Subcellular Biochemistry: Volume 6 [1 ed.] 978-1-4615-7947-2, 978-1-4615-7945-8

Citation preview

Subcellular Biochemistry Volume

6

ADVISORY EDITORIAL BOARD J. ANDRE Laboratoire de Biologie Cellulaire, 4 Faculte des Sciences, 91 Orsay, France D. L. ARNON Department of Cell Physiology, Hilgard Hall, University of California, Berkeley, California 94720, USA

J. BRACHET Laboratoire de Morphologie Animale, Faculte del Sciences, Universite Libre de Bruxelles, Belgium

J. CHAUVEAU Institut de Recherches Scientifiques sur Ie Cancer, 16 Avenue VaillantCouturier, 94 Ville Juif, Boite Postale 8, France C. de DUVE Universite de Louvain, Louvain, Belgium and The Rockefeller University, New York, NY 10021, USA M. KLINGENBERG Institut fUr Physiologische Chemie und Physikalische Biochemie, Universitiit Miinchen, Goethestrasse 33, MUnchen 15, Germany A. LIMA-de-FARIA Institute of Molecular Cytogenetics, Tornavagen 13, University of Lund,Lund,Sweden O. LINDBERG The Wenner-Gren Institute, NorrtulIsgatan 16, Stockholm, VA, Sweden V. N. LUZIKOV A. N. Belozersky Laboratory for Molecular Biology and Bioorganic • Chemistry, Lomonosov State University, Building A, Moscow 117234, USSR H. R. MAHLER Chemical Laboratories, Indiana University, Bloomington, Inlliana 47401, USA M. M. K. NASS Department of Therapeutic Research, University of Pennsylvania School of Medicine, Biology Service Building, 3800 Hamilton Walk, Philadelphia, Pennsylvania 19104, USA A. B. NOVIKOFF Department of Pathology, Albert Einstein College of Medicine, Yeshiva University, Eastchester Road and Morris Park Avenue, Bronx, NY 10461, USA R. N. ROBERTSON Research School of Biological Sciences, P. O. Box 475, Canberra City, A.C.T. 2601, Australia P. SIEKEVITZ The Rockefeller University, New York, NY 10021, USA F. S. SJOSTRAND Department of Zoology, University of California, Los Angeles, California 90024, USA A. S. SPIRIN A. N. Bakh Institute of Biochemistry, Academy of Sciences of the USSR, Leninsky Prospekt 33, Moscow V-71, USSR D. von WETTSTEIN Department of Physiology; Carlsberg Laboratory, Gl. Carlsbergvej 10, DK-2500, Copenhagen, Denmark V. P. WHmAKER Abteilung fiiI Neurochemie, Max-Planck Institut fUr Biophysikalische Chemie, D-3400 Gottingen-Nikolausberg, Postfach 968, Germany A Continuation Order Plan is available for this series. A continuation order will bring delivery of each new volume immediately upon publication. Volumes are billed only upon actual shipment. For further information please contact the publisher.

Subcellular Biochemistry Volume

6

Edited by

Donald B. Roodyn University College London London, England

PLENUM PRESS • NEW YORK AND LONDON

ISBN 978-1-4615-7947-2 DOI 10.1007/978-1-4615-7945-8

ISBN 978-1-4615-7945-8 (eBook)

Library of Congress Catalog Card Number 73-643479

This series is a continuation of the journal Sub-Cellular Biochemistry, Volumes 1 to 4 of which were published quarterly from 1972 to 1975

© 1979 Plenum Press, New York

Softcover reprint of the hardcover 18t edition 1979 A Division of Plenum Publishing Corporation 227 West 17th Street, New York, N.Y. 10011 All rights reserved No part of this book may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher

Contributors Mark S. Boguski Laboratory of Molecular Biology, Baltimore Cancer Research Center, DCT, NCI, NIH, Baltimore, Maryland 21201, U.S.A. Frederick L. Crane Department of Biological Sciences and Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907, U.S.A. Hans Goldenberg Department of Biological Sciences and Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907, U.S.A. Niels Peter Hundahl M0lIer Institute of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmark; Present address: Institute of Medical Microbiology, University of Aarhus, DK-8000 Aarhus C, Denmark Timothy P. Karpetsky Laboratory of Molecular Biology, Baltimore Cancer Research Center, DCT, NCI, NIH, Baltimore, Maryland 21201, U.S.A. U. C. Knopf IPRIP, University of Neuchatel, 2001 NeucMtel, Switzerland N. Lakshminarayanaiah Department of Pharmacology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, U.S.A. Giorgio Lenaz Institute of Biochemistry, Faculty of Medicine and Surgery, University of Ancona, Ancona, Italy Carl C. Levy Laboratory of Molecular Biology, Baltimore Cancer Research Center, DCT, NCI, NIH, Baltimore, Maryland 21201, U.S.A. Hans Low Department of Endocrinology, Karolinska Hospital, S-10401 Stockholm, Sweden Niels Peter Hundahl M0lIer Institute of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmark D. James Morre Department of Biological Sciences and Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907, U.S.A. v

vi

Contributors

Jens Steensgaard Institute of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmark Peter A. Whittaker Biology Department, St. Patrick's College, Maynooth, County Kildare, Ireland

