Equilibrium sedimentation

59,400

54,000

33,000

53,200

50,800

53,300

4

5

6

13,600

-

-

33,000

17,300

11,350

5,570

49,000

33,160

16,100

-

5,850

54,800 55,000

50,300 52,000

30,000

17,500 12,500

8,000

-

53,000 53,000 65,000

50,000 60,000

25,000 28,000 36,000

12,500 12,500

58,500

54,000

38,500

31,000

Beef heart Knowles and Penefsky, (1972b)

Capaldi (1973) R a t liver Senior and Brooks (1971) Lambeth and Lardy (1971) Catterall and Pedersen (1971)

-

10,500 8000-9000 -

7,500 -

Yeast Tzagoloff (1971a) a

Values shown are the molecular weights.

12,000

-

116

RIVKA PANET A N D D. RAO SANADI

analysis, (3) gel filtration, and (4) equilibrium sedimentation. In the determinations by the last two methods, high concentrations of guanidine or urea were present together with DTT (Knowles and Penefsky, 1972a) to keep the subunits in solution. The values by these methods were very similar. Subunit 1 with a molecular weight of 53,000 obtained by Lambeth and Lardy (1971) might be unresolved subunits 1 and 2. With subunit 4 the variations among different reports are larger: by SDS gel electrophoresis, values between 12,500-13,500 daltons were obtained with beef heart and rat liver, whereas the subunit 4 of yeast FI was much higher in molecular weight (see Table 111). Knowles and Penefsky (1972b) noted higher molecular weights for subunit 4 (16,000-1 7,500) with their other measurements compared to their SDS gel measurements. The molecular weight determination by SDS gel electrophoresis mobility is less accurate for the smaller peptides and might, thus, explain these variations. 3. STUDIESON

THE

ACTIVESITEOF F1 (AND CF1)

Soluble beef heart mitochondria1 ATPase contains a total of eight sulfhydryl groups and two disulfide bonds per 360,000 gm. Two of the sulfhydryl groups are freely accessible to iodoacetate or Ellman’s reagent (Senior, 1973). The ATPase activity is remarkably resistant to reagents that block sulfhydryl groups in proteins such as N-ethylmaleimide, mercurials, and Ellman’s reagent (Pullman et al., 1960; Senior, 1973). Penefsky (1967) studied the effect of low iodine on F1 activities and found that loss of ATPase activity was accompanied by disappearance of -SH groups. The iodinated F1 lost almost entirely the ability to bind adenine nucleotides but retained coupling activity with N particles. The inhibition of ATPase activity by iodine was not affected by subsequent treatment with - S H compounds such as DTT or mercaptoethanol. Thus, it would appear that IZ reacted with other groups, such as tyrosine (Penefsky, 1967), at the active center of the ATPase. The recent paper of Hilborn and Hammes (1973) shows that the -SH derivative of ADP, 6-mercapto9D-ribofuranosyl purine-5-diphosphate (SHDP), which is known to form disulfides with protein - S H groups, failed to label F1, in agreement with the conclusion that no - S H is present at the ATPase active center. It was shown by Senior (1973) that tetranitromethane and iodoacetate inactivated the F1-ATPase rapidly, and the inactivation was prevented by ATP. When the inactivation by tetranitromethane was complete, in the presence of ATP, a total of 9 to 10 tyrosine residues were nitrated; ATP reduced the amount of inactivation by half and the amount of tyrosine residues nitrated to 6-7. Based on the preceding results, Senior (1973)

MEMBRANE ATPases

117

suggested that tyrosine may be involved in the binding and hydrolysis of ATP. Ferguson et (11. showed that NBD-Cl, which is known to react with tyrosine and -SH groups (Gosh and Whitehouse, 1968), inactivated beef heart Fl-ATPase (Ferguson et al., 1974). From spectral properties of the reaction product, they suggested that the modified group is tyrosine and not 4 H . Bacterial ATPase (Nelson et al., 1974) as well as CFI (Deters et al., 1975) were also found to be inactivated by NBD-Cl. Xelson el al. and Deters et al. claimed that since the inhibition was completely reversed by DTT, the NBD-C1 reacts with -SH at the active site. They observed that the spectral data were not in agreement with a NRD-sulfur derivative but consistent with NBD-tyrosine; nevertheless, they claim that the microenvironment of the chromophore on the protein might change its spectral properties. From its chemistry, NBD-C1 is known to be more reactive with -SH than with tyrosine (Gosh and Whitehouse, 1968), but the spectral properties of NBD bound to ATPases from beef heart, chloroplast, as well as bacterial ATPase are consistent with NBD-tyrosine. The suggestion that -SH group is functional a t the active site contrasts with the finding that the ATPase is resistant to reagents that block -SH groups. It is, thus, necessary to examine how DTT relieves NBD-Cl inhibition. If, indeed, NBD-Cl reacts with -SH in ATPase, it should be possible to demonstrate this directly by -SH titration. D. Summary

I n the last few years, there has been some progress in unraveling the molecular and enzymatic properties of F1. The subunits of the enzyme have been isolated and their molecular weights and amino acid composition determined. Some additional information has become available on the amino acid(s) a t the active site. Isolation of the subunits of F1 holds promise for the elucidation of their role, although the major difficulty may be that they have been isolated in the presence of urea to keep them in solution. If they are denaturated, other methods may have to be devised for their isolation. It is still to be proved unequivocally that tyrosine is, indeed, the amino acid a t the active site and not -SH. It is not known on which subunit it is located. Studies with specific inhibitors, such as aurovertin, Pullman inhibitor, quercetin, and AMP-PNP show clearly that F1 may have two catalytic sites specialized, respectively, for ATP synthesis and ATP utilization. This raises the interesting possibility that the two opposite pathways could be controlled separately. This hypothesis might lead to the identification of two different active groups functional in opposite directions.

118

RIVKA PANET AND D. RAO SANADI

111.

OLIGOMYCIN-SENSITIVE ATPase

The OS-ATPase complex is firmly associated with the inner mitochondrial membrane and could be “solubilized” only by surface-active reagents. Detergents dissociate lipid-protein complexes and, therefore, different detergents produce complexes varying with respect to their phospholipid content. Tzagoloff has published excellent reviews on the properties of this complex (Tzagoloff, 1971a; Tzagoloff, 1973). A. Enzymatic Properties

1. OLIGOMYCIN SENSITIVITY

The OS-ATPase, isolated either from beef heart mitochondria (Kagawa and Racker, 1966a, b; Tzagoloff et al., 1968a, b; Kopaczyk et aZ.,1968) or from yeast (Tzagoloff, 1969a; Tzagoloff and Meagher, 1971), is sensitive to oligomycin, rutamycin, trialkyltin chloride, and DCCD, but FI is resistant. The oligomycin-binding site is probably located in the OS-ATPase complex. Beef heart mitochondria are more sensitive to oligomycin than yeast mitochondria. Twenty times higher concentrations of oligomycin are needed to inhibit the yeast mitochondria than the beef heart enzyme. The yeast OS-ATPase also is less sensitive to rutamycin than the beef heart enzyme reflecting the sensitivity of the mitochondria (Tzagoloff, 1969a). It has been reported that when yeast F1 is bound to beef SMP, the reconstituted hybrid ATPase is inhibited by oligomycin at the same concentrations that inhibit beef ATPase (Schatz et al., 1967). The degree of oligomycin sensitivity depends on the phospholipid composition (Bulos and Racker, 1968b) and amount (Tzagoloff, 1969a) of the complex (see also Sections 111, A, 4 and 111, C). OS-ATPase isolated from oligomycin-resistant yeast mutants could still be inhibited by oligomycin but the concentration required to give 50% inhibition increased thirty-fold compared to the wild-type OS-ATPase. This again reflects the sensitivity of the intact wild-type and mutant yeast cells (Broughall et al., 1973). The OS-ATPase complex isolated with Triton X-100 from yeast (Tzagoloff and Meagher, 1971) is inhibited at higher concentrations than the complex isolated by deoxycholate extraction (Tzagoloff, 1969a), presumably due to the presence of the detergent. The same phenomenon appears when an excess of phospholipid is added to the OS-ATPase complex, more oligomycin being needed for inhibition due to competition by excess lipid (Kagawa and Racker, 1966b; Tzagoloff, 1969a). Compounds DCCD, rutamycin, and tri-n-butyltin chloride (which are all

MEMBRANE ATPaser

119

believed to act at the same site or very close to each other in the overall scheme of ATP synthesis; see Fig. 1) inhibit OS-ATPase from beef heart (Tzagoloff et al., 1968b) or yeast (Tzagoloff, 1969a; Tzagoloff and Meagher, 1971). All the F1 inhibitors including aurovertin, Pullman inhibitor (Tzagoloff el al., 1968b) and antiserum against F1 (Tzagoloff and Meagher, 1971) inhibit the OS-ATPase also. 2. COLDSTABILITY The OS-ATPase is stable at 0°C in contrast to the cold lability of Fl (Tzagoloff et al., 1968b; Kopaczyk et al., 1968). Factor F1isolated from the complex shows the usual cold lability (Tzagoloff et al., 1968b; Kopaczyk et al., 1968).

3. NUCLEOTIDE AND DIVALENT CATIONSPECIFICITY The purified OS-ATPase complex exhibits enzymatic properties similar to those of SMP ATPase; it cleaves nucleoside triphosphates other than ATP. GTP and ITP are hydrolyzed at rates 26 and 17%, respectively, compared to that of ATP, whereas other triphosphates are cleaved at less than 50/, of the rate with ATP (Tzagoloff et aZ., 1968b; Kopaczyk et aZ., 1968). Thus, its specificity to triphosphate is more restricted than that of F1, which cleaves all triphosphates except CTP (see Section 11, B, 5). The OS-ATPase is stimulated by Mg2+ in the same manner as F1-ATPase; cation Mg2+could be substituted by Mn2+ (80%) or Go2+ (3oyO), whereas Ca2+ is almost ineffective. FrhTPase, on the other hand, cleaves ATP well when Mg2+is substituted by Ca2+ (Tzagoloff et al., 196813).

4. PHOSPHOLIPIDS AND THEIR POSSIBLE ROLE Phospholipids are essential components of the OS-ATPase complex and they markedly stimulate ATPase activity (Kagawa and Racker, 1966a; Bulos and Racker, 196813; Kopaczyk et al., 1968; Tzagoloff, 1969a), whereas F,-ATPase activity is not affected by phospholipids. The degree of activation by phospholipids depends on the intrinsic amount of phospholipids already bound to the particular preparation. Kagawa and Racker’s CFo-Fl preparation has very low phospholipids and almost no ATPase activity; activity could be induced by adding phospholipids (1966b). Kopaczyk et al. (1968) purified OS-ATPase with roughly 10% phospholipids from beef heart mitochondria and found that it could be activated up to ten-fold by adding phospholipids. Saturation was obtained with 30% phospholipids. The yeast OS-ATPase isolated using deoxycholate and salt precipitation is also markedly (ten-fold) stimulated by adding phospho-

120

RIVKA PANET AND D. RAO SANADI

lipids (Tzagoloff, 1969a). The beef heart OS-ATPase isolated by Tzagoloff et al. (1968b) has 30% phospholipids by weight and is not stimulated further by phospholipids. The OS-ATPase isolated from yeast mitochondria by a new purification using Triton X-100 (Tzagoloff and Meagher, 1971) is not stimulated by adding phospholipids although it contains only 10% phospholipids (in addition to the detergent present in the complex). It would appear that detergents are able to substitute for phospholipids in the OS-ATPase (Kagawa and Racker, 1966b). Kopaczyk et al. (1968) suggest that the activation of the ATPase by detergent is achieved by making the small amount of phospholipids in the complex more effective. The phospholipid requirement of the OS-ATPase is not specific. Different preparations of phospholipids from different sources, as well as fatty acids or detergents, could induce activity in the complex. In a recent paper, Cunningham and George (1975) showed that acidic phospholipids such as cardiolipin, phosphatidylglycerol, and phosphatidic acid activate the OS-ATPase to higher levels than do the neutral phospholipids such as lecithin and phosphatidylethanolamine. They measured the apparent K , for activation of different phospholipids and showed that it varied from 30 to 100 p M . No correlation was found between the relative affinity of the enzyme for phospholipids and the V , value (Cunningham and George, 1975). The oligomycin sensitivity of the complex, on the other hand, appears more dependent on the nature of the phospholipids (Kagawa and Racker, 1966b; Kopaczyk et al., 1968; Tzagoloff, 1969a). Cunningham and George (1975) could show that oligomycin is a competitive inhibitor with respect to all phospholipids tested except phosphatidylethanolamine and phosphatidylglycerol. It was proposed that ergosterol might have an important role in producing oligomycin sensitivity (Broughall et al., 1973). However, Griffiths and Houghton (1974) showed that there is no correlation between oligomycin resistance and the ergosterol concentration of yeast cells, yeast mitochondria, or mitochondria1 ATPase. Isolation of the proteolipid subunit that binds DCCD might give more information about the site of DCCD and oligomycin binding and the role of the lipid in the inhibition (see Section 111, C).

B.

Molecular Properties

1. MOLECULAR WEIGHTAND SIZE The OS-ATPme complex tends to aggregate if the bile salts are removed. By adding phospholipids to the complex, the amorphous aggregates are

MEMBRANE ATParer

121

converted to vesicular membranes. The fact that low concentrations of Triton X-100 maintain the yeast complex in a dispersed form has made it possible to estimate its molecular size (Tzagoloff and Meagher, 1971). The sedimentation of the complex and of F1 was carried out in a sucrose gradient containing 0.2% Triton. The s2Olw of the yeast complex was found to be 15.3 S and that of F1, 12.1 S. Knowing the phospholipid content and the specific volume of phospholipids, Tzagoloff and Meagher arrived a t a molecular weight of 520,000 for the complex and 340,000 for F1. Despite the limitations of this measurement, it shows that the complex is not very much larger than Fl. Electron microscopy revealed that the complex from yeast is an oval particle with dimensions of 100 X 150 A (Tzagoloff and Meagher, 1971 ; Kopaczyk et al., 1968). Tzagoloff proposed that a small unit, roughly 50-70 A, is attached to Fl to give the oval shape to the complex. 2. SUBUNIT STRUCTURE OF OS-ATPase The OS-ATPase isolated from beef heart (Tzagoloff et al., 1968a; Kopaczyk et al., 1968) and yeast mitochondria (Tzagoloff, 1969a; Tzagoloff and Meagher, 1971) consists of phospholipid and eight or nine different peptides of varying molecular weight. The protein subunits consist of three functional components: (1) F1; and ( 2 ) a membrane factor that is esscmtial for the oligomycin sensitivity; ( 3 ) a small prptide that has been claimed to act as a link between F, and the mrmbrane factor called OSCP (oligomycin sensitivity conferring protein) or F,. a. Factor 1 . The five subunits of Fl and OSCP are components, as shown by gel electrophoresis of beef heart (Tzagoloff et al., 1968a; MacLennan and Tzagoloff, 1968; MacLennan et al., 1968; Capaldi, 1973) and yeast (Tzagoloff and Meagher, 1971) complexes (see also Section 11,C, 2 ) . Extraction of submitochondrial particles with NaBr, which is known t o solubilize F1-ATPase, removes subunits 1 and 2 of the complexes (the major subunits of F1) completely; 3, 4, and 8 only partially; and subunits 5, 6, 7, and 9 that are specific to the complex are not extracted a t all (Tzagoloff and Meagher, 1971). b. OSCP. Compound OSCP is a peptide that, together with a membrane fraction, is essential for expression of oligomycin sensitivity. It is claimed to be the stalk connecting F1to the membrane piece seen in electron micrographs. It has been isolated from the beef heart OS-ATPase complex (MacLennan and Tzagoloff, 1968) and from SMP (MacLennan and Asai, 1968). The OSCP has been extracted from OS-ATPase by dilute ammonium hydroxide and purified to near homogeneity (MacLennan and Tzagoloff, 1968). Bulos and Racker (1968a) isolated from beef heart mitochondria a factor, which they denoted F, or FO1, with properties similar to those of

122

RIVKA PANET A N D D. RAO SANADI

OSCP. Later the F, was shown to be very crude (Senior, 1971) by SDS gel electrophoresis. It has also been purified from yeast mitochondria (Tzagoloff, 1970). The yeast OSCP appears almost identical with beef heart OSCP, although they are not fully interchangeable in hybridization experiments. The term OSCP can lead to misunderstanding; it does not confer oligomycin sensitivity to F, alone but only to a complex of F1and a membrane fraction (MacLennan and Tzagoloff, 1968; Bulos and Racker, 1968a). The complex has much less ATPase activity than the F1 used to prepare the complex. There is evidence indicating that the hydrolytic site in the complex is probably different from the active site of F1. The OSCP also stimulates the P,-ATP exchange activity of ammonia particles and phospholipid particles (MacLennan and Tzagoloff, 1968). Seen in the electron microscope, OSCP has a cylindrical shape, 50-55 A in length, 30-35 A in diameter, and tends to form tetrads (MacLennan and Asai, 1968). It is a strongly basic protein with an isoelectric point of 9.3. Its molecular weight is 18,000 by SDS gel electrophoresis (MacLennan and Asai, 1968; Senior, 1971). It may be identical with subunit 7 of OS-ATPase according to Tzagoloff and Meagher (1971), or to subunit 6, according to Capaldi (1973). The amino acid analysis of OSCP shows 17 lysine and 9 arginine residues per 18,000 molecule weight and only 1.3 histidine (Senior, 1971). c. The Membrane Factor. In addition to F1 and OSCP, the OS-ATPase complex of yeast or beef heart contains three or four other peptide subunits that together have been named the membrane factor (Tzagoloff, 1973) because of its low solubility in aqueous media and its solubilization with detergents (Tzagoloff and Meagher, 1971; MacLennan et al., 1968). The membrane factor is essential for the conferral of oligomycin sensitivity as was shown by the reconstitution of ATPase activity of mitochondrial membranes depleted of F1and OSCP (Tzagoloff, 1970; Bulos and Racker, 1968a). Table IV shows the subunits of yeast and beef heart OS-ATPase compared to F1 subunits. The five subunits of F1 and OSCP are present in both complexes together with at least three more subunits constituting the membrane factor. The molecular weights of the subunits of the membrane factor are quite similar in yeast (Tzagoloff and Meagher, 1971) and beef heart (Capaldi, 1973) (Table IV) . By double-labeling experiments, Tzagoloff et al. (1972) showed that subunits 1, 2, 3, 4, and 8 are common to F1and to OS-ATPase, whereas subunits 5, 6, 7, and 9 are present only in the complex. Subunit 7 is OSCP. Tzagoloff has shown that subunits of the membrane factor are synthesized by the mitochondrial protein-synthesizing system (Tzagoloff, 1969b; 1971b; Tzagoloff and Meagher, 1972; Tzagoloff et al., 1972). The studies on

TABLE IV SUBUNIT STRUCTURE OF OS-ATPase, F,,AND Yeast (Tzagoloff and Meagher, 1971)

Subunit No.

PROTEIN SYNTHESIZED BY MITOCHONDRIA~

Beef heart (Capaldi, 1973)

F1

OS-ATPaseb

Fi

OS-ATPaseb

58,500 54,000 38,500 31,000

58,500 54,000 38,500 31,000 29,000 22,000 18,500 12,000 7,500

55,000 52,000 30,000

55,000 52,000 30,000 29,000 20,000 19,000 12,500 10,000 8,000

-

12,000 a

THE

Values shown are the molecular weights. OSATPase, oligomycin-sensitiveATPase.

-

12,500

-

8,000

Mitochondria1 product Tzagoloff and Akai (1972)

(45,000)

29,000 21,000 12,000 7,000

Thomas and Williamson (1971) 48,000 33,000 28,000

15,000 11,000

124

RIVKA PANET AND D. RAO SANADI

the biosynthesis of mitochondrial ATPase have been reviewed recently (Tzagoloff, 1973) and will not be repeated here. We shall limit ourselves only to the structure of OS-ATPase complex, which can be understood better in the light of the experiments on its biosynthesis. By specificlabeling of the products of mitochondrial synthesis, Tzagoloff and Akai (1972) showed that five subunits are synthesized within yeast mitochondria (see also Table IV). The labeled products have molecular weights of 45,000 (major peak), 29,000, 21,000, 12,000, and 7,800. The 45,000-dalton subunit appears to be a polymeric form of the smallest subunit (7800 daltons) . By pretreatment with organic solvents or depolymerization with SDS under alkaline conditions, the 45,000-dalton subunit undergoes reduction in size and is converted to the 7800-dalton subunit (Tzagoloff and Akai, 1972). Thus, the authors have proposed that the major product of mitochondrial protein synthesis is a small peptide that resists depolymerization under the standard conditions used in SDS gel electrophoresis. This smallest peptide was shown in a later paper (Sierra and Tzagoloff, 1973) to be identical with subunit 9 of OS-ATPase complex. It was purified and its amino acid composition determined. It has an extremely large amount of nonpolar residues which may account for its solubility in organic solvents. In Table IV it may be seen that the four proteins synthesized by mitochondria in vitro (excluding the 45,000-dalton protein which is a polymer of the smallest subunit) have molecular weights close to the molecular weights of the membrane factor (Tzagoloff and Akai, 1972) present in the OS-ATPase complex. They could be identical with subunits .5, 6, 8, and 9. Subunit 8 probably contains two peptides of roughly the same molecular weight-one from F1 and one from the membrane factor. Except for subunit 9, which was shown to be the major product of mitochondrial protein synthesis and identical with the smallest subunit of the complex, it remains t o be established firmly that the other three minor proteins synthesized by mitochondria in vitro are identical with the three in the OS-ATPase complex. Thomas and Williamson (1971) found that five subunits are synthesized by the yeast mitochondria (see Table IV), some with molecular weights different from those reported by Tzagoloff. They noticed that the low molecular weight band is more sensitive to inhibition of mitochondrial synthesis than the others. In contrast to Tzagoloff’s results showing that one major product results from mitochondrial protein synthesis in yeast, isolated rat liver mitochondria synthesized different types of proteins (Kadenbach and Hadvary, 1973; Burk and Beattie, 1973). In agreement with Tzagoloff, Kadenbach and Hadvary (1973) and Burk and Beattie (1973) also found proteolipid

MEMBRANE ATPares

125

synthesis by rat liver niitochoridria. The 40,000-dalton protrolipid (Burk and Beattir, 1973) might also hr a polymeric form of Tzagoloff’s subunit 9. Capaldi (1973) has noted that beef hrart CFo-Fl (Iiagawa and ltackrr, 1966a, b ) contains a 45,000-dalton subunit that, is absent in Tzagoloff’s (1969a) beef heart complex. When th r rrlationship bct w e n th r polymrric and the monomeric forms of the mitochondria1 product is hettcr undrrstood, much of this ineonsistcncy and confusion might bc rrsolvrd. C. The Oligomycin and Dicyclohexylcarbodiimide Site of Action

The mechanism of ATPase inhibition by oligomycin and related compounds is still not understood. The insensitivity of F1to oligomycin suggests that the binding site of oligomycin resides in one or more of the protein subunits associated with OS-ATPase. Kagawa and Racker (1966b) found that radioactive rutamycin interacts with CFo and not, with F,. Bulos and Racker (1968a) showed that exposure to heat or trypsin produced loss of oligomycin sensitivity. They showed also that the DCCD site of action is in the submitochondrial particles, which are resolved in F1 and OSCP and contain the membrane factor. Later, more depleted SMI’ resolved in F,, FO1( = OSCP), and FCz were shown to interact with DCCD (Knowles et al., 1971). Experiments with yeast mutants resistant to oligomycin also showed that the resistance is controlled by the membrane factor. Hybrids of F1 either from oligomycin-resistant mutants or from the wild type produce an oligomycin-sensitive complex only with the wild-type membrane (Criddle et al., 1973). I n the last few years, there has been considerable progress in identifying more specifically the membrane subunit that interacts with DCCD. It seems that a low molecular weight protein with proteolipid characteristics (soluble in chloroform-methanol) synthesized by mitochondria is the site of DCCD action, and it has been suggested that this proteolipid also binds oligomycin (based on the assumption that the two inhibitors act a t the same site) (Cattel et al., 1971; Stekhoven et al., 1972). Cattel et al. (1971) purified a labeled proteolipid of 10,000 daltons from beef heart mitochondria which had been treated with labeled DCCD. The DCCD-binding protein was shown t o be a component of beef OS-ATl’ase complex by Stekhoven et al. (1972) who reported a molecular weight of 14,000. This proteolipid isolated from beef heart mitochondria or from the OS-ATPase complex appears to be similar to Tzagoloff’s subunit 9 (see Section 111, B, 2, c), but this remains to be shown directly. Cattel et al. (1971) found that cardiolipin is the major lipid in the purified proteolipid fraction using Sephadex LH2.

126

RIVKA PANE1 AND D. RAO SANADI

D. Summary

Considerable progress has been achieved in the last few years in defining the molecular properties of OS-ATPase. The OS-ATPase subunits were separated and their molecular weights were determined. On the other hand, there has been little progress in understanding the mechanism of oligomycin inhibition. The proteolipid shown to bind DCCD (and presumably also oligomycin) may be an important clue to explaining the mechanism of oligomycin inhibition. The oligomycin-resistant yeast mutant provides a good tool for the study of the mechanism of oligomycin action. Since no differences were found in subunit mobilities with the mutant on SDS gel and in lipid composition, the lesion would appear to be a small change that could not be detected by conventional methods. The isolation of the specific proteolipid that binds DCCD in the wild type from the oligomycin-resistant mutant should provide better understanding of the mechanism of DCCD inhibition. IV.

CHLOROPLAST COUPLING FACTOR 1

A. Reaction Catalyzed by Chloroplast Coupling Factor 1

1. COUPLING ACTIVITY AND ATP SYNTHESIS

Coupling factor 1 from spinach chloroplasts isolated by washing the chloroplasts with EDTA (Avron 1963) or by acetone extraction (Vambutas and Racker, 1965; McCarty and Racker, 1966) stimulates cyclic photophosphorylation in the presence of phenaeine methosulfate (PMS) (Avron, 1963) and noncyclic photophosphorylation in the presence of ferricyanide (Avron, 1963; Vambutas and Racker, 1965; McCarty and Racker, 1966) as electron acceptor. McCarty and Racker (1967) established the identity of the factors isolated by the two different methods (Avron, 1963; Vambutas and Racker, 1965). Using subchloroplast particles (SCP) sonicated in the presence of phospholipids or washed with EDTA, they showed that both the EDTA extract of Avron and their CFI (Vambutas and Racker, 1965) stimulated cyclic and noncyclic photophosphorylation. Chloroplasts are capable of forming a limited amount of ATP without illumination by changing the pH from acid (pH 4.0) to base (pH 8.0) (Jagendorf and Uribe, 1966). This reaction is inhibited by a specific antiserum against CFI (McCarty and Racker, 1966) which shows that CFI takes part in ATP synthesis by chloroplasts. The ATP-stimulated H+

MEMBRANE ATParer

127

uptake by chloroplasts in the dark is also inhibited by antiserum against C F I (McCarty and Racker, 1966). Direct evidence of the involvement of CF1 in H+ uptake by chloroplasts was produced by Lynn and Straub (1969a,b) who found that CF1 stimulated light-dependent H+ uptake by chloroplasts using ferricyanide or ubiquinone as electron acceptors. 2. I-’,-ATP EXCHANGE

One striking difference between oxidative phosphorylation and photophosphorylation systems is that the former is reversible. High rates of ATPase, P,-ATP exchange, and ATP-dependent, reversed electron transport can be readily observed in mitochondria1 particles. Attempts to demonstrate similar reactions in chloroplasts, on the other hand, have been unsuccessful. Special treatments are needed in order to activate the ATPase and P,-ATP exchange in chloroplasts. The P,-ATP exchange could be demonstrated in chloroplasts under some of the conditions that induce also ATPase (Carmeli and Avron, 1966). Brief illumination of chlorop1:rsts in the presence of high concentrations of D T T induced P,-ATP exchange activity which was linear for at least 20 minutes in the dark. The results were confirmed by McCarty and Racker (1968) who found that by increasing DTT concentration up to 20 mM, the light could be omitted and high rates of I-’,-ATP exchange could be induced in chloroplasts. The reaction in the presence of light may represent reversal of overall oxidative phosphorylation. Cation Mg2+ and PMS (Carmeli and Rvron, 1966) or pyocyanine (McCarty and Racker, 1968) that are needed for photophosphorylation are essential for the light-triggered P,-ATP exchange. Photophosphorylation inhibitors and uncouplers inhihit the light (and DTT) -triggered P,-ATP exchange. Atebrin, octylguanidine, and phlorizin (Carmeli and Avron, 1966), Dio-9, and NH4+ (McCarty and Racker, 1968) all inhibit the exchange if they are present during the exposure to light and DTT. From the foregoing results, it has been proposed that light exposes a high-energy intermediate to DTT and/or causes some conformational changes in the enzyme(s), inducing the reverse reactions and P,-ATP exchange. The role of CF1 in the P,-ATP exchange was examined by McCarty and Racker, who found that the light-DTT-induced exchange in chloroplasts is inhibited by an antiserum against CF1 (1968). Removal of CF1 from the chloroplasts that were illuminated in the presence of DTT, by washing with EDTA, inhibited the P*-ATP exchange. I t has not been shown yet that added CF1 stimulates or induces an exchange in deficient chloroplasts. Although the evidence is indirect, it could be concluded from these results

128

RIVKA PANET AND D. RAO SANADI

that CF1 bound to the chloroplast membrane participates in the Pi-ATP exchange. 3. Ca2+ AND Mgz+-ATPase ACTIVITY Chloroplast factor 1 could be isolated in a latent form with no ATPase activity and then its ATPase activity could be induced. This is similar to the latent ATPase or Factor A of mitochondria (Andreoli et al., 1965; Warshaw et al., 1967). Alternatively, the ATPase activity could be first induced on the membrane and then the active CF1-ATPase could be removed from the membrane. Illumination of the chloroplasts in the presence of --SH compounds activates the ATPase as well as the Pi-ATP exchange (Marchant and Racker, 1963; Hoch and Martin, 1963; Petrack et al., 1965; McCarty and Racker, 1968). ATPase activity could also be induced in the latent form of CF1 by incubating it with -SH compounds (McCarty and Racker, 1968) or by trypsin or heat treatment (McCarty and Racker, 1968; Vambutas and Racker, 1965; Farron, 1970; Farron and Racker, 1970). a. ATPase Activation by DTT. Extraction of chloroplasts with EDTA after illumination and DTT treatment yielded a soluble active ATPase (McCarty and Racker, 1968). On the other hand, extraction of untreated chloroplasts gave CF1 with low-ATPase activity that could be increased by incubation with DTT. Other -SH compounds such as thioglycerol and /3-mercaptoethanol similarly unmasked the ATPase of CF1 but were less effective than DTT (McCarty and Racker, 1968). The DTT-CFI isolated from activated chloroplasts, or isolated in a latent form and then activated, retains its coupling activity (see below). The DTT-CF1 binds to deficient chloroplasts, and by binding to the membrane its ATPase is inhibited. b. Heat and Trypsin Activation of CF1-ATPase. ATPase could be induced in the isolated CFl by a short period of heating in the presence of ATP which protects against complete inactivation (Vambutas and Racker, 1965; Bennun and Racker, 1969; Farron, 1970). Heat activation is also a property of F1-ATPase (see Section 11, A, 1 ) . Farron and Racker (1970) showed that treatment of chloroplasts with N-ethylmaleimide (NEM) or iodoacetamide prevents the heat or trypsin activation of CF 1-ATPase. When the enzyme was previously activated by heat or trypsin, its catalytic activity was not affected any further by NEM treatment. It may be concluded that some - S H group(s) are involved in the activation of the hydrolytic site but are not essential for the catalytic activity. By heat or trypsin activation, the enzyme loses its ability to rebind t o deficient chloroplasts (McCarty and Racker, 1968; Vambutas and Racker, 1965) and as a result loses its coupling activity (Vambutas and Racker, 1965).

MEMBRANE ATParer

129

This effect is different from the stimulatory effect of heat-activated Factor A on urea SMP (Andreoli et ol., 196.5). Heat treatment of CF1 in the presence of digitonin maintains its coupling activity. The presence of digitonin is essential during the heating (Nelson et al., 1972a). The heat activation of CF,-ATPase may explain the ten-fold increase in ATPase rates between 22" to 37°C observed by Karu and Moudrianakis (1969). They isolated the enzyme in a latent form with low-ATPase activity and, by incubation a t 37"C, apparently measured two phenomena-heat activation plus the normal higher rate at higher temperatures. Vambutas and Racker (1965) first showed that incubation of SCP with trypsin stimulated Ca2+ATPase. Later it was shown (McCarty and Racker (1968) that this trypsin treatment of the SCP caused also detachment of CFI from the chloroplast membrane. McCarty and Racker actually measured the activity of the soluble enzyme. For maximal ATPase activation in SCP, prolonged exposure and relatively large amounts of trypsin are needed (Vambutas and Racker, 1965). Lynn and Straub (1969a) showed that brief incubation of chloroplasts with trypsin results in marked activation of Mg-ATPase if the chloroplasts were previously illuminated. The light probably induces some conformational changes in the chloroplasts that expose the hydrolytic site of CF, for trypsin action (as well as for the DTT effect) and resultant ATPase activation. Anti-CF1 inhibited the aforementioned activity of the chloroplasts (Lien and Racker, 1971). McEvoy and Lynn (1973) showed that the incubation of CF1 with trypsin under conditions that activate the latent ATPase caused selective digestion. The a subunit was the one most susceptible to the trypsin digestion, whereas the p subunit was stable. When they incubated chloroplasts with trypsin following illumination and then removed the CF1 from the chloroplasts, the CF1 had all the normal subunits seen in the untreated CF1 as judged by gel electrophoresis. It seems from the above results that the effect of trypsin on CF, is different from its effect on CF1 attached to the chloroplast membrane. B. Enzymatic Properties

1. SPECIFICITY TOWARD DIVALENT CATIONS

Unlike Fl which requires Mg2+ for its activity either in the soluble or the membrane-attached form, soluble CF1 is preferentially activated with Ca2+, whereas the chloroplast membrane-bound ATPase requires Mg2+ (Vambutas and Racker, 1965; McCarty and Racker, 1968; Karu and Moudrianakis, 1969) ; Mg2+ inhibits the Ca2+dependent ATPase. A controversy arose and confusion ensured when Vambutas and Racker (1965) found that

130

RIVKA PANET AND D. RAO SANADI

heat or trypsin treatment of chloroplasts induced high Cazf-dependent ATPase activity, whereas the Mg2+-ATPase was much lower (Vambutas and Racker, 1965; Karu and Moudrianakis 1969). One should remember that photophosphorylation is Mgz+dependent and is inhibited by Ca2+. Treatment of chloroplasts with heat or trypsin, which is known to solubilize CF1 from the membrane, activates Ca2+-dependent ATPase (Vambutas and Racker, 1965), whereas illumination of chloroplasts in the presence of DTT activates Mg2+-dependentactivity (Carmeli and Avron, 1966; McCarty and Racker, 1968) and retains the CFI on the chloroplast membrane. Moreover, it was found that when the CF1-Mg2+-ATPase is extracted from chloroplasts previously illuminated in the presence of DTT, its soluble ATPase activity was about 6 times higher with Ca2+than with Mg2+ (McCarty and Racker, 1968). When latent CF1 is exposed to heat, trypsin digestion, or DTT, the resulting ATPase is Caz+-dependent under the normal assay conditions. Other divalent cations such as Ni2+, Mg2+, Mn2+, Co2+, and Sr2+ (at 10 mM) were less than 3% as effective as Caz+. The ATPase activity of the soluble enzyme is inhibited approximately 50% by 0.3 mM Mg2+ (Vambutas and Racker, 1965; Karu and Moudrianakis, 1969). The finding that brief exposure of preilluminated chloroplasts to trypsin yields Mg2+-dependentATPase (Lynn and Straub, 1969a) might merely indicate that under these mild conditions the CF1 is still attached to the chloroplasts membrane, but this theory remains to be proved. Nelson et al. (1972a) examined the assay conditions for the Mg2+-dependent ATPase and showed that under the right conditions, it is as high as the Ca2+dependent activity. The presence of carboxylic acids and optimal Mgz+ concentration were the main factors determining the rate of Mg2+ATPase activity. At low pH (pH 6.0), sodium maleate accelerated the Mg2+-dependent ATPase of CF1 up to 30 times higher, where@ at high pH (pH 8.0), bicarbonate was more effective than maleate as an activator. The optimal Mg2+concentration is 8 mM at low pH and only 2 mM at the high pH. Higher concentrations of Mg2+inhibited the reaction. The Mgz+-dependent ATPase activated by the carboxylic acids was comparable to the Ca2+-dependent activity. Activation of Mg2+-ATPase by carboxylic acids is in agreement with the finding that photophosphorylation is accelerated by these acids. 2. SPECIFICITY TOWARD NUCLEOTIDES

CF1-ATPase is most active with ATP; GTP and ITP were hydrolyzed at 25% of the rates compared to the rate with ATP, whereas CTP and UTP were ineffective (Vambutas and Racker, 1965).