Aims and Scope SUBCELLULAR BIOCHEMISTRY aims to bring together work on a wide range of topics in subcellular biology in the hope of stimulating progress towards an integrated view of the cell. In addition to dealing with conventional biochemical studies on isolated organelles, articles published so far and planned for the future consider such matters as the genetics, evolution, and biogenesis of cell structures, bioenergetics, membrane structure and functions, and interactions between cell compartments, particularly between mitochondria and cytoplasm and between nucleus and cytoplasm. Articles for submission should be sent to Dr. D. B. Roodyn, Department of Biochemistry, University College London, Gower Street, London WCIE 6BT, U.K., and are best sent in the period February to April inclusive of each year. There are no rigid constraints as to the size of the articles and in general they should be between 9,000 and 36,000 words, with an optimum size of about 20,000 words. Although articles may deal with highly specialized topics, authors should try as far as possible to avoid specialist jargon and to make the article as comprehensible as possible to the widest range of biochemists and cell biologists. Full details of the preparation of manuscripts are given in a comprehensive Guide for Contributors which is available from the Editor or Publishers on request.

vii

Preface This volume continues the tradition of SUBCELLULAR BIOCHEMISTRY of trying to break down interdisciplinary barriers in the study of cell function and of bringing the reader's attention to less well studied, but nevertheless useful, biological systems. We start with an extensive article by T. P. Karpetsky, M. S. Boguski and C. C. Levy on the structure, properties and possible functions of polyadenylic acid. Apart from revealing a general lack of appreciation of many important aspects of the chemical properties of polyadenylic acid, the literature also shows that there is a great gulf between those who study the biological role of polyadenylic acid. and those who study its physicochemical properties. The article by Karpetsky and his colleagues is an attempt to overcome this lack of communication and to present an integrated view of the subject. The authors go into the subject in full detail and the more biologically inclined reader may on occasion have to reread his nucleic acid physical chemistry notes! However, the effort is worthwhile and the article is a timely reminder that we cannot treat nucleic acids as mere abstractions, but that they are complex organic macromolecules capable of equally complex, but nevertheless important, interactions. The next article is by J. Steensgaard and N. P. Hundahl M0ller and deals with computer simulation of density gradient centrifugation systems. From the rather early "hit and miss" bulk fractionation schemes, preparative centrifugal fractionation of cell homogenates has now developed into a rigorous and technically sophisticated discipline. The fact that many centrifugation procedures can now be accurately represented by computer models is a great advance. Apart from the convenience it provides of being able to carry out "dummy" runs without actually wasting precious biological material, the fact that the major parameters in the system can be quantified and handled in this way puts centrifugal fractionation on a much more sound theoretical basis. It is to be hoped that Steensgaard and Hundahl M0ller's article will stimulate as much as it simulates. The next article by U. C. Knopf deals with crown-gall tumors in ix

x

Preface

general as well as the specific role of Agrobacterium tumefaciens. From relatively obscure beginnings, the subject is becoming more generally recognized as a most interesting experimental system for studying the induction of tumors. It is also becoming clear that there are some similarities between the processes involved in the action of Agrobacterium tumefaciens and of the nitrogen-fixing bacteria, particularly the Rhizobia. The famous root nodules of the nitrogen-fixing legumes are in fact "benign" nodules, perhaps different only in degree from the massive crown-galls. We thus have a most interesting comparative system of "benign" and "malignant" growth, and we can only hope, with the author, that the medical implications will not be lost just because the systems are found in plants and not in mice. We next tum to another system that has only recently achieved the full interest and recognition that it deserves. This is the so-called "petite" mutation in yeast, and it is described in detail by P. A. Whittaker. Although the existence of "petites" was known for years amongst yeast geneticists, it was only with the explosive development of research in mitochondrial biogenesis in the last five years that the full significance of the "petite" mutation became widely appreciated. Mitochondrial genetics has now become a major discipline in its own right, with its own terminology and expertise. It is to be hoped that Whittaker's article will help to guide the reader through some of the intricacies of this new methodology. The next article by G. Lenaz looks at some fundamental aspects of the role of lipids in cell membranes. In a detailed and extensive article, the author identifies five major roles for lipids in biomembranes. They act as binding surfaces for proteins, they are needed to separate aqueous compartments so as to allow vectorial processes to take place, they provide a hydrophobic milieu for reactions that require one, they are required for the formation of membranes from dissociated subunits, and finally they can act as modulators of membrane-bound enzymes. As with the article by Karpetsky and colleagues, this article delves into fundamental physicochemical aspects of the molecular entities involved and the reader is continually reminded that membranes are not abstractions but are made from very real molecules that have their own inherent chemical properties, a knowledge of which can greatly help in our understanding of the behavior of biological membranous assemblies. and H. The next article is by F. L. Crane, H. Goldenberg, D. J. ｍｯｲｮｾＬ＠ Low and deals with the dehydrogenases of the plasma membrane. As the detailed tables presented by the authors clearly show, there is now overwhelming evidence for the existence of a range of dehydrogenases in plasma membranes isolated from a variety of cell types. Just as it took some time to appreciate the fact that the mitochondrial membrane is not the

Preface

xi

only site for linked respiratory activity and the "microsomal" respiratory chain is a complex system in its own right, so we must now generalize the picture even further, and think in terms of other membrane systems having bound respiratory enzymes, performing functions specific to the membrane in which they are found. The authors discuss several interesting ways in which plasma membrane dehydrogenases can act, for example, in redox control of the formation or breakdown of cyclic nucleotides. From primitive views of membrane-bound respiratory enzymes being solely involved in mitochondrial processes, we must now develop much more sophisticated attitudes toward the cellular role of "intrinsic" dehydrogenases, perhaps almost to the point of believing that the mitochondrion is really only a "special case" in which bound redox systems happen to be linked to the production of ATP. The last article is by N. Lakshminarayanaiah and is entitled "Transport Processes in Membranes: A Consideration of Membrane Potential across Thick and Thin Membranes." Here we are hoping to bridge yet another unfortunate interdisciplinary gap, namely that between biophysicists and biochemists. One of the most important consequences of the famous chemi-osmotic theory of Mitchell is that biochemists have come to realize that the ion transport and membrane potential phenomena studied so eruditely and mathematically by biophysicists are in fact closely interconnected with the respiratory and bioenergetic properties of cell membranes. It is as if there has been a sudden realization that the term "membrane" as used by biophysicists is not some abstract concept or barrier, but refers in reality to actual membranes in real cells. Unfortunately, the mathematical rigor of the biophysical approach has not yet fully spilled over into biochemical membranology. Perhaps the phenomena under study are too complex to be represented by formal equations. Nevertheless, any attempt to propagate rigorous attitudes in cell biochemistry is surely to be encouraged, and it is to be hoped that Lakshminarayanaiah's article will demonstrate the remarkable extent to which "classical" physicochemical theory can be applied to the study of biomembranes. As in previous volumes of SUBCELLULAR BIOCHEMISTRY we end with an account of recent books in cell biochemistry and biology . We discuss a number of texts in membrane research, organelle biochemistry, and plant biochemistry as well as some educational texts and once again hope that we are of some use in guiding the reader through the very extensive literature currently published in the overall field of cell biology. D. B. Roodyn London

Contents

Chapter 1 Structures, Properties, and Possible Biological Functions of Poly adenylic Acid Timothy P. Karpetsky, Mark S. Boguski, and Carl C. Levy 1. Introduction ........................................... 2. Isolation and Detection of Poly(A) . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Methodology ...................................... 2.2. Determination of the Size of Poly(A) Segments ........ 3. Messenger RNA and the 3'-Terminal Poly(A) Sequence..... 3.1. Occurrence of Poly(A) in Living Organisms ........... 3.2. Poly(A) Sequences in Prokaryotes . . . . . . . . . . . . . . . . . . . . 3.3. Messenger RNA Lacking Poly(A) .................... 3.4. Complexes of Poly(A) with Amino Acids and Proteins. . 4. Possible Biological Functions of Poly(A) .................. 4.1. Covalent Linkage of Poly(A) RNA ................... 4.2. Transport of mRNA from the Nucleus to the Cytoplasm 4.3. Poly(A) and the Stability of mRNA . . . . . . . . . . . . . . .. . . . 4.4. Poly(A) Involvement in the Binding of mRNA to Membranes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. 3'-Terminal Poly(A) Sequences of mRN A and Protein Synthesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. Summary......................................... 5. Structure of Poly(A) .................................... 5.1. Poly(A) at Neutral pH .............................. 5.2. Acidic Forms of Poly(A) .......... . . . . . . . . . . . . . . . . . . 5.3. Effect of Substituents on Poly(A) Structure. . . . . . . . . . . . 5.4. Synthesis of Analogues of Poly(A). . . . .. . . . . . . . . . . . . . . 5.5. Influence of Metal Ions on the Structure of Poly(A) xiii

1 2 2 5 9 9 9 15 18 24 24 27 32 40 41 42 43 43 49 58 64 66

Contents

xiv

6. Interaction of Poly(A) with Monomers and Polymers. . . . . . . . 6.1. Complexes of Low-Molecular-Weight Organic Compounds and Poly(A) ............................ 6.2. Complexes of Poly(A) and Complementary Monomers . . 6.3. Interaction of Poly(A) with Poly(U) and Other Complementary Polynucleotides ..................... 7. Conclusions ........................................... 8. References ............................................