MEMBRANE ATParer

131

The K , for ATP was found to be 0.4ri m M a t 37°C and 0.82 mM a t 22°C for the Ca2+-dependent activity (Karu and Moudrianakis, 1969) and 0.11 m M for the h!Ig2+-ATPase (Nelson et al., 1972a). Sodium maleate increases the VIn of the Mg2+-ATPase thirty-fold but decreases the affinity of CF1 to ATP by increasing the K , by a factor of 10 (Nelson et al., 1972a). The Mg2+-dependent activity showed the same nucleotide specificity as Ca*+-ATPnse; ATP was hydrolyzed 6 times more rapidly than GTP, ITP, or U T P (Nelson et al., 197%). ADI’ inhibits both Ca*+- (Vambutas and Racker, 1965; Nelson et al., 1972a) and Mg*+-ATPase (Nelson et al., 1972a). The inhibition of CF1-ATPase by ADI’ is specific; other nucleoside diphosphates such as IDP, GDP, and C D P are weak inhibitors (Vambutas and Racker, 1965). The ADP inhibition of CF1-ATPase surprisingly is not simply competitive with ATP; ADP altered the K,, and the V,,,,, for ATP. It changes also the saturation curve of ATP from hyperbolic to sigmoid shape, and the apparent reaction order from 1.0 to 2.3 (Nelson et al., 1972a). This interesting observation indicates that ADP has an allosteric effect on the CF1 enzyme.

3. BINDING OF NUCLEOTIDES BY CF1 Roy and Moudrianakis (1971a) found two ADP-binding sites on their 13 S coupling factor from spinach chloroplasts (which is analogous to CF1) with dissociation constants of 2 X 10-6M and 3.5 X 10-SM. The binding of ADP is slow and reaches maximum aftcr 1 to 2 hours. To release the bound ADP, perchloric acid, urea, or formamide are needed. The binding of ADP, like the inhibition of ATI’ase by ADP, is quite specific; other diphosphates do not compete with ADP. After binding ADP to CF1, nucleotides ADP, AMP, and ATP were detected as bound products on the enzyme. The AMP, although bound when it is produced from ADP, could not be bound directly to CFI even at high concentrations. Roy and Moudrianakis showed with double-labeling experiments that the enzyme is able to carry out a stoichiometric transphosphorylation reaction on its surface. They proposed that the ,&phosphate of one ADP is transferred to the P-phosphate of another ADP molecule in an adenylate kinase type of reaction. Whereas the AMP and ATP produced from ADP are loosely bound to the enzyme and could be recovered by gel filtration, ADP is tightly bound t o CFI, and only drastic treatments promote its release. They showed also that by illumination of chloroplasts, 3H AMP is converted to bound ADP and could be recovered in the CF1 isolated from these chloroplasts. The authors proposed that ADI’ is photosynthetically generated from

132

RIVKA PANET A N D D. RAO SANADI

AMP, Pi, and the energy derived from illumination. They claimed that ADP is the high-energy intermediate of photophosphorylation, and ATP and AMP are formed by an adenylate kinase-like reaction. Evidence has been presented that this preparation is free from contamination by the classic adenylate kinase. They showed also that the formation of ADP-CF1 complex is dependent on photoinduced electron transport, AMP, and Pi. The reaction is sensitive to arsenate and sulfate. The bound ADP exchanged with exogenally added ADP. Roy and Moudrianakis (1971b) propose the interesting mechanism shown in Fig. 2 for photophosphorylation. Yamamoto et al. (1972) found that ADP binding to the Rhodospirillum rubrum chromatophore is very tight, and there was no exchange between bound ATP and free ADP. The ADP is photophosphorylated to ATP. In addition to tightly bound ADP, they reported also a loosely bound ADP; binding is claimed to be oligomycin-sensitive. Consistent with the proposal of Roy and Moudrianakis, Forti et al. (1972) showed that isolated CF1 carries out incorporation of inorganic phosphate into the phosphate of ADP. The experiment involved addition of ADP and Pi to their purified to the @-phosphateof ADP. During CFI, which led to incorporation of 32Pi this incubation, ATP is also formed with the label in the @-phosphate.

+ AMP + Pi

1. MembraneSCF

//photoinduced

’I-+

ADP

2.

3.

Membrane * CF

electron transport

+ HzO ADP

/ADp MembraneaCF

\

ADP

I 4.

/AMP Membrane. CF

\ 5. MembranemCF

Membrane-CF

ATP

1 + ATP + AMP

+ AMP + Pi + ADP

light

Membrane .CF

+ AMP + ATP + HzO

FIG.2. Mechanism of ATP formation in chloroplasts. Roy and Moudrianakis (1971b).

MEMBRANE ATParer

133

However, they could not detect 32Piincorporation into the y-phosphate of ATI’. Their interpretation of the data is t,hat CFI preserved its high-energy state during the purification procedure. Their proposal for the photophosphorylation mechanism is very similar to that of Roy and Moudrianakis (see Fig. 2 ) . 4. COLDLABILITY

The ATPase activity of CF, is cold-labile whether the hydrolytic activity is induced by trypsin, heat (McCarty and Racker, 1966), or by DT T (McCarty and Racker, 1968). The coupling activity of CF1 is also coldlabile. It dissociates into inactive subunits in the cold, as shown by gel electrophoresis (McCarty and Racker, 1966). ADP and ATP protect CF1-ATPase from the cold inactivation (just as for F1-ATPase) . Untreated CFI acquires cold stability on binding to deficient subchloroplast particles. Lipids obtained from chloroplasts also confer some degree of cold stahility. However, the same subchloroplast particles do not confer cold stability to the heat-activated CF,-ATI’ase. I t would seem that heat- or trypsintreated CF,-ATPase loses its capacity to bind to the chloroplast membrane (Rennun and Racker, 1960). Cold inactivation of the enzyme renders the solution cloudy. Some degree of reactivation could be produced by dilution and warming. Addition of glycerol to the dilution medium to prevent nonspecific aggregation restores up to 65% of the original activity (Lien et aE., 1972). 5. INHIBITORS OF CF1-ATPase

a. Dio-9 and Phlorizin. McCarty et nl. (1965) found that Dio-9 inhibited oxygen evolution accompanying ferricyanide reduction in coupled chloroplasts in the presence of ADP, P,, and Mg2+.Only electron transport that is tightly coupled to photophosphorylation is inhibited by Dio-9. Chloroplasts uncoupled by NH4+ are not inhibited by it. Dio-9 inhibits both cyclic and noncycric photophosphorylation, but NH4+ relieves the inhibition (McCarty et aE., 1965). The site of 130-9 inhibition is believed to be the reaction(s) catalyzed by CFl based on the following findings: (1) Phosphorylation accompanying an acid-base transition is inhibited by Dio-9 (McCarty et al., 1965) (for the role of CFI in this phosphorylation, see Section IV, A) ; ( 2 ) Dio-9 inhibits membrane-bound ATPase that has been activated by DTT in the same manner as it inhibits the light-DTTtriggered Pz-ATP exchange; ( 3 ) Dio-9 inhibits the soluble ATPase activity of CF I induced by trypsin or by D T T (McCarty and Racker, 1968); ( 4 ) Dio-9 inhibits the Ca-ATPase more than the Mg-dependent activity

134

RIVKA PANET AND D. RAO SANADI

(Nelson et at., 1972a). The maximal inhibition of CF1-ATPase activity by Dio-9 is only SO%, whereas photophosphorylation is inhibited up to 80-90%. In general, Dio-9 is a more potent inhibitor of the particulate ATPase than soluble CF1-ATPase (McCarty and Racker, 1968). Based on the model of Dio-9 inhibition, McCarty and Racker (1968) proposed that CF1 catalyzed the last step of photophosphorylation, namely, the transphosphorylation to ADP: X P ADP + X ATP. This same reaction has been proposed for F1 in mitochondria. Phlorizin, a muscle phosphorylase inhibitor, inhibits cyclic and noncyclic photophosphorylation, as well as coupled electron transport, in the presence of Pi and ADP (Izawa et al., 1966). As in Dio-9 inhibition, uncouplers of photophosphorylation relieve phlorizin inhibition of the electron transport. In the absence of Pi and ADP, phlorizin does not inhibit electron transport in chloroplasts (Izawa et al., 1966). Like Dio-9, phlorizin inhibits DTTlight-induced Pi-ATP exchange and the ATPase of chloroplasts (Carmeli and Avron, 1966). Although more information is needed on the effect of phlorizin on isolated CF1, it would seem that it inhibits at the same site as Dio-9 does, which is the terminal reaction of photophosphorylation catalyzed by CFl. b. The CF1-ATPase Specific Inhibitor. Chloroplasts, in contrast to mitochondria, have no ATPase activity and catalyze no Pi-ATP exchange or reversed electron flow, under the conditions that permit photophosphorylation to occur. It appears, therefore, that in chloroplasts one of the phosphorylation steps is not readily reversed. It was suggested by several groups that in mitochondria the specific inhibitor of F1 modifies it in such a manner that reversed reactions with ATP are prevented, but the forward reactions with ADP are able to proceed (see Section 11, B, 4). Based on the preceding suggestion, attempts were made to isolate a specific CF1 inhibitor from isolated CF1, but these were unsuccessful. Moreover, no differences were detected between native CF1 and the heat-activated CF1-ATPase in amino acid composition (Farron, 1970). This finding implies that by inducing ATPase in CF1no protein is separated from CFI, but it is possible that an inhibitor is inactivated by the heat but not removed from CF1. Later, it was shown that, indeed, such an inhibitor exists firmly bound to CF1 and could be removed by heat treatment only in the presence of digitonin, followed by fractionation with Sephadex G-200 (Nelson et al., 1972b). The inhibitor is probably a subunit of CF1, as in the case of FI. In F1, it is readily dissociated, whereas the CF1 inhibitor is very hydrophobic and soluble only in the presence of urea or detergent. Heat, trypsin, and incubation with DTT probably remove the inhibitor from the active site of

-

+

+

MEMBRANE ATPases

135

CF1-ATl’ase. Like the mitochondria1 ATPasc-specific inhibitor (Pullman and Monroy, 1963), the CF, inhibitor is sensitive to trypsin, thus, explaining the ATPase activation by trypsin digestion. The inhibitor isolated by Nelson et al. (197%) was identified with the smallest of the five subunits of CF,; its estimated molecular weight is 13,000. It is specific to chloroplast A’I‘l’ase and does not inhibit other ATI’ases . c. Eflect of Photophosphorylation Uncouplers on CFl-ATPase. Few compounds are known that uncouple photophosphorylation. Among them are NH4+, which is the most popular, carhonylcyanide-m-chlorophenylhydr:tzone (CCP) , and n-butyl-3,5-diiodo-4-hydroxybenzoate. The NH4+ increases photohydrolysis of ATP in chloroplasts in the presence of lipoic acid (Petrack et al., 1965) up to 0.74mM and, in this concentration range, it inhibits ATP synthesis by chloroplasts. In 3 mM concentration, NH4+ inhibits photohydrolysis and photosynthesis of ATP (Petrack et al., 1965). The other uncouplers (CCP and n-butyl-3,5-diiodo-4-hydroxybenzoate) stimulate photohydrolysis to the same extent as NH4+. Increasing the concentration of all the uncouplers inhibits photohydrolysis. Vambutas and Racker (1965) showed that trypsin- and heat-activated CF,-ATPases are inhibited by the uncouplers NH4+, CCP, and n-butyl3,5-diido-4-hydroxybenzoate. They measured only the effect of high uncoupler concentration that inhibited also the ATPase of chloroplasts (McCarty and Racker, 1968). Since the ATPase was already activated, it is likely that the activation of ATPasc :it lower uncoupler concentrations was not seen. The inhibition of CF1-ATPase by uncouplers was no more than 50% compared to almost 100% inhibition of ATI’ photohydrolysis by chloroplasts (McCarty and Racker, 1968). d . Eflect of DCCD on Photophosphorylation and ATPase. It was found that the light-DTT-induced Mg2+-ATl’ase of chloroplasts is inhibited by DCCD in concentrations that inhibited photophosphorylation. However, the Ca2+-dependent ATI’ase activity of soluble CFI is resistant to DCCD (McCarty and Racker, 1967). C. Molecular Properties

1. MOLECULAR WEIGHTAND SIZE

The sedimentation value for CF1 measured in the analytical ultracentrifuge was 13.8 S in both the latent and active ATPase form. The molecular weight by this method wits 325,000 or 358,000 (Farron, 1970), which is very similar to that of F1 from different sources (see Section 11, C, 1 ) . The purified CF1 is a sphere, roughtly 90 A in diameter (Moudri-

136

RIVKA PANET AND D. RAO SANADI

anakis, 1964; Vambutas and Racker, 1965). By electron microscopy, these

90-A spheres appear attached to the outer membrane of SCP (Vambutas and Racker, 1965; Lien and Racker, 1971), and the resolved particles contain only few of the membrane-attached spheres. Silicotungstate, which solubilizes F1 from mitochondria, also removes CF1 from SCP. The treatment removes the 90-& spheres from the SCP (Lien and Racker, 1971). 2. SUBUNIT STRUCTURE OF CF1 In the presence of 5 M guanidine-HC1, the 325,000-dalton enzyme is dissociated into subunits of 62,000 daltons, whereas by amino acid analysis the minimal molecular weight is 28,000 (Farron, 1970). As in F1, the subunit structure of CF1 is more complicated. The existence of nonidentical subunits was first shown by immunoelectrophoresis which revealed the existence of at least two antigenically distinct subunits (Lien et al., 1972). By disc electrophoresis in the presence of SDS, the CF1 revealed two, major, slow-moving bands and three more rapidly moving bands that had not been seen in all the preparations of CF1. The molecular weights of the for the two major bands, and five subunits were 59,000(a) and 56,000 37,000 (y), 17,500 (a), and 13,000 (e), for the three minor bands (Nelson et al., 1973). A similar pattern was obtained by McEvoy and Lynn (1973) with their preparation of CF1 (Table V) . A comparison of the CF1subunits with the subunits of F1 is shown in Table V, the similarity is quite remarkable. Using urea and mercaptoethanol to dissociate the enzyme, Nelson et al. (1973) separated the five subunits of CF1 by DEAE cellulose chromatography. They determined their amino acid composition and prepared antibodies against each one of the five CF1 subunits, which provides a useful

(a),

TABLE V

MOLECULAR WEIQHTSOF CFI

Subunit 1 2 3 4 5 6

AND

Fl SUBUNITS

Nelson el al. (1973)

McEvoy and Lynn (1973)

Brooks and Senior (1971)

59,000 56,000 37,000 17,500 13,000

62,000 57,000 38,000 21 ,000 14,000

53,000 50,000 25,000 12,500 10 ,500 7,500

-

MEMBRANE ATParer

137

tool to study their role. They were able to extract the two low molecular weight subunits from the CF1using pyridine and to purify the CF1 inhibitor from the extract. The smallest subunit, 13,000 daltons, has been identified as a potent inhibitor of CFl-ATPase (Nelson et al., 1973), resembling the inhibitor of mitochondria1 ATPase (see Section 11, B, 4).

3.

I’OSSIBLE

ROLEOF CF1 SUBUNITS

The role of CF1 subunits has been examined using specific antibodies prepared against each one of the five subunits. a. The 6 and t Subunits. Nelson el al. (1973) found that CFl, after extraction with pyridine to remove the two smallest subunits, retained activity with only three subunits ( a , 0, y). Both the ATPase and the coupling activity were unaffected by removing the two smallest subunits. They proposed that the latter may have a regulatory role but are not essential for CF1 activity (Nelson et al., 1972b, 1973). b. The a, 0, and y Subunits. Nelson et al. (1973) have examined the role of these three subunits using specific antibodies to each one of them. Their results may be summarized as follows: (1) antibodies against the a and y subunits strongly inhibited photophosphorylation and light-triggered Mg-ATPase in chloroplasts when added separately; (2) anti-a inhibited also the stimulation of Hf uptake by AT]’ into chloroplasts; ( 3 ) the antibodies against a and 0 agglutinated subchloroplast particles; (4) none of the antibodies inhibited CF1-ATPase when added singly. However, a combination of a and y together inhibited the ATPase to the same extent as anti-native-CF,. From these results they conclude that the a and y subunits are involved in the coupling activity of CFI. The E subunit appeared to be the ATPase inhibitor which might play a regulatory role, but more data are needed to confirm this assumption. Considerable caution is warranted in interpreting data or activity changes obtained with antibodies to individual subunits. It seems quite likely that an antibody to a subunit could modify the activity of the complex by inducing configurational alterations in the entire complex although the subunit is not directly involved in the activity. In dealing with coupling activity, it has to be recognized that some of the subunits may be present in the membrane and capable of associating with the complementary subunit, although unavailable to the antibody. In a recent paper, Deters et al. (1975) claim that the a and 0 subunits are involved in CFL-ATPase activity (and not a and y as claimed by Nelson et al., 1973), based on the following effects of trypsin. a. They treated CFl with trypsin (free of chymotrypsin) and obtained a preparation that, according to amino acid analysis, appeared to contain a

138

RIVKA PANET AND D. RAO SANADI

mixture of a and j3 subunits, although they could not always separate the two. Anti-CF1 inhibited the activity of the trypsin-treated CF1 but antibodies against a and j3 subunits did not inhibit the ATPase activity, casting doubt on their conclusion that intact a and j3 subunits were present in the trypsin-treated preparation. b. They showed that NBD-Cl, which inhibits the ATPase of CF1 and of the trypsin-treated preparation, was incorporated into the B subunit of the enzyme. It seems a little premature to make any conclusions regarding the role of CF1 subunits from the above results, and probably the system is not so simple. McEvoy and Lynn (1973) have examined the effects of trypsin on the subunits of CFl and on ATPase activation, and found by SDS gel electrophoresis that the highest molecular weight subunit (a)is more susceptible to trypsin, whereas the j3 subunit is stable. The three minor subunits were almost unaffected. The mobility of proteins in SDS gel electrophoresis is not the most sensitive method to detect the small changes that could occur by tryptic digestion, and thus one cannot reliably conclude from their results that the hydrolytic site is on the a subunit. The Mg2+-ATPase, activated by exposure of chloroplasts to trypsin and recovered by EDTA washing, showed a normal pattern on SDS gel electrophoresis. According to Nelson et al. (1973), trypsin causes inactivation of the isolated CF1-ATPase inhibitor, but this has not been shown with the inhibitor attached to CF1. 4. THE--SH CONTENT OF CFl

A total of twelve -SH groups were titrated per mole CF1 after reduction. Only eight -SH groups out of the twelve are present if the reduction is omitted. Similar results were obtained with F1 (see Section 11, C, 3). The CF1-ATPase activity is not affected by blocking its -SH groups as in F1. Once the ATPase activity is induced by trypsin or heat, its catalytic activity is not affected by incubation with N-ethylmaleimide (NEM) or iodoacetamide (Farron and Racker, 1970). The -SH groups are probably involved in the ATPase activation of CFI, isolated or attached to the membrane (see Section IV, A, 3, a). The distribution of the -SH groups in the five subunits WM studied by McEvoy and Lynn (1973). After reduction with mercaptoethanol, they incubated the CF1 with NEM-W. The alkylated CFl was then analyzed by SDS gel electrophoresis for distribution of radioactivity. They found 9.75 -SH per mole CF1 (mol w t 325,000) in the a subunit, 3.25 in the B subunit, and only 1.35 in the y subunit. Based on the finding that photophosphorylation catalyzed by chloroplasts is inhibited by NEM only in light but not in the dark (McCarty

MEMBRANE ATPorer

139

et al., 1972), McCarty and Fagan (1973) showed that light markedly enhanced the radioactive NEM incorporation into the CF1 of chloroplasts previously incubated with unlabeled NEM in the dark. The CFl isolated from these treated chloroplasts showed that only the y subunit (out of the five subunits) contained most of the radioactivity. According to Nelson et al. (1973), the y subunit was one of the two subunits involved in CFl activity (see the previous section). D. Effect of light on Conformational Changes in Chloroplast Coupling Factor 1

There is considerable evidence in the literature suggesting that light causes conformational changes in the CF1 attached to illuminated chloroplasts. Lynn and Straub (1969a) showed that by illumination of chloroplasts, even a brief exposure t,o a small amount of trypsin activated the Mg-ATPase. This is in contrast to the long incubation with trypsin needed to activate ATPase in the dark (Vambutas and Racker, 1965; Lynn and Straub, 1969a). The ATPase activation by trypsin in illuminated chloroplasts is blocked by uncouplers, suggesting that trypsin may be acting on an energized “intermediate” or an energized state (Lynn and Straub, 1969a). lZyrie and ,Jagendorf (1971) have carried out experiments designed to look for conformational changes in chloroplasts associated with energy coupling. Their finding may be summarized as follows. 1. Chloroplasts were illuminated briefly in the presence of tritiated water, MgZ+,ADP, P,, and the electron carrier pyocyanine; then CF1 was removed from the membrane by EDTA washing in the presence of ATP to prevent cold inactivation. The protein isolated from illuminated chloroplasts was labeled, but that from chloroplasts kept in dark was not. The labeling of CF1 by tritium was very low despite the high specific radioactivity in the medium. 2. Tritium uptake occurred during illumination in the presence of pyocyanine (cyclic phosphorylation) or ferricyanide (noncyclic phosphorylation). It occurred also during the acid-base transition. They concluded that tritium uptake by CF1 is concomitant with the formation of a highenergy intermediate or state in the chloroplasts. 3. The ADP and P,, when added together, decreased the CF1 tritiation, but there was no effect when they were added separately, indicating that phosphorylation is not needed for CF, labeling. 4. Uncouplers (such as NH4+, CCP, hexylamine) and electron-flow inhibitors inhibited CF1 tritiation.

140

RIVKA PANET AND D. RAO SANADI

5. Dio-9 and phlorizin did not inhibit the incorporation of tritum into CFl in the presence or absence of ADP and Pa. They proposed that parts of the protein become exposed on illumination, get tritiated, and subsequently refold into regions of the molecule inaccessible to solvent hydrogen. From the foregoing results, it seems that the conformational state undergoing labeling occurs prior to the entry of Pi into the sequence, since it is not needed for the labeling. The labeling is also independent of the transphosphorylation to ADP since Dio-9 and phlorizin do not affect it. From the effect of uncouplers, it would appear that the conformational change permitting labeling is coupled to electron transport. The hypothesis that a conformational change is produced by light in CFI is consistent with the ideas about similar conformational changes in F1, based on the fluorescence of the F1-aurovertin complex. Also, ADP, ATP, and Pullman inhibitor all seem to produce conformational changes in F1 (on the membrane or in the isolated state), as indicated by their effect on the fluorescence of the F1-aurovertin complex (for details, see Sections 11, B, 4, b and c). E. Summary

Recently, the subunits of CF1 have been separated and their molecular weight and amino acid composition determined, but their specific role in ATPase or coupling is uncertain. There is general agreement that the subunit is a specific CF1-ATPase inhibitor. The confusion is regarding which subunit(s) are involved in the coupling activity, and which in the ATPase. The experiments with antibodies against each subunit might provide some clues when isolated CF1 is used and its activity without the membrane is examined. With membrane-bound CFI, however, antibody interaction could yield misleading results since accessibility to antigen could be impaired by the membrane. Labeling of the enzyme with the specific ATPase inhibitor could give direct information on the regulation of the active site of the enzyme. In a similar manner, the /3 subunit was labeled by NBD-C1 (Deters et al., 1975), and most likely it is the subunit carrying the active center of ATPase but this still needs to be confirmed. The isolation of CF1 preparations lacking one or more subunits would be a useful approach for determining their role (see Futai et al., 1974 and Section V, C) . The inhibitor of CFI by NBD-C1 has promise of yielding information on the location of the active center of CF1 and on the nature of the active group reacting with it.

141

MEMBRANE ATPases

The experiments demonstrating conformational changes in CFI during photophosphorylation are consistent with the recent hypothesis of Boyer (1973).

V.

BACTERIAL ATPace

This section deals with soluble ATPases from different bacteria, their enzymatic and molecular properties, comparison with the ATl’ases from mitochondria and chloroplasts, and their role on the membrane. Other aspects of bacterial energy conservation have been reviewed recently (Harold, 1972; Abrams and Smith, 1974; Cox and Gibson, 1974; Kovac, 1974). A. Reactions Catalyzed by Bacterial ATPase and the Use of Mutants

1. OXIDATIVEPHOSPHORYLATION A N D TRANSHYDROGENATION

It is now accepted that the bacterial RTPase is analogous to the ATPase in mitochondria and chloroplasts and that it catalyzes the terminal step in oxidative phosphorylation. Indeed, Rogin et al. (1970) have shown that coupling factors of mammalian and bacterial origin could be interchanged although the activities in the assay system were quite low. It was shown that ATPase isolated from Escherichia coli stimulated ATP-driven energydependent transhydrogenase reactions in particles deficient in ATPase (Bragg and Hou, 1972). Mutants defective in oxidative phosphorylation are just beginning to be used for defining the precise role of the ATPase and for analyzing other aspects of energy conservation. Direct evidence for the participation of ATPase in bacterial oxidative phosphorylation comes also from the recent isolation of E. coli mutants with greatly reduced levels of membrane ATPasc activity (Bultin et al., 1971; Cox et a!., 1971; Kanner and Gutnick, 1972a, b ; Scharier and Haddock, 1972; Rosen, 1973; Bragg and Hou, 1973). These mutants, although capable of oxidizing lactate, fail to couplo such oxidation to the phosphorylation of ADP (Bultin et al., 1971). The inhibition of bacterial ATPase by Dio-9 (Harold et al., 1969) and by azide (Munoz et al., 1969; Johansson et al., 1973; Hanson and Kennedy, 1973) also support the conclusion that this enzyme catalyzes the phosphorylation of ADP to ATP. Both Dio-9 and azide are known to inhibit soluble and membrane-bound yeast F1 (see Section 11, B, 4). Dio-9 inhibits also CF1 (see Section IV, B, 5). Vesicles from the mutants of E. coli with reduced levels of ATPase (Bultin et al., 1971; Kanner and Gutnick, 1972a;

142

RIVKA PANET AND D. RAO SANADI

Bragg and Hou, 1973) cannot carry out the ATP-dependent transhydrogenase reaction as in the parent strain. The vesicles, however, retain their ability to catalyze the transhydrogenase driven by respiration (Kanner and Gutnick, 1972a). These findings attest to the presence of the primary energy-transducing reaction coupled to respiration in the mutants, but the ability either to make ATP or to utilize ATP for energy-dependent reactions is lost. 2.

ROLEOF ATPase

IN

ACTIVETRANSPORT

ATPase is present in E. coli, grown either aerobically or anaerobically, and in Streptococcus faecalis which does not ordinarily derive metabolic energy from aerobic oxidative phosphorylation. I n both cases and in others as well, it appears to be the single major ATPase as judged by purification and inhibition studies (Hanson and Kennedy, 1973). The role of this ATPase in the transport of K+, amino acids, and phosphate was first suggested by the finding that an inhibitor of membrane-bound ATPase, DCCD, inhibits glycolysis-dependent uptake of K+ ions (in exchange for intracellular Hf or Na+) , alanine, and phosphate. Furthermore, Dio-9 and chloroheximide inhibit bacterial ATPase as well as net uptake of K+ by exchange for Na+ and Hf (Harold et al., 1969). It was proposed that the ATPase mediates between the ATP synthesized by the cytoplasm and the membrane to communicate energy for the membrane functions, such as active transport. The finding that the amount of membrane ATPase in S. faeculis nearly doubles when the organism is grown at K+ levels low enough to limit the growth rate, and that in these cells the rates of net K+ and *Rb uptake is increased during glycolysis (Abrams and Smith, 1971) support the idea that the bacterial ATPase plays a role in cation transport. Experiments of Pavaslova and Harold (1969) indicated that the E. coli ATPase might function in the active transport of galactosides also. They reported that in cells grown anaerobically, thiomethylgalactoside accumulation was blocked by uncouplers of oxidative phosphorylation, although ATP was present at normal levels and could be used for other reactions. Direct evidence for the involvement of ATPase in galactoside transport comes from the finding that mutants of E. coli lacking the Mg2+-Ca2+activated ATPase could not catalyze the active transport of galactosides (Scharier and Haddock, 1972; Rosen, 1973). In summary, it seems that the bacterial ATPase, like FI, catalyzes the last step of oxidative phosphorylation and is involved in the ATPdriven transhydrogenase activity and in the active transport of K+, amino acids, inorganic phosphates, and galactosides.

MEMBRANE ATParcr

B.

143

Enzymatic Properties

1. ACTIVATIONOF ATPase

The ATPase activity from different bacteria could be unmasked by trypsin (Ishikawa, 1966; Munoz et al., 1969; Milton et al., 1972), heat (Adolfsen and Moudrianakis, 1971b), or DNP (Gurraia and Peck, 1971). Many preparations exhibit considerable activity without the need for any unmasking (Abrams, 1965; Evans, 1969; Evans, 1970; Gross and Coles, 1968; Guarraia and Peck, 1971; Harold et al., 1969), presumably because the activation is produced during the isolation. In one case it is reported that ATPase can be isolated from E. coli in two forms-one needs trypsin activation and the other does not (Milton et al., 1972). The bacterial ATPase is cold-labile, like F1 and CFI, whereas the membrane-bound enzyme is cold-stable (Ishida and Mizushima, l969a, b ; Evans, 1970; Mirsky and Barlow, 1971) (see also Tahlt. V I ) . 2. SPECIFICITY TOWARD NUCLEOTIDEY

All purified ATPases isolated from different bacteria cleave purine nucleoside triphosphates with different degrees of effectiveness (see Tables VI and VII) (Abrams, 1965; Mirsky and Barlow, 1971; Hanson and Kennedy, 1973). The K,,, reported for Mg2+-ATP is in the range of 0.1 to 2.5 mM (see Table VI). The ATl’ases isolated from different hacteria are all inhibited competitively by ADP, with a K i= 0.7 mM for the S. faecalis ATPase (Schnebli and Abrams, 1970) (see also Table VI), and noncompetitively by Pi (Schnebli and Abrams, 1970; Hanson and Kennedy, 1973). CATIONS 3. MONO-AND DIVALENT Bacterial ATPase is completely dependent on divalent cations for its activity. Cation Mg2+ is the common activator (see Table VI), but Ca2f could replace Mg2+ for the ATPase from E. coli, Alcaligenes faecalis, Micrococcus lysodeikticus, and Bacillus megaterium. In the last two bacterial ATPases, the Ca2+-dependent activity is much higher than the Mg2+ATl’ase, reminiscent of CFI-ATPase. In these cases, Mg2+inhibited the Ca2+-ATPase (Munoz et al., 1969). The concentration curve for activation by Ca2+described by Munoz el al. (1969) with the M . lysodeikticus ATPase is sigmoidal, whereas with Mg2+it is bell-shaped. The ATPase isolated from Rhodospirilluin rubrum chromatophores is activated by Ca2+ alone, and Mg2+, Mn2+, CoZ+, or Zn2+ inhibit the Ca2+-dependent activity. The effects are similar to those seen with CFI (Johansson et al., 1973).

d

P P

TABLE

VI

COMPARISON OF ENZYMATIC AND MOLECULAR PROPERTIES OF ATPase Characteristics

E. coli

S . faeculis

MgZ+ or Mn2+, Activation by cations Mg2+ or Caz+, no activation by Na+ Caz+ ineffective or K+

M . lysodeikticus

ATP, GTP, ITP; ADP inhibits

K, for ATP is 0.1

ADP inhibits

Inhibitors

Nitrate, C1-, acetate, azide, Pi

Die9 and chlorohexidine

Azide

Cold 1ab;lity

Cold labile

No activation is needed

A . faecalis

Mg", Caz+, Cdz+; K+ stimulates at low ATP

ATP, GTP, UTP, K, for ATP is 2.5 mM

-

B. megaterium CaZ+ or less so Mg2+

ATP, GTP, ITP, K, for Mg-ATP is 0.29 mM

Latency of activity

DIFFERENT BACTERIA

CaZ+ and less Mg2+, Mg*+inhibits Caz+ ATPase, high Na-or K+ inhibit

Specificity toward nucleotides

-

FROM

mM

-

$ 3

3> Z

P

P

Cold stable Activation by trypsin

P

Cold labile Activation is needed

0

S value and molecular weight

S

Subunits

60,000 (a), 56,000 (19, 36,000 (Y), 13,000 (6) or 58,000 (a), 52,000 (81, 31,000 (Y), 31,000 (a), 20,000 (f)

EM shows hex-

Evans (1970); Davies and Bragg (1972); Hanson and Kennedy (1973); Bragg and Hou (1973); Futai et al. (1974)

Abrams (1965); Abrams and Baron (1967); Harold et al. (1969); Redwood et al. (1969); Shnebli et al.