72 72 80 83 90 91

Chapter 2 Computer Simulation of Density-Gradient Centrifugation

Jens Steensgaard and Niels Peter Hundahl M011er 1. Introduction ........................................... 2. Some Aspects of the Basic Theory of Gradient Centrifugation 3. The Indirect Approach to Simulation of Gradient Centrifugation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 4. The Compartmental Approach to Simulation of Gradient Centrifugation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 5. The Analytical Approach to Simulation of Gradient Centrifugation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 6. General Discussion ..................................... 7. References ............................................

117 118 122 127 132 138 139

Chapter 3 Crown-Gall and Agrobacterium tumefaciens: Survey ofa Plant-CellTransformation System of Interest to Medicine and Agriculture U. C. Knopf

1. Introduction ........................................... 2. Overview of the Process of Plant-Cell Transformation by Agrobacterium tumefaciens . . . . . . . . . . . . . . .. . . . . . . . . . . . . .. 3. Conditions for Plant-Cell Transformation by Agrobacterium tumefaciens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 3.1. Dicotyledonous Host Plants or Gymnosperms ......... 3.2. A Temperature below 30°C. . . . . . . . . . . . . . . . . . . . . . . . .. 3.3. A Wound or Wound Stimulus. . . . . . . . . . . . . . . . . . . . . . .. 4. Properties and Products of Agrobacterium tumefaciens ..... 4.1. Induction of Crown-Galls ........................... 4.2. General Properties and Classification .................

143 145 145 146 146 146 149 149 150

Contents

5.

6.

7.

8. 9.

4.3. Differential Ability to Use Unusual Amino Acids as Sole Nitrogen Source ................................... 4.4. Production of Plant Growth Substances .............. \ 4.5. Production of Polysaccharides ....................... 4.6. Production of Vitamins ............................. 4.7. Production of Antibiotics. . . . . . . . . . . . . . . . . . . . . . . . . . .. Molecular Components, Genetic Systems, and Search for the Tumor-Inducing Principle (TIP) of Agrobacterium tumefaciens 5.1. DNA and DNA Plasmids. . .. ... . .. .. . . . . .. . .. .. . . . .. 5.2. An RNA Polymerase and Its Components. . . . . . . . . . . .. 5.3. RNA ............................................. 5.4. Ribosomes and Their Components ................... 5.5. Bacteriophages and Their Components. . . . . . . . . . . . . . .. Attempts to Define the Crown-Gall Tumor Cell ............ 6.1. Transplantability ................................... 6.2. Presence of Unusual Amino Acids ................... 6.3. Autonomy......................................... 6.4. Accelerated Growth Rate. . . . . . . . . . . . . . . . . . . . . . . . . . .. 6.5. Limited Capacity for Differentiation. . .. . . . . . . . . . . . . .. On the Genetic Basis ofthe Formation ofthe Crown-Gall Tumor Cell. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 7.1. Experiments on the Reversion and Suppression of the Tumorous State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 7.2. Experiments Directed to the Detection of Bacterial and Bacteriophage Genes and Gene Products in Crown-Gall Tumor Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Medical and Agricultural Interest in Crown-GalV Agrobacterium Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. References ............................................

xv

151 152 152 153 153 153 153 157 157 157 158 159 159 159 161 162 163

163 163

164 166 168

Chapter 4 The Petite Mutation in Yeast Peter A. Whittaker

1. Discovery and Initial Characterization. . . . . . . . . . . . . . . . . . . .. 1.1. Introduction ....................................... 1.2. Discovery......................................... 1.3. Genetic and Biochemical Characterization. . . . . . . . . . . .. 2. Cytology and Ultrastructure of Petite Mutants ............. 3. Mitochondrial DNA in Petite Mutants. . . . . . . . . . . . . . . . . . . .. 3.1. Grande Yeast Mitochondrial DNA ...................

175 175 176 176 182 183 183

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Contents

3.2. Petite Yeast Mitochondrial DNA. . . . . . . . . . . . . . . . . . . .. 3.3. Mitochondrial DNA Synthesis ....................... 4. Mitochondrial RNA in Petite Mutants. . . . . . . . . . . . . . . . . . . .. 4.1. Grande Yeast Mitochondrial RNA...... . . . . . .. . .. .... 4.2. Petite Yeast Mitochondrial RNA ... . . . . . . . . . . . . . . . . .. 5. Mitochondrial Proteins in Petite Mutants .................. 5.1. Synthesis of Mitochondrial Proteins .................. 5.2. Tricarboxylic Acid Cycle and Other Enzymes ......... 5.3. Respiratory-Chain Components ...................... 5.4. Mitochondrial Adenosine Triphosphatase. . . . . . . . . . . . .. 5.5. Mitochondrial Transport Systems .................... 6. Induction of the Petite Mutation. . . . . . . . . . . . . . . . . . . . . . . . .. 6.1. Temperature and Nutritional Effects. . . . . . . . . . . . . . . . .. 6.2. Inhibitors of Mitochondrial Macromolecular Synthesis .. 6.3. Miscellaneous Chemical Mutagens. . . . . . . . . . . . . . . . . . .. 6.4. Additional Mutagenic Treatments .................... 6.5. Spontaneous Mutation. . . . . .. . . . . . . . . . . . . . . . . . . .. . .. 6.6. Antagonists of Petite Mutation . . . . . . . . . . . . . . . . . . . . . .. 7. Petite Mutants and Mitochondrial Genetics ................ 7.1. Suppressiveness.................................... 7.2. Petite Deletion Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 7.3. Petite Marker Rescue. .. . . . . . . . . .. . . . . . . .. . . . . . . . . .. 8. Petite-Negative Yeasts .................................. 9. The Petite Mutation: A Broader View. . . . . . . . .. . . . . . . . . . .. 10. Appendix: Abbreviations and Terms. . . . .. . . . . .. . . . . .. . . .. 11. References ............................................

184 188 189 189 191 194 194 195 195 197 198 199 199 200 201 202 208 209 210 210 212 213 213 215 218 219

Chapter 5 The Role of Lipids in the Structure and Function of Membranes

Giorgio Lenaz 1. Introduction 2. Properties of the Lipid Bilayer .......................... . 2.1. Lamellar Systems ................................. . 2.2. Thermotropic Phase Changes and Phase Separations ... . 2.3. Lipid Viscosity ................................... . 2.4. Summarizing Concepts ............................. . 3. Lipid-Protein Interactions and Lipid Organization in Membranes ........................................... . 3.1. Lipid-Protein Interactions .......................... . 3.2. Asymmetry of Membrane Components ............... . 3.3. Protein Mobility ................................... .

233 235 235 236 239 242 243 243 248 252

Contents

4. Effects of Lipids and Their Physical State on the Properties of Biomembranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 4.1. Means Employed to Investigate the Effects of Lipids in Membrane Functions ............................... 4.2. Permeability and Transport .. , . . . . . . . . . . . . . . . . . . . . . .. 4.3. Lipids and Enzyme Activity. . . . . . . . . . . . . . . . . . . . . . . .. 4.4. Effects of Lipids on Hormonal Response ............. , 4.5. Lipids and Other Membrane Properties ............... 4.6. Coenzymatic Function of Lipids ..................... 5. Roles of Lipids in Membrane Functions . . . . . . . . . . . . . . . . . .. 5.1. Lipids Represent a Binding Surface for Proteins. . . . . . .. 5.2. Latency and Compartmentation . . . . . . . . . . . . . . . . . . . . .. 5.3. Lipids Provide a Hydrophobic Medium or a Binding Interface .......................................... 5.4. Molecularization and Membrane Formation ........... , 5.5. Conformational Role of Lipids. . . . . . . . . . . . . . . . . . . . . .. 6. Summary.............................................. 7. References ............................................

xvii

256 256 258 264 280 283 283 285 286 288 292 299 301 315 317

Chapter 6 Dehydrogenases of the Plasma Membrane

Frederick L. Crane, Hans Goldenberg, D. James Morn!, and Hans Low 1. Introduction ........................................... 2. Extrinsic Dehydrogenases ............................... 2.1. Glyceraldehyde-3-phosphate Dehydrogenase .......... , 2.2. Lactic Dehydrogenase .............................. 2.3. Other Dehydrogenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 3. Intrinsic Dehydrogenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 3.1. NADH Dehydrogenases ............................ 3.2. Selective Inhibition of Plasma Membrane NADH Dehydrogenase .................................... 3.3. NADPH Dehydrogenases ........................... 3.4. Xanthine Oxidase .................................. 3.5. Other Dehydrogenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 4. Relationship of Dehydrogenases to Membrane Function . . . .. 4.1. Energy-Linked Transport ........................... 4.2. Metabolic Conversions. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 4.3. Peroxide or Superoxide Generation. . . . . . . . . . . . . . . . . .. 4.4. Redox Control of Plasma Membrane Functions ........ 5. Conclusions ........................................... 6. References ............................................

345 347 348 349 351 352 352 365 367 369 371 372 372 375 375 376 381 382

xviii

Contents

Chapter 7 Transport Processes in Membranes: A Consideration of Membrane Potential across Thick and Thin Membranes

N. Lakshminarayanaiah 1. Introduction ........................................... 2. Biological and Lipid Bilayer Membranes .................. 2.1. Chemical Constituents and Physical Structure. . . . . . . . .. 2.2. Properties of "Undoped" Bilayer Membranes and Biomembranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 2.3. Properties of "Doped" Bilayer Membranes and Biomembranes .......................... '. . . . . . . . . .. 3. Membrane Potential .................................... 3.1. Donnan Potential. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 3.2. Diffusion Potential ................................. 3.3. Theories of Membrane Potential ..................... 3.4. Distribution, Surface, or Interfacial Potentials. . . . . . . . .. 3.5. Applications of the Gouy-Chapman Double-Layer Theory ............................................ 3.6. Adsorption Approach to Membrane Potential. . . . . . . . .. 4. Summary.............................................. 5. Appendix: Mathematical and Electrochemical Terms and Symbols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 6. References ............................................