Source

12.9; 365,000-390,000 Z = ~

S*

= 13.4;

s m = 14-13

385,000

agonal array

(1970)

>

2 100,000, 379,000

S%

= 13

z W

D

rn Z

EM shows 9O-.k

E M shows 100-d spere with central subunit encircled by 6 additional units. Two subunits: 62,000 and 60,000

Two subunits: 68,000 and 65,000 in equal proportion

Yamashita and Ishikawa (1965); Munoz et al. (1968, 1960) ; Milton et al. (1972)

Ishida and Adolfsen and Mizushima Moudrianakis (1969a,b); hlirsky (1971b, 1973) and Barlow (1971, 1973)

sphere

3 -0

2 2

146

RIVKA PANET AND

D.

RAO SANADI

TABLE VII SPECIFICITY OF E. Coli ATPase FOR METALSA N D NUCLEOTIDES~ Metal MIX* Mg* Mg* Mg* Mg" Ca2+ Co2+ Ni2+ Mn* None 0.

Nucleotide ATP GTP ITP UTP CTP ATP ATP ATP ATP ATP

Relative rates 100 61 33 11

3 108 19 14 5 0

Data from Hanson and Kennedy (1973).

Monovalent cations such as Kf or Na+ are known to activate membrane-bound ATPase (Hafkenscheid and Bonting, 1969; Adolfsen and Moudrianakis, 1971a). On removing the enzyme from the membrane, Kf or Na+ activation is lost (Hafkenscheid and Bonting, 1969; Davies and Bragg, 1972). High concentrations of Naf or K+ (100 mM) were found, in one case, to inhibit the ATPase (Munoz et al., 1969). Activation of ATPase by K+ occurs with the intrinsic ATPase activity, before activation by trypsin, and might represent a more natural property of the enzyme (Adolfsen and Moudrianakis, 1973).

4. INHIBITORS OF BACTERIAL ATPase Like CF1 and yeast F1, bacterial ATPase is inhibited by Dio-9 (Harold et al., 1969). Azide, which is known t o inhibit the mammalian and yeast FI, also inhibits bacterial ATPase (Munoz et al., 1968; Hanson and Kennedy, 1973). Oligomycin inhibits neither the membrane-bound nor the soluble bacterial ATPase (Sato et al., 1971). On the other hand, DCCD is a potent inhibitor of membrane-bound S. faecalis ATPase, but does not inhibit the soluble enzyme (Harold and Barda, 1969). The fact that the bacterial enzyme is released easily from the membrane provides a good tool for the study of the site of action of DCCD. Harold and Barda (1969) showed that when they released the ATPase from the previously inhibited S. faecalis membrane, the ATPase activity was restored. Conversely, sensitivity to DCCD was restored by reconstituting the ATPase membrane complex from the solubilized active ATPase and the depleted membrane.

MEMBRANE ATParer

147

From these results they concluded that the DCCD inhibits the bacterial ATPase indirectly by reacting with a membrane component(s) with which the enzyme is associated rather than with the enzyme itself. Similar results have been obtained with F1 and CF1 (see Sections 111, C and IV, €3, 5). The DCCD-binding membrane component(s) is probably a hydrophobic protein, since hydrophobic carbodiimides were more potent than the water-soluble carbodiimides (Abrams and Baron, 1970). The inhibition of a soluble ATPase preparation from E. coli by DCCD (Evans, 1970) might be explained by the fact that Evans solubilized the enzyme using detergent, which can extract membranous components. Abrams et al. (1972) isolated by direct selection S. jaeculis mutants resistant t o DCCD, and by reconstitut,ion experiments they showed that the sensitivity t o DCCD requires membrane ATPase. They showed that when the complex was reconstituted with mutant ATPase, mutant nectin (Baron and Abrams, 1971) , and wild-type-depleted membranes, the enzyme was normally sensitive to DCCD. However, when mutant membranes were used for reconstitution, the attached ATPase was insensitive to DCCD, indicating that the mutation affecting DCCD binding was on the membrane and not in the ATPase or nectin (Abrams et ul., 1972). Purified ATPase isolated from E. coli was found to he inhibited by NBD-Cl; this inhibition is reversed by DTT, and the diazole was found preferentially associated with the 0 subunit of the enzyme (Nelson et ul., 1974). From this finding the author concluded that there are -SH group(s) at the active site of E. coli ATPase, which is contrary to what has been accepted for Fl- and CF1-ATPases (for more details, see Section 11, C, 3 ) . C. Molecular Properties

Structurally the ATPase preparations from different bacteria closely resemble the F1 and CF1. I,n the electron microscope the enzyme appears nearly spherical with 100 A diameter, but it contains a hexagonal array of six subunits arranged around a central subunit (Munoz et al., 1968; Redwood et al., 1969; Schnebli et al., 1970) (see also Table VI). The enzyme is a highly acidic protein (Abrams and Baron, 1967). The sedimentation constant is in the range of 12.9 to 15.0 S for different preparations, and the molecular weight 360,000-385,000. The bacterial ATPase has been reported to contain five (Bragg and Hou, 1972; Bragg et al., 1973; Futai et al., 1974) or only four (Hanson and Kennedy, 1973; Nelson et al., 1974) nonidentical subunits. The preparation of Hanson and Kennedy (1973) consisted of only four subunits but had a low specific ATPase activity. Recently, Nelson et al. (1974) purified this enzyme to a

148

RIVKA PANET A N D D. RAO SANADI

homogeneous state with the highest specific activity reported so far and found also only four subunits. Preparations of E . coli ATPase with only four subunits lost their coupling activity, but the five subunit preparations retained the coupling activity. Futai et al. (1974) showed that the four and the five subunit enzymes have the same ATPase activity, but only the five subunit enzymes could stimulate ATPdriven transhydrogenase in deficient membranes. They suggested that the 6 subunit, which is missing in the four subunit preparation, is required for the binding of the enzyme to the membrane. The two large subunits (aand p ) appear to be sufficient for the ATPase activity of the soluble enzyme since three preparations of bacterial ATPase have been reported with only these two subunits (Schnebli et al., 1970; Milton et al., 1972; Mirsky and Barlow, 1973). Nelson et al. (1974) were able to convert by trypsin digestion the four-subunit ATPase to one with two subunits (a and p ) with retention of its activity. D. Comparison of Bacterial ATPase with FI and CF,

Bacterial ATPase resembles F1 and CF1 in its morphology and enzymatic and molecular properties. They all have latent ATPase activity that needs to be activated; their activity is cold-labile and DCCD-insensitive in the soluble form, and cold-stable and DCCD-sensitive when bound to the membrane. From an evolutionary standpoint, it is not surprising that the terminal ADP phosphorylation enzyme complex is quite similar to that in mammals, plants, and microorganisms. E. Summary

Despite a late start, studies on bacterial ATPase are quite advanced. The role of the enzyme in active transport and oxidative phosphorylation has been established using mutants lacking this enzyme. The use of different mutants offers a powerful tool in studying the role of the ATPase, its enzymatic and molecular properties, and its role in energy-linked reactions. At present, it would seem that the a and/or /3 subunits are needed for ATPase activity since some preparations of bacterial ATPase were reported to contain only these two subunits. But it has yet to be settled whether one or both are needed. The finding that the j3 subunit gets labeled by NBD-C1 (Nelson el al., 1973) probably locates the active site of the ATPase, but additional evidence would be needed before it can be accepted fully.

149

MEMBRANE ATPares

Very important is the finding of Futai et al. (1974) that preparations lacking the 6 subunit have no coupling activity, whereas preparations with this small subunit have full ATPase and coupling activities. Mutants resistant t o DCCD could be used to identify the membrane components that are needed for this sensitivity.

VI.

GENERAL CONCLUSIONS AND PERSPECTIVE

I n the intact organelle (or membrane system in bacteria) , the electrontransport process and ATP production are tightly coupled. Oxidation does not seem to occur unless the energy is utilized purposefully. There is evidence that the (nonphosphorylated) energized state can also be coupled directly t o processes such as membrane transport. Once ATP is formed, it does not seem t o be broken down (resulting in ATPase activity) unless the energy is utilized for a specific function. Thus, the high ATPase activity of membrane fractions derived from mitochondria are clearly artifacts. They are useful artifacts for designing experiments to understand some aspects of the mechanism of action of the enzyme system. The information obtained by examining the ATP (and other nucleotide) binding, regulation of the binding properties, and identification of the subunits concerned with the binding and their regulation, are bound to be of immense value. Progress on this aspect may be fast since the standard methods in enzymology and protein chemistry can be applied readily. However, the hydrolytic step or process is of little value, and, in fact, may be a hindrance, in understanding the mechanism of coupling of oxidative energy to ATP synthesis. To the best of our knowledge, the coupling process occurs in a hydrophobic environment where the hydrophobic site seen in F1 may not even exist, or if it exists, it may not be accessible to water and may well have quite a different conformation. In this connection, an interesting question to explore in the future may be the relationship between the different forms of the ADP phosphorylation enzyme, namely, FI, Factor A (with its low ATPase activity), and the membrane-bound state. Techniques for the fractionation of hydrophobic membrane proteins are currently being developed, and there is reason to be optimistic that rational methods may soon become available. However, the more challenging problem of how t o study these interactions in the hydrophobic membrane phase shows little progress. The next phase in research on ADP phosphorylation enzyme complexes will no doubt deal with the questions: How does the membrane ATPase “pick up” energy from the electron-transport complexes? What are the subunits acting in the coupling process? and

150

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How is ATP produced? Current detailed mechanistic studies on how ATP is broken down may conceivably have but little bearing on this synthet,ic process and should be interpreted with caution. ACKNOWLEDGMENT This work was supported by Grant No. GM 13641 from the National Institutes of Health. REFERENCES Abrams, A. (1965). The release of bound adenosine triphosphatase from isolated bacterial membranes and the properties of solubilized enzyme. J . Biol. Chem. 240,3675-3681. Abrams, A., and Baron, C. (1967). The isolation and subunit structure of streptococcal membrane adenosine triphosphatase. Biochemistry 6, 225-229. Abrams, A., and Baron, C. (1970). Inhibitory action of carbodiimides on bacterial membrane ATPase. Biochem. Biophys. Res. Commun. 41, 858-862. Abrams, A., and Smith, J. B. (1971). Increased membrane ATPase and K+ transport rates in Streptococcus fuecalis induced by K+ restriction during growth. Biochem. Biophys. Res. Commun. 44, 1488-1495. Abrams, A., and Smith, J. B. (1974). Bacterial membrane ATPase. I n “The Enzymes” (P. Boyer, ed.), Vol. 10, pp. 395-429. Academic Press, New York. Abrams, A., Smith, J. B., and Baron, C. (1972). Carbodiimide-resistant membrane adenosine triphosphatase in mutant of Streptococcus fuecalis. I. Studies of the mechanism of resistance. J . Bio2. Chem. 247, 1484-1488. Adolfsen, R., and Moudrianakis, E. N. (1971a). Kinetics characterization of oxidative phosphorylation in Alcaligenes faeca2is. Biochemistry 10,434-440. Adolfsen, R., and Moudrianakis, E. N. (1971b). Purification and properties of two coupling factors of oxidative phosphorylation from Alcaligenes faeculis. Biochemistry 10, 2247-2253.

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Sanadi, D. R., Sani, B. P., Fisher, R. J., Li, O., and Taggart, W. V. (1971). Soluble energy-transfer factors of the mitochondrial oxidative phosphorylation system. In “Energy Transduction in Respiration and Photosynthesis” (E. Quagliariello el al., eds.), pp. 89-107. Adriatica Editrice, Bari, Italy. Sato, V. L., Levine, R. P., and Neumann, J. (1971). Photosynthetic phosphorylation in Chlamydomonas reinhard. Effect of mutation altering an ATP-synthesizing enzyme. Biochim. Biophys. Acta 253, 437-448. Scharier, H. V., and Haddock, B. A. (1972). 0-galactoside accumulation in a Me*-, Ca+Z-activated ATPase deficient mutant of E. coli. Biochem. Biophys. Res. Commun. 48, 544-551. Schatz, G., Penefsky, H. S., and Racker, E. (1967). Partial resolution of the enzymes catalyzing oxidative phosphorylation. Interaction of purified mitochondrial adenosine triphosphatase from bakers’ yeast with submitochondrial particles from beef heart. J. Biol. Chem. 242,2552-2560. Schnebli, H. P., and Abrams, A. (1970). Membrane adenosine triphosphatase from Streptococcus faecalis. Preparation and homogeneity. J . Biol. Chem. 245, 1 1 15-1 121. Schnebli, H. P., Vatter, A., and Abrams, A. (1970). Membrane adenosine triphosphatase from Streptococcus faecalis. Molecular weight, subunit structure and amino acid composition. J. B i d . Chem. 245, 1122-1127. Selwyn, M. J. (1967). Preparation and general properties of a soluble adenosine triphosphatase from mitochondria. Biochem. J. 105, 279-288. Senior, A. E. (1971). On the relationship between the oligomycin-sensitivity conferring protein and other mitochondrial coupling factors. J. Bioenerg. 2, 141-150. Senior, A. E. (1973). Relationship of cystein and tyrosine residues to adenosine triphosphate hydrolysis by mitochondrial adenosine-triphosphatase.Biochemistry 12, 3622-3627. Senior, A. E. (1974). The structure of mitochondrial ATPase. Biochim. Biophys. Acta 301, 249-277. Senior, A. E., and Brooks, J. C. (1970). Studies on the mitochondrial oligomycininsensitive ATPase. An improved method of purification and the behavior of the enzyme in solutions of various depolymerizing agents. Arch. Biochem. Biophys. 140, 257-266. Senior, A. E., and Brooks, J. C. (1971). The subunit composition of the mitochondrial oligomycin-insensitive ATPase. FEBS (Fed. Eur. Biochem. Soc.) Lett. 17, 327-329. Sierra, M. F., and Tzagoloff, A. (1973). Assembly of the mitochondrial membrane system. Purification of a mitochondrial product of the ATPase. Proc. Natl. A d . Sci. U.S.A. 70, 3155-3159. Sone, N., and Hagihara, B. (1966). A coupling factor of oxidative phosphorylation without adenosine triphosphatase activity from beef heart mitochondria. J. Biochem. (Tokyo) 60, 622-631. Sone, N., Furuya, E., and Hagihara, B. (1969). Purification and properties of mitochondrial adenosine triphosphatase from yeast, Endomyces magnusii. J. Biochem. (Tokyo) 65, 935-943. Stekhoven, F. S., Waitkus, R. F., and Moerkerk, H. Th. B. V. (1972). Identification of the dicyclohexylcarbodiimide-bindingprotein in the oligomycin-sensitive adenosine triphosphatase from bovine heart mitochondria. Biochemistry 11, 1144-1149. Thomas, D. Y., and Williamson, D. H. (1971). Products of mitochondrial protein synthesis in yeast. Nature (London) 233, 196-199. Tzagoloff, A. (196%). Assembly of the mitochondrial membrane system. Characterization of some enzymes of the inner membrane of yeast mitochondria. J. Biol. Chem. 244, 50204026.

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Tzagoloff, A. (196913). Assembly of the mitochondrial membrane system. Synthesis of the mitochondrial adenosine triphosphatase, F,. J. B i d . (‘hem. 244, 5027-5033. Tzagoloff, A. (1970). Assembly of the mitochondrial membrane system. Function and synthesis of the oligomyciii sensitivity-conferring protein of yeast mitochondria. J . Biol. Chem. 245, 1545-1551. Tzagoloff, A. (19714. Structure and biosynthesis of the membrane adenosine triphosphatase of mitochondria, Ciirr. Top. M r m b r . Trump. 2, 157-205. Tzagoloff, A. (1971b). Assembly of the mitochondria1 membrane system. Role of mitochondrial and cytoplasmic protein synthesis in the biosynthesis of the rutamycin-sensitive adenosine triphosphatase. J . Biol. Chem. 246, 3050-3056. Tzagoloff, A. (1973). Biosynthesis of niitochondrial enzymes. Biochim. Biophys. Aclu 301, 71-104. Tzagoloff, A., and Akai, A. (1972). Assembly of the mitochondrial membrane system. Properties of the products of mitochondria1 protein synthesis in yeast. J . B i d . Chem. 247, 65174523. Tzagoloff, A., and Meagher, 1’. (1971). Assembly of the mitochondrial membrane system. Properties of a dispersed preparation of the rutamycin-sensitive adenosine triphosphatase of yeast mitochondria. J. Biol. Chem. 246, 732&7336. Tzagoloff, A., and Meagher, P. (1972). Assembly of the mitochondrial membrane system. Mitochondria1 synthesis of subunit proteins of the rutamycin-sensitive adenosine-triphosphatase. J . Biol. Cheni. 247, 594403. Teagoloff, A., MarI,ennan, D. H., and Byington, I<. H. (1968a). Studies on the mitochondrial adenosine triphosphatase system. Isolation from the oligomycin-sensitive adenosine-triphosphatase complex of the factors which bind F1 and determine oligomycin sensitivity of bound F,. Biochemistry 7, 1596-1602. Tzagoloff, A , , Byington, I<. H., and MacLcnnan, 11. H. (1968b). Studies on the mitochoiidrial adenosine triphosphatase system. The isolation and characterization of an oligomycin-sensitive adenosine triphosphatase from bovine heart mitochondria. J . Rial. Chem. 243, 2405-2412. TzagolofT, A., Akai, A., and Sierra, M. F. (1972). iZssembly of the mitochondrial membrane system. Synthesis and integration of F1 subunits into the rutamycinsensitive adenosine triphosphatase. J . Biol. Chem. 247, 651143516. Vambutas, V. K., and Racker, E. (1965). Partial resolution of the enzymes catalyzing photophosphorylation. Stimulation of photophosphorylation by a preparation of a latent, Ca++dependent adenosine triphosphatase from chloroplasts. J . B i d . Chem. 240, 2660-2667. Van de Stadt, It. J., and Van Dam, I<. (1974a). The equilibrium between the mitochondrial ATPase (F,) and its natural inhibitor in submitochondrial particles. Biochim. Biophys. Acta 347, 240-252. Van de Stadt, R. J., and Van Dam, I(.(1974b). Binding of aurovertin in phosphorylating submitochondrial particles. Biochim. Biophys. Actu 347, 253-263. Van de Stadt, It. J., De Boer, B. I,., and Van Dam, K. (1973). The interaction between the mitochondrial ATPase (F,) and the ATPase inhibitor. Biochim. B i o p h p . A d a 292, 338-349. Van de Stadt, R. J., Van Dam, I<., and Slater, 15. C. (1974). Interaction of aurovertin with submitochondrial particles, deficient in ATPase inhibitor. Biochim. Biophw. A c h 347, 224-239. Warshaw, J. B., Lam, K. W., Nagy, B., and Sanadi, D. R. (1967). Studies on oxidative phosphorylation. Latent adenosine triphosphatase activity of factor A. Arch. Bwchem. Biophys. 123, 385-396.

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Yamamoto, N., Yoshimura, S., Higuti, T., Nishikawa, K., and Horio, T. (1972). Role of bound ADP in photosynthetic ATP formation by chromatophores from Rhodospirillum rubrum. J. Biochem. (Tokyo) 7 2 , 1397-1406. Yamashita, S., and Ishikawa, S. (1965). A heahtable protein coupling factor from Micrococcus lysodeiklicus. J. Biochem. (Tokyo) 57, 232-234. Zalkin, H., Pullman, M. E., and Racker, E. (1965). Partial resolution of the enzymes catalyzing oxidative phosphorylation. Formation of a complex between coupling factor 1 and adenosine diphosphate and its relation to the 1.C-adenosine diphosphate-adenosine triphosphate exchange reaction. J . Biol. Chem. 240, 4011-4016.

Competition, Saturation, and Inhibition-Ionic Interactions Shown by Membrane Ionic Currents in Nerve, Muscle, and Bilayer Systems* ROBERT J . FRENCH AND WILLIAM J . ADELMAN, J R . Laboratory of Biophysics, IR P National Institute of Neurological and Communicative Disorders and Stroke National Institutes of Health, Department of Health, Education and Welfare Marine Biological Laboratory Woods Hole, Massachusetts

I. Perspectives. . . . . . . , . . . . . . . . A. Adsorption and Enzymes. . . , , . . . . . . B. The Independence Principle . . , , , . . . . . 11. Saturation Phenomena , . . . . . . , , , . , A. Channels in Lipid Bilayer Membranes , . . . . , . B. Divalent Ion Currents . . . . . . . . . , . C. Do Nerve Axon Currents Show Saturation Behavior? . . 111. Blocking and Competition . . , . , . . , . . . A. Blocking of Divalent Ion Currents . . . . . . . . B. Blocking of Nerve Axon Currents , . . . . . . , C. Anionic Currents . . , , , , . , . . . . . IV. Models and Analyses . , . . , . . . . . . . . A. Single-File Diffusion Theory . . , . . . . . . . B. Single-Occupancy Models . , . . . . . , . , C. Qualitative Models-Molecular Architecture of Ionic Channels V. Concluding Remarks . . . . . , . . , . . . . References , . . . . . , . . . . . . . . .

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185 166 166 167 168 169 169 171 184 189 189 192 197 198 200

PERSPECTIVES

Ionic transport processes in living cells may be broadly subdivided into two classes: those in which the transported species flows down the gradient of its own electrochemical potential, and those for which there is a n

* This article was prepared a t Woods Hole, Massachusetts and Bethesda, Maryland, with sponsorship of the National Institutes of Health. Reproduction of a single copy for the purposes of the United States Government is permitted. 161

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immediate requirement for metabolic energy. Systems belonging to the latter class would cease to transport as soon as the immediate energy source (ATP, for example) was depleted. The ionic currents that flow during action potentials in excitable membranes seem to belong exclusively to the first category (Taylor, 1974; Ulbricht, 1974) even though they are ultimately dependent on energy-requiring pump systems that maintain the ionic gradients across the membrane of the living cell (e.g., Hodgkin and Keynes, 1955a; Schwartz et al., 1972). This discussion will focus on the passive systems responsible for excitability in nerve and muscle membranes and the analogies that may be drawn with some lipid bilayer systems. No attempt will be made to present an exhaustive bibliography of the literature on ionic interactions. Rather, we will mention selected papers that will serve to outline the chronological accumulation of experimental evidence documenting ionic interaction effects on transmembrane currents and we will discuss in somewhat more detail recent papers that we feel are of particular interest. Various nonlipid components added to lipid bilayer membranes enhance their ability to conduct an ionic current under the influence of a transmembrane electric field. Structural and functional properties are consistent with the idea that some of these substances form pores or channels through the membrane lipid whereas others act as carriers, complexing ions and then diffusing across the lipid phase (see Iirasne et al., 1971; Jain, 1972, Chapter 6; Lieb and Stein, 1974a, b). Presumably it is also the nonlipid molecules that confer upon biological membranes their selective permeabilities to different ions (Bretscher, 1973). I n the case of excitable membranes, conductances are voltage-dependent, a property that has been attributed to a voltage-controlled gating process that opens and closes the conducting channels. Both excitable (Armstrong, 1969; Hagiwara and Takahashi, 1967; Hille, 1975; Woodhull, 1973) and nonexcitable (Hladky and Haydon, 1972a) systems have yielded experimental data that can be plausibly interpreted as due to competition between ions for occupancy of a channel or a site within the channel. Some other cases of inhibition of ionic currents in excitable systems seem to be caused by an interference with the normal gating process rather than competition for the conducting pathway itself (Begenisich and Lynch, 1974). Perhaps the simplest type of ionic interaction is that in which ions of a single species compete with each other for a given site. If only a finite number of sites is available, there will come a point, as one increases the concentration of ions, at which all sites are continuously occupied and one can increase neither the fraction of bound sites nor the rate of the process occurring at the site by increasing the concentration of ions. The rate

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process has then saturated with respect to concentration. However, if there is another component to the driving force of the reaction, such as the voltage or temperature in the case of ionic diffusion through putative channels, it may be possible to speed up the process by manipulation of one of these other factors. Even so, the process may be termed “saturating” with respect to increasing concentration. If ions of more than one species can occupy the same site, but only one ion can occupy the site a t a given instant, then there will be competition for the site among the species. For a conducting channel in which two ionic species can bind a t a site in the channel, but only one can pass through, such competition will result in an inhibition of the ionic currents across the membrane (Adelman and Senft, 1966; Armstrong and Binstock, 1965; Chandler and Rfeves, 1965a, b ) . Blocking of this type may be voltagedependent to such an extent that a region of negative resistance is produced in the current-voltage relation (Adelman, 1971; Bezanilla and Armstrong, 1972). Already, valuable information about the nature of the c*onducting channels has been deduced from the systematic study of competitive ionic bloc.king. Within the framework of a well-defined model, one may estimate the position of the blocking site and obtain a measure of the affinity of the blocking ion to the site (Woodhull, 1973; Strichartz, 1973).One would hope that, in future, sequences of binding affinities obtained for various ions might allow deduction from selectivity theory (Diamond and Wright, 1969; Eisenman, 1961, 1965) of a mow detailed picture of the electrostatic nature of the binding site. The various ionic. species that have marked effects on the magnitude of ionic currents across excitable mrmbranes frequently modify the time course of conductance changes. Such modifications can arise in a t least two ways. The reaction of the modifying ion with the channel may be essentially instantaneous and lead to a change in the speed of the gating process (Begenisich and Lynch, 1974), or the rraction rate of the modifying ion with the site might be the rate-determinirig step (Armstrong, 1969; Vierhaus and Ulbricht, 1971; Schwarz el al., 1973). I n other instances the mechanism by which time-course changes occur is not clear and may provide a fruitful subject for further experiments. A number of theoretical approaches to competition and saturation have been applied with some success to experimental data from bilayer, nerve, and muscle preparations. These analyses have aImost invariably been based on reaction rate theory. We will compare the details of some of these approaches and discuss the restrictions on th r models that are demanded by the cxperimental data from various systems.

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A. Adsorption and Enzymes

Molecular processes showing saturation properties have long been of interest, Michaelis and Menten ( 1913) derived their well-known expression for the rate of an enzyme reaction as a function of substrate concentration assuming that the reaction forming the enzyme-substrate complex was a t equilibrium. An equation of identical form may be obtained using the more general assumption of a steady state. In this case, it is assumed only that the concentration of the enzyme-substrate complex is not changing with time, and the only difference is that there is a slight change in the meaning of the estimated “dissociation constant’’ for the enzyme-substrate complex. This modification, suggested in a note by Briggs and Haldane (1925) , is discussed together with a number of other elaborations of enzyme kinetic theory by Dixon and Webb (1958). The fraction of sites occupied when molecules are adsorbed onto a surface may also be described by an expression analogous t o the Michaelis-Menten equation. Indeed, the Michaelis-Menten view of an enzyme reaction is essentially that of a first-order dissociation of the enzyme-substrate complex, proportional in rate to the fraction of enzyme molecules accommodating an “adsorbed” substrate molecule. In the following equation, f may be interpreted as the fraction of occupied sites, if applied to an adsorption process, or as the fraction of maximum reaction velocity attainable by varying substrate concentration in the presence of a fixed enzyme concentration, if applied to describe the rate of an enzyme-catalyzed reaction :

CSl =

[S]+ K

Here [S] represents the concentration of the adsorbed or reacting species, and K is the dissociation constant. The problem of adsorption from a gas was first tackled by Langmuir (1916), and adsorption from both liquids and gases was discussed by Adam (1941). In considering experimental data on transmembrane ionic currents, different authors have used the notions of ionic flux as a directed, ongoing chemical reaction of the ions with a limited number of membrane sites and of adsorption of the ions on to sites a t the surface as a necessary step in the transport process. These views immediately call to mind the ideas of Michaelis-Menten kinetics and Langmuir adsorption. Treatments of experimental data from both points of view show many similarities, but subtle differences in the deductions that one may make about the structure of ionic channels also emerge.

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IONIC COMPETITION IN MEMBRANE CHANNELS

6. The Independence Principle

I n their historic analysis of the ionic currents across the squid axon membrane, Hodgkin and Huxley (1952) showed that the observed dependence of the magnitude of the early inward current on the external sodium concentration could be described under the assumption that the probability of any individual ion crossing the membrane in a given time interval was independent of the presence of any other ions. They derived an expression, which we present here in a slightly more general form, for the relat,ive currents a t different sodium concentrations:

IN:INS

-

[NaJ’ - [Nali’ exp(FE/RT) [Nalo - [Nali exp(FE/RT)

Here IN^ and INa‘ represent the contribution of Naf ions to the current at the different concentrations, and E is (Ei- E 0 ) ,the potential inside the axon with respect to the external solution. As written here the expression allows for variation of both internal and external concentrations. As Hodgkin and Huxley pointed out, the coincidence of the observed currents with the independence principle predictions does not place very stringent restrictions on the possible physical mechanisms of transport. Such diverse schemes as the “constant field” electrodiffusion system first discussed by Goldman (1943) and a carrier mechanism (provided, in the latter case, that only a small fraction of the carrier molecules were occupied a t the concentrations in question) would still be possible. Frankenhaeuser (1962) compared the delayed currents in a Xenopus laeuis node of Ranvier over a range of external potassium concentrations from 2.5 t o 114.5 m M with the independence principle predictions and concluded t ha t data and theory were in agreement. On careful scrutiny, the outward delayed currents that he observed do appear to fall consistently below the independence principle predictions by margins greater than the indicated range of reproducibility for points of the control curve, although the deviations are quite small. A more stringent test of the independence principle is made possible by the use of tracers to measure one-way ion fluxes during current flow. The independence principle expression for the ratio of one-way ionic fluxes derived by Hodgkin and Huxley (1952) may be rewritten

Ci exp Mi-, -- - Mo-i

Co

(g)

(3)

where Mi, and M,-i represent the unidirectional efflux and influx, respectively, and Ci and C, represent the internal and external concentra-

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ROBERT J. FRENCH AND WILLIAM J. ADELMAN, JR.

tions. The sign convention for E is the same as in Eq. (2). This expression is analogous to that used by Ussing (1949) to provide a criterion to distinguish between active transport and diffusion. Hodgkin and Keynes (195513) found the potassium ion influx/efflux ratio to be a much steeper function of the gradient of electrochemical potential than the independence principle predicted. This would be expected if the.potassium ions moved in single file through narrow transmembrane channels without the possibility of one ion passing another in the channel. Net flux in one direction would tend to inhibit unidirectional flux in the other direction, just as the data appear to indicate. By contrast, Keynes (1951) showed that during the net influx of sodium under repetitive stimulation at 100/second the sodium influx rose 18 times and the efflux 22 times above resting values. Although they were not able to study the sodium influxlefflux ratio during activity as a function of the membrane potential, Hodgkin and Keynes (1955a) did obtain an influxlefflux ratio for total movements during a period of repetitive stimulation. The observed ratio was about 2. In the following paper (Hodgkin and Keynes, 1955b), they note that there could not have been interaction between sodium ions approaching the extent to which it occurred between potassium ions, since the observed influxlefflux ratio for sodium ions was so close to 1 during a period of large net influx. Thus a t that time, there was no compelling evidence against the application of the independence principle to the early inward current flow carried by sodium ions, although tracer experiments did indicate significant deviations from independence by potassium ions carrying the delayed currents. There are, of course, ways other than single-file flow of ions that could lead to deviations from independence, and we will explore some of these in greater detail in later sections.

II. SATURATION PHENOMENA A. Channels in Lipid Bilayer Membranes

The clearest examples of saturation of currents as ionic concentrations increase have been found in studies of channel-forming agents in lipid bilayer membranes. Hladky and Haydon (1970; 1972a, b) observed that the single-channel conductance of gramicidin A a t 100 mV applied voltage clearly approaches limiting values in both KC1 and NaCl solutions a t concentrations greater than 1M . These authors suggested that electrostatic repulsion prevents more than one ion from entering the channel at any given time. Further, Myers and Haydon (1972) present evidence that

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IONIC COMPETITION IN MEMBRANE CHANNELS

chloride carries negligible current when sodium or potassium is the cation, thusit islikely that the saturating conductance is due to a genuine competition of ions of a single species for occupancy of the channels. Following Pant and Conran’s (1972) report that keyhole limpet hemocyanin markedly reduces the resistance of lipid bilayer membranes, Alvarez et al. (1975) have recently found evidence that the hernocyanin forms discrete channels through the membrane. Latorre and Alvarez (personal communication) have now found that currents through the hemocyanin channel also show saturation at increasing concentrations of KCl. Saturation occurs a t concentrations somewhat lower than for gramicidin A. Lauger (1973) used the single occupancy constraint in a rate theory channel model that mimics much of the behavior seen in experimental studies of gramicidin-treated bilayers. No specific binding site for ions within the channel was invoked in this model, consistent with the argument by Myers and Haydon that the selectivity data for gramicidin could not readily be explained by notions of selective binding.

B. Divalent Ion Currents 1. CALCIUM CURRENTS IN GIANTBliRNACLE

hrUSCLE

Hagiwara et al. (1964) and Hagiwara and Naka (1964) describe the generation of Ca2+-dependent action potentials in barnacle muscIe fiber under conditions of low internal Ca2+ concentration produced by the injection of EDTA into the fiber. These Ca2+spikes were not affected by the external Na+ concentration and were not blocked by tetrodotoxin ( T T X ) . Cations Ba2+ and S?+ could substitute for Ca2+allowing spike generation in its absence. The potential reached by the overshoot of the spike past zero was determined by the concentration of Ca2+in the external medium. Some data presented by Hagiwara and Nakajima (1966) suggest that the rate of rise of the action potential saturates with increasing external Ca2+ concentration. Hagiwara and Takahashi ( 1967) further examined the relation between the Ca2+ currents generating the spike and the ionic composition of the external medium. By using the maximum rate of rise of the spike as a n indication of the relative magnitude of the peak Ca2+ current, they varied the external Ca2+ concentration under conditions for which the maximum rate of rise always occurred a t about the same membrane potential. The linear plot of the reciprocal of the maximum rate of rise against the reciprocal of the extwnal Ca2+concentration, analogous to the standard reciprocal plots of mzymo kinetics, indicated that the inward Ca2+current was a saturating function of external Ca2+concentration with a dissociation constant bctwwn 25 and 40 mM. The authors postu-

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ROBERT J. FRENCH A N D WILLIAM J. ADELMAN, JR.

lated, on this basis, that the Ca2+ions, as a first step in permeation through the membrane, had to be adsorbed onto fixed sites on the membrane surface. A t high concentrations of Ca2+,then, the sites could all be expected to be occupied, and Ca2+currents would approach a maximum value. 2. SLOWINWARD CURRENTS IN CARDIAC MUSCLE Trautwein (1973) has reviewed the recently unfolding evidence for two distinguishable components of inward current during the cardiac action potential (see also Orkand and Niedergerke, 1964; Peper and Trautwein, 1968; Rougier el al., 1968; Besseau and Gargouil, 1969). The earlier faster component is normally carried by Na+ ions and is blocked by TTX. Turning on more slowly is a component that continues throughout the plateau phase and is inhibited by millimolar concentrations of Mn2+. The slow current itself under normal circumstances probably has components carried by sodium and by calcium ions (Rougier el al., 1969; Ochi, 1970). However, in sheep and calf ventricular trabeculae, Scholz (1969) observed no differences between the slow depolarization in Tyrode’s solution with TTX and in Na+-free Tyrode’s solution. Furthermore, Scholz showed that the maximum rate of rise during the slow depolarization was related to Ca2+ concentration in the bathing medium. Although drawing no comment from the author, Scholz’s data for maximum rate of rise, presumably proportional to maximum inward current, do appear to be approaching a saturating level as Ca2+ concentration increases. Taking these data, although limited, a t face value, one can estimate from a standard reciprocal plot a dissociation constant of about 3 mM for Ca2+binding. Vereecke and Carmeliet (1971a, b) studied the slow inward current in bovine Purkinje fibers in Nrt-free solutions containing Sr2+and also in Sr2+ and Ca2+mixtures. The strontium current, as measured by the maximum rate of rise, saturates at high Sr2+concentrations consistent with a dissociation constant of 7 mM. In this preparation, Ca2+ appeared to carry very little current and, in fact, competitively inhibited the currents carried by strontium. C. Do Nerve Axon Currents Show Saturation Behavior?

Although Frankenhaeuser ( 1962) concluded that his measurements of delayed K+ currents in Xenopus node of Ranvier were generally in agreement with the independence principle predictions, there do appear to be small deviations. At least for outward currents the deviations are consistently in the direction that one would expect for partial saturation of the channels.

IONIC COMPETITION IN MEMBRANE CHANNELS

169

Recently, Hille ( 1975) reported reproducible small deviations from independence of sodium currents in the frog node of Ranvier a t various external Na+ concentrations. He concluded that these observations were consistent with saturation behavior. In earlier publications he has noted that various cations including lithium, thallium, and hydroxylamine, as well as guanidine and some of its derivatives, carried smaller currents through the sodium channel than would be estimated using the independence principle with permeabilities estimated from reversal potentials in ion-substitution experiments (Hille, 1971, 1972). Again, the behavior was consistent with the notion of saturation, that is, ions remained in the channel for a sufficiently long time so that they excluded, presumably by electrostatic repulsion, other ions from entering.

111.

BLOCKING AND COMPETITION

From the experimental data just discussed, it seems clear that ions of a single species, do compete with each othcr for occupancy of, and passage through, transmembrane channels. What examples arc there in which ions of two species compete with each othcr? And what is sufficient evidence for such a claim? A. Blocking of Divalent Ion Currents

1. CALCIUM CURRENTS IN INVERTEBRATE MUSCLE Hagiwara and colleagues have made extensive studies of the Ca2+-mediated spikes that are generated by barnacle muscle under conditions of low internal Ca2+ concentration. Hagiwara and Nakajima (1966) draw a parallel between the currents of the rising phase of the barnacle muscle action potential and those responsiblc for t,he plateau of the frog ventricular action potential. Both were blocked by Mn2+ ions but not by TTX or procaine. Hagiwara et al. (1969) have demonstrated that K+ currents and Ca2+ currents in barnacle muscle may br: selectively blocked by procaine and Co2+,respectively. A long list of ions that block the Ca2+ current was presented by Hagiwara and Takahashi (1967). They examined the effects of La3+, UOZ2+,Z$+, Co2+,Fez+, Mn2+,Ni2+as well as Mg2+and Ca2+,and they present kinetic data from which they argue that the blocking is genuinely competitive. Evidence for the similarity of Ca2+entry pathways in a variety of species and preparations is mounting. Kidokoro et al. (1974) showed that the

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action potential of striated muscle from the glochidium larva of the mollusk Anodonta implicata is caused by an increase in Ca2+permeability of the membrane and is blocked by La3+ a t 1 mM. In the protochordate Amphioxus, although the myotomal muscle spike is generated normally by an increase in Na+ permeability of the membrane, Hagiwara and Kidokoro (1971) concluded that there is also an independent permeability increase to Ca2+ions. In the presence of TTX to block Na+ currents and procaine to prevent increase in permeability to potassium, the membrane responds to a short current pulse with a regenerative change in potential. The overshoot of the spike so produced is dependent on external Ca2+concentration, and the data show some tendency toward saturation of the response a t 30 mM Ca2+in the external medium. This Ca2+-dependentresponse is reversibly suppressed by Co2+and La3+ions and could also be obtained when Ba2+or Sr2+were substituted for Ca2+. There is a striking similarity, which will continue to emerge in the following section, between Ca2+currents seen in invertebrate muscle and the Ca2+component of the inward current in vertebrate cardiac muscle. 2. CALCIUM CURRENTS IN VERTEBRATE CARDIAC MUSCLE

Numerous experimental preparations have provided evidence that there are two inward components of current during the cardiac action potential: a fast component, specifically blocked by TTX or lowered Na+ concentration in the external medium, and a slower component inhibited by millimolar levels of Mn2+ions. Manganese inhibition of the slow component has been shown in frog ventricle (Hagiwara and Nakajima, 1965), frog atrium (Rougier et al., 1969; and Tam, 1971), guinea pig papillary and trabecular muscle (Ochi, 1969), and mammalian Purkinje fibers (Vereecke and Carmeliet, 1971a; Vitek and Trautwein, 1971). Vereecke and Carmeliet (1971b) present convincing evidence that Sr2+ and Ca2+ compete for membrane sites. I n trying to formulate general ideas on selectivity in permeation of ionic channels, it is important to note that a single ionic species may frequently display both blocking and conducting properties in the same channel. A special case of this is the saturation behavior discussed in the previous section. To a certain extent, increasing the number of available chargecarrying ions increases the current. As the concentration becomes higher and higher, the ions begin to compete with each other more and more for the channels, and, finally, increase in concentration produces no further increase in current. The conducting ions, then, are acting in a sense both as charge carriers and as blocking ions. The same may be seen in interspecies competition. Vereecke and Carmeliet (1971a, b) show that Sr2+can carry

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the slow inward current, and, if Ca2+ is then added, a decrease in the current is observed. Calcium, the normal charge carrier, can thus block the passage of Sr2+.An interesting observation in a similar vein was made by Ochi (1970). Manganese, hitherto portrayed only as a blocker, was seen to act as a charge carrier through the slow channel when Na+ and Ca2+were both absent from the external medium. These observations have significant implications for a general theory of pore selectivity (see Sections 111, B, 1 and IV, B). One further point should be made. Neither in binding of blockers, nor in passing of current are the channels carrying the inward current absolutely selective. I n addition to the observations of Ochi (1970), Tarr (1971) stated that a t least in some frog atrial preparations the slow inward current appeared to be carried by Na+, even though the weight of his evidence favored Ca2+as the normal charge carrier. He also noted that, whereas the slow current is certainly more sensitive to hInZ+,a t concentrations of 10 mM both fast and slow currents were abolished. This observation is not surprising in the light of evidence that many di- and trivalent ions inhibited Na+-dependent currents and action potentials in nerve (see, for example, Woodhull, 1973; Hartz and Ulbricht, 1973). B. Blocking of Nerve Axon Currents

1. THESODIUM CHANNEL

Development of techniques of internal perfusion for the squid giant axon provided a powerful tool for the detailed study of the effects of ionic interactions on membrane currents. Thus, the experimenter could determine a t will the ionic environment a t both inner and outer surfaces of the membrane. A number of such studies will be mentioned here, and the reader who is so inclined may consult Adelman (1971) for a more extensive review of studies on internally perfused axons. Chandler and Meves (1965a) estimated the relative permeabilities of the alkali cations Li+, Na+, I<+,Rbf, and Csf through the early channel of squid giant axon and also observed the effect of perfusion with various cations, They noted that the early channel currents fell below those predicted by the independence principle when the axon was perfused with a solution containing 150 mM I<+ and 150 mhf of either choline, Na+, Rb+, or Cs+. A following paper by Chandler el al. (1965) stated that with I<+ as the only cation inside, late currents with 300 and 24 mM internal concentrations were consistent with the independence principle. The experiments of Adelman and Senft (1966) indicated that internal Cs+ also affected the time course of the early current by inhibiting the

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inactivation process. In a later paper (Adelman and Senft, 1968), they found that K+, Rb+, and Cs+ added to the external solutions, decreased the amplitude of the early current, and caused a negative shift in reversal potential. This reversal potential shift was particularly puzzling in that, if the ions were moving independently through the channel, one would expect the addition of even poorly permeating ions to cause a shift of the reversal potential in the positive direction; or one would expect no shift if the added ions did not penetrate at all. Two possibilities seem open. First, if the Tris ion used to make up the ionic strength of the control solution did have a finite permeability compared to the poorly permeating alkali cations, substitution might lead t o the negative shifts. This, however, seems unlikely since various measurements of the K+ permeability relative to Na+ have produced quite consistent values, and there is no indication that Tris can permeate (see Hille, 1971, 1972). A second alternative is that K+, Rb+, and Cs+ block access of Na+ ions to a fraction of the channels from the outside but allow them to enter the channels from the inside and pass through relatively freely. This would lead to reversal potential shifts in the observed direction with a magnitude depending on the relative strength of binding of the K+, Rb+, and Cs+ t o the channel. A brief discussion by Hladky (1965) of a “beveled screw hole model” proposes a mechanism of this type in a different context. The results of Binstock and Lecar (1969) imply a dual role of the ammonium ion as a charge carrier and a partial blocker. By using squid axon internally perfused with ammonium A uoride solution, they observed both inward and outward currents well below the independence principle predictions. This is true in spite of the fact that added ammonium ions can carry sufficient current to restore axon excitability in sodium-free solution. Hille (1971,1975a) notes two different observations that point to blocking of currents through the sodium channel by thallium ions. When all external sodium was replaced by thallium, both inward and outward early transient currents were reduced. As the internal sodium concentration is presumably unchanged, it seemed probable that the reduction of the outward currents was due t o the blocking of a fraction of the channels by thallium ions. When early transient currents were measured in preparations having half of the external sodium replaced by thallium or by the totally impermeant ion Tris, currents were lower in the case when thallium was present. Again, the only explanation seems to be that thallium partially blocks the component of current carried by the sodium ions present. How else could substitution of the somewhat permeant thallium ions for the impermeant Tris lead to a reduction of current? A discussion of Hille’s quantitative treatment of these data is deferred to Section IV, B.

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Sodium channels of nerve axons are also blocked by increased external concentrations of hydrogen and calcium ions. Complicating the analysis of the data, however, is a second action of these ions. They appear to screen the effect of negative charges a t the surface of the membrane and so produce a shift in the positive direction along the voltage axis of the various voltage-dependent conductance parameters (Gilbert, 1971 ; Gilbert and Ehrenstein, 1969; Blaustein and Goldman, 1968; D’Arrigo, 1973, 1974). By using frog node of Ranvier, Woodhull (1973) found convincing evidence for a voltage-dependent blocking action of both H+ and Ca2+after taking into account the shift in the sodium conductance-voltage relation due to changes in the surface potential (see Frankenhaeuser and Hodgkin, 1957; and Hille, 1968). In a recent paper, Drouin and Neumcke (1974) gave estimates of the charge density and pK for both “specific” charges in the sodium channel and “unspecific” charges on the general membrane surface. Calcium ions also appear to compete with various drugs for binding sites in or near the Na+ channel. Blaustein and Goldman (1966) noticed competitive effects of Ca2+on procaine action; Henderson et al. (1973) were able to reduce saxitoxin (STX) binding to a solubilized membrane extract, apparently containing a t least some components of Na+ channel, by increasing the Ca2+concentration. The complexity of the action of some divalent ions is further underlined in recent papers by Begenisich and Lynch (1974) and Armstrong and Bezanilla (1974). Begenisich and Lynch found that 1 mM Zn2+reduced the Na+ current and slowed the time to peak as well as markedly slowing the rate of rise of the I<+current. They suggest that the gating protein of the potassium channel is highly susceptible to binding of Zn2+.Armstrong and Bezanilla (1974) demonstrated that Zn2+not only reversibly blocks the Na+ current but also simultaneously abolishes the gating current. The message for those interested in the mechanism of ionic permeation across excitable membranes is clear. There are a variety of qualitatively different ways of inhibiting ionic currents across membranes. Direct competition among ions for occupancy of ionic sites in the conducting pathways appears to be one of those ways, and we feel that a study of such competition can yield useful information about the nature of the conducting channels. The investigator is, however, always faced with the task of evaluating the evidence as to whether or not an observed inhibition of ionic currents is due to some directly competitive ionic interaction, some effect on the voltagedependent channel gating system, or perhaps some nonspecific modification of the field within or near the membrane. Table I summarizes the various postulated mechanisms by which added ions may modify the function of the sodium and the potassium channels.

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TABLE I SOMEMECHANISMS BY WHICHADDEDIONS MAYMODIFYSODIUM AND POTASSIUM CHANNEL FUNCTION ~~

Postulated mechanism

Experimental observation

Exampleso

Rapid binding of ion in the channel

Instantaneous I / V curves show voltage dependent reduction of currents

Blocking of K channel by Cs+, Na+ and Li+; blocking of Na+ channel by T1+, H+, Caw and small organic ions

Blocking ion binds to channel with ratelimiting reaction

Change in time course of conductance change; reduction of currents

Blocking of K channel by quaternary ammonium ions

Modification of membrane Gating parameters show parallel shifts along the surface charge density, voltage axis changing intramembrane electric field

Effects of hydrogen, divalent and trivalent cations on sodium and potassium conductances

Direct effect on the gating Parallel reduction in ionic and gating currents. process Modification of time course of conductance change

Effect of Zn++on sodium ionic and gating currents

a

Further details and specific references are given in Section 111, B.

2. THEPOTASSIUM CHANNEL-BLOCKAGE BY ALKALI CATIONS In early studies on internally perfused squid axons, Baker et al. (1962) found that the resting potential was made less negative and the action potential was increased in duration upon changing from an internal KzSOc solution t o an internal CsaSOc solution, Tasaki et al. (1962) reported repetitive firing and also noted increased action potential duration during internal perfusion of the squid axon with Cs+ ions. In 1965, Chandler and Meves reported that several alkali cations interfere with the flow of potassium ions through the channels that conducts the delayed membrane current in the voltage-clamped axon. They showed that outward potassium currents recorded from squid giant axons internally perfused with solutions having equal concentrations (150 mM) of potassium and a given test ion (Na, Rb, or Cs) were of lower amplitude than expected on the basis of independence principle (Hodgkin and Huxley, 1952) predictions. The ratios of the observed to the calculated (from independence) values were 0.76 for Na+, 0.53 for Rb+, and 0.03 for CS+.

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Pickard et al. (1964) had previously concluded that Cs+ ions did not act as carriers of charge through the potassium channel, because external Cs+ had no influence on the amplitude of outward potassium currents as might be expected for an increased external concentration of an ion permeant through the potassium channels. The severe inhibition of outward potassium ion flow by internal cesium ion was soon confirmed by Adelman and Senft (1966). These authors also showed that deviations from independence were greater for large depolarizations than for small depolarizations. Adelman and Senft ( 1966) stated, “AS the effect also seems to be voltage-dependent this suggests that there may be a competition between cesium arid potassium for conductance sites which are also voltage-dependent.” I n addition to the inhibitory effect of internal Cs+ ion on outward current flow through potassium channels, external Cs+ ions inhibit inward potassium current flow (Adelman and Senft, 1968; Adelman, 1968; Rezanilla and Armstrong, 1972). In 1973, Adelman et al. published the results of a study of the kinetics of potassium ion accumulation in the squid axon periaxonal space (Frankenhaeuscr and Hodgkin, 1956). This study showed that the ionic componrnt of inward-flowing current tails following outward potassium currents was carried across the membrane exclusively by potassium ions. Upon stepping up the membrane potential from a depolarized value to a more hyperpolarized value, the potassium current instantaneously changes to a new value which then declines exponentially with time (Hodgkin and Huxley, 1952). In 1968, Adelman and Senft demonstrated that external Cs+ decreases these current tails manyfold (in the range from 100 to 200 mM Cs+, the reduction was about one order of magnitude). Inasmuch as the experiments of Adelman and Senft (1968) attempted to demonstrate external Cs+ inhibition of inward K+ currents in external solutions containing significant Na+ concentrations, there was a possibility that external Na ions contribute to this inhibition. However, Bezanilla and Armstrong (1972) concluded, on the basis of their experiments comparing the effects of Na+ and Cs+ on inward potassium currents, that external Na has a qualitatively different effect from external Cs+, i.e., Na+ does not block potassium channels externally. The same finding was made on frog single myelinated fibers (Hille, 1973). Thus, Cs+ ion inhibition of potassium channels is dependent of the direction of current flow. Internal cesium ions do not block inward potassium currents, nor do external cesium ions block outward potassium current flow (Adelman and Senft, 1968; Adelman, 1968). Sjodin (1966) also studied the effect of internal Cs+ ion on action potential duration and potassium and cesium ion effluxes (measured with radioisotopes) in squid axon. He confirmed the finding of Baker el al.

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(1962) that the action potential duration was increased by internal Cs+ ions. He also noted that resting potassium ion effluxes were not altered by internal Cs+ ions but that extra potassium effluxes associated with repeatedly stimulated action potentials were greatly reduced below values measured in normal axons. With respect to Cs+ ion effluxes,Sjodin (1966) observed that, for roughly equal internal concentrations of K+ and Cs+, resting cesium ion effluxes were 34% of the resting potassium ion effluxes. However, the extra cesium ion efflux per impulse determined during repetitive stimulation was only 8% of the extra potassium ion efflux per impulse. Sjodin concluded “that the squid giant axon membrane discriminates markedly between potassium and cesium ions during an action potential.” The inhibition of the expected extra potassium effluxes during the action potential noted by Sjodin (1966) is consistent with the voltage-dependent inhibition of the potassium conductance seen in the voltage clamp experiments of Adelman and Senft (1966). Upon considering the prolongation of the action potential and taking normal K+ loss values per impulse (Brinley and Mullins, 1965; Caldwell and Keynes, 1960), Sjodin estimated that the membrane potassium current was reduced to 14% of that normally associated with an action potential. The prolonged action potential, recorded by Sjodin from axons containing roughly equal concentrations of K+ and Cs+, had plateaus with the membrane potential remaining almost steady at +30 mV for a few milliseconds. The results of Adelman and Senft (1966) indicate that a t +30 mV the steady-state value of the potassium current is only 12% of the normal value. Thus, there is rather close agreement between the flux measurements and the voltage clamp results on the inhibitory effect of internal cesium ions on outward membrane potassium flow. Following the initial suggestion of Chandler and Meves (1965a) that replacement of a fraction of the internal squid axon [K+] with Na+ resulted in a reduction of outward potassium current flow below values predicted from the independence principle, Bergman (1970) was able to show that internal accumulation of Na+ ions inhibited outward potassium currents in voltage-clamped myelinated nerve fibers. Adelman and Senft ( 1971) demonstrated in squid axons that internal perfusion with 200 mM K+ and 240 mM Na+ solution reduces outward potassium currents to only one-tenth of the value predicted from the independence principle. All of these findings are clear examples of ionic inhibition of potassium channels. Squid giant axons internally perfused with 300 mM K F and 100 mM CsF solution exhibited steady-state current-voltage relations that develop a negative slope conductance at clamped membrane potentials more positive than +20 mV (Adelman 1968, 1971). Figure 1 illustrates this

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IONIC COMPETITION IN MEMBRANE CHANNELS

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phenomenon. Bezanilla and Armstrong (1972) compared the effects of internal Li+, Na+, Rb+, and Cs+ on potassium currents in voltage-clamped squid axon. All of these ions, when present inside the axon, reduce the outward current through the potassium channel. In Fig. 2, we have plotted the ratios of currents in the presence and absence of each of the blocking ions as a function of the membrane potential from the isochronal' currentvoltage relations presented by Bezanilla and Armstrong (1972), The internal concentration of K+ was the same in each case, and from the independence principle only current ratios of 2 1 would be expected. Currents are clearly less than the independence principle prediction for most of the points shown. For Rb+ the current ratio increases steadily with increasing potential, whereas for sodium and cesium the current ratio becomes less and less as the potential increases, i.e., for Na+ and Cs+ the 1 For the isochronal Z/V relations, currents measured at a fixed time a few milliseconds after the beginning of the depolarieing pulse were used.

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FIG.2. Ratios (ordinate) of isochronal potassium channel currents obtained upon internally perfusing squid axons with 300 mM K+ solution plus any one of several blocking ions, to currents obtained with 300 mM K+ only, plotted against membrane potential (abscissa). Open circles, K+ 100 mM Na; half-filled circles, K+ 100 mM Rb+; filled circles, K+ 50 mM Cs+. A current ratio of 1 (upper horizontal line) would indicate identity of currents obtained with and without an added ion. (Data obtained from Figs. 2,6, and 8 of Bezanilla and Annstrong, 1972.)

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FIG.3. Normalized instantaneous potassium channel currents plotted against the membrane potential (A) and against the difference between the membrane potential and the reversal potential (B). In all cases squid giant axons were perfused with 300 mM K+ solution. Filled circles, control curves (K+only); open circles, K+ 100 m M Rb+; open squares, K+ 100 mM Na+; half-filled circles, K+ 100 m M Li+; filled triangles, K+ 50 mM Cs+. To normalize the currents, the values for the control (K+ only) curves at E = 100 mV were set equal to 1. Notice that the points for the control curves from all four axom cluster around the same line. (Data obtained from Figs. 4,6,7,and 8 of Bezanilla and Armstrong, 1972.)

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ROBERT J. FRENCH A N D WILLIAM J. ADELMAN, JR.

and Armstrong (1972) found that the instantaneous potassium currentvoltage relation also developed a region of negative slope when squid giant axons were internally perfused with (K+ Na+) or (K+ Cs+) solution. Figure 3 plots normalized values of instantaneous potassium currents against membrane potential. The data used to derive this figure were obtained from Bezanilla and Armstrong (1972, Figs. 4, 6, 7, 8). In Fig. 3A the I / V curves are only normalized so as to scale all the I / V relations obtained upon perfusing axons with 275 mM K F solution to a relative current value of 1 a t a membrane potential of 100 mV. All other current values were then normalized to this standard current. In Fig. 3B, the normalized currents were plotted against the difference between the transmembrane potential and the reversal potential to give an empirical picture of the current as a function of the net driving force. It is easy to see how one might obtain a region of negative slope in the instantaneous I / V relation in terms of the simple single-site kinetic model detailed in Section IV, B. If the blocking and current-carrying ions compete for a site that is part way through the membrane potential field, but the blocking ion cannot pass that site, i.e., there is an infinite energy barrier preventing its passage to the outside, then there is the possibility of seeing negative slope conductance. A potential gradient tending to drive ions into the channel from the axoplasm will tend both to increase the rate a t which the binding of the blocking ions with the site occurs and to decrease the rate of their dissociation from the site. As the voltage increases, a greater and greater proportion of the sites will be occupied by the blocking ions and the current will eventually begin to decrease with an increase in applied voltage. In other words, the dissociation constant for the reaction of the blocking ion with the site decreases markedly with voltage (see Eq. 7, Section IV, B) . The actual voltage at which the current begins to decrease rather than increase with increasing applied voltage, and the steepness of the negative slope region, are dependent on the dissociation constant for the blocking ion a t zero applied voltage and on the fraction of the membrane potential drop that occurs between the blocking site and the side of the membrane from which the ion enters the channel (French, Lecar, and Ehrenstein, unpublished). The only additional restriction that must be placed on this model to mimic the negative slope in the instantaneous current-voltage relation is that the reaction of the ions with the site must be fast compared with the speed of the voltage clamp. This is implicit in the assumption of a steady state for the reaction of the ions with the site in the analysis of Section IV, B. One might, quite arbitrarily, use as an index of blocking effectiveness the voltage a t which the current attains its maximum value and the negative

+

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slope region begins.2 More potent blockers would cause decreasing currents a t smaller applied voltages. As Itb did not cause negative slope conductance to appear, it may reasonably be included in the list as the least effective blocker. The order of blocking potency would then be Cs+ > Na+ > Li+ > Rb+. This order is the same regardless of whether the criterion is applied to Fig. 3A or Fig. 3B. Rough estimates of thc dissociation constants from the single-site model give a sequence consistent with this. The sequence of dissociation constants a t zero applied voltage is Cs+ < Na+ 5 Li+ (French, Lecar and Ehrenstein, unpublished). As might be expected, the most effective blocker is the most strongly bound ion. Notice that, in Fig. 3, Csf is present at only half the concentration of the other blocking ions to produce the effect indicated. It is also worth noting that the shifts in reversal potential from the “K+ only” to “I< plus blocking ion” curves, are, in order of decreasing magnitude of the shift, Csf > Na+ > Li+ > Rbf. If the reversal potential shift is primarily due to differences in the I<+ accumulation in the periaxonal space during the prepulse preceding the determination of points for the instantaneous I / V relations, as Bezanilla and Armstrong (1972) suggest, and if also the prepulses used with the different solutions were of about the same magnitude and duration, then the sequence of reversal potential shifts should correspond to the relative blocking effects a t the prepulse potential. All of these criteria give the same sequence of blocking potency. It is likely, then, that this is the sequence of binding affinities of these ions for the site a t the inner mouth of the potassium channel, provided the ions do, indeed, all bind a t the same site. If they did not, the differing effects of voltage on the binding of each ionic species would immensely complicate the interpretation.

3. THE POTASSIUM CHANNEL-BLOCKAGE BY QUATERNARY AMMONIUM IONS Many interesting inferences about the structure of the potassium channel may be drawn from studies of the effects of quaternary ammonium (&A) ions on nerve action potentials and membrane currents. In 1957, Tasaki and Hagiwara reported that, in response to electrical stimulation, squid axons injected with tetraethylammonium chloride fired long-duration action potentials reminiscent of those seen with cardiac muscle preparations. They noted that during the plateau phase of the action potential, the membrane resistance was approximately a t the resting level, and that under voltage clamp, potassium currents were much reduced from those seen in control axons. More recently, Bergman et al. (1968) showed that the analogy 2 Although not shown in the figures we present in this article, Li also showed a slight tendency to produce negative slope conductances a t high positive voltages.

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between suitably treated nerve and cardiac muscle may be carried a step further. Single nodes of Ranvier bathed by Ringer’s solution with lowered calcium concentration (0.05 to 0.1 mM) and containing 5 mM tetraethylammonium (TEA) showed sustained spontaneous repetitive firing of cardiaclike action potentials. This behavior is presumably due to two separate ionic interaction effects-first, reduced calcium competition for the sodium channel allowing residual sodium current to produce a “pacemaker” potential, and, second, a reduction of the potassium currents due to blocking by TEA ions causing a slow, plateaulike repolarization phase. Armstrong and Binstock (1965) performed a series of voltage clamp experiments on squid giant axon injected with TEA. When the external medium was normal artificial seawater (ASW) , outward steady-state potassium currents approached leakage current values. With high potassium concentration in the external solutions (100 and 400 m u ) , the instantaneous current-voltage ( I / V ) relation showed a rectification such that inward potassium currents were approximately normal while outward currents were greatly reduced compared to those in axons not injected with TEA. Armstrong and Binstock noted that the rectification seemed to be a function of the potassium current rather than the absolute value of the membrane potential. They suggested that the TEA ions entered the channel in the direction of current flow and that blocked channels could be cleared by inflowing K+ ions. The preceding work was followed up by much more extensive studies by Armstrong (1966,1969, and 1971) using TEA and a variety of related &A derivatives. This work is summarized in a current article (Armstrong, 1975) and here we present only a brief outline of the key points. 1. Quaternary ammonium ions can only enter and block the channels when the normal voltage-dependent n4 gates are open. This is an implication of kinetic studies in which potassium currents in the presence of &A ions were seen to begin rising with normal time course before reaching a maximum and then declining to a low steady-state value. 2. Binding of the &A ions in blocking position may be stabilized by hydrophobic bonding in the region of the inner channel mouth. All the most effective blockers tried were of the form R-N (CzHs) a+. The &A ions block at lower and lower concentrations as the chain length of the R group, a straight-chain alkyl group, is increased, at least up to Ca. A similar observation was made for R groups with a benzene ring of the form Ph-(CHZ).-. With the Ph- only, or with Ph-CH2-, the compounds produce but slight blocking. Addition of one or two -CHgroups greatly increases the blocking effectiveness, and the Ph-(CHz) a- compound is a more effective blocker than the &A ion with a straight-chain Cg R group.

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3. Clearing of &A ions from the channels when the membrane is repolarized is caused by the actual flux of incoming K+ ions rather than just the reversal of membrane potential, although this in itself probably also has some effect.Conclusive evidence for this statement is found in the speeding up of the unblocking reaction produced by a raise in the external I(+ concentration. A simple competition effect would change the steady-state blocking but would not alter the rate of unblocking if it were a unimolecular dissociation reaction. 4. The n4activation gates of QA-blocked channels do not readily close. Inward tail currents on repolarization after a voltage clamp in high external K+ show a hump before declining to the base line, rather than having a simple exponential form. This observation is also consistent with the suggestion that incoming R+ ions sweep the &A ions out of the channel. 5. The effect of hyperpolarization on channel clearing is complex. Initially the rate a t which QA ions leave the channel appears to be speeded up-perhaps, partly by the simple reversal of the field and partly by the sweeping effect of incoming I(+ ions. Later phases of clearing are slowed. Armstrong suggests that this is due to a trapping effect because n4 gates that do manage to close around &A ions prevent those QA ions from leaving the channel. Armstrong concluded from this work that the potassium channel is gated

at the h e r end, with a relatively wide inner mouth to the channel, which is capable of accepting either a hydrated K+ ion or a TEA (or some other &A) ion. The site that the QA ions occupy is within the gate and their binding is stabilized by binding of the hydrocarbon tail of the &A molecule to a hydrophobic region of the membrane. The competition between K+ ions and the &A ions arises because of the similarity in size between the hydrated K+ ion and TEA (or the triethyl end of some larger QA ion). To cross the membrane, the K+ ion is presumed to shed most of its water of hydration (see also Hille, 1973) to pass through a narrower ‘Ltunnel”section of the channel. Unable to shed its outer shell in like manner, TEA is impermeant. Although some of the larger &A ions do slowly penetrate the membrane, this is presumably because their greater lipid solubility offers them a n alternative route through the lipid phase. A kinetic scheme proposed by Armstrong (1969, 1971, 1975) is able quantitatively to duplicate the experimental observations in considerable detail. In contrast to squid axon, the potassium currents in the amphibian node of Ranvier are blocked by &A ions present in the external bathing solution (Schmidt and Stampfli, 1966; Hille, 1967; Koppcnhofer, 1967). The doseresponse curve for external TEA suggests that 1 molecule of TEA reacts with one membrane site to depress the conductance, with a dissociation constant of approximately 0.4 m M (Hille, 1967). Internal TEA also

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blocks K+ currents (Koppenhofer and Vogel, 1969), and more detailed experiments by Armstrong and Hille (1972) provide evidence that the inner TEA-binding site in the frog node of Ranvier is in all respects similar to that inside the squid axon. By contrast, binding of TEA from the external solution is independent of the channel-gating mechanism and is not time- or voltage-dependent. Furthermore, the more hydrophobic &A ions are less effective blockers outside the node than is TEA. It, thus, seems likely that there are two different kinds of TEA-binding sites in the frog node, one accessible from each side of the membrane. C. Anionic Currents

1. ANIONICCURRENTS IN FROG SKELETAL MUSCLE Anions, usually chloride, account for a measurable fraction of the electrical conductance of many biological membranes. A great deal of effort has been expended in experimentally defining the anionic contributions to membrane currents, and there are many papers in the literature to which we cannot give due credit here. Although chloride ions do not, in general, play as direct a role in membrane excitability as Na+, K+, or even Ca2+, there is one report of chloride-dependent action potentials (Fukuda, 1974), and the chloride conductance of frog muscle does show some time and voltage dependence (Hutter and Warner, 1972; Warner, 1972). For the most part, the role of the chloride ion seems to be that of a modifier of membrane electrical responses (for example, see Takeuchi and Takeuchi, 1967; Horowicz, 1964). The review by Horowicz (1964) gives a comprehensive account of the earlier work in this field. Here we discuss only selected papers that give examples of ionic interaction effects that modify anionic fluxes and currents. Following is an outline of results obtained using frog skeletal muscle, which is by far the most actively studied preparation, and these results will be used as a basis for comparison with those obtained in other experimental systems. That chloride is responsible for about two-thirds of the conductance of the resting membrane of frog skeletal muscle in normal Ringer’s solution was shown by Hodgkin and Horowicz (1959). Their results were consistent with the hypothesis that resting muscle behaved as a Donnan system in which potassium and chloride ions contributed about 100 pmho/cm2 and 200 pmho/cm2, respectively, to the membrane conductance. A comparison of flux data from the literature with calculated values using their estimate of chloride permeability revealed discrepancies consistent with the singlefile diffusion postulate put forward by Hodgkin and Keynes (1955b) for potassium fluxes in the squid axon. Keynes (1954) and Adrian (1961)

IONIC COMPETITION IN MEMBRANE CHANNELS

185

argued that, on the basis of conductance measurements, fluxes were too low to explain the observed conductance values if influx and efflux were assumed to be independent. Carrying the investigation of chloride movements a step furthcr, Harris (1963) was only able to explain his efflux data on the basis of two “intracellularl’ compartments. One of these compartments was accessible to sodium and the “nonpenetrating” anions, methyl sulfate and bicarbonate, and washout of chloride from this compartment was independent of the presence of foreign anions and of chloride influx. Harris tentatively identified this compartment, with the eridoplasmic reticulum noting, however, that the calculated volume, 15-35%, of the intracellular water was greater than would be expected from the available electronmicroscopic data. He assumed that this compartment was in free communication with the external solution. The second compartment, presumably the truly intracellular one, was loaded in high-potassium solutions and showed efflux times dependent on external chloride concentratkm. Both chloride influx and the presence of external chloratc ion inhibited the efflux from this compartment. Earlier work by Harris (1958) showed that chloride efflux was reduced when various penetrating anions were added to the external solution, the diminishing effect being in the order 13r- < NO3- < I- < ClO, < C NS-. Reduction of efflux also occurred in the presence of the iionpenetratirig ions, sulfate, phosphate, and bicarbonate. In 1965, Harris reported that the concentration dependence of chloride efflux could only be described by the constant field equation (with the implicit assumption of the independence principle) in the absence of “foreign” blocking anions. Hutter and Padsha (1959) estimated the effect on membrane resistance of substituting various anions for chloride in the external solution, finding ionic conductances in the sequence: C1- > Br- > NO,- > I-. They also noted that nitrate a t low concentration seemed to have a disproportionately large cffect on the conductance and suggested the possible implication that only a limited number of membrane sites were involved in anion permeation. Hutter and Warner ( 1967c) found that various ionic interaction effects were markedly pH-dependent. Chloride efflux was much faster a t p H 9.8 than a t p H 5.0, and inhibition of the efflux was much more marked at the high pH. This was true both for the blocking action of the divalent cations, Cu2+,Zn2+, and U022+,and for inhibition by the anion I-. Stanfield’s (1970) observation that 2.5 mM Zn2+ increased the resting resistance from 3350 cm2 to 6830 ohm cm2 may well be due to zinc’s blocking action on the chloride conductance. Cations Ni2+,Co2+,Pb2+,Ce3+,and La3+ produced no detectable effect at 10-4 M , and the same was true of Ca2+over a “wide” range. Apparently related to the iodide inhibition of chloride efflux was an enhancement of efflux from iodide-loaded muscles when chloride was