401 403 403 410 418 427 428 429 430 439 449 463 468 471 474

Some Recent Books in Cell Biochemistry and Biology. . . . . . . . . . . .. 495 1. 2. 3. 4.

Molecular Biology and Cell Organelles .................... Membrane Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Plant Biochemistry and Morphology. . . . . . . . . . . . . . . . . . . . .. Educational Texts ......................................

496 500 505 506

Index ........................................ " ....... , ....... 511

Chapter 1

Structures, Properties, and Possible Biologic Functions of Polyadenylic Acid Timothy P. Karpetsky, Mark S. Boguski, and Carl C. Levy Laboratory of Molecular Biology Baltimore Cancer Research Center, DCT, NCI, NIH Baltimore, Maryland 21201

1.

INTRODUCTION

Our original interest in preparing this review lay in the fact that no one had presented a thorough examination of the topic, with particular attention to the several possible biological functions of poly adenylic acid [poly(A)). However, as we scrutinized the literature, one point cropped up repeatedly: those engaged in research efforts aimed at clarifying the physiological significance of poly(A) did not make full use of the current body of knowledge concerning the chemical properties of the homopolymer. Similarly, results of experiments that clarify aspects of the physical nature of poly(A) were never interpreted in terms of intracellular functions. Thus, two vast bodies of literature exist in roughly equal proportions, one conThe MEDLINE computer service of the National Library of Medicine, Bethesda, Maryland, and the CHEMCON data base (BRS, Inc., Schenectady, New York) were utilized to compile the initial bibliography on poly(A), consisting of articles published between 1966 and February 1977. The literature survey for this review was completed in July 1977. Abbreviations used in this chapter: (CD) circular dichroism; (cDNA) complementary DNA; (DEAE) diethylaminoethyl; (DMSO) dimethylsulfoxide; (ESR) electron spin resonance; (HnRNA) nuclear heterogeneous RNA; (mRNA) messenger RNA; (mRNP) ribonucleoprotein complex; (NMR) nuclear magnetic resonance; [oligo(dT)] oligodeoxythymidylic acid; (ORD) optical rotatory dispersion; [poly(A)] polyadenylic acid; [poly(C)] polycytidylic acid; [poly(dT)] polydeoxythymidylic acid; [poly(G)] polyguanylic acid; [poly(U)] polyuridylic acid; (rRNA) ribosomal RNA; (SDS) sodium dodecyl sulfate; (tRNA) transfer RNA. 1

Timothy P. Karpetsky, Mark S. Boguski, and Carl C. Levy

2

cerning the biochemistry of poly(A) , the other dealing with more physically oriented considerations, and the amount that either group draws on the knowledge or experience gained by the other is small. It was therefore obvious to us that a deficit was present in the field of poly adenylic acid research: no source of knowledge concerning all facets of the biology and chemistry of poly(A) existed. Consequences of the lack of discussion between chemists and molecular biologists engaged in work involving poly(A) were manifest. Experimental results based on quantitation of poly(A) . polyuridylic acid (poly(U)] hybrids prepared under conditions wherein the triplex [poly(A)· 2 poly(U)] may exist is one example. Another is the attitude with which the structure of poly(A) is approached by most biologists. Few papers dealing with the molecular biology of poly(A) give consideration to the different structural forms that the polymer may assume. Despite overwhelming evidence from chemical and physical studies that this polymer is unique in many respects and that an alteration in experimental conditions may induce a radical change in polymeric structure, little consideration is given to this information. As a final example, the researchers attempting to define the nature of the poly(A)-binding proteins appear to be unaware of classes of enzymes that interact with poly(A) and are not cognizant of the consequences of the partially stacked structure of the polymer relevant to amino acid and protein binding. Thus, the knowledge regarding the many aspects of poly(A) chemistry and biochemistry are, in our opinion, in need of organization and presentation in one place. We feel that such an effort will be of importance to both the biochemist and the chemist, since no review of the chemical and physical properties of poly(A) has been published in more than a decade, and the last ten years have seen the most significant advances in knowledge concerning the structure ofpoly(A) and its interactions with cations, low-molecular-weight organic compounds, and macromolecules. Accordingly, we have divided our manuscript approximately in two; the first part deals with the biochemical and subcellular aspects of poly(A), and because of the biochemical importance ofthe structure of poly(A) , the second half concentrates on this topic-but includes, as well, sections on metals, complementary monomers, and polymers that interact with poly(A).

2.

2.1.

ISOLATION AND DETECTION OF POLY(A) Methodology

Poly(A) and poly(A)-containing RNA are most commonly isolated by some form of affinity chromatography. But before material containing

Polyadenylic Acid

3

poly(A) is separated from nucleic acids that lack this sequence, preparations are usually deproteinized either chemically or enzymatically. Of these two basic techniques, enzymic deproteinization by broad-spectrum proteases (Wiegers and Hilz, 1971, 1972; Faust, C. H., et al., 1973) has the advantage of being considerably milder than chemical treatment, but because of its impracticality on a large scale, has only limited use. Chemical deproteinization is the most general method employed and is accomplished usually with phenol either alone or in combination with such organic solvents as chloroform or isoamyl alcohol. The stability and efficiency of separation of poly(A) and poly(A)-containing RNA varies with the conditions under which phenol extraction is performed. Phase separation, for example, at room temperature between phenol and near-neutral-pH buffer containing sodium dodecyl sulfate (SDS) results in a considerable loss of poly(A) sequences when messenger RNA (mRNA) is extracted from polyribosomes (Perry et al., 1972b). Loss and cleavage of poly(A) sequences during phenol extraction is, apparently, a process that requires the participation of specific protein(s) present in the polyribosomes (Perry et al., 1972b). The loss can be avoided with techniques that evoke a more effective deproteinization of the poly(A)-protein complexes, such as extraction with chloroform-phenol (Perry et al., 1972b). When phenol is used in the presence of tris-HCl buffer (pH 7.6), the poly(A)-protein complexes are trapped in the nonaqueous phase. Recovery of the poly(A) sequences can be affected by reextraction of the nonaqueous phase with pH 9.0 tris-HCl buffer (Brawerman et al., 1972). For some purposes, a partial deproteinization may be adequate or even desirable. It has been shown, for example, that centrifugation of poly somes through cesium sulfate gradients, in the presence of dimethylsulfoxide (DMSO) (Greenberg, 1977), will dissociate them into ribosomal subunits and mRNP particles. Nearly all of the protein is removed from the ribosomal RNA (rRNA), and the resulting particles are separated according to buoyant density. This technique was used to study the association of specific proteins and mRNA (Greenberg, 1977). A number of procedures have been developed for the detection and isolation of poly(A) and poly(A)-containing RNA from eukaryotes. In general, depending on the principle used for isolation, they can be divided into two categories. The most widely used takes advantage of the hydrogen-binding properties of the adenylic acid residues of poly(A) to affect hybridization (via base-pairing) with such complementary homopolymers as poly(U) and polydeoxythymidylic acid [poly(dT)). These homopolymers can be immobilized on an insoluble matrix and are often available as commercial preparations for affinity chromatography. Poly(U), for example, has been coupled to Sepharose (Adesnik et al., 1972), to cellulose (Kates, 1970; Sheldon et al.. 1972), to glass-fiber filters (Sheldon et al ..

4

Timothy P. Karpetsky, Mark S. Boguski, and Carl C. Levy

1972), and to mica (Pulkrabek et ai., 1975). Poly(dT), on the other hand, has been linked to cellulose (Gilham, 1964; Edmonds and Caramela, 1969; Nakazato and Edmonds, 1972; Aviv and Leder, 1972). Some success has been attained with preparations of poly(U) that when hybridized directly with poly(A), enabled one group (Greenberg and Perry, 1972) to isolate the hybrid by hydroxylapatite chromatography. The second type of isolation procedure involves hydrophobic or basestacking interactions, or both, between poly(A) and a variety of substances, including unmodified cellulose* (Sullivan and Roberts, 1973; De Larco and Guroff, 1973; Kitos et ai., 1972, 1974; Kitos and Amos, 1973), nitrocellulose (Millipore) filters (Brawerman, 1976), benzolated cellulose (Roberts, 1974), methylated albumin kieselguhr (Miller, A. O. A., et ai., 1976), and polystyrene (Lim et ai., 1969; Lim and Canellakis, 1970). These techniques have the disadvantage of being specific, not for poly(A) but for polypurines in general (Kitos et ai., 1972, 1974) and, to a lesser extent, single-stranded DNA (Kitos et ai., 1972; Kitos and Amos, 1973). All the techniques referred to above differ in terms of their efficiency [lower size limit of poly(A) bound], capacity, recovery, degree of nonspecific binding, fractionation potential, and whether they are more suitable for analytical or preparative purposes. At least one comparative study has been done, and in this definitive work, it was determined that of the various materials for mRNA isolation examined, oligo(dT) cellulose was the best for general purposest (Mercer and Naora, 1975; see also Bantle et ai., 1976). To detect poly(A) sequences, and to estimate their length, one method that has been developed is that of annealing unlabeled poly(A)-containing material with radioactive poly(U) (Brawerman, 1976). There is danger in this method, however, since simply mixing equimolar quantities of the two homopolymers may, besides forming the complex poly(A) . poly(U), also result in the formation of considerable amounts of the triplex poly(A) . 2 poly(U). Estimates of the amount of poly(A) present in mixtures of these complexes can, of course, result in considerable error, so great care should be taken to minimize triplex formation. The use of a derivatized form of poly(U) that interacts with poly(A) to form the duplex exclusively would seem desirable (Zmudzka and Shugar, 1970). A technique has been developed for identifying and mapping poly(A) *Poly(A) does not interact with cellulose itself but with polyaromatic lignins, which exist as minor constituents in plant celluloses and persist as contaminants in cellulose preparations. tApparently, oligo(dT)-cellulose and poly(U)-Sepharose cannot efficiently bind large (on the order of 2 x 106 daltons) mRNA molecules (Deeley et aI., 1977; Gordon et al., 1977). This problem has been overcome by using 9 S poly(U) bound to Sephadex G-I0 as an affinity matrix.