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ROBERT J. FRENCH AND WILLIAM J. ADELMAN, JR.

substituted for iodide in the external solution. Both of these observations would be consistent with the idea that iodide binds to the anion-channel sites in the membrane blocking the passage of other ions, and one would expect the flux of iodide to be a visibly saturating function of iodide concentration within the normal working range. The pH effects on the anionic conductance of frog muscle pose a number of interesting questions that still await definitive answers. Hutter and Warner (19674 showed that the resting conductance increases in alkaline solutions and decreases in acid solutions with the steepest pH dependence in the neighborhood of neutrality. Changes in pH had little effect on conductance when impermeant methyl sulfate was used to replace the chloride in the external solution. Tracer flux measurements (Hutter and Warner, 1967b) confirmed that the increase in conductance with an increase in pH was primarily due to an increase in the membrane’s permeability to chloride. One possible mechanism required that the density of positive charge on the membrane sites was actually reduced a t low pH due to the displacement of divalent calcium by hydrogen ions. This, however, appeared unlikely in the light of the observed lack of dependence on calcium concentration of the observed fluxes. In a later synthesis of available data, Hutter et al. (1969) attempted to reconcile the experimental observations with the field strength theory of selectivity of Eisenman. By that time, Hutter and Warner (1968) had shown that not only did the chloride permeability fall when the pH was lowered, but anion permeability sequence changed from C1- > Br- > NO3- 1 I- at pH 9.8 to the reverse, C1- 5 Br- 5 NO3- < I-. Hutter et al. (1969) postulated that (1) cationic field strength of the sites increased as the pH of the bathing solution decreased, and ( 2 ) the permeability of an ion through the membrane is inversely related to the strength of its binding to membrane sites. This hypothetical framework thus provided a fairly straightforward rationale for the observations on frog muscle, at least in a qualitative sense. However, it requires some extension to cope with results from crustacean muscle, which we will mention below, and the more recent data from frog muscle presented by Woodbury and Miles (1973). These authors found that, as well as “chloride-like” ions whose conductance decreases with a decrease in pH, there are also a number of “benzoate-like” ions whose conductance increases with a decrease in pH. The permeability and conductance sequences were identical. The complete sequence a t pH 7.4 was as follows: C1- > Br- > NO3- > I- > trichloroacetate 2 benzoate > valerate > butyrate > propionate > formate > acetate 2 lactate > benzenesulfonate 2 isethionate > methylsulfonate > glutamate 2 cysteate. “Benzoate-like” anions are those from benzoate through acetate in the sequence. The correlation between the sizes of their hydro-

IONIC COMPETITION IN MEMBRANE CHANNELS

187

phobic moieties arid their sequence of conductance, together with the unexpected pH dependence of their conductances, led to the postulate that interaction with a hydrophobic region of the membrane near the site of a limiting energy barrier aids penetration of these ions through the membrane. We mention further details of the proposed model in Section IV, C. 2. A COMPARATIVE LOOKA T ANIONICINTERACTIONS

Studies of anion permeation in different preparations have revealed a diversity of properties with respect to pH dependence, interactions among different anions, and selectivity. These diff ercnces doubtless correspond to significant differences in the molecular architecture of sites a t which anions penetrate the different membranes. The fraction of the total membrane conductance for which anions arc responsible is highly variable. Table I1 summarizes estimates made on a number of Preparations. As already mentioned, anion conductance in frog skeletal muscle decreases with decreasing pH, having an apparent pK = 7.7. Qualitatively similar behavior was reported by Hagiwara and Takahashi (1974) for elasmobranch muscle, the one notablc difference being that the pK = 5.3. A somewhat more complex picture has emerged for the giant barnacle muscle fiber. Hagiwara et al. (1968) observed a steep rise in chloride conductance as the external p H was lowered below 5. A corresponding increase in tracer efflux (apparent pK = 4.5) was also seen by DiPolo (1972) a t low external pH. However, DiPolo’s results show tracer efflux a t a minimum a t pH = 5.5, with a further gradual increase as p H was raised up to 9. Hagiwara and Takahashi (1974) speculated on the analogy between this last-mentioned behavior and the increasing conductance with increasing pH seen in frog arid clasmobranch muscle. Brief reports by Reuben et al. (1962, and DeMello and Hutter (1966) showed that crayfish muscle chloride conductance decreased with an increase of pH from 4.5 to 8.5 and above. The results mcntioned leave a number of possibilities open. It is likely that different charged groups limit the chloride permeability a t diffcrent pH values. I n barnacle muscle there mag be two charged groups each having a n important influence a t different pH ranges. Moreover, it is possible, although not yet experimentally tested, that the presence of high concentrations of urea normally bathing calasmobranch muscle modify the pK of membrane sites (for discussion, see Hagiwara and Takahashi, 1974). The reversal of selectivity sequence exhibited by frog muscle with a change in pH has not been seen in other preparations. For barnacle muscle a t pH = 3.9, Hagiwara et al. (1971) reported the permeability sequence: C1- < C103- < Br- < NO3- < CNS. Except for the reversal of C1- and C103-, the sequence was unchanged a t pH 7.7. Another crustacean muscle,

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ROBERT J. FRENCH AND WILLIAM J. ADELMAN, JR.

TABLE I1 RELATIVE CONDUCTANCES DUETO CHLORIDE IONS IN DIFFERENT EXPERIMENTAL PREPARATIONS ~

Preparation

Estimate of the relative chloride conductancea

Source

Barnacle muscle (a) pH 7.7

1@

gK

1 1 = 8-7

Hagiwara et al. (1968)

(b) pH 4.0 Crayfish neuromuscular junction inhibitory synapse

Takeuchi and Takeuchi (1967)

Elasmobranch muscle (stingray)

Hagiwara and Takahashi

Frog skeletal muscle

Hodgkin and Horowicz

(1974)

(1959)

Cardiac muscle (sheep and dog Purkinje fibers)

Hutter and Noble (1961)

a In all preparations the major anion present is chloride. Where chloride conductance is given relative to potassium conductance, potassium is the predominant currentcarrying cation. Relative conductancea are expressed here in the same terms that were used in the original papers.

that from crayfish, showed no reversal of relative conductances due to C1and NO,- when pH was shifted from 4.5 to 8.5 (Hutter et al. 1969). It is interesting to note that the conductance sequence for barnacle muscle, also obtained by Hagiwara et al. (1971), is not the same as the sequences of permeabilities calculated from shifts in resting potential. The sequence of relative conductances, again at pH 3.9, was CNS- < NO*- < ClOs- < C1- < Br-. Differing sequences for permeabilities and conductances were observed by Takeuchi and Takeuchi (1971) for the inhibitory postsynaptic membrane of the crayfish neuromuscular junction. The sequence of permeabilities was C1- < NOa- < I- < CNS-, whereas the sequence of conductances was NOa- < I- < CNS- < C1-. Hagiwara and Takahashi (1974) also found different sequences for anionic conductances and permeabilities in stingray muscle.

IONIC COMPETITION IN MEMBRANE CHANNELS

189

A thread of evidence strongly suggesting a dual role-bot#h blocking to other ions, and carrying current-for thiocyanate has been uncovered in several preparations. This is the so-called anomalous mole-fraction dependence of conductance when thiocyanate is used to replace chloride in the bathing solution. With small quantities of thiocyanate added the conductance decreases, but after reaching a minimum when about one-quarter to one-half of the chloride is replaced by thiocyanate, the conductance rises steadily again as more thiocyanate is introduced into the medium. Similar observations have been made on frog skeletal muscle (Hutter and Padsha, 1959) crayfish neuromuscular junction inhibitory synapse (Takeuchi and Takeuchi, 1971), and stingray skeletal muscle (Hagiwara and Takahashi, 1974). In Takeuchi and Takeuchi’s results, there is a hint of the same type of behavior with nitrate and iodide as well. Clearly, from the observations summarized here, anionic selectivity sequences exhibited by natural membranes do not always follow the simple lyotropic series corresponding to hydration energies of the ions. Changes in pH have different effects on conductivities in different preparations and, indeed, on different ions in the samc preparation. Differences between permeability sequences determined from reversal potentials and ionic conductance sequences in the same preparation and from ionic blocking effects have been clearly demonstrated. Both of the latter observations imply deviations from the independence principle and are analogous with observations already presented for cation currents. The task remaining is to fit this diversity of functional properties into a rational mechanistic framework. For convenience we summarize a number of experimentally determined selectivity sequences in Table 111. Only studies in which numerical values for the relative permeabilities or conductances have been determined are included in the table; some others have been mentioned in the text.

IV.

MODELS AND ANALYSES

A. Single-File Diffusion Theory

We have already mentioned the earliest analysis of excitable membrane currents in terms of ionic interaction effects: the brief treatment used by Hodgkin and Keynes (1955b) to interpret their tracer studies of ionic fluxes during electrical activity in nerve. This work stimulated, after an incubation period of 10 years, a series of theoretical studies exploring in detail the implications of single-file diffusion through narrow channels.

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ROBERT J. FRENCH AND WILLIAM J. ADELMAN, JR.

TABLE 111 ANIONSELECTIVITY SEQUENCE0I N

Preparation

Frog skeletal muscle

Quantity determined

VARIOU0 PREPARATION0

Selectivity sequence

Source

Membrane resistance on ionic substitution

Cl-:Br-:N03-: I- = 1.0:1.5:

Hutter and Padsha

Relative anionic conductances

Cl-:Br:NO,-: Woodbury and Miles I-: trichloro(1973) acetate :benzoate:valerate: lactate :benzene sulfonate, isethionate: glutamate:cysteate =

(1959)

2.0:2.3

1:0.53:0.44: 0.29 :0.17 :0.15 : 0.13 :0.07:0.05 :

0.02:o.o

Elosmobranch (stingray muscle)

Barnacle muscle

Relative permeabilities from resting potential shifts on ionic substitution

C1O-s:I-:Br, C1-: N03-:CNS- =

Hagiwara and Takahashi (1974)

0.3-0.4:0.5-0.7: 1.0: 1.5-1.7 :3-5

Relative permeabilities from resting potential shifts (pH 3.9)

C1- :C108-:B r : NOs-:CNS- =

Relative anionic conductances

CNS-: Nos-: ClOa-: Cl-:Br- = 0.62:

Hagiwara et al. (1971)

1.0: 1.3: 1.8:2.0: 4.8

0.7:0.8:1.0:1.02

Crayfish neuroPermeabilities muscular junction from reversal inhibitory synapse potential shifts Relative anionic conductances

C1- :NO, :I- : CNS- = 1:1.32: 1.54 :1.60

NOa-:I- :CNS-: C1- = 0.67: 0.86:0.95: 1.0

Takeuchi and Takeuchi (1971)

191

IONIC COMPETITION IN MEMBRANE CHANNELS

Hladky (1965) gave a formal generalization of the Hodgkin-Keynes analysis and in an example briefly considered the possibility of rectification produced by a blocking ion, Papers by Heckmann (1965a, b, 1968), Heckmann et al. (1969), and Heckmann and Vollmerhaus (1970) have probed the theoretical consequences of single-file diffusion for measurements of permeability, unidirectional tracer flux, and net movement of particles across a membrane. The results of this work are collected together in a n English language paper by Heckniann (1972). Properties of single-site pores-not allowing a bimolecular “knock-on” reaction-are compared with those of long pores containing a t least two sites and having a “no passing’) restriction on the particles in the pores. Even with a single-site pore, one might expect to see saturation, inhibition, and even, if the barriers on either side of the site are unequal, flux asymmetry when the concentration gradient is reversed. Single-file long pores may also exhibit these properties, but only single-file long pores can show the following behavior: ( 1 ) unidirectional fluxes that increase in proportion to Cn (n > l ) , where C is the concentration on the side from which the flux is coming; ( 2 ) positive coupling between the fluxes of one species and the driving forces on another species (no coupling would be expected for a single site) ; (3) net flux permeability passing through a minimum value a t equilibrium (whereas for a single site it depends only on the mean of the two external concentrations) ; and ( 4 ) for correlation functions, j = P*/Po

<1

where P* is the unidirectional tracer permeability, and Po the limiting value of the net flux permeability a t equilibrium. This difference arises since a n individual tracer molecule traversing the pore must jump every barrier, whereas there can be net transfer across a long pore simply by shifting each of the particles in the pore across one barrier. The notion that functionally narrow long pores may exist thus gives a rationale for a variety of complex flux-force relations and flux-flux interrelations. Our emphasis here is on the behavior of channels and channel models, but the possibilities within this framework should not be allowed to obscure the fact that under some circumstances a quite different explanation for flux coupling seen in experimental data may be acceptable or even preferable. Properties of carrier and electrodiffusion models have been reviewed by Adrian (1969). Horowicz et aE. (1968) apply a carrier model to their data on fluxes across frog striated muscle membrane, although Arrnstrong (1975) shows that their observations are also consistent with a channel model allowing a bimolecular knock-on interaction between the ions. Heckmann and Vollmerhaus (1970) show that i t is possible, under certain conditions, for the flux equation for a pore along which particles

192

ROBERT J. FRENCH AND WILLIAM J. ADELMAN, JR.

progress by hopping into adjacent vacant sites to be identical with those from a knock-on model. Any further resolution of details of mechanism must then be achieved by a more appropriate experiment. B. Single-Occupancy Models

Current-voltage relations for single gramicidin channels, grading from upward curving a t high concentrations to downward curving (i.e., ascending less and less steeply) a t low concentrations, prompted the following interpretation by Hladky and Haydon (1972a, b) . First, to give an upward curving I / V relation, there must be fewer than ten energy barriers to be surmounted during an ion’s passage through the channel (see Woodbury, 1971, for a detailed argument of this point). Second, for an I / V curve that flattens at high voltages and low concentrations, there must exist a ratelimiting step that is not affected by the applied potential, and this they suggested could be a pair of barriers at each end of the pore across which there was negligible drop of the applied potential. Lauger (1973) compared the predictions of a rate theory pore model of this type with the data obtained from the gramicidin channel by Hladky and Haydon. No unique binding site in the channel was assumed. Saturation of currents a t high concentrations resulted from the assumption that, due to electrostatic repulsion, only one free ion at a time could enter the dipole-lined channel. With a symmetric barrier profile having uniform barrier heights within the pore and larger barriers at the ends (ratio of internal jump rates to those across the ends taken to be equal to 3.4), Lauger calculated I/ V relations quite closely approximating those observed. Rate parameters and binding constants calculated for different ions then allowed prediction of biionic potentials (analogous to reversal potentials determined for zero current through channels in excitable membranes). Here, agreement between experiment and theory was not so good. Not only were there quantitative differences, but the experimental biionic potentials (Myers and Haydon, 1972) for KCI/NaCI and for NH&I/NaCl systems increased with increasing concentration. Lauger ( 1973) points out that biionic potential for his single-occupancyrate theory model is not dependent on absolute concentrations-the predicted value is not changed when all concentrations are multiplied by a constant factor. In other words, the biionic potential or reversal potential is in general independent of any saturation effects for the single-occupancy rate theory model (see also Hille, 1975; French et al., 1974). A much simpler and more direct analytical approach was taken by Hagiwara and Takahashi (1967) and by Hagiwara et al. (1974) in describ-

IONIC COMPETITION IN MEMBRANE CHANNELS

193

ing competition and saturation behavior shown by currents through the Ca2+ channels of barnacle muscle fibers. In the earlier paper, maximum rate of rise of the action potential was taken as an indicator of the relative value of the maximum inward current and, in the later article, voltage clamp currents were measured directly. After taking pains to point out that the maximum rate of rise or the maximum currents occurred a t about the same voltage under the various conditions described in each paper, the authors presented linear plots of the reciprocal of the maximum rates of risc of the action potentials (or maximum inward current for the voltage clamp experiments) against the reciprocal of the concentrations indicating that the currents could be described by an equation of the form

( V ) is the maximum current attainable a t voltage V by a n Here Ic, increase of the Ca2+concentration, and Kca ( V ) is the dissociation constant for the reaction of Ca2+ions with sites in the membrane, a reaction assumed to be necessary for the Ca2+ t o permeate the membrane. If we set f = Ica (V)/Zca ( V )and a = 1, then Eq. (4) may be readily rearranged into the form of Eq. (1), and the analogy with other saturating processes may be clearly seen. Factor a is equal to unity if only Ca2+ions are present externally but becomes [l [M2+],/KM2+ ( V ) ] if some other ion, M2+, which also binds to the membrane sites but does not pass through the CaZ+ channels, is present. Thus, the ratio of currents in an experiment in which currents were successively measured with equimolar concentrations of Ba2+ and Ca2+bathing the muscle would be

+

where C is the common concentration of Ca2+ and Ba2+ in the bathing medium. Hagiwara et al. (1974) call thc first factor on the right-hand side of this expression (the ratio of I,,, valuchs) the “mobility” term, and the second factor the “affinity” term. Thcse twins show a curious interaction in thc presence and absencfk of Co2+ions (0 and 20 mM). In the absencc. of Co2+,which was shown by Hagiwara and Takahashi (1967) to block competitively the Ca2+current, the scqucmx. of maximum currents was IB, > Isr 2 Ica. When Co2+ ions were present, the sequence reversed, i.e., Ica > Isr > I B ~ .Hagiwara et nl. (1974) proposed that this apparent reversal of the selectivity sequence for Ca2+, Ba2+, and S P was due to a shift in dominance of the current ratio expression from the mobility term, in the absence of Co2+,to the “affinity” term, in the presence of Co2+.I n

194

ROBERT J. FRENCH AND WILLIAM J. ADELMAN, JR.

other words, in the absence of Co2+competing for the channel sites, relative currents depend only on how quickly the ions are able to pass through the open channels. However, when Co2+ions are present and occupy a large fraction of the sites, the Ca2+ion that binds more strongly than Ba2+is better able to compete with Co2+ for the sites and, thus, to obtain an opportunity to pass through the membrane. The analyses of divalent ion currents in cardiac muscle by Scholz (1969) and by Vereecke and Carmeliet (1971b) are basically similar in approach to that of Hagiwara and co-workers just described, although somewhat less detailed in application. In the treatment of Hagiwara et aE. (1974)) it was stated that the mobility term, I,,,, and the dissociation constant, K , were both functions of voltage. Although this was acknowledged, no attempt was made either theoretically or experimentally to determine the values of either function a t more than one voltage. Both Woodhull (1973) and Strichartz (1973) have approached the problem of determining the binding constant for a blocking ion acting on the sodium channel. Blocking by H+ and Ca2+, investigated by Woodhull, and blocking by quaternary lidocaine derivatives, in the work of Strichartz, both showed voltage dependence. From the voltage dependence of the blocking, these authors were able to estimate the position of the binding site in the transmembrane potential field on the assumption that the membrane field modified the rate constants for the blocking ion-site reaction by appropriate Boltzmann factors. Cations H+ and Ca2+appeared to enter the channel from the outside and block a t a point about one-quarter of the way down the membrane potential drop, whereas the lidocaine derivatives seemed only to enter the open channels and to do so from the inner end blocking a t a point about halfway along the transmembrane potential gradient. Heckmann et al. (1972) put forth the suggestion that ionic blocking and unblocking of pores might provide the physical mechanisms for the normal voltage-dependent conductance changes of excitable membranes. There does not a t this point appear to be any compelling experimental evidence for this notion, and i t will be implicitly assumed in the treatment that follows that we are describing ionic fluxes through open channels. Some independent knowledge of the voltage and time-dependent action of the channel gates would be required to reconstruct a complete current-voltage curve from first principles. The model does, however, provide a basis for expressing saturation and blocking effects in terms of the voltage-dependent rate parameters for the reaction of ions with the channel sites. The approach that we outline follows that of Hille (1975a,b) and of French et al. (1974) and owes much to the pioneering work of Eyring and collaborators (see Parlin and Eyring, 1954), A similar analytical formalism was also used by Hill and Chen (1971) in a wide-ranging exploration of the proper-

195

IONIC COMPETITION IN MEMBRANE CHANNELS

0

I

FIG.4. Energy profile for a simple single-site channel model. The ionic binding site S is represented by an energy well. The ai and a. are the association rates of ions with the site S from inside (i) and outside (o), respectively, a t unit concentration and zero applied voltage; the ai and a. are dissociation rates toward the inside and outside, respectively, a t zero applied voltage.

ties of one-site, two-site, and multisite channel models. Not only blocking ions but also ions passing through the channel are assumed to bind transiently to a common site in the channel. The passage of an ion through the channel may then be represented by the following equations and the barrier profile depicted in Fig. 4. At zero voltage, where M is a univalent positive ion, ai

L

Mi + S S M S = M . di

f S

(10

When there is an applied voltage

V

=

( E ; - E,) F / R T

the rate parameters become ai (V) = a;exp (pV/2)

di (V) =

ai exp (- pV/2)

a, (V) = a, exp (-[I

do ( V ) = 8, exp ([I

PI V/2) - PI Vl2) -

( 6)

where p is the fraction of the total potential drop between the inside and the site. If the ion is only present on one side of the channel or if it can react with the site S from only one side, the dissociation constant K M(V) may be defined by

&(V) a; K u ( V ) = -- - exp(-pV) a d V ) a;

(7)

Under the assumption that the reaction between ions and site is in the

196

ROBERT J. FRENCH AND WILLIAM J. ADELMAN, JR.

steady state, one can derive a current-voltage relation of the general form as that used by Hagiwara et al. (1974). Hille (1975a) has used a slightly more complex model with several barriers in order to make the instantaneous current-voltage relation more linear. He did, however, retain in his treatment of Na channel currents, the single-occupancy restriction and assumed one of the energy wells was significantly deeper than the rest. The latter assumption means that it is probable that any ion dwelling for a significant time in the channel would do so a t the same site. Hille was able to fit sodium current-voltage relations for a wide range of concentrations (0.13-2 times normal Ringer solution concentrations) over which there were significant systematic deviations from independence principle predictions. These deviations from independence he attributed to small but significant saturation effects; he estimated the dissociation constant for Na+ in the channel to be approximately 400 mM. Other ions whose currents fell more obviously below independence principle predictions yielded much lower dissociation constants within the concentration range of ions in normal physiological solutions (values for thallium and hydroxyguanidine fall in the 20-40 mM range). Still, there remain complications in the behavior of real membranes that do not seem to be reconcilable with a simple single-occupancy model of an ionic channel. It is a common feature of all the physical models so far discussed that the interactive effects of different ions arose purely from competition for unoccupied channel sites. Once occupied by one ion, a channel site was assumed immune to any interaction with other ions until the first ion spontaneously leaves the site in a first-order dissociation reaction. There are observations on both the sodium and the potassium channels of nerve preparations that appear qualitatively inconsistent with this relatively simple view. Examples already mentioned are those of the reversal potential behavior of the sodium currents of squid axon (Adelman and Senft, 1968) and the biionic potentials of the gramicidin bilayer (observations-Myers and Haydon, 1972; rate theory model predictionsLauger, 1973). The observation by Armstrong (1971) that an increase in external K+ concentration speeds the rate of unblocking of K+ channels that have been exposed to &A ions is another case. Such behavior cannot be obtained from a single-site model unless one allows incoming ions to react with a n occupied pore. This is the basis of a “knock-on, knock-off” type of model proposed by Armstrong (1975). An alternative possibility that explains the unblocking 8 Since the preparation of this manuscript, a lucid and detailed account of a fourbarrier model for the sodium channel has appeared in print. We refer the reader to Hille (1975b).

IONIC COMPETITION IN MEMBRANE CHANNELS

197

effect of potassium and better fits the voltage dependence of action of some blocking ions is the two-site model described by Hille (1975a). C. Qualitative Models-Molecular

Architecture of Ionic Channels

An imaginative set of experiments by Hillc (1971) using a series of small organic ions enabled him to suggest limits on the internal dimensions of the sodium channel. Other studies of the interaction of relatively complex organic ions with excitable membranes have yielded a diverse collection of clues about the structure of ionic channels. The effects of agents such as TT X , STX, and &A ions cannot be attributed simply to a n elcctrostatic interaction between the ionic center of charge and the channel; the more detailed structure of these compounds is clcarly important. Kao (1966), Hille (1970), and Evans (1972) have discussed the pharmacological effects of T T X and STX. Both toxins possess a guanidinium group, and, since the free guanidinium ion passes through the sodium channel fairly readily ( P g u n n i d , n i u m / P ~ s = 0.13) (Hille, 1971), it is reasonable to suggest that the guariidinium groups of TTX and STX enter the sodium channel when these molecules bind to the membrane and block the sodium current. However, the ionic interactions of the simple guariidinium ion with the channel give no explanation of the tremendous potency of these drugs that bind with dissociation constants in the nanomolar range (Cuervo and Adelman, 1970; Hille, 1970; Henderson et al., 1973; Schwarz et al., 1973). One must, therefore, postulate interactions between other parts of the molecule with components of the chanriel or the adjacent membrane. I n a n analogous manner, although TEA ions are probably able to block the potassium channels in nerve and muscle because of the similarity in size between the hydrated K+ ion (with a single hydration shell) and TEA, the experimental evidence of Armstrong (1969, 1971) indicates that the more hydrophobic tails of other &A ions of the form R - N ( C Z H ~ ) ~ + enhance greatly their binding in the blocking position. Thus, there is presumably a n accessible hydrophobic region of the membrane in the immediate vicinity of the inner mouth of the potassium channel. At the outer end, at least in frog node, this is apparently not true as the more hydrophobic &A ions are less potent blockers of the I<+current than TEA (Armstrong and Hille, 1972). Woodbury ( Woodbury and Miles, 1973) postulated that hydrophobic interactions also play a role in determining the conductance sequence of the anion channel in frog skeletal muscle. He hypothesized that for those ions that are functionally benzoate-like, namely, benzoate, valerate, butyrate, propionate, formate, acetate, the rate of passage through the membrane is

198

ROBERT 1. FRENCH AND WILLIAM J. ADELMAN, JR.

limited by an energy barrier. Moreover, this barrier is effectively lowered by binding of the hydrophobic moiety of the ion to a hydrophobic site adjacent to the point of the rate-limiting step in permeation. Aside from the anomalous position of formate with respect to acetate, the conductance sequence (see Section II1,C) is qualitatively consistent with this hypothesis. Formate, of course, can hardly be considered to have a significant hydrophobic moiety and, hence, one might suppose that the details of its ionic interactions with the channel are more important in fixing its exact position in the conductance sequence. I n future, such studies may well throw further light, not only on the nature of molecular components in and around ionic channels, but also on the quantitative interrelation between binding and barriers in determining ionic selectivity of membranes.

V.

CONCLUDING REMARKS

It seems worth making a couple of general observations in retrospect. First, relatively simple models may be used to analyze data from carefully designed experiments to yield a t least a semiquantitative description of some channel properties. Second, new models need to be worked out in detail if we are to rationalize quantitatively even the data on hand. The analyses by Hagiwara and co-workers of the Ca2+channel currents in barnacle muscle (Hagiwara and Takahashi, 1967; Hagiwara et al., 1974) provide an apt illustration of the first point. Although not attempting to delve analytically into the subtleties of the voltage dependence of I,,, or Kpr+, these authors were able to extract binding sequences to the membrane sites for various divalent ions. The theory of equilibrium binding selectivity has been extensively investigated by Eisenman and co-workers (see Eisenman, 1961, 1965; Diamond and Wright, 1969). The ionic current data thus provide useful information about the membrane sites when considered in the light of binding selectivity theory, although this would not have been possible had the authors not been careful to obtain their affinity sequences at a constant membrane voltage. More information about the channel and the sites might certainly be and K M Hparamobtained by studying the voltage dependence of the Imax eters, which would provide a complement and an extension to the analysis of the simpler model rather than negating its contribution. I n a similar spirit, the negative slope resistance shown by K+ channel currents in the presence of other alkali cations (Bezanilla and Armstrong, 1972) may be analyzed in terms of a single-site channel model to give estimates of

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dissociation constants of the blocking ions and of the site position (French, Lecar, and Ehrenstein, unpublished!. As already mentioned, however, the simple single-site model is inadequate to describe all of the available data. One should also be aware that “dissociation constants,” estimated under the assumption of a single, physically unique site in the channel from, say, the concentration dependence of conductance, take on a different significance if one is actually studying a long pore system. The half-saturation concentration then becomes a somewhat more general function, depending on the probability of occupancy of the various pore sites, rather than the property of a distinct type of site (see, for example, the series of papers by Heckmann) . The exciting experimental approach of Henderson et al. (1973) provides a direct approach to measuring the properties of presumed channel sites from the competitive effects of various ions on STX binding to a solubilized membrane extract. Binding constants for H+, Ca2+,and Tl+ ions estimated in this way are remarkably close to the values estimated from their effects on the current through the sodium channel in physiological preparations. The focus throughout this paper has been on the effects of ion binding in channels. Data showing clear evidence of ionic binding affecting transmembrane currents have been reviewed. A central problem that remains is to sort out the relative influences of energy barriers and energy wells (binding sites) on the selectivity exhibited by biological membranes. In a number of different preparations, it has been experimentally demonstrated that there is no single selectivity sequence for a given channel. The sequences of relative permeability estimated from resting or reversal potential shifts, and the relative ionic conductance sequences are not identical (for data on frog node, see Hille, 1971, 1972; for crayfish neuromuscular junction, see Takeuehi and Takeuchi, 1971). I n frog muscle, the sequence of conductances of anions appears not to be identical with the sequence of binding constants (Hestenes and Woodbury, 1973) , although the conductance and permeability sequences are the same (Woodbury and Miles, 1973). What, then, is the importance of barriers and binding sites? The question has been tackled directly by Hestenes and Woodbury (1972, 1973) and by Woodbury and Miles (1973), and it would be interesting to see the predictions of the model pursued and tested in quantitative detail. One can devise kinetic models of channels in which selectivity is totally determined by barriers or selective exclusion (see Armstrong’s appendix in Bezanilla and Armstrong, 1972; and Armstrong, 1975) and this may well be the basis for the all-important selection between Na+ and K+ by the channels of nerve membrane. Still, there is strong experimental evidence that binding can limit currents and modify apparent channel selectivity in other cases.

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An important function of a working model is to provide a plausible physical interpretation of selectivity sequences as indicated by different parameters, such as conductance and permeability, as well as to suggest further experimental tests. Perhaps a complete model of any channel will require the development, following Hille’s (19754 suggestion, of a theory of selectivity by energy barriers along the general lines of Eisenman’s theory of selective binding. Nonetheless, we feel that studies of ionic binding as indicated by saturation and competition phenomena have played and will play a significant role in extending our underst,anding of the mechanism(s) of ionic permeation through excitable membranes. ACKNOWLEDGMENTS We would like to thank Drs. Gerald Ehrenstein and Harold Lecar for their critical comments in the early stages of preparation of this article, Drs. Terrell Hill and Adrian Parsegian for reading the manuscript and making critical comments, and Mrs. Sandra Cotter for her assistance in producing the typed copy. REFERENCES Adam, N. K. (1941). “The Physics and Chemistry of Surfaces.” Oxford Univ. Press, London. Adelman, W. J., Jr. (1968). In the squid axon, potassium current blockade by internal cesium is dependent on membrane potential and current direction. Annu. Meet. Biophys. SOC.Abstr., Idth, 1968 133a (Abstract). Adelman, W. J., Jr. (1971). Electrical studies of internally perfused squid axons. In “Bio,physics and Physiology of Excitable Membranes” (W. J. Adelman, Jr., ed.), pp. 274-319. Van Nostrand-Reinhold, Princeton, New Jersey. Adelman, W. J., Jr., and Senft, J. P. (1966). Voltage clamp studies on the effect of internal cesium ion on sodium and potassium currents in the squid giant axon. J. Gen. Ph,ysiol. 50, 279-293. Adelman, W. J., Jr., and Senft, J. P. (1968). Dynamic asymmetries in the squid axon membrane. J. Gen. Physiol. 51, 102s-114s. Adelman, W. J., Jr., and Senft, J. P. (1971). In the voltage clamped squid axon internal sodium inhibits potassium ion membrane currents. Fed. Proc., Fed. Am. Soe. Ezp. Biol. 30, 665. Adelman, W. J., Jr., Palti, Y., and Senft, J. P. (1973). Potassium ion accumulation in a periaxonal space and its effect on the measurement of membrane potassium ion conductance. J. Membr. Biol. 13,387410. Adrian, R. H. (1961). Internal chloride concentration and chloride efflux of frog muscle. J. Physiol. (London) 156,623-632. Adrian, R. H. (1969). Rectificat,ion in muscle membrane. Prog. Biophys. Mol. Biol. 19, Pt. 2, 341-361. Alvarez, O., Diaz, E., and Latorre, R. (1975). Voltage dependent conductance induced by hemocyanin in black lipid films. Bioehim. Biophys. Acta 389, 444-448. Armstrong, C. M. (1966). The time course of TEA+-induced anomalous rectification in squid giant axons. J. Gen. Physiol. 50,491-503.

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Properties of the Glucose Transport System in the Renal Brush Border Membrane R. K I N N E Max-Planck- Instit ut jur Bio physik Frankfurt, Germany

. . . . . . . . . . . . . . . . . I. Introduction 11. Isolation and Characterization of Plasma Membranes from Proximal Tubular Epithelium . . . . . . . . . . . . . . . A. Isolation of Brush Border Fragments, Single Microvilli, and BasalLateral Plasma Membranes . . . . . . . . . . . . B. Chemical and Biochemical Composition of Brush Border Membranes and Basal-Lateral Plasma Membranes . . . . . . . . . 111. Interaction of D-Glucose with Isolated Renal Plasma Membranes . . . A. Glucose Transport by Isolated Brush Border Microvilli and Isolated Basal-Lateral Phsma Membranes. . . . . . . . . . . B. Other Studies on the Interaction of Glucose with Renal Plasma Membranes . . . . . . . . . . . . . . . . . IV. Interaction of Phlorizin with Isolated Renal Plasma Membranes . . . A. High-Affinity Phlorizin-Binding Sites in the Brush Border Membrane. B. Interaction of Phloriain with Low-Affinity Binding Sites in Renal PlasmaMembranes . . . . . . . . . . . . . . V. Molecular Characteristics of the Sugar Transport System in the Brush Border Membrane . . . . . . . . . . . . . . . . A. Stereospecificity of the Sugar-Binding Site . . . . . . . . . . . B. Cation-Binding Site. . . . . . . . . C. Aglucon-Binding Site . . . . . . . . . . . . . VI . Conformational Response of the Glucose Transport System . . . . VII. Relation of Renal Glucose Transport System to Enzymes Interacting . . . . . . . . . . with Carbohydrates . . . . VIII. One or Several Glucose Transport Systems in the Brush Border Membrane? . . . . . . . . . . . . . . . . . . IX. Summary and Conclusions . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . Note Added in Proof 1.

209 21 1 213 214 218 222 230 233 233 243 245 245 250 250 23 1 2.56 2.57 2.59 2.X 267

INTRODUCTION

Uptake of sugars by cells or cell-like organisms is a phenomenon observed in a variety of biological systems. In principle two types of uptake might be 209

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distinguished. In nonpolarized cells, such as bacteria, muscle, adipose tissue, and erythrocytes, the uptake of sugars is destined to provide sufficient substrate for the intracellular utilization. In polarized epithelial cells, however, the cellular uptake can be considered as an intermediate step in the transcellular transport that involves the transfer of sugars through the two cell membranes arranged in series. The transport of sugars can either be active or passive. A transport is defined thermodynamically as active if it is observed under conditions where all physical driving forces, such as electrochemical potential, pressure, temperature, and, in addition, water flow, across the membrane are zero. The active transport of a solute can be coupled either directly t o the cell metabolism via ATP or other energy-rich substrates, resulting in a so-called primary active transport, or can be coupled to the flux of another substance whose transport in this case is directly dependent on the cellular metabolism. This so-called cotransport can be either a symport when both substances move in the same direction or an antiport if the directions are opposite. The term passive is used for all other transports that do not fulfill the foregoing requirements. In bacteria, active transport dominates and involves three systems: the phosphoenolpyruvate-dependent sugar phosphotransferase system (85, 1 1 1 ) , active transport systems similar to the Escherichia coli lactose system ( 5 , 5 5 ,57,58),and active transport systems sensitive to cold osmotic shock (51).In mammalian cells, both active and passive transport of sugars have been observed. In liver (@), muscle (98), adipose cells ( l o g ) , eye lens (54), and erythrocytes (84,148),the transport is passive and facilitated by the presence of specific carrier systems in the membrane [for a recent review, see Morgan and Whitfield ( 9 7 ) ] ,some of them, as in muscle (98) and adipose cells ( 9 ) , are subject to hormonal regulation, especially by insulin. Examples of tissues transporting sugars actively are the mucosa of the small intestine ( 1 , 23, 24, 26, 26, 48, 131) and the proximal convoluted tubule of the kidney. Investigations on renal sugar transport have begun to define the overall capacity of the whole organ and its regulation by various physiological parameters. With the aid of micropuncture techniques, the proximal tubule has been identified as the main site of sugar reabsorption (42, 110). The phenomenological description of sugar transport across this epithelium demonstrated two components-a passive and an active one. The latter shows saturation kinetics with respect to D-glucose and is inhibited by low concentrations of phlorizin (88, 89). In parallel with these studies, kidney slice experiments were the first to provide information on substrate specificity and electrolyte requirement of the sugar transport systems in the tubular cells (76, 77, 118, 119). The results obtained by those techniques have been reviewed recently (96, 99).

GLUCOSE TRANSPORT IN KIDNEY BRUSH BORDER MEMBRANE

21 1

During recent years, an increasing number of studies have utilized isolated plasma membrane fractions to investigate the mechanism of renal sugar transport a t the subcellular level. The purpose of the present contribution is to review these investigations and to discuss present knowledge of the molecular mechanism of sugar transfer across the epithelial plasma membranes in the proximal tubule of the kidney.

II. ISOLATION AND CHARACTERIZATION OF PLASMA MEMBRANES FROM PROXIMAL TUBULAR EPITHELIUM

Epithelial cells of the proximal tubule show an asymmetric organization of their plasma membranes that face the lumen of the tubule and the interstitial fluid (Fig. 1 ) . The luminal or apical surface is covered by the brush border membrane, which consists of numerous extensions of the plasma membrane, the so-called microvilli. The contraluminal or basallateral face is characterized by the invaginations and interdigitations of the basal-lateral plasma membrane which, as a whole, form the basal infoldings of the tubular epithelium (35). The analysis of biochemical aspects of membrane function in the transport process required isolation of the luminal and contraluminal membranes separately. nuclei

brush border

7

basal -

lateral plasma membrane basement ‘membrane

FIG.1. Schematic representation of the morphological appearance of epithelial cells in renal proximal tubules. Note the different organization of luminal plasma membrane (brush border) and contraluminal plasma membrane (basal-lateral plasma membrane). Modified after Yoshimura and Nakamura, Folia. Anut. Jpn. 41, 121 (1965).

212

R. KINNE

GLUCOSE TRANSPORT IN KIDNEY BRUSH BORDER MEMBRANE

213

A. Isolation of Brush Border Fragments, Single Microvilli, and Basallateral Plasma Membranes

The brush border membrane and the basal-lateral plasma membrane are different regions of the same cellular envelope. Although different in function, they have very similar lipid and protein content (see in following). Therefore, the membranes exhibit only small differences in buoyant density and cannot be separated effectively on that ground. Two other propesties of the membrane fragments have been utilized instead: the shape of the fragments and their different surface charges. The shape of the fragments is the dominating discriminatory factor for the isolation of coherent clusters of microvilli (the so-called brush border fragments). Brush border fragments were first isolated from the intestine by Miller and Crane (94). In the kidney these fragments can be obtained essentially by three methods : (i) by low-speed differential centrifugation, followed by sucrose density gradients and further differential washings (4,68, 145) ; (ii) by first using a sucrose density gradient for the subfractionation of the homogenate and then washing the membranes by an additional series of centrifugation steps (8, 107, 140) ; and (iii), most recently, by successfully applying the two-phase polymer system of Albertsson to the separation of brush border fragments (46). The typical morphological appearance of such fragments is shown in Fig. 2A. The fragments form a spherelike structure with an average diameter of 5 pm, and the well-preserved microvilli extend in all directions. Thin sections (Fig. 2B) show a high number of pseudovesicular elements representing sections through different planes of the microvilli. The enrichment of marker enzymes for the brush border membrane (alkaline phosphatase, maltase, and trehalase) in brush border fragments prepared by the different isolation procedures are compiled in Table I. All membrane fractions are devoid of significant contamination by nuclei, mitochondria, endoplasmic reticulum, Iysosomes, and cytoplasmic enzymes. They contain, however, to a varying degree basal-lateral plasma membranes as can be concluded from the enrichment of the Na-K-ATPase, a marker enzyme for contraluminal membranes in the kidney (60, 116). This contamination of brush border fragments with basal-lateral plasma membranes arises most probably because during the homogenization of the renal cortex the cells of FIG.2. (A) Morphological appearance of a brush border fragment isolated according to Kinne and Kinne-Saffran (68). The membranes were fixed in glutaraldehyde and placed directly on the copper grid for electron-microscopical analysis. Magnification : X23,OOO. (B) Thin section through a brush border fragment isolated according to Kinne and Kinne-Saffran (68). Magnification: X24,900.

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TABLE I ENRICHMENT VALUESREPORTED BY VARIOUS AUTHORS FOR ENZYMES IN KIDNEY BRUSHBORDER FRAQMENTB'

Enzyme Alkaline phosphatase Maltase Trehalase Mg-ATPase Na-K-ATPase

KK Rat 12

1.4 4.4

WN Rat

GG Rat

16

7.1 12.5

2

5 4

Authorb BS QR GK CL Rabbit Rabbit Rabbit Rabbit 4.5 14 12

-

19 16 14 3.7 5

15 22 15 5.5 4

4.9 9.2 9.2

-

-

a Enrichment is expressed as the ratio of the specific activity in the brush border to that of the kidney cortex homogenate. -, Not determined. b Key to authors: KK, Kinne and Kinne-Saffran (1969); WN, Wilfong and Neville (1970); BS, Berger and Sacktor (1970); QR, Quirk and Robinson (1972); GK, George and Kenny (1973); CL, Chertok and Lake (1972); GG, Glossmann and Gips (1974).

the proximal tubule rupture in planes below the junctional complexes (140) and, therefore, part of the basal-lateral plasma membranes remains linked to the brush border fragments. When higher shearing forces are used to break up the cells, predominantly single microvilli are formed. They cosediment in the heavy microsomal fraction with fragments of basal-lateral plasma membranes. The former can be separated from the latter by free flow electrophoresis owing to a difference in electrophoretic mobility (60, 69). As shown in Fig. 3, the microvillar fraction obtained after electrophoresis is characterized by a high enrichment of alkaline phosphatase and a reduction of Na-K-ATPase activity as compared to the starting material. The basal-lateral membrane fraction, however, shows a high specific activity of Na-K-ATPase and a low activity of alkaline phosphatase. In both fractions, no enrichment of marker enzymes for other cellular elements is observed (60),indicating a high degree of purification. The morphological appearance of the two fractions separated by free flow electrophoresis is depicted in Fig. 4: the microvillar fraction consists of single microvilli with the typical glycocalyx at the membrane surface, whereas the basal-lateral membrane fraction contains big sheets of plasma membranes with a smooth surface. B. Chemical and Biochemical Composition of Brush Border Membranes and Basal-lateral Plasma Membranes

Determination of the components of single microvilli and basal-lateral plasma membranes revealed, as shown in Table 11, only minor differences in

GLUCOSE TRANSPORT IN KIDNEY BRUSH BORDER MEMBRANE

215

FIG.3. Activity of alkaline phosphatase (clear) and Na-K-ATPase (shaded) in rat kidney cortex homogenates, unfractionated plasma membranes, brush border microvilli, and basal-lateral plasma membranes purified by free flow electrophoresis. The data (n = 5 ) are expressed as enrichment compared to the homogenate. Specific activity is expressed as unita/mg protein as stated below t,he bars.

the composition of the membranes with respect to carbohydrates, sialic acid, and phospholipids. However, the enzyme composition of the two membranes differs markedly. Table I11 compiles the data obtained so far on the distribution of membrane-bound enzymes in the apical and basal regions of the cellular envelope of the epithelial cell of the proximal tubule. The brush border membrane contains hydrolases acting on phosphate esters (alkaline phosphatase, 5-nucleotidase, Mg-ATPase and HC03ATPase) , on peptides (aminopeptidases and 7-glutamyltranspeptidase) , and on disaccharides (trehalase and maltase) . Also a cyclic AMP-dependent protein kinase, which might be involved in the regulation of phosphate transport by parathyroid hormone, was found in the apical membrane. In the basal-lateral plasma membranes, a Na-K-ATPasc, a Ca-ATPasc, and a parathyroid hormone-sensitive adenylate cyclase have been described. Corresponding to the discrepancies in the enzyme content, the protein pattern of the two membranes obtained after polyacrylamide gel electrophoresis is diverse (Fig. 5). At the moment, only a few bands of the whole spectrum can be related to an enzymatic activity or transport function. The main bands of the microvillar proteins in the region of molecular

216

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weights between 140,000 and 160,000 are most probably identical with aminopeptidase M (137) and alkaline phosphatase (137, I.&), respectively. Other hydrolases, such as aminopeptidase A (60),with similar molecular weights migrate also into this part of the gel. The function of the majority of the peptides separated by gel electrophoresis, however, is as yet undefined.

GLUCOSE TRANSPORT IN KIDNEY BRUSH BORDER MEMBRANE

217

FIG.4. (A) Morphological appearance of brush border microvilli isolated by free flow electrophoresis. In the upper part, a thin section and, in the lower part, a negatively strained specimen are shown. A t higher magnification (see inset) the glycocalyx covering the microvilli is clearly visible. Reprinted from Heidrich el a2. (60) by kind permission of the Rockefeller University Press. (B) Morphological appearance of basal-lateral plasma membranes isolated by free flow electrophoresis. In the upper part a thin section and in the lower part a negatively stained specimen are shown. Reprinted from Heidrich el al. (60) by kind permission of the Rockefeller University Press.

218

R. KINNE

TABLE I1

CHEMICAL COMPOBITION OF BRUBH BORDER MICROVILLI AND BABAGLATERAL PLABMA MEMBRANES IBOLATED FROM RATKIDNEYCORTEX BY FREEFLOWELECTROPHOREBIBa-b

Compound Lipids : Phospholipids (nmoles/mg protein) Sphingomyelin Phosphatidylcholine and 1ysophosphatidylethanolamine Phosphatidylethanolamine Phosphatidylserine and phosphatidylinositol Cholesterol and choleaterolester (nmoles/mg protein) Carbohydrates (nmoles/mg protein) : Hexosamine Sialic acid

Basal-lateral plasma membranes

Brush border microvilli

470 f 75 22.5% f 1.4

395 f 104 28.9% f 0.5

24.8% f 2.0 22.0% f 1.0

18.9% f 2.7 23.5% f 0.9

32.4% f 2 . 0

28.1% f 3.6

356 f 30

264 f 17

156 f 5 29 f 2

201 f 12

29f2

Data from Kinne et al. (71). The mean values and standard deviation derived from 3 to 5 debrminations are given. a

111.

INTERACTION OF D-GLUCOSE WITH ISOLATED RENAL PLASMA MEMBRANES

There is increasing evidence from studies on the transport properties of a variety of cells that the nonelectrolyte (and probably also the electrolyte) permeation through the plasma membrane is determined both by the composition of the membrane lipids and by the presence or absence of specific transport systems ($9, 147). The permeation of the compounds across the lipid phase is controlled largely by the solute-membrane partition coefficients and the molecular size of the solutes ($9). For large lipophilic solutes, the partition coefficient appears to be the predominant factor, whereas, for small polar solutes, molecular size seems to be more important. Specific transport systems increase the permeability of the membranes by order of magnitude above the permeability observed in artificial lipid bilayer membranes. This increase in permeability is accompanied by an increased stereospecificityof the permeation. In the case of sugar transport,

GLUCOSE TRANSPORT IN KIDNEY BRUSH BORDER MEMBRANE

219

TABLE I11 OF ISOLATED BRUSHBORDER MEMBRANES AND ENZYMATIC ACTIVITIES BASALLATERAL PLASMA MEMBRANES AS DETERMINED BY VARIOUS AUTHORS

Brush border membranes (Ref.)

Basal-lateral plasma membranes (Ref.)

Esterases: Alkaline phosphatase (8, 19, 44, 46, 107, Na-K-ATPase (60,69)

146)

5'-Nucleotidase (46, 69) Mg-ATPase (44, 68, 69, 107) HCOa-ATPase (76)

Ca-ATPase (74)

Disaccharidases: Maltase (8, 18, 44, 45, 46, Trehalase (8, 18, 44, 107)

lm, 133)

Aminopeptidase M (44, 69) Aminopeptidase A (44, 69, 60) yGlutamyltranspeptidase (46) -

-

-

Peptidases:

-

Cyclases: Parathyroid hormonesensitive adenylate cyclase (93, 122, 123) Kinases :

CAMP-dependent protein kinase (73, 122)

-

no difference in the uptake of D- and L-glucose into erythrocyte lipids waa observed (86), whereas the glucose transport system of the intact red blood cell shows a high degree of stereospecificity ( 8 4 ) .Also, the glucose permeability of lipid bilayer membranes has been shown to be some lo6 times lower than that of the human red cell (146). All specific transport systems isolated so far are polypeptides (38, 53, 54, 65, 103, 104). The protein nature of the systems is also obvious from the sensitivity of transport processes against side group-specific reagents, such as SH-group reagents that modify side chains of membrane-associated proteins, and is supported by the observation that transport systems are susceptible to proteolytic digestion. In studies on the properties of a transport system using isolated plasma membranes, we need to consider which reactions between the membranes and the solute can take place and which are investigated under the given experimental conditions. D-Glucose, for example, can be bound to the membranes and, if the membranes are vesiculated, subsequently can be

220

R. KINNE

FIQ.5. Protein composition of basal-lateral plasma membranes and brush border microvilli isolated from rat kidney cortex by free flow electrophoresis. The membranes were solubilieed by treatment with 0.21% sodium dodecyl sulfate (SDS) (ratio protein/ detergent, 2: 1) for 10 minutes at 25'C. After centrifugation for 10 minutes a t 15,000 g, aliquota of the supernatant containing 100 pg protein were placed on polyacrylamide gel slabs (7.5% acrylamide, 0.1% SDS), and the proteins were separated by application of 75 mA for 30 minutes followed by 150 mA for 2 hours. After separation the proteins were stained with comassie blue. The front is marked by the internal reference protein, cytochrome c.

transported into the vesicles (see also 85,95). These two phenomena can be distinguished by the different sensitivity of the processes to the osmolality of the incubation medium. Since the intravesicular space is surrounded by a selectively permeable membrane, increased concentrations of nonpermeable substances outside the vesicles reduce the intravesicular space and also decrease the amount taken up by the membranes at equilibrium. This phenomenon is illustrated in Fig. 6 for the uptake of D-glucose by

22 1

GLUCOSE TRANSPORT IN KIDNEY BRUSH BORDER MEMBRANE

cpm

t

cpm

[1 I OSmOl]

t

.

6 Zmin

,Om,n} Incubation

[1 /Osmol]

FIG.6. Effect of increasing osmolality of the incubation medium on the uptake of D-glucose by isolated brush border microvilli (left) and isolated basal-lateral plasma membranes (right). The osmolality was increased by the addition of sucrose to the standard incubation medium, the uptake was determined as described previously (79).

brush border microvilli. This is one of the strongest arguments for vesicular transport, provided that the osmotically active substance does not interfere directly with the transport systems. In the case just demonstrated, it has been shown by independent experiments on phlorizin binding that sucrose does not influence the binding characteristics of the transport system. Other indications for transport rather than binding are the observation of countertransport and the inhibition of efflux by specific inhibitors ( 2 , 72). I n the reports published so far on the interaction of glucose with isolated renal membranes, the contribution of binding can be estimated to be very low. All studies employed filtration techniques to separate the membranes from the incubation medium whereby the samples are diluted prior to filtration and the membranes are washed additionally after collection on the filter. These procedures introduce a disequilibrium and reduce, due to the reversible nature of the binding process, the amount of radioactivity associated with the membranes, especially if a low-affinity system such as the glucose transport system is investigated. Thus transport of sugar into membrane vesicles rather than binding to the membranes must have occurred. The amount transported by the vesicles as measured by the filtration technique is also influenced by the dilution and washing procedures. During the time required for the transfer of the membranes to the filter and for the washing procedure, solutes from the inner space of the vesicles are lost. This loss of material cannot be neglected for the relatively leaky plasma membrane vesicles. Therefore, kinetic constants derived from such experiments

R. KlNNE

222

have to be interpreted with great caution. They result from several processes: some related to the transport properties of the membranes and some introduced by the experimental procedures. All of these processes take place simultaneously as well as sequentially. The most favorable technique for uptake studies seems to be the centrifugation procedure developed by Klingenberg and his associates (79) in which the intravesicular concentration is influenced as little as possible by the method used to separate the vesicles from the incubation medium. The kinetics of vesicular uptake can be further complicated by the metabolic conversion of substrate inside the vesicles. Indeed, some membrane preparations have been shown to contain an appreciable amount of glycolytic enzymes and coenzymes that catalyze the conversion of glucose to glycolytic metabolites (16) 62).Moreover, the results can be influenced by the presence of more than one population of membrane vesicles in the fraction studied. This is especially important for membrane fractions derived from polarized epithelial cells where the luminal and the contraluminal membranes are supposed to contain transport systems with different properties (78, 101, 128, 136). This complexity of the membrane system and of the experimental procedures can explain at least partially the contradictory results obtained so far on the interaction of D-glucose with isolated renal plasma membranes (Table I V ) . A. Glucose Transport by Isolated Brush Border Microvilli and Isolated Basal-lateral Plasma Membranes

Recently, a more defined study on D-glucose transport of isolated brush border microvilli and isolated basal-lateral plasma membranes was carried out in our laboratory (70, 72). In these experiments, membranes separated by free flow electrophoresis were used. These membranes were only slightly cross-contaminated M judged by analysis of marker enzymes; in other words, the brush border microvillar fraction contained few basal-lateral plasma membranes and vice versa. Thus relative homogeneous membrane populations with respect to the cellular origin were utilized. Furthermore, the extent of glucose leakage out of the vesicles after termination of the incubation was minimized by the use of a low-temperature (“ice cold”) “stop solution” that in addition contained phlorizin, the specific inhibitor for glucose transport in kidney and intestine (SO, $1) 90). In Fig. 7 the typical characteristics of the transport of D- and L-glucose by the brush border microvilli are shown. At a concentration of 1 mi44 each, D-glucose is taken up by the microvilli about 10 times faster than L-glucose

CHARACTERISTICS OF THE ~

0

TABLE IV IXTERACTION BETWEEX SUGARS A N D RESALPLASMA MEMBRASES

E

k

2rn

~~~

Study No. and Sourre

Method

I. Busse el al. (1.5)

Rapid filtration and extensive washing with 4 x 10ml saline bicarbonate acetate buffer

Membrane preparation (Ref.) Rabbit kidney plasma membranes ( 5 2 ) , suspended in saline bicarbonate buffer

-4

Results Two uptake systems: System A. K , = 3 mM, VmaX= 2.2 X 10-lo moles/mg protein/2 min; inhibition of D-glucose uptake by D-galactose > 3-o-methyl-~-glucose > D-fructose > mannose > 2-deoxy-~-glucose > L-sorbose; phlorizin (0.1 mM) inhibits 27%; replacement of sodium by Tris or lithium inhibits 21 % System B. K , = 0.07 mM, VmaX= 50 X lo-'* moles/ mg protein/2 min; inhibition of D-glUCOSe uptake by mannose > D-fructose > L-sorbose > 2-deoxy-~glucose > 3-o-methyl-~-glucose > D-galactose; phlorizin (0.1 mM) inhibits 35%; replacement of sodium by Tris or lithium inhibits 53%

11. Chesney et al.

Rapid filtration and extensive washing with 5 X 5 ml Krebs-Ringer bicarbonate buffer

Rabbit kidney brush border fragments (8) suspended in KrebsRinger bicarbonate buffer

Two uptake systems: System 1. K , = 0.67 X M , VmBX= 1.4 X moles/mg protein/min; competitive inhibition by phlorizin ( K i = 2 x M ) ; replacement of sodium by Tris inhibits SO%, no change after replacement of sodium by choline or lithium System 2. K , = 20 ma!, Vmax = 21 nmoles/min/mg protein; replacement of sodium by Tris inhibits 50%, no change after replacement of sodium by choline or lithium; noncompetitive inhibition by phlorizin

(Continued)

P 5

f 7

z 6

E

TABLE IV (Continued)

Study No. and Source

Method

Membrane preparation (Ref.)

Results GGlucose uptake is not affected by sodium replacement and phlorizin; tglucose system is not saturable

Rabbit kidney brush One system; kinetic data derived from equilibrium (16 border fragments (18) min incubation); KD = 4 X 10-6 M, maximal “binding” capacity = 264 pmoles/mg protein; inhibisuspended in TrisMg-NaC1 b d e r ;for tion by phloriein (1mM) 80%; inhibition of =glucose studies on sodium uptake by 3-o-methyl-=glucose > D-mannose > dependence, wash in 2-deoxy-~-glucose > D-galactose > D-arabmose = sodium-free solutions =ribose > =fructose; replacement of sodium by (sodium replaced by potassium reduces uptake max. 55% potassium)

111. Chertok and Lake (19)

Iy.

Aronson and Sacktor (9)

Rapid filtration and rapid washing with cold buffer

Rabbit kidney brush border fragments (8) suspended in saline

-

One system; saturation phenomenon above 5 mM D-glucose; K, 1 mM; inhibition of D-glucose uptake by 3-o-methy1-~-g1ucose > D-galactose >

P

2

z,

V. Kinne el al. (70,721

Rapid filtration and rapid washing with cold phlorizin-containing isotonic solution

phosphate buffer; for studies on sodium dependence, wash in choline instead of sodium-containing buffers

D-mannose > 2-deoxy-~-glucose > a-methyl-Dglucoside; inhibition by phlorizin (1 mM) 40%; stimulation of uptake by sodium gradient SO%, by sodium 16%

Brush border microvilli (50) suspended in mannitol-Tris-Hepes buffer

Uptake of D-glucose and bglucose, initial rate of uptake for D-glucose about 10 times faster than for bglucose; inhibition of D-glucose uptake by 0.1 mM phlorizin So%, by 0.05 mM phloretin 10%; stimulation of D-glucose uptake by a sodium gradient about 4000/,; overshoot phenomenon for D-glucose and 3-o-methylD-glucose; half-maximal stimulation of uptake by sodium at a concentration of 29 meq

Basal-lateral plasma membranes (50) suspended in mannitol-Tris-Hepes buffer

Uptake of D-glucose 3 times faster than bglucose uptake; inhibition of D-glucose uptake by 0.1 mM phlorizin 35%, by 0.05 mM phloretin 25%; Stimulation by sodium gradient 65%; no overshoot

226

R. KINNE

O-gbm L-glucose

{

0

6

lWmM NaCl l W M KCI 100mM NaCl4.lmM phlaizin lOOmM NaCl

150

0

1

2

3 L 5 incubation time

25

[minl

FIQ.7. Transport of D-glucose and >glucose by isolated brush border microvilli. The membranes were prepared in 100 mM mannitol-1 mM Tris-Hepes buffer, pH 7.4, and incubated at 25OC in the same buffer containing in addition 1mM D- and 1 mM >glucose and the compounds indicated on the figure. The uptake waa determined aa previously described by rapid filtration methods (79).Mean values and standard errom of mean derived from four experiments are given aa percent of the equilibrium value.

in the initial phase. Transport of D-glucose but not of L-glucose is characterized by an overshoot phenomenon. Between 1 and 5 minutes of incubation, the amount of D-glucose found in the vesicles is greater than the amount found after 25 minutes. This value is identical to the equilibrium because longer incubation leads to no further change in the glucose retained by the vesicles. Since volume changes of the vesicles under these conditions are very unlikely [for further discussion, see Kinne et al. (72)], the overshoot indicates a transient accumulation of D-glucose in the microvilli above the concentration present in the incubation medium. The overshoot disappears when the membranes are incubated in a sodium-free solution in which sodium is replaced by potassium, or if phloriain at a concentration of 1 X M is present in the incubation mixture. Under both conditions, the initial uptake is inhibited about 80% (Fig. 7 ) . These data show that the brush border microvilli contain a stereospecific, sodium-dependent, phloriain-sensitive transport system for D-glucose. The sodium dependence of the glucose transport in the microvilli may

GLUCOSE TRANSPORT IN KIDNEY BRUSH BORDER MEMBRANE

227

be due both to the Na gradient (medium to vesicles) and to an interaction of Na with the membrane system facilitating D-glucose movement even if the sodium gradient is absent. Figure 8 shows the uptake of L- and D-glucose into microvillar vesicles preincubated in a sodium-free solution or in a solution containing 100 mM NaC1. In both cases, there was a greater uptake of D-glucose compared to L-glucose; however, in the absence of a sodium gradient, no overshoot was observed. The absence of an overshoot under conditions of equal sodium concentration on both sides of the membrane suggests that the sodium gradient is responsible for the transitory accumulation of glucose inside the vesicles. If the sodium dependence of D-glucose transport is studied further by stepwise substitution of Na+ by K+, the initial uptake rate (i.e., the uptake in 15 seconds) increases as a function of the sodium concentration. Doublc reciprocal plots lead to an apparent K , for sodium of 29 mM (72). To investigate further the cation site involved in glucose uptake, the

t

incubat ion time

FIG.8. Transport of D-glucose and tglucose by isolated brush border microvilli in the and in the absence ( 0),. of a sodium gradient across the membrane. presence ( 0 ,0) The gradient was abolished by preincubation of part of t,he vesicles in a solution containing 100 mM mannitol-1 mM Tris-Hepes buffer, pH 7.4, and 100 mM NaCl for 30 minutes a t 25°C. The control vesicles were preincubated for the same period in buffer from which sodium was omitted. The uptake was determined as described previously (72).

228

R. KINNE

TABLE V EFFECTOF CATIONS ON D-GLUCOSE UPTAKE BY ISOLATED BRUSHBORDER MICROVILLI

Cation Na+

K+

cs+ Li+ Rb+ Na+ + 0.1 mM phloriein

Glucose uptake in yoof uptake in the presence of sodiuma 100 31 =k 5

30 =k 12 26 f 10 21 Z k 12 30

a The amount taken up after 1 minute is given. The values represent mean values and standard error of mean derived from three experiments (78).

initial rate of D-glucose uptake was measured in the presence of other alkali metals, as shown in Table V. The sequence obtained was Na > K = Cs = Li = Rb, i.e., the acceleration of glucose uptake is sodium-specific. Since D-glucose uptake by brush border microvilli is related to the presence of a concentration gradient of sodium, it is possible that sodiumdependent glucose entry into the brush border particle is in part anion restricted. Figure 9 shows uptake data when a highly permeable anion such as thiocyanate (SCN) and a less permeable anion such as sulfate replaced chloride. The presence of SCN accentuated the D-glucose overshoot, and the presence of sulfate abolished the overshoot. These results can be explained by the assumption that the transfer of sodium coupled to the glucose transfer is electrogenic in nature, i.e., not charge compensated by the symport of an anion or antiport of a cation and, therefore, dependent on the transmembranal potential of the vesicle. This, in turn, is determined by the concentration and relative permeability of the cations and anions. Further evidence for this hypothesis can be derived from experiments in which the potential across the membranes was artificially increased. This can be accomplished by preloading the vesicles with potassium in the presence of an impermeable anion such as sulfate and by increasing the membrane permeability to potassium by the addition of the ionophore, valinomycin. As shown in Fig. 10, valinomycin induces an overshoot uptake of D-ghlCOSe into the potassium-preloaded vesicles. The same observation has been made with intestinal microvillar vesicles. There maneuvers such as using uncouplers, together with proton gradients, accelerate glucose uptake (100). Evidence for electrogenic transfer of

GLUCOSE TRANSPORT IN KIDNEY BRUSH BORDER MEMBRANE

n

.s L

229

3.0-

ea

. m

E

u)

%

.

b

-

C

I

A

2.0-

A-SCNA- so:-

E

U c

'

A

al

Y

0

c

n

=

1.0-

Q)

u)

-8

l?

n

0-.... 0

I

1

1

2

I

L

1

I

I

6 incubation time

t

8

I

10

[min]

FIG.9. Influence of anions on D-glucose transport by isolated brush border microvilli. Uptake waa measured in the presence of NaSCN, NanSO,, and NaCl with sodium kept a t 100 mM. I n the case of NaSO,, osmolality of the incubation medium was kept constant by increasing the mannitol concentration.

0

2

L

I

6

-

8 [min]

incubation time

FIG.10. Induction of D-glucose accumulation in isolated brush border microvilli by hyperpolarization of the intravesicular space. The microvilli vesicles were preloaded with K2SO. and uptake was measured in an incubation medium containing Na804. Addition of valinomycin (+ Val) renders the microvillar membrane permeable to K+ and induces a diffusion potential (intravesicular space negative compared to the incubation medium). For further detail see Murer and Hopfer (100).

230

R. KINNE

sodium via the glucose transport system is also adduced by the depolarization of the luminal membrane, which is observed when, in microperfusion experiments, glucose is added to the sodium-containing solution used for intratubular perfusion (43). Calculation of transfer numbers for the glucosedependent sodium transport yields a sodium-to-glucose ratio close to 1. For the intestine, Goldner et al. (48) obtained the same results from influx measurements of sodium and 3-o-methyl-D-glucose across the brush border membrane. Sodium-dependent electrogenic transport is also observed for neutral amino acids in the kidney (36, 43, 142) and intestine (102, 124). In addition, mutual interaction of amino acids and sugars has been described for the uptake by cells and isolated vesicles (1, 102, 120). According to Murer et al. ( l o g ) , this phenomenon is best explained by changes in transmembranal potential in the presence of the inhibitory species. The knowledge that the potential is a denominator of the uptake raises some criticism of the experiments performed with intact cells to determine the number and stereospecificity of sugar transport systems in the kidney. Inhibition of n-glucose transport does not necessarily mean a direct interaction of the compound tested with the transport system but could also be due to a change of transmembranal potential induced by the substance. Figure 11 gives the time course of sugar uptake into isolated basal-lateral plasma membrane vesicles. n-Glucose uptake in the first 30 seconds was about 3 times faster than that of L-glucose and was inhibited about 30% by 0.1 mM phlorizin. The replacement of sodium by potassium reduces D-glucose uptake to about the same degree as phlorizin. Microvilli are very sensitive to phlorizin inhibition of glucose transport and less sensitive to phloretin, whereas the basal-lateral membranes show a lower sensitivity t o phlorizin but a higher sensitivity to phloretin (Table VI). Thus the basal-lateral membranes also seem to contain a stereospecific glucose transport system, which, in contrast to the microvillar system, is only moderately phlorizin-sensitive or sodium-dependent.

B. Other Studies on the Interaction of Glucose with Renal Plasma Membraner

The results obtained by other authors on the interaction of glucose with isolated renal membranes are compiled in Table IV. Only the studies by Aronson and Sacktor (study IV) are in general accordance with the results reported in the preceding. The difference in the stimulatory potency of sodium and in the degree of inhibition by phlorizin can be explained if it is

23 1

GLUCOSE TRANSPORT IN KIDNEY BRUSH BORDER MEMBRANE

%

150

i

0

0

100mM NaCl

B

100mM NoCI+O.lmMph(arizin lOOmM NaCl

L-glucose

1

2

3

L

5

25

[min]

incubation time

FIG.11. Transport of D-glucose and L-glucose by isolated basal-lateral plasma membranes. Mean values and standard errors of mean derived from four experiments are given as percent of the equilibrium value. (For further details, see legend of Fig. 7.)

assumed that both transport into brush border microvilli and transport into basal-lateral plasma membranes occurred. This is because the brush border fragments investigated in study IV are said to contain a considerable amount of Na-K-ATPase (8),the marker enzyme for basal-lateral plasma membranes. The reports (studies I and 11) on the presence of two different transport systems in the membranes may be due to the inhomogeneity of TABLE V I EFFECT OF PHLORIZIN AND PHLORETIN ON D-GLUCOSE UPTAKEBY ISOLATED BRUSHBORDERMICROVILLI AND BASAL-LATERAL PLASMA MEMBRANESO

% ' Inhibition

Inhibition Phlorizin (M) 1 5 1

x x x

10"

10-6 10-4

Brush border

Basallateral

59.3 76.2 79.2

26.5 29.8 33.8

Phlore t in (MI 5 x 10-6 2.5 X loW6 5 x 10-5

Brush border

Basallateral

0 0 9.2

0

0 24.3

a Mean values of two experiments are given. Results were obtained a t a *glucose concentration of 1 mM after 1 min incubation under standard conditions (72).

232

R. KINNE

the membrane fraction. In study I1 the high-affinity transport system was found t o be caused by bacterial contamination of the fraction (94a), a problem that sometimes can also occur in glucose-"binding" studies with intestinal membranes. The kinetic data found by various authors for the transport systems differ markedly. These differences are probably more a reflection of the different experimental conditions than an indication of the existence of a variety of transport systems for glucose in the brush border membrane. Only the saturation phenomenon observed above 5 mM and the apparent K, value in the range of 1 mM for D-glucose, EW estimated for brush border fragments by Aronson and Sacktor ( d ) , seem to be a reasonable estimation in view of the data obtained by microperfusion in the proximal tubule (43, 88). There seems to exist also a great discrepancy concerning the sodium dependence of the transport. Studies IV and V (Table IV) have shown that marked effects of sodium on the glucose uptake by microvilli are observed only if a concentration gradient for sodium across the brush border membrane is present. Such a gradient is probably absent in experiments I and 11; this difference in experimental conditions may explain the apparent lack of sodium dependence in the study of Chesney et al. Again, due to the difference of the transport system in the microvilli and the basal-lateral plasma membranes with respect to sodium, the sodium response of a membrane fraction markedly depends on the number of functionally intact brush border vesicles. I n view of the difficulties discussed before, the stereospecificity of the systems in the plasma membrane fractions can hardly be considered to describe properly the stereospecificity of the sugar transport system in the brush border membrane. The experiments performed by Aronson and Sacktor and by Kinne et al. can be regarded as relatively well-defined transport studies since they exhibit sensitivity against medium osmolality, countertransport, and phlorizin-induced efflux inhibition. These studies demonstrate that the stereospecific, sodiumdependent, phlorisin-sensitive sugar transport system of the luminal cell pole is preserved in the isolated brush border membranes. They also show that an electrochemical potential difference for sodium across the brush border membrane seems to be a sufficient condition for an uphill transport of D-glucose. Because of the complexity of the system under study, however, the contribution of the transport studies to the characterization of the molecular properties of the transport system is as yet limited. It proved possible to simplify the system by using phlorizin instead of D-glucose to study the first reaction in the overall transport process, i.e., the binding of the substrate to t,he transport system. Studies of the effects of this competitive inhibitor of renal sugar transport in isolated plasma membranes are treated in the next section.

GLUCOSE TRANSPORT IN KIDNEY BRUSH BORDER MEMBRANE

IV.

233

INTERACTION OF PHLORlZlN WITH ISOLATED RENAL PLASMA MEMBRANES

Phlorizin, a phloretin 2’-glucoside (chemical structure shown in Fig. 12), is known t o inhibit sugar transport in the kidney and in the intestine in low concentrations by competing with the sugar for a binding site of the transport system localized in the luminal membrane (30,31, 90). Perfusion experiments in the kidney showed that phlorizin poisoning of sugar transport is rapidly reversible, indicating that the main action of phlorizin is restricted t o the cell surface (SO). This inference was supported further by radioautographic studies on mucosal cells in the intestine which demonstrated that the radioactive label of phlori~in-~H did not penetrate the cell (134). The high affinity of phlorizin for the sugar transport system and the lack of appreciable penetration of the membranes rendered phlorizin very suitable for studies of the glucose-binding protein in the kidney brush border, since it permitted to investigate how the transport inhibitor is bound to isolated membranes. Similar studies cannot be done in the intestine because of the presence of a phlorizin hydrolase in the membranes (32, 91); this enzyme, however, is absent from renal brush border membranes (20, SS, 47). A. High-Affinity Phlorizin-Binding Sites in the Brush Border Membrane

Figure 13 shows the concentration dependence of the interaction of phlorizin with purified brush border fragments, as determined by incubation of brush border membranes at 25°C for 5 minutes followed by centrifugal

FIG.12. Structure of phlorizin a.~ proposed by Diedrich (1.9).

234

R. KINNE

r

1

Bound Phlorizin . [lo-" moles/mg protein]

FIG.13. Phlorizin binding to brush border fragments from rat kidney. Each point represents the mean and standard deviation value from 32 to 41 separate observations a t each concentration of phlorizin tested. Solid line, phlorizin binding with no additions; broken line, phlorizin binding in the presence of 0.4 or 0.6 M D-glucose. The inset illustrates the data representing the high-affinity phloriain-binding component; each point represents the difference between the broken-line values and the composite phlorizin-binding curve in the main figure along each of the diagonals intersecting at the origin. Reprinted from Bode et al. (11) by kind permission of the North Holland/ Elsevier Company.

separation of membranes and incubation medium. During the centrifugation the equilibrium between free and bound phlorizin is not disturbed, and, therefore, a kinetic analysis, as described by Scatchard, can be applied (113). The Scatchard plot presented in Fig. 13 shows a curve (solid line) that gives the binding of phlorizin alone and another curve (broken line) for the data obtained in the presence of 0.4M D-glucose. The solid curve can be considered to be composed of two or more lines, each representing a different receptor population. Thus the brush border membrane fraction used in this study seems to contain several receptor populations with different affinities and capacities. D-Glucose inhibits high-affinity receptors but not low-affinity receptors (see broken line). If the difference between the results obtained in the presence and absence of D-glucose is calculated and plotted, one obtains the nearly straight line shown in the insert. This

GLUCOSE TRANSPORT IN KIDNEY BRUSH BORDER MEMBRANE

2 35

indicates that mainly one receptor type is titrated with D-glucose. Kinetic analysis of this line representing D-glucose-sensitive phlorizin receptors yields a number of receptor sites of 27.5 pmoles/mg membrane protein and an apparent dissociation constant for the phlorizin-receptor complex of 0.Sl p M . Almost identical values have been found by Frasch et al. (41) using the same brush border fraction derived from rat kidney cortex and by Glossmann and Neville (47) in their studies with rat kidney brush border fragments isolated by a different procedure. When N-ethylmaleimide (NEM) instead of glucose was used to differentiate between high-affinity and low-affinity sites (11) or a simple extrapolation of the Scatchard plot was employed (21), somewhat higher values for KD and n were obtained (Table VII). This might be due to an underestimation of the contribution of low-affinity sites in the high-affinity region. Moreover, the experiments of Chesney et al. (21) may also reflect a species difference. That the inhibition of phlorizin binding by D-glucose is of the competitive type is shown in Fig. 14. These data yield a Ki value for D-glucose of about 1 m M for rabbit renal brush border (21) and in the range of 2.8 to 13 m M for rat renal brush border (41, 47). Other sugars also compete with phlorizin for the high-affinity binding site. According to Glossmann and Neville (47) (Table VIII), the relative potency of the sugars decreases in the order: D-glucose > 8-methyl+glucoside > 3-o-methyl-D-glucose. D-Mannose and several other sugars do not influence high-affinity binding. L-Glucose, on the other hand, stimulates the binding of phlorizin to the high-affinity receptor; as demonstrated by Frasch et al. (41) it increased the affinity of the receptors rather than the number of binding sites. It should be pointed out that, whereas in the intestine L-glucose is taken up via the D-glucose transport system (16'), L-glucose is not absorbed by the proximal tubule (141). Therefore one would not expect L-glucose to interact with the D-glucose transport system a t the surface of the renal microvilli nor phlorizin binding to be inhibited. On the other hand, L-glucose secretion has been observed in the proximal tubule. This secretion is inhibited by the intratubular application of phlorizin and stimulated by the presence of D-glucose ( 7 ) . These findings have been interpreted as evidence for a countertransport of D- and I,-glucose ( 7 ) . Thus, the stimulatory effect of L-glucose on the binding of phlorizin to isolated brush border membranes might be explained by the assumption that L-glucose, which (as shown in the transport studies reported previously) is able to enter the intravillous space by a phlorizin-insensitive mechanism, interacts with the sugar system at the inside of the membrane. This interaction could be envisaged to influence the affinity of the glucose transport system a t the outside of the membrane by a conformational change. I n turn, this suggests that the transport system behaves allosterically .

TABLE VII CHARACTERISTICS AND STEREOSPECIFICITY OF HIGH-AFFINITY PHLORIZIN-BINDING SITESIN BRUSHBORDERMEMBRANES

Source Bode et al. (11)

Method used for binding studies Centrifugation

Membrane fraction (Ref.) Rat kidney brush border fragments (68)

Results n = 2.4 X moles/mg protein; KD = 2.0-3.4 X 10-6 M; the high-affinity sites are inactivated by

N-ethylmaleimide and pchloromercuribenzoate Frasch et at. (41)

Centrifugation

Rat kidney brush border fragments (106)

n = 0.87 X 10-10 moles/mg protein; KD = 0.23 X 10-6 M; &glucose inhibits the binding competitively (Ki 15 X 10-3 M) ;L-glucose stimulates the binding by increasing the affinity for phlorizin (KD in the presence of 0.3 M L-glucose = 0.08 X 10-6 M); sodium stimulates binding by increasing the affinity of the binding site, apparent a i t y constant for sodium is 13 meq

-

w

5

z

A

Bode et al. (19)

Centrifugation

Rat kidney brush border fragments (106)

Glossmann and Neville (47)

Chesney et al. (21)

Rapid filtration and rapid washing

Rapid filtration and rapid washing

Rat kidney brush border fragments (146)

Rabbit kidney brush border fragments (8)

n = 0.27 X 10-lo moles/mg protein; KD = 0.81 X 10-6 M; no inhibition by mannose; slight inhibition by Zdeoxy-&galactose; competitive inhibition by phloretin-2’-galactose, 4methoxyphlorizin, 4’-deOxyphlOrizin, and deoxycorticosterone glucoside; phloracetophenone and phlorizin chalcone without effect; phloretin inhibits noncompetitively n = 0.45 X moles/mg protein; KD = 0.25 X 10-6 M; relative potency to replace phlorizin from the binding site is D-glucose > pmethylglucoside > a-methylglucoside > D-galactose > 2-deoxy-~-glucose > 3-o-methyl-~-ghcose; K i for D-glucose = 2.8-15 mM; L-glucose and D-fructose increase binding; sodium increases affinity n = 0.8 X 10-10moles/mg protein; KD = 8 X 10-6 M; D-glucose inhibits the binding competitively (Ki = 1 mM); sodium increases affinity of the system; phloretin inhibits competitively

-Z a W

c v, I

238

R. KINNE

t

[.!o'*moles-']

7 1

1

-

,', -2

! 0

,

, 2

,

, 4

,

, 6

,

, 0

,

, 10

FIG 14. Competitive inhibition of phloriein binding to high-affinity sites in the brush border by -glucose. All data obtained for phlorizin binding in the absence of D-glucose (A) and in the presence of 0.04 M D-glucose ( 0 )are plotted according to LineweaverBurk. Reprinted from Frasch et al. (41) by kind permission of Springer Verlag, Heidelberg.

Binding of phlorizin has been found by all authors (see Table VII) to depend on sodium ions. As shown in Table IX, sodium increases the affinity of the receptors without any concomitant change in the number of binding sites. The increase in affinity reported varies from six- to thirty-fold. A change in affinity can also be detected with respect to the binding of D-glucose to the receptor. The apparent Ki of D-glucose increases about 2.5 times when sodium is replaced by potassium in the incubation mixture. Thus, the affinity of the receptor for phlorizin and for D-glucose is increased by sodium. The different degree of stimulation for phlorizin and glucose might indicate that, in addition to the glucose-binding site, the agluconbinding site is altered in the presence of sodium. Interestingly the sodium concentration needed for half-maximal stimulation of phlorizin binding is

239

GLUCOSE TRANSPORT IN KIDNEY BRUSH BORDER MEMBRANE

TABLE VIII

INHIBITION OF PHLORIZIN-aH BINDINGBY VARIOUSSUGARS AND SUGARDERIVATIVE SO^^ ~~

Substance tested

Concentration (mM)

Inhibition

D-Glucose >Glucose D-Galactose D-Mannose D-Fructose Daorbose 2-Deoxy-~-glucose 2-Deoxy-D-galactose &Deoxy-~-galactose 6-Deoxy-~-galactose 3-0-Methylglucose D-Arabinose >Ara binose D-Ribose PUeoxy-D-ribose D-Xylose ~Xylose D-Glucosamine D-Galactosamine D-Mannosamine Sucrose Maltose Sorbitol Mannitol Mannuheptulose Glucoheptose a-Methylglucoside 8-Methylglucoside Phenyl-a-~-glucoside Pheny l-p-D-glucoside p-Ni trophen yl-8-D-glucoside p-Ni trophen yl-p-D-galactoside

3-GOO 3-600 3-GOO 50-600 GOO 600 GOO

Yes Nonee Yes None Nonec None Yes None Slight None Slight None Nonec None None None None None None None None Slightd None None None None YeS Yes Slight Yes YeS Yes

A00

600 GOO 600 GOO GOO GOO GOO GOO 600 200

200 200 GOO 600 600 600 600 150 GOO

GOO 3 3 3 3

Data from Glossmann and Neville (47). table summarizes the results obtained by studying inhibition of phlorizin binding to kidney brush border membranes by different sugars and sugar derivatives a t low phlorizin concentrations (0.15-0.47 p M ) . For further details see text. Yes = 20-95oJ, inhibition a t the highest concentration of inhibitor used; slight = less than 20% inhibition at the highest concentration of inhibitor used; none = no significant inhibition. Stimulation of binding. Due to glucose liberated by maltase. (1

* The

h)

P 0

TABLE I X

INFLUENCE OF KA+, CA*+,M G ~ +AND , K+ ON THE DISSOCIATION CONSTANTS FOR MEMBRANE BINDINGOF PHMRIZIN AND -GLUCOSE (KD-,~)AND ON THE NUMBER OF BINDING SITES~JJ

ZfSD

KD-,I (lo-' M)

M)

Kphl

Cations in the incubation medium

N

ZfSD

p

N

n (1O-l2 moles/EM) ZfSD

p

N

p

Na+ (30 meq/liter)

0.41 f 0.23

Na+ (150 meq/liter)

0.25 f 0.10 20

14.7 f 6.3

Naf (150 meq/liter) Cs4+(10 mM) Na+ (150meqfiter) Mg*+ (5 mM) Na+ (5 meq/liter) K+ (150 meq/liter)

0.26 f 0.05

1

5 N.s."

17.2 f 5.2

1

0.22 f 0.09

4 N.S.~

16.8 f 4.9

4 N.s.c

1.67 f 0.70

3 N.s."

42.7 f 8.7

3 N.S."

Na+ (5 meq/liter)

1.37 f 1.20

24.9 f 11.7

1.26 f 0.75

4'

~N.s.


<0.010

Data from Frasch et al. (41). N = number of experiments; N.s. = not significant; n Statistical evaluation from paired data.

=

8 .<0.05 16,

N.s.

0.97 f 0.11

,N.s.

<0.005

1.57 f 0.48 20

5 <0.10"

number of binding sites. P

GLUCOSE TRANSPORT IN KIDNEY BRUSH BORDER MEMBRANE

24 1

about 13mM, as the sodium binding site is apparently saturated a t 100 mM NaCI. These values agree well with the results obtained in microperfusion studies on the sodium dependence of active sugar transport in the proximal tubule. I n these experiments a half-maximal stimulation of sugar transport was observed a t an intratubular sodium concentration of 15 mM (141). The reports on the influence of other cations on the phlorizin binding are equivocal. According to Frasch et al. ( 4 1 ), addition of 10 mM CaClz did not change the binding characteristics, whereas Chesney et al. (21) observed a 67% inhibition of high-affinity binding when calcium was omitted from the incubation medium. Magnesium ions seem to interfere with the binding slightly, if a t all (21, 41). The inhibitory potency of phlorizin analogs on high-affinity phlorizin receptors was studied in great detail by Bode et al. ( 1 2 ) who used compounds synthetized by Diedrich. With respect to the sugar moiety, replacement of D-glucose by D-galactose reduces the affinity of the analog to 16-22 pM in accordance with the lower inhibitory effect of D-galactose on phlorizin binding. The other derivatives provided mainly information on the structural requirements for the attachment of the aglucon part of the molecule. Phlorizin-like glucosides, such as 4’deoxyphlorizin, 4-methoxyphlorizin, and deoxycorticosterone glucoside inhibit binding of phlorizin in a competitive manner with decreasing potency (see Table VII). This indicates that changes in the functional grouping of the 0 tail of phlorizin (see structural formula in Fig. 12) considerably reduce the affinity of the phlorizin glucoside to the high-affinity receptor. This notion is further supported by the result that phloracetophenone 2’-glucoside, which has no p-tail ring a t all, does not inhibit phlorizin binding. Due to the restricted solubility of the compound, it proved impossible to attain the concentrations needed for the replacement of phlorizin by D-glucose (about 10 mM at least). Such high concentrations of phloracetophenone 2’-glucoside should inhibit the phlorizin binding by interaction with the glucose binding site. However, as pointed out earlier by Diedrich (SO, 3 1 ) , not only the exact spacing of the phenol hydroxyl group on the 0 ring is essential but also the steric arrangement of the different planes of the molecule and their mobilities are of importance. Comparison of phlorizin with its chalcone demonstrates this fact quite remarkably. Although both compounds have identical functional groupings, in the chalcone, which is an a,P-unsaturated ketone, the aromatic rings are coplanar and the molecule is relatively flat. In the phlorizin molecule the two aromatic rings are independently mobile. The finding that the chalcone does not inhibit the phlorizin binding led Diedrich (12, 31) to the hypothesis that the sugar moiety of phlorizin is bound in a plane nearly perpendicular to that which binds the phenolic 0 ring of the molecule. The phenolic ring due to its hydrophobic nature

R. KINNE

242

probably interacts with a hydrophobic part of the phlorizin receptor or with the phospholipids in which the receptor is embedded. The aglucon phloretin inhibits the phlorizin binding noncompetitively 10 p M ) . One in the rat (12) and competitively in the rabbit (21) ( K i could visualize that the binding of phloretin to the aglucon-binding site changes the conformation of the glucose transport system. This would lead to noncompetitive and pseudocompetitive inhibition. In the preceding paragraphs the term binding was used for the kind of interaction observed between phlorizin and the isolated membranes. One piece of evidence for the validity of this assumption was derived from physiological and radioautographical studies. In the isolated membrane system the tightness of the membrane against phlorizin is also preserved. The binding of phlorizin to the high-affinity site is not influenced by a change in the osmolality of the incubation medium. This strongly contrasts with the behavior of the transported D-glucose demonstrated in Section 111. High-affinity binding sites can almost certainly be located in the brush border membranes. The evidence for this statement comes from brush border membrane enrichment experiments showing that the marker enzyme, alkaline phosphatase, parallels the number of high-affinity receptors (41, 4 7 ) . Also multiple indicator dilution experiments performed by Silverman et al. indicate that the high-affinity sites are located along the brush border of the proximal tubule (127). Treatment of isolated brush borders with proteolytic enzymes, such as trypsin and papain or a mixture of carboxypeptidase A and B, reduces the D-glucose-sensitive binding of the brush border membranes (Table X) , whereas phospholipase C and neuraminidase do not alter the binding properties of the membrane. Furthermore, it has been demonstrated that SH-group reagents such as p-chloromercuribenzoate, mersalyl, and N-ethyl-

-

TABLE X EFFECTOF PROTEASES ON D-GLUCOSESENSITIVE PHLORIZIN BINDINGTO BRUSHBORDER MEMBRANES" Control Trypsin (1 mg/lO mg membrane protein, 15 min at 37" C) Papain (1 mg/25 mg membrane protein, 15 min at 37' C) Carboxypeptidase A and B (1 mg/25 mg membrane protein, 15 min at 37" C) (I

Data from Glossmann and Neville (47).

100% 43 % 16%

61%

243

GLUCOSE TRANSPORT IN KIDNEY BRUSH BORDER MEMBRANE

maleimide inhibit the binding of phlorizin to the high-affinity site (11, 61) 47) and the transport of D-glucose by brush border membrane vesicles

( 2 , 15).The sensitivity of the high-affinity site to N-ethylmaleimide seems to be higher than the sensitivity of low-affinity sites (11). This fact has been used to label the high-affinity site (136, 139). From these results, it may be concluded that phlorizin and glucose interact specifically with a protein component of the brush border microvilli and that this protein component facilitates glucose membrane transfer. B. Interaction of Phlorizin with low-Affinity Binding Sites in Renal Plasma Membranes

In all studies reported so far on the binding of phlorizin to isolated plasma membrane fractions, an interaction of the compound with low-affinity sites was observed in addition to its binding to high-affinity sites (see Fig. 13 and Table XI). The low-affinity sites can be divided into at least two groups. According to Bode et al. (12 ) and Glossmann and Neville (47) one kind of receptors has an apparent KDbetween 1-100 p M , adsorbs mannose, 2deoxy-~-galactose,D-fructose, and L-arabinose in preference to D-glucose, and has a high affinity for phloretin. It remains to be clarified whether this receptor site is located in the brush border membrane [mannose site of Silverman? (129)] or whether it represents binding of phlorizin to basallateral plasma membranes. The latter are present in the brush border fragments utilized in the studies reported in the foregoing. The inhibition of binding by phloretin and by 2-deoxy-~-galactose would point to a localization of this receptor population in the basal-lateral plasma membranes. The sugar transport across the basal cell side has been shown to be sensitive to phloretin and to include 2-deoxy-n-galactose as substrate (78). Furthermore, in the rat proximal tubule, no reabsorption of mannose can be demonstrated by micropuncture techniques (141) . It is unfortunate that the inhibitory effect of mannose and phloretin was not investigated (105) in binding studies on a plasma membrane fraction that predominantly contained the basal-lateral plasma membranes. A second kind of low-affinity receptor has been described by Bode et al. (11) and Chesney et al. ( 2 1 ) . The apparent KD for this population lies between 0.4 and 2.5 mM. It has been suggested by Chesney et al. that this binding site might be related to a phlorizin transport system present in the basal-lateral plasma membranes. Another possibility is that under these conditions phlorizin is adsorbed to the Na-K-ATPase in the basal-lateral plasma membranes. This enzyme is known to be inhibited by high concentrations of phlorizin (14) 108). Moreover, the distribution of phloriein into )

h)

P P

TABLE XI CHARACTERISTICS OF LOW-AFFINITY PHLORIZIN-BINDING SITESIN RENALPLASMA MEMBRANES

~~

Membrane fraction (Ref .)

Method

Source ~~~~~

~

~~

~

~

~~

~~

Results ~

Bode et al. (11)

Centrifugation

Rat kidney brush border fragments (68)

n = 2.0 x 10-8 moles/mg protein; KD = 3.9 x 10-4 M; the low-afiinity receptor is inhibited by p-chloromercuribenzoate but not by N-ethylmaleimide

Bode et al. ( l e )

Centrifugation

Rat kidney brush border fragments (106)

Two low-affinity sites, only one characterized; KD = 1-100 x M, high a f h i t y for phloretin and phlorizin chalcone; adsorbs mannose and 2-deoxy-Dgalactose in preference to glucose

Glossmann and Neville (47)

Rapid filtration and rapid washing

Rat kidney brush border fragments

n = 60 pmoles/mg protein; KD = 6.6 X M; inhibited by mannose, D-fructose, and karabinose

(I@) Chesney et al. (21)

Oi?egovid et al. (105)

Rapid filtration and rapid washing

Rabbit kidney brush border fragments (8)

n = 1.2 nmoles/mg protein; KD = 2.5 d ; noncompeti-

Centrifugation

Rat kidney plasma

No inhibition by D-glucose (100 mM) and resorcinol (100

membranes (39)

tive inhibition by D-glucose and D-galactose; noncompetitive inhibition by phloretin

W

-Z %

5

GLUCOSE TRANSPORT IN KIDNEY BRUSH BORDER MEMBRANE

245

the lipid phase of the membranes could contribute to the observed lowaffinity association.

V.

MOLECULAR CHARACTERISTICS OF THE SUGAR TRANSPORT SYSTEM IN THE BRUSH BORDER MEMBRANE

The active center of the glucose transport system localized in the brush border may be considered as composed of three different sites adjacent to each other: a sugar-binding site; a cation-binding site; and a binding site for aromatic rings, the aglucon-binding site. A. Stereorpeciflcity of the Sugar-Binding Site

Studies of the stereospecificity of the glucose transport system in the renal proximal tubule have been impaired for a long time because no distinction can be made between transfer occurring a t one or the other of the poles of the epithelial cell. Thus, in studies on the accumulation of sugars in kidney cortex slices, several pathways of sugar uptake with apparent different stereospecificity have been described (76, 7 7 ) . These can now be attributed to different cell membranes (78) and, in part, may be explained by intracellular sugar consumption. A multiple-indicator dilution technique has been applied by Silverman (125-129) to study in wivo the interaction of sugars with the membrane surfaces of the dog kidney. With this technique it proved possible to discriminate between interaction a t the luminal and the contraluminal nephron membranes. There remains, however, some uncertainty with respect to the nature of the interaction and the part of the tubule where interaction takes place. In discussing the stereospecificity of the glucose transport system in the brush border membrane of the proximal tubule, therefore, we will concentrate mainly on micropuncture studies performed in the proximal tubule of rat kidney and on phlorizin-binding studies in which rat renal brush border fragments were investigated (see Table VIII) . In microperfusion experiments, active sugar transport is measured and sodium dependence and phlorizin sensitivity are considered as indicating a transport of the monosaccharide studied via the 11-glucose transport system (Table XII) . Results concerning the inhibition of u-glucose uptake by other sugars must be regarded with caution because of the possibility of mutual interaction of transport systems at the level of membrane potential, as previously discussed. I n binding studies, the degree of displacement of phlorizin from

TABLE XI1 A. Z a O NET FLUX C~NCENTEATION DIFFERENCEB AC (Cinterstithm - Clumsn) O F HEXWEE ACTIVELY TRANSPORTED BY RAT F'IIOXIMAL CONVOLUTION, THEIRDEPENDENCE ON LUHINALNA+ CONCE-TION, AND SENEITIVI~~ AQAINBT PIILORIZZN' Low sodium

High sodium

Ac

Sugar

D-Glucose

Chemical Na+ concentration (meq/Iiter) (mmoled fSE liter) lumen 5.0

gMethyl-D-glucoaide

5.0

a-Methyl-D-gludde

5.0

D-GalactOse

5.0

3-0-Methyl-D-glucwe

n-Allwe

10.0 2.5

% fSE

147.0 f 1.0 83.2 f 3.3 n - 3 n = 15 (140) 85.3 f 3.1 n = 6 135.3 f 1.6 73.0 f 2.4 n=14 n=21 138.8 f 1.6 64.3 f 4.0 n - 8 n 10 (140) 15.2 f 2.4 n - 8 (140) 17.8 f 3.6 n - 8

-

~ ~ o r i 10-4 d n

Ac

Ac

mmoles/ liter 4.16

Na+ (mesfliter) fSE lumeu

3.21 1.52 0.45

mmolea/ liter

9.6 f 1.2 38.2 It 4.5 n-22 n-22

1.91

10.6 f 1.2 n-15 10.9 f 2.3 n = 8 13.0 f 3.3

14.6 f 4.8 n-15 8.7 f 2.9 n - 8 -0.7 f 2.2

0.73

n = l l

n=11

13.2 f 1.05

9.4 f 5.8 n - 6

-

4.26 3.65

%fSE

n - 6

-

M

Na+ (merlfliter) fSE lumen (140)

-

0.44

-0.07 0.23

%

f SE

--

15.6 f 4.8 12

n

17.6 f 1.4 n = 6 136.7 f 1.9 2.4 f 2.2 n - 7 n - 8 142.5 f 2.6 -8.1 f 1.7 n - 8 n = 8 (140) 5.3 1.5 n = 6 (140)

> 10% andAc = 100 corresponds to 5 mmolea/liter exeapt with ~ a l l o s ewhere it was 2.5 mmolesfliter and 3-o-methyl-wglucose,

*

mmolea/ liter 0.78

0.86 0.12 -0.81 0.26

where i t wan10 mmolea/liter. According to Ullrich d d.(141) ACis a meaaure of the active transport rate. Valuea in parentheses represent the initial sodium concentration in the tubular perfusate.

*m

$

i

B.

ZERU

NET FLUX

CONCENTRnllON

-

DIFX-ERENCES A C (Cinterstitium Clumn) OF S U G A R S NOT ACTIVELYTRANBPORTED BY RAT (Ac < f 8%) (FOR COMPARISION. MANNITOLIS ALSOINCLUDED)

PROnaraL

CONVOLUTION

n

% m 4

High aodium

Sugar L-GlUCOse

Chemical concentration (mmoles/liter) 5.0

D-MallllOM!

5.0

2-Deoqr-~-glucose

5.0

136.4 f 1.3 n = 9 130.8 f 4.1 n - 8

-

142.6 f 2.0 16 143.4 f 0 . 8

n

% ' f SE 4.5 f 0 . 0 n = 13 1.2 f 3.8 n - 8 -0.2 f 3.5 n = 16 4.5 f 2.6

n-Fructoae

5.0

ffilucoaamine

5.0

(140)

n-Fucoae (6-deory-D-galactose)

5.0

(140)

n-Ribose

5.0

(140)

5.3 f 1.0

n-Mannitol

5.0

(140)

4.7 f 2.4 n = 8

n = 8

mmolee/liter

Na+ (meq/liter) (medliter)

0.23

-

0.06

-

-0.01 0.23 -0.42 0.02

n = 8 n - 8

0.26 0.24

-

19.8 f 2.7 14 13.5 f 1.6 n = 15 n

n - 8

-8.4 f 3.7 n - 7 0.4 f 3.0

5

Ac

Ac Na+ (rneq/liter) f SE lumen

$!

Low eodium

-

14.9 f 2.2 n - 9

-3.7

0

g -I

% f SE

mmoledliter

-

-

8

-0.18

u,

f 2.5

n = 14

--

2.9 f 2.9 n 15

-5.1 f 4.4 n u 9

0.14

-0.25

0

I

4 ID W

c I

ID

g

x W

B ID

P

m Z

248

R. KINNE

its high-affinity binding sites is taken as criterion for the interaction of the sugar in question with the sodium-dependent phlorizin-sensitive D-glucose transport system of the brush border membrane. One essential structural requirement for a sugar to be transported actively by the proximal tubule is the presence of a pyranose-like ring system; pentoses such as D-ribose are not transported and do not inhibit phlorizin binding. The binding is also unaffected by arabinose and xylose. Another critical factor is the orientation of the hydroxyl groups, which in the normal C-1 configuration of D-glucose are all oriented equatorially to the plane of the molecule. The change into an axial position is most effective at C-2. Mannose shows no active transport and no inhibition of phlorizin binding. Axial orientation at C-3 (allose) decreases the active transport by 80%; the corresponding alteration at C-4 reduces the interaction with the D-glucose transport system only moderately. Replacement of equatoridly oriented hydroxyl groups by other substituents also influences the affinity of the sugar. Thus an exchange of the hydroxyl group at C-2 by an amino group (D-glucosamine) abolishes transport and the interaction with the phlorizin-binding site. A very drastic decrease in affinity is also observed after methylation of the hydroxyl group at C-3. The transport of 3-0methyl-D-glucose is only 20% of the transport observed for D-glucose. Accordingly, phlorizin binding is inhibited much less by 3-o-methyl-~glucose than by D-glucose. Methylation at C-1, leading to a fixation of the glycosidic bond in the a or B position, does not influence the interaction between the sugar and the transport system. Thus the spatial arrangement at C-1 does not seem to be crucial for transport. In the case of the deoxy sugars, results are equivocal. Micropuncture studies have failed to reveal an active, sodium-dependent transport of 2deoxy-~-glucose.However, replacement of phlorizin by 2-deoxy-~-glucose has been shown to occur in rat kidney brush border membranes. Moreover, transport studies with isolated brush border membrane vesicles have demonstrated that the sodium-independent uptake of 2-deoxy-~-glucose lacks the overshoot phenomenon (R. Kinne, 1975 unpublished). From these findings one might speculate that 2-deoxy-~-glucosecan react with the D-glucose-binding site and is subsequently transferred by the sugar transport system across the brush border. However, due to the absence of the hydroxyl group a t C-2, a receptor-sugar complex is formed that is unable to react with sodium, and, therefore, neither sodium-dependent active transport nor sodium-dependent intracellular accumulation (76) is observed. Also, the role of the hydroxyl group at C-6 has not been elucidated completely. When D-galactose, which is transported at a rate corresponding to approximately 70'% of the D-glucose transport, is reduced at C-6, yielding 6-deoxy-~-galactose,the transport drops to zero, &s does the ability

GLUCOSE TRANSPORT IN KIDNEY BRUSH BORDER MEMBRANE

249

to displace phlorizin. On the other hand, 6-deoxy-D-glucose inhibits tubular a-methyl-D-glucoside uptake by 42%. These findings could mean that D-glucose attachment at C-4 and C-6 involves the combined action of the equatorially oriented C-4 hydroxyl and the freely mobile C-6 hydroxyl group. When the C-6 hydroxyl is removed from the D-glucose molecule, the sugar might still be able to interact with the carrier because of the proper configuration a t C-4. In the case of 6-deoxy-~-galactose, however, the equatorial C-4 hydroxyl is lacking and, therefore, the affinity is very low. It is also possible that 6-deoxy-~-glucosewithout being transported itself simply inhibits the binding of a-methyl-D-glucoside to the transport system, thus behaving like 6-deoxy-~-galactose. I n conclusion the sterical arrangement of the groups a t carbons 2, 3, 6, and 4 seems t o determine in decreasing order the affinity of the pyranoside for the receptor. All appear to contribute to the formation of a loose complex. The nature of the interaction between the sugar and the receptor is not yet clarified; probably stereospecific hydrogen bonding is involved. The donors and acceptors for the protons on the receptor are also still unknown, and there is need for further studies such as experiments with side group-specific reagents on the isolated protein. The apparent affinity of the glucose transport system in the rat kidney proximal tubule for D-glucose is 0.6-1 mM in microperfusion studies (43, 88, 141) and 2.3-16 mM in the binding studies (20, 41, 47). It is very unlikely that this discrepancy is due to an inactivation of the binding protein during isolation of the membranes, because the in vivo and in vitro affinities for phlorizin and its analogs are in good agreement (12, 143). Probably the reason for the lower affinity for glucose in the in vitro studies is the manner by which constants are obtained, i.e., as inhibitory constants with respect to the phlorizin interaction. Since phlorizin not only interacts with the sugar-binding site of the glucose transport system but also with the aglucon-binding site, higher concentrations of D-glUCOSe might be needed for the reversal of phlorizin binding than for the half-maximal saturation of the glucose site in the absence of phlorizin. In this context mention should also be made of the very high sugar concentrations (up to 0.6 M ) that are used in phlorizin-binding studies t o distinguish between high- and low-affinity sites. Such concentrations can effect physicochemical changes of the membrane, e.g., dehydration, which, in turn, might affect the properties of membrane-bound receptors. Moreover in vivo and in vitro derived constants describe different molecular events. Direct comparisons, therefore, are not possible [for further discussion see Schultz and Curran (117)and Schachter (lid)]. Accordingly, the constants found in transport studies with vesicles may under appropriate conditions come closer to the constants determined in wivo than the binding constants.

250

R. KINNE

B. Cation-Binding Site

Sodium increases the affinity of the sugar-binding site toward phlorizin ( 2 1 , 4 l ,47) and D-glucose (41) ;it also increases the affinity of the D-glucose transport system in vivo (141). These findings provide strong evidence for the presence of a specific cation-binding site in the active center of the glucose transport system. The cation site has a high specificity for sodium; other cations cannot replace sodium to an appreciable degree. Transport and binding studies have yielded an apparent affinity constant of 13-29 meq. The interaction of sodium with the cation site changes the affinity of the sugar-binding site, probably by inducing a conformational change of the binding protein. The nature of this site is not yet clear. Studies of the type performed by Shamoo et al. (121) probably would be very useful in determining whether part of the sugar transport system has the property to increase the conductance of artificial bilayers to sodium in the presence of actively transported sugars. There is only one report in the literature about experiments designed to characterize the cation site involved in coupled transport of sodium and organic solutes. Schaeffer et al. (115) observed that p-chloromercuriphenyl sulfonate (PCMBS) reduced the cation sensitivity of the sodium-dependent phenylalanine transport system in the intestine. They postulated that, in the hypothetical sequence of events, namely, binding of the amino acid to the carrier, unmasking of a cation site, and interaction of sodium with the cation site to form a ternary carrier-sodium-amino acid complex, PCMBS reacts with a so-called inhibitory site that regulates the exposure of the cation site. The sequence of the reaction of sugars and sodium with the carrier has not yet been'determined in the kidney. From studies of Goldner et al. (48) in the intestine there are two possibilities: a primary reaction of the carrier with sodium or with sugar. C. Aglucon-Binding Site

From studies on phlorizin analogs, Diedrich postulated the presence of a membrane locus that interacts with the 4-hydroxyphenyl group of phlorizin (12,SO). This locus is located at a defined distance (12-15 8) from the glucose-binding site, equivalent to the intramolecular spacing between the oxygen at position 4 and the pyranoside moiety of the glucoside. Deoxycorticosterone-P-D-glucoside can also interact with this membrane locus, most likely because the spacing of the carbonyl oxygen to the pyranoside moiety is also about 14-16 8. The linkage probably involves hydrogen bonding, with a membrane constituent serving as hydrogen donor.

GLUCOSE TRANSPORT IN KIDNEY BRUSH BORDER MEMBRANE

25 1

The binding site may be hydrophobic, allowing noncovalent bonding due to van der Waals and other hydrophobic interactions. When the aglucon phloretin is attached to this binding site, the affinity and/or the number of binding sites for phlorizin and sugars is reduced. This suggests a distortion of the sugar transport system by phloretin, which, depending on the experimental conditions, may reflect either competitive or noncompetitive inhibition. It is not yet clear whether there exists only a glucose transport system in the brush border membrane or whether a mannose transport system exists in addition. Phlorizin-insensitive mannose absorption independent of D-glucose transport has been observed in the dog kidney ( l a g ) , whereas microperfusion studies in rat kidney have provided no evidence for active mannose transport in the proximal tubule (141). Possibly, D-mannose absorption occurs in a segment of the canine nephron that is distal from the proximal tubule. Currently the interaction of mannose with medium-affinity phlorizin-binding sites of isolated renal membranes appears to be related more to the transport system in the basal-lateral membranes than to a reaction a t the luminal cell side.

VI.

CONFORMATIONAL RESPONSE OF THE GLUCOSE TRANSPORT SYSTEM

As yet we understand only very incompletely the molecular events that take place when glucose binds to the outside of the brush border membrane, is transferred across it, and released inside the cell. This is owing to the fact that until now it has not been possible to investigate the protein in the isolated state, although procedures are now emerging that allow purification of the protein, as by affinity chromatography ( 3 7 ) .Only one parameter is known from analytical studies with polyacrylamide gel electrophoresis, namely the molecular weight (- 30,000 daltons) of t,he smallest subunit related t o glucose-sensitive phlorizin binding (1%). Depending on the extraction procedure used, dimcrs of this subunit which are linked by disulfide bonds (136), have been isolated. Studies on the interaction of side group-specific reagents with the membrane-associated binding protein suggest some conclusions about the properties of the binding site. As shown in Fig. 15, NEM inhibits the binding of phlorizin to high-affinity sites. Kinetic studies have shown that the inhibition is noncompetitive, as the number of binding sites is reduced by preincubation with NEM, whereas the apparent affinity of the sites is not changed (139) (Table XIII) . There are several reports in the literature

252

R. KINNE

TABLE XI11 EFFECTOF N-ETHYLMALEIMIDE (NEM) ON PHLORIZIN-JH BINDINGTO ISOLATED BRUSHBORDERS”~~ Brush border

A

B

Incubation with phlorizin-*H Incubation with 0.5 mM NEM for 30 min; for 30 min thereafter incubation (control) with phloriein-SH for 3 min

I

c Incubation with 0.5 mM NEM together with phlorizin-aH for 30 min

Data from Thomas et al. (139).

* The number of binding sites

(nphl) and the dissociation constant of the phlorih receptor complex ( K p h l ) were determined in a binding study; n is expressed in df. moles/mg brush border protein, K p h l in

to indicate that the effect of side group-specific reagents on erythrocyte sugar transport can be enhanced or inhibited (6, 10, 13, 28, 80, 81, 82). Thus, when brush border membranes are treated with 0.5 mM NEM in the presence of phlorizin, the number of binding sites and the affinity of the receptor remain unchanged (Table XIII) . Phlorizin exhibits a substrate protection effect as described for many enzymes (22) and for the M protein (40). This substrate protection effect made it possible to determine that the number of NEM molecules attached moles NEM to the phlorizin receptor during inactivation was 2.4 X bound per milligram protein (Table XIV). The same experimental conditions prevented the binding of 0.82 X 10-lo moles phlorizin. Hence, on the average, three NEM-sensitive sites are protected by 1molecule of phlorizin. These groups are probably SH groups, because PCMB completely inhibits the binding of NEM as demonstrated in Table XV. D-Glucose (0.45 M ) protects about 12 X 10-10 moles NEM-sensitive groups. Some of these groups are identical with those groups protected by phlorizin because phlorizin and D-glucose show no additive effect (Table XVI) and sites protected first by D-glucose can be protected subsequently by phlorizin

253

GLUCOSE TRANSPORT IN KIDNEY BRUSH BORDER MEMBRANE

I

b [ ' 10'" moles] ..

I

0

10

20

30

40

50

60

70

80

FIG.15. Inhibition of phlorizin binding to brush border fragments by N-ethylmaleimide (NEM). The membranes were preincubated for 30 minutes a t 37°C in the presence of 20 mM NEM, thereafter the binding of phloriein w&s determined. The data are taken from Bode el al. (11).

(Table XVII). Thus, the sites protected by phlorizin seem to be related to the interaction of the glucose moiety of phlorizin with the binding protein, rather than to the attachment of the aglucon moiety. Interestingly, phloretin has an effect opposite to that of phlorizin: NEM binding of the isolated membranes is actually enhanced by phloretin, as compared to the unequivocal reduction caused by phlorizin (139). The substrate protection effect can be explained either by assuming that the essential residues labeled by NEM belong to the active center of the binding protein or that binding of the substrate to the transport system induces a conformational change that leads to the masking of NEM-reactive groups. Such a conformational change has been postulated to occur in the intestine on the basis of the observation that Bdeoxy-~-galactoseinhibits the sodiumdependent sugar transport competitively but is not transported by the system. The sugar probably forms an abortive complex with the transport system that lacks the conformation required for the translocation (f7).There is also increasing evidence that the facilitated diffusion of sugars through the erythrocyte membrane is mediated by a system composed of several subunits, which as a whole are not mobile but promote

R. KINNE

254 TABLE XIV

DETERMINATION OF N-ETJ3YLMALEIMIDE-14cBINDING [N(NEMJ4C)] TO PHLORIZIN-SENSITIVE SITES OF ISOLATED BRUSHBORDER' Brush border 1

1

Pretreatment with 0.5 mM NEM (unlabeled) together with 0.005 mM phloriain (unlabeled) for 70 min at 28"C, followed by centrifugation and washing procedures B

C Incubation with 0.5 mM NEM-l4C and 0.08 mM phloriain for 30 min at 37"C, followed by washing procedures

Incubation with 0.5 mM NEM-"C for 30 min a t 37"C, followed by washing procedures

1

1 N(NEM-"C) 24.3 i 7.6

22.2 f 6.6

AN(NEMJ4C) = 2.4 f 1.2 (P = 0.005) 4 The N(NEM-W) is given in 10-10 moles/mg brush border protein. The mean values of 24 experiments are given with the standard deviations. The AN(NEM-"C) and P are derived from paired data. NEM, N-ethylmaleimide.

sugar movement by a series of conformational changes (10,80, 87). The subunits located at the outside of the membrane are assumed to differ in their properties from those located at the inside; this leads to an asymmetry of the transport system. If similar systems exist in the renal brush border, then the first transport step, binding, would already be controlled by the proper conformation of that part of the transport system that faces the outside of the membrane. This conformatim would be regulated by the sodium-binding site; occupancy of this site increases the affinity of the sugar-binding site to phlorizin and sugars. The attachment of the monosaccharide to the sugar-binding site would then induce a further conformational change that might be relat.ed to the translocation step. In analogy with Singer's model (ISO), a dimer can be imagined that spans the brush border membrane and

255

GLUCOSE TRANSPORT IN KIDNEY BRUSH BORDER MEMBRANE

TABLE XV INHIBITION OF N-ETHYLMALEIMIDE (NEM) BINDINGTO BRUSHBORDER FRAGMENTS BY ~CHLOROMERCURIBENZOATE (PCMB). Concentration of PCMB (M) 0 3 3 3 3

x x x x

%

Amount of NEM bound (10-'0 moles/mg protein)

of control

14.2

100

10-7

13.9

10-6 10-5 10-4

11.3 4.7 . 0.6

98 79

33

Brush border membranes were preincubated with 0.5 mM NEM (unlabeled) together with 5 p M phlorizin for 70 min a t 28°C. After several washings, they were incubated in triethanolamine-NaCl-EDTA buffer with the concentration of PCMB as indicated in the table for 10 min a t 28°C. Thereafter radioactive NEM was added to the incubation mixture to give a final concentration of 3 X 10-4 M, and the incubation was continued for another 5 min. The amount of NEM bound to the membranes was determined as described previously (139).

operates via a series of conformational changes. Indeed, preliminary results of the molecular weight determination of the purified, brush border glucose-binding protein suggest that the glucose transport system exists as a dimer in the membrane. The high number of NEM-reactive sites protected by phlorizin and D-glucose may also constitute an argument in favor of the hypothesis that no less than two and no more than four monomers are protected by the conformational response induced by the interaction of D-glucose and sodium with the transport system. TABLE XVI

PROTECTION OF N-ETHYLYALEIMIDE (NEM) BINDINGSITES BY PHLORIZIN A N D D-GLUCOSE' Substance present in the incubation mixture Phlorizin D-Glucose D-Glucose and phlorizin

AN(NEM-"C) (10-'0 moles/mg protein)

2.4 f 1.2 12.6 f 5 . 0 12.2 f 3 . 5

m 24 6 6

Determination of NEM binding according to the procedure described by Thomes et al. (139).

R. KINNE

256 TABLE XVII

PROTECTION OF PHLORIZIN-SENSITIVE

N-ETHYLMALEIMIDE-14c-BlNDIN0

SITES BY D-GLUCOSE [AN(NEM-14C-G~uc)l.*b Brush border

I

Pretreatment with 0.5 mM NEM (unlabeled) together with 0.005 mM phlorizin (unlabeled) for 70 min at 28"C, followed by centrifugation and washing procedures

I

I

B

C

,

I

min together with 0.45 M D-glucose a t 37"C, followed by washing procedures

min a t 37"C, followed by washing

I

I

I

Incubation with 0.08 mM phlorizin and 0.5 mM NEM-1%

Incubation with 0.5 mM NEM-

IuI Incubation with 0.5 mM NEM14c

I

24.9 i 6.2

AN (NEM-"C) 1.1 i 0.8.

Incubation with 0.08 mM phlorizin and 0.5 mM NEM- "C

I

I

e

N (NEM-"C)

I

4*6 0.01

I

I

25.4 f 5.5

> P > 0.005

AN(NEM-"C-gluc)

5

21.9 f 4.4

7 3.5 f 1.2

2.4

Data from Thomas et al. (139). The N(NEM-l4C) is expressed as 10-10 moles N-ethylmaleimide-1% per mg protein. The mean values of 5 experimenb are given with the standard deviations. AN(NEM-14C) and P are derived from paired data, NEM, N-ethylmaleimide.

VII.

RELATION OF RENAL GLUCOSE TRANSPORT SYSTEM TO ENZYMES INTERACTING WITH CARBOHYDRATES

It is thought that several enzymes interacting with sugars and phlorizin are involved in sugar uptake by the proximal tubule. These include brush border components, such as alkaline phosphatase (92) and trehalase (112), M well as enzymes, such as mutarotase (3, 4, 66, 61, 62, 63, 64) and glucose-6-phosphatase ( I S ) , whose activity is not increased during the

GLUCOSE TRANSPORT IN KIDNEY BRUSH BORDER MEMBRANE

257

isolation of brush border membranes. When the apparent affinities of the enzymes for D-glucose and other sugars are compared to the apparent affinity of the glucose-binding protrin, all systrms (23, 27) show a low affinity for sugars in a similar range of conccntrations. I n this respcct all of them differ from bacterial transport systems for which the affinity constant is as high as lo6 M-I. The most striking difference, however, brtwccn the aforementioned enzymes and the glucose transport system is the high affinity of the latter for phlorizin. Half-maximal inhibition of mutarotase (63), alkaline phosphatase (92), and glucose-6-phosphatase (132) is observed only when concentrations in the millimolar range are used, whereas the D-glucose transport is reduced 50% in the presence of 0.2 p M phlorizin (14 3 ) . Other evidence helps to indicate which membrane components cannot be part of the glucose-binding site. Studies on the spatial arrangement of the glucose transport system in the brush border membrane, performed in vivo by multiple indicator dilution techniques, have led to the conclusion that maltase and trehalase are not involved in the reabsorption of monosaccharides, since cleavage of disaccharides continues under conditions where D-glucose reabsorption is completely blocked by phlorizin ( 1 2 5 ) .Also during successive digestion of isolated membranes, disaccharidases can be released from the membrane without concomitant extraction of the phlorizin-binding site (13 8 ) . Alkaline phosphatase and the phlorizin receptor also behave differently during these solubilization experiments. It is interesting to refer to the hypothesis of Kimmich (65, 66, 67) that a close relation exists between Na-K-ATPase and the sodiumdependent sugar transport system. Since this enzyme, as outlined in the foregoing, is not present in the brush border membrane, it cannot be involved directly in sugar transfer across the brush border membrane. Furthermore, an electrochcmical potcntial diff crence for sodium across the mcmbranr in the absence of metabolic energy seems to be a sufficient condition for isolated brush border membrane vesicles to accumulate sugars. Since this accumulation is not ATPdependent, an ATPase-system does not appear to be necessary for the transformation of chemical energy into osmotic work. In view of these results, Kimmich’s model does not seem to apply to renal sugar transport.

VIII.

O N E O R SEVERAL GLUCOSE TRANSPORT SYSTEMS IN THE BRUSH BORDER MEMBRANE?

In the preceding sections the attention was focused mainly on the glucose transport system, which is sodiumdependent and phlorizin-sensitive.

258

R. KINNE

Sodium dependence and phlorizin sensitivity were used to distinguish this system from other modes of glucose transfer across the brush border membrane. Careful inspection of the data obtained for sugar uptake by isolated brush border vesicles reveals that, even in the absence of sodium and in the presence of phlorizin, there is an uptake of D-glucose that is higher than the Gglucose uptake (see Fig. 7 ) . Also in the micropuncture studies compiled in Table XI1 there might be some indication that part of D-glucose and p-methyl-D-glucoside transport is phlorizin-resistant. Studies devoted especially to the existence of two types of carrier-mediated transfer for D-glucose in brush border vesicles were recently performed by Busse et al. [Biochim. Biophys. Acta Pol, 231-243 (1975)l. Based on different phlorizin sensitivity and pH dependence of the sodium-dependent and sodium-independent path, the authors concluded that two types of carrier exist. These two types of carrier were thought to represent two interchangeable conformational states of a single carrier rather than two transport systems working independently. The latter concept resembles the kinetic analysis of sugar transport in the intestine by Goldner et al. (48). These authors assumed that the carrier system X can exist in the free form (X), as a complex with sodium (XNa) , as a complex with glucose (XS), and as a ternary complex (SXNa). The affinity of X for glucose is lower than the affinity of XNa for glucose. As shown directly in phlorizin-binding studies with renal brush borders, also the affinity of X for phlorizin is lower than that of XNa. If XS and SXNa are able to move across the membrane, the aforementioned findings would easily be explained. The movement of XS would be responsible for the non-sodium-requiring, phlorizin-insensitive component of glucose flux, whereas SXNa movement would represent the sodium-dependent highly phlorizin-sensitive flux. On the other hand, especially in the studies with isolated membrane vesicles the purity of the membrane fractions has t o be considered because the glucose transport system in the basal-lateral plasma membranes is not sodium-dependent nor phlorizin-sensitive (see Fig. 11). Thus, a contamination of the brush border membranes with basal-lateral plasma membranes would simulate the presence of two different transport systems. There is a third possibility to explain the preceding observations. It might be possible that in contrast with the membrane-bound enzymes, some transport systems are not asymmetrically distributed in the cell envelope. Thus it could be envisaged that the sodium-independent phlorizin-insensitive transport system is present both in the basal-lateral plasma membranes and the brush border membranes. The brush border membrane as the more specialized structure would then contain in addition the sodium-dependent phlorizin-sensitive system which enables the cell to reabsorb sugars actively.

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SUMMARY AND CONCLUSIONS

The brush border membrane of the proximal tubule epithelial cell hm been shown to contain a stereospecific sugar transport system that is inhibited competitively by phlorizin and whose affinity is increased by the presence of sodium ions. The transport system in addition is capable of accumulating sugars across the brush border membrane, provided there exists an electrochemical potential difference for sodium. This indicates that a t the cellular level D-glucose entry across the luminal cell membrane is coupled to a symport of sodium and that the energy for intracellular accumulation and transepithelial sugar transport is derived from the electrochemical potential difference for sodium between the intra- and extracellular fluids. This potential difference is maintained with energy input aided by the action of the Na-K-ATPase. The active center of the sugar transport system seems to be composed of three binding sites: a sugar-binding site, a cation-binding site, and an aglucon-binding site. For the interaction of a monosaccharide with the sugar-binding site to occur requires a D-glucose-like orientation of the hydroxyl groups at C-2 and, to a lesser extent, a t C-3, C-6, and C-4. The cation-binding site is sodium-specific, whereas the aglucon-binding site interacts relatively unspecifically with aryl residues of the aglucon. The sugar transport system possesses essential SH groups (about 2-4) which change their affinity to NEM during the formation of the sodium-glucose“carrier” complex. These findings suggest that, during binding to and transfer of sugars through the brush border membrane, conformational changes of the transport system occur. The transfer of sugars across the basal-lateral plasma membranes is also facilitated by transport systems, which, however, lack sodium sensitivity and sodium-dependent accumulation. Also their sensitivity to phlorizin is much lower than the sensitivity of the transport system located in the brush border. Thus, a t the contraluminal cell side, sugars leave the cell via facilitated diffusion, with the concentration difference for the sugars established by the sodium-dependent transport system a t the luminal cell pole. ACKNOWLEDGMENTS

I would like to thank Drs. K. J. Ullrich, €1. Murer, J. Kaplan, and E. Kinne-Saffran for valuable discussions during the preparation of the manuscript. The author is also indebted to ME. G. Zimmerschied for her help in compiling the bibliography, and to Miss M. Becker for typing the manuscript. REFERENCES 1. F. Alvarado, Intestinal transport of sugars and amino acids independence or federalism? Am. J . Clin. Nutr. 23, 824-828 (1970).

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124. K. SigrisbNelson, H. Murer, and U. Hopfer, “Active” alanine transport in isolated brush border membranes. J. Biol. Chem., 250, 5674-5680 (1975). 125. M. Silverman, Brush border disaccharidases in dog kidney and their spatial relationship to glucose transport receptors. J. Clin. Invest. 52, 2486-2494 (1973). 126. M. Silverman, The chemical and steric determinants governing sugar interactions with renal tubular membranes. Biochim. Biophys. Acta 332, 248-262 (1974a). 127. M. Silverman, The i n viuo localization of high-affinity phlorizin receptors to the brush border surface of the proximal tubule in dog kidney. Biochim. Biophys. Actu 339, 92-102 (1974b). 128. M. Silverman, M. A. Aganon, and F. P. Chinard, D-glucose interactions with renal tubule cell surfaces. Am. J. Physiol. 218,735-742 (1970a). 129. M. Silverman, M. A. Aganon, and F. P. Chinard, Specificity of monosaccharide transport in dog kidney. Am. J. Physiol. 218,743-750 (1970b). 130. S. J. Singer, The molecular organization of membranes. Annu. Rev. Biochem. 43,805-833 (1974). 131. D. H. Smyth, Sodium-hexose interactions. Phil. Trans. Roy. SOC.London, Xer. B 262, 121-130 (1971). 132. J. F. Soodsma, B. Legler, and R. C. Nordlie, The inhibition by phlorizin of kidney microsomal inorganic pyrophosphate-glucose phosphotransferase and glucose 6-phosphatase. J. Biol. Chem. 242, 1955-1960 (1967). 133. F. K. Stevenson, The disaccharidase activity of a membrane fraction obtained from the rabbit renal cortex. Biochim. Biophys. Acta 266, 144-153 (1972). 134. C. E. Stirling, High-resolution radioautography of phlorizin-3H in rings of hamster intestine. J . Cell Biol. 35, 605-618 (1967). 135. T. Takenawa and T. Tsumita, Myo-inositol transport in plasma membrane of rat kidney. Biochim. Biophys. Acta 373, 106-114 (1974). 136. L. Thomas, Isolation of N-ethylmaleimide-labelled phlorizin-sensitive D-glucose binding protein of brush border membrane from rat kidney cortex. Biochim. Biophys. Acta 291,454-464 (1973). 137. L. Thomas and R. Kinne, Studies on the arrangement of aminopeptidase and alkaline phosphatase in the microvilli of isolated brush border of rat kidney. Biochim. Biophys. Acta 255, 114-125 (1972a). 138. L. Thomas and R. Kinne, Studies on the arrangement of a glucose sensitive phlorizin binding site in the microvilli of isolated rat kidney brush border. FEBS Lett. 25, 242-244 (1972b). 139. L. Thomas, R. Kinne, and P. P. Frohnert, N-ethylmalehide labeling of a phlorizin-sensitive D-glucose binding site of brush border membrane from the rat kidney. Biochim. Bwphys. Acta 290, 125-133 (1972). 140. L. Thuneberg and J. Rostgaard, Isolation of brush border fragments from rat and rabbit kidney cortex. Ezp. Cel2 Res. 51, 123-140 (1968). 141. K. J. Ullrich, G. Rumrich, and S. Kloss, Specificity and sodium dependence of the active sugar transport in the proximal convolution of the rat kidney. PjZuegers Arch. 351,3548 (1974a). 142. K. J. Ullrich, G. Rumrich, and S. Klijss, Sodium dependence of the amino acid transport in the proximal convolution of the rat kidney. PjZuegers Arch. 351, 4 9 4 0 (1974b). 143. H. Vick, D. F. Diedrich, and K. Baumann, Reevaluation of renal tubular glucose transport inhibition by phlorizin analogs. Am. J. Physiol. 224, 552-557 (1973). 144. E. D. Wachsmuth, and K. Hiwada, Alkaline phosphatase from pig kidney. Method of purification and molecular properties. Biochem. J . 141, 273-282 (1974).

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145. It. F. Wilfong and D. M. Neville, Jr., The isolation of a brush border membrane fraction from rat kidney. J. Biol. Chem. 245, 6106-6112 (1970). 146. R. E. Wood, F. P. Wirth, Jr., and H. E. Morgan, Glucose permeability of lipid bilayer membranes. Biochim. Biophys. Acta 163, 171-178 (1968). 147. E. M. Wright and R. Pietras, Routes of nonelectrolyte permeation acros9 epithelial membranes. J. Membr. Bid. 17, 293-312 (1974). 148. H. Zipper and R. C. Mawe, The exchange and maximal net flux of glucose acroa the human erythrocyte. Biochim. Bwphys. Acta 282, 311-325 (1972). NOTE ADDED I N PROOF After completion of the manuscript the rcsults o n the electrogenic transport of u-glucose across the brush border membrane were confirmed by Beck and Sacktor in rabbit kidney [J. C. Beck and B. Sacktor, Energetics of the Na+-dependent transport of u-glucose in renal brush border membrane vesicles. J . B i d . Chem. 250, 8674-8680 (1 975) 1.

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A A, protein, of myelin

see Encephalitogenic protein Active transport ATPase in, 142 cation-binding site affinity and, 84-90 Aglucon-binding site, in renal brush border membrane, 250-251 u-Allose, renal transport of, 246 Anionic currents, in ionic interactions, 184-1 89 Arginine, methylated, in encephalitogenic protein, 4 ATl’, hydrolysis of, by sodium pump, 68-77 ATPase(s), 99-160 bacterial, 141-149 chloroplast coupling factor 1, 126-141 complex of, in endoplasmic reticulum, 13 inhibitors of, 107-110 mitochondria1 coupling factor 1,100-117 oligomycin-sensitive, 118-126 of sarcoplasmic reticulum, 19 sodium-dependent, 75-76 solubilized, dissociation constants of, 112 Aurovertin, as ATPase inhibitor, 107-108 Azide, as ATPase inhibitor, 107

B Bacteria, ATPases of, 99-160 Bacterial ATPase, 141-149 in active transport, 142 cation requirements of, 143, 144, 146 cold lability of, 144 comparison with other ATPasos, 148 enzymatic properties of, 143-147 inhibitors of, 144, 146

molecular properties of, 147-148 nucleotide specificity of, 143, 144 in oxidative phosphorylation, 141-142 reactions catalyzed by, 141-142 in transhydrogenation, 141-142 Basal-lateral plasma membranes, glucose transport by, 222-230

C Calcium currents in giant barnacle muscle, 167-168 in invertebrate muscle, 169-171 Cardiac muscle calcium currents in, 170-171 slow inward currents in, 168 Cation-binding sites affinity changes in, for active transport, 84-90 interactions among, 78-79 metabolite-binding site interaction with, 79-84 of sodium pump, 3 1 4 0 cation-site complexes 33-34, 35-37 inner and outer, 37-68 interactions, 34-35 Cationic fluxes apparent uncoupling between partial reactions and, 72-77 kinetics of, 4 4 4 8 equilibrium treatment, 48-65 overall rate equation for, 46-48 steady-state treatment, 6 5 4 8 Chloroplast coupling factor 1, 126-141 cold lability of, 133 divalent cation specificity of, 129-130 enzymatic properties of, 129-135 inhibitors of, 133-135 light effects on, 139-140 molecular properties of, 135-139 269

270

SUBJECT INDEX

M.W. and size of, 135-136 nucleotide specificity and binding of, 130-133 reactions catalyzed by, 126-129 -SH content of, 138-139 subunit role of, 137-138 subunit structure of, 136-137 Chloroplasts, ATPases of, 99-160 Circular dichroism of brain proteolipid protein, 13 of myelin basic protein, 8 Citrulline, in myelin protein fraction, 17-18 Coupling factor 1, see Mitochondrial coupling factor 1 Cytochrome ou3, 100 Cytochrome bs, physical properties of, 20 Cytochrome b-c, 100

Fatty acid, in proteolipid apoprotein P7, 12-13 Frog skeletal muscle, anionic currents in, 184-189 D-Fructose, renal transport of, 247 D-Fucose, renal transport of, 247

D

H

Danielli-Davson model, of proteins, 1 DCCD, effect on ATPase, 135 2-Deoxy-~-glucose,renal transport of, 247

5-Hydroxytryptamine, encephalitogenic protein binding site for, 6

4,4’-Diisothiocyano-2,2’-ditritiostilbene sulfonate, as nonpenetrating agent, 23 Dio-9, as ATPase inhibitor, 133-134 Divalent ion currents blocking of, 169-171 in ionic interactions, 167-168 DM-20 protein, from myelin, 18

E Electron transport system, complexes of, 100 Encephalitogenic protein (of myelin), 3-7 amino acid sequence of, 6 phosphorylation of, 6-7 Enzymes in ionic interactions, 164 in renal glucose transport system, 256257 N-Ethylmaleimide, effect on phlorizin binding, 252-256 Experimental allergic encephalomyelitis (EAE), protein induction of, 3

F FI coupling factor, see Mitochondrial coupling factor 1

0 D-Galactose, renal transport of, 246 D-Glucose, interaction with renal plasma membranes, 218-232, 246 cGlucose, renal transport of, 247 Glucose transport system in renal brush border membrane, 209267 conformational response of, 251-256 enzymes of, 256-257 mechanism, 245-251

I Independence principles, in ionic interactions, 165-166 Inhibitors, of sodium pump, 36 Ion transport, mechanism of, 77-91 Ionic interactions, 161-207 adsorption and enzymes in, 164 anionic currents, 184-189 blocking and competition in, 169-189 independence principles in, 165-166 models and analysis of, 189-198 molecular architecture of ionic channels in, 197-198 saturation phenomena in, 166-169 single-file diffusion theory of, 189-192 single-occupancy models of, 192-197

K K systems, in cation-binding sites, 34, 35 Kidney brush border membrane, glucose transport system in, 209-267

1 Lactic dehydrogenase, multistability of, 19 Light, effects on chloroplast coupling factor, 139-140

271

SUBJECT INDEX

Lipid bilayer of myelin, 21 structure of, 2 Lipid bilayer membranes, channels in, 166-167 Lipophilin, N-2 myelin protein as, 18 Liposome, freeze fracture of, 22

encephalitogenic type, 3-7 localization of, 21-24 of multiple sclerosis, 23 N-2 protein, 13-16 proteolipid fraction of, 2, 10-16

M

N-2 protein (of myelin) electron micrographs of, 16 physical properties of, 13-16, 21 possible function of, 20 NADH dehydrogenase, 100 Ncrve axon currents blocking of, 171-184 saturation behavior of, 168-169 Nucleoside, binding to F,, 111-1 13

o-Mannitol, renal transport of, 247 D-Mannose, renal transport of, 247 Membrane factor, in oligomycin-sensitive ATPase Membrane proteins conformational changes in, 20 structure of, 1 Membranes, models of, 1-3 Metabolite-binding sites, cation-binding site interaction with, 79-84 3-O-i%thyl-~-glucose, renal transport of, 246 a-Methyl-o-glucoside, rend transport of, 246 8-Jlethyl-D-glucoside, renal transport of, 246 Mitochondria, ATPases of, 99-160 Mitochondria1 coupling factor 1, 100-117, 121 activation of, 105-107 active site of, 116-117 ATPase activity of, 101-103 chemical modification of, 103-104 cold lability of, 104-105 enzymatic properties of, 104-113 function and properties of, 100-117 inhibition of, 107-110 molecular properties of, 114-1 17 h1.W. of, 114 nucleoside binding to, 111-1 13 i n oxidative phosphorylation, 101-104 resistance to oligomycin and DCCD, 105 sedimentation coefficient of, 114 specificity of, 110-1 11 subunit structure of, 114-116 Moffitt parameter, of N-2 protein, 14 Multiple sclerosis, myelin proteins of, 23 Multistability, of proteins, 19 Myelin protein(s) basic, physical structure of, 7-10 DM-20 fraction, 18

N

0 Oligomycin-sensitive ATPase, 118-126 cation sensitivit,y of, 119 cold stability of, 119 dicyrlohexylcarbodiimide site of action in, 125 enzymatic properties of, 118-120 molecular properties of, 120-125 M.W. and size of, 120-121 oligomycin site of action in, 125 phospholipid stimulation of, 119-120 subunit structure of, 121-125 Optical rotary dispersion, of myelin basic protein, 7-8 “Overlapping transport,” as active-transport” mechanism, 66 Oxidative phosphorylation bacterial ATPase in, 141-142 F, action in, 101-104

P P7 proteolipid apoprotein, isolation of, 12 Phlorixin as ATPase inhibitor, 133-134 interaction with renal plasma membranes, 233-245 Phosphorylation, of encephalitogenic protein of, 6-7 Photophosphorylation uncouplers, as ATI’ase inhibitors, 135

272

SUBJECT INDEX

Plasma membranes, from proximal tubular epithelium, isolation of, 211-218 Potassium channel in nerve-axon-current blockage, 174-181 quaternary ammonium ion blockage of, 181-184 Potassium transport, sequential and simultaneous models for, 29-97 Proline-rich sequence in encephalitogenic protein, 4 Proteins of membranes, 1-3 multistability of, 19 Proteolipid protein fraction of myelin, 2, 10-16 physical properties of, 13-16 Proximal tubular epithelium, plasma membrane isolation from, 211-218 Pullman inhibitor, as ATPase inhibitor, 109-110 Pyruvate kinase, multistability of, 19

0 Quercetin, as ATPase inhibitor, 110

R Radius of gyration, of myelin basic protein, 8 Renal brush border membrane chemical and biochemical composition of, 214-218 enzymatic activities of, 219, 256-257 glucose transport system in, 209-267 mechanism, 245-251 isolation of, 213-214 phloriain interaction with, 233-245 Red cells, cation flux studies on, 45 Rhodopsin, physical properties of, 19-20 D-Ribose, renal transport of, 247 Rotary dispersion, of brain proteolipid fraction, 13-14

S Sarcoplasmic reticulum, proteins of, 19 Saturation phenomena,. in ionic interactions, 166-169 Semlicki forest virus, spikes on, 20 Sequential models, for sodium and potassium transport, 29-97 Sodium channel, in nerve-axon-current blockage, 171-173 Sodium pump ATP hydrolysis by, 68-77 cation-binding sites of, 3 1 4 0 occluded forms of, 63-65 phosphorylation and dephosphorylation of, 69-71 structure of, 4 M 1 as V system, 77-84 Sodium transport sequential and simultaneous models for, 29-97 Theorell-Chance kinetics of, 66-68 Succinate dehydrogenase, 100 Sugars, inhibition of phloriain binding by, 239 Sulfhydryl groups, in chloroplast coupling factor 1, 138-139 Surface tension studies, on myelin basic protein, 9

T Theorell-Chance kinetics, of active transport, 66-68 Threonine, in encephalitogenic protein, 4 Transhydrogenation, bacterial ATPase in, 141-142 Transphosphorylation, sodium : sodium exchange and, 72-75 Tryptophan, in encephalitogenic protein, as active site, 4-5

U Ubiquinone, function of, 100 A

6

8 7

c a

D 9 E O F 1 G 2

H 3 1 4 J

5