Polyadenylic Acid

5

stretches in nucleic acids by electron microscopy (Bender and Davidson, 1976). In this method, short lengths of poly(dT) are polymerized onto nicked simian virus 40 (SV40) DNA and then hybridized with nucleic acids containing poly(A). Poly(A) stretches are marked because they are attached to easily recognized SV40 duplex circles. 2.2.

Determination of the Size of Poly(A) Segments

Before the size of polydispersity of poly(A) tracts can be measured, a population of mRN A molecules must be selected for study. Details of the techniques for separating poly(A)-containing mRNA from other types of RNA are discussed in Section 2.1. It is important, however, to recall that not all methods of mRNA-poly(A) purification yield identical products. Thus, it is possible to separate mRNA-poly(A) from total RNA by different isolation techniques and obtain samples of mRNA having distinct size distributions of 3' -terminal poly(A) segments. Millipore filters retain, for example, mRNA having large tracts (more than 70 nucleotides) ofpoly(A), whereas mRNAs having shorter segments are not retained. On the other hand, poly(A) tracts containing as few as 12 nucleotides form stable helices with, and bind to, poly(U) columns (Schumm and Webb, 1974; Eaton and Faulkner, 1972; Niyogi, 1969). Depending on the experimental conditions utilized, it is possible to select for poly(A) segments containing more than 20-30 adenylic acid residues by chromatography on oligo(dT)cellulose columns (Wong-Staal et at., 1975; Ohta et at., 1975). Finally, extraction of mRNA with phenol at neutral pH and in the absence of chloroform results in considerable loss ofthe poly(A)-containing RNA from the aqueous phase, and in cleavage of poly(A) from mRNA (Schumm et ai., 1973; Perry et ai., 1972b). These considerations should serve to emphasize the importance that must be attached to the mode of purification of poly(A)-containing mRNA, if this material is to be used to determine the size or polydispersity of poly(A) tracts. Lengths of3'-terminal poly(A) segments may be measured using either intact mRNA or mRNA that has been hydrolyzed to remove the nonpolyadenylated portion of the molecules. In this latter case, hydrolysis is achieved by treatment of the mRNA with bovine pancreatic RNase under conditions of high ionic strength. Although, in general, this treatment has been reported to result in hydrolysis of the nonpolyadenylated moiety of the mRN A with no concomitant degradation of the polypurine segment (Schwartz, H., and Darnell, 1976; Wong-Staal et aI., 1975; Hirsch and Penman, 1974), some care must be exercised in the exact choice ofhydrolytic conditions, since it is known that even at high ionic strength, a small amount of enzymic degradation of poly(A) may occur (Iqbal, 1975). Several

Timothy P. Karpetsky, Mark S. Boguski, and Carl C. Levy

6

investigators, in addition, supplement the degradative mixture with RNase Tl (Sakamoto et at., 1975; Hirsch and Penman, 1974), which hydrolyzes at guanylic acid residues. After hydrolysis, a distribution of the various-sized poly(A) segments may be brought about by such techniques as diethylaminoethyl (DEAE)-Sephadex chromatography or by polyacrylamide gel electrophoresis (PAGE). In either case, the size of a poly(A) segment is usually obtained by comparison of the mobility ofthe unknown sample with that of a standard of known molecular weight. The choice, therefore, of a proper standard is essential in obtaining accurate size information on the poly(A) segments under study. In this connection, it should be stressed that because of the physical properties of poly(A), this homopolymer behaves, in many respects, quite differently from any randomly selected heteropolyribonucleotide. The utilization, therefore, of transfer RNA (tRNA) or 5 S rRNA as molecular-weight markers might be expected to lead to erroneous values for the sizes of poly(A) segments (Pinder and Gratzer, 1974). That this is in fact the case was shown recently when size estimates obtained by comparison of electrophoretic mobilities of poly(A) fractions of known length (determined by intrinsic viscosity measurements) with those of 4 S and 5 S RNA overestimated the actual size of the poly(A) segments (Burness et at., 1975, 1977; Morrison et at., 1973). The use of denaturing concentrations of formaldehyde (2.2 M) did not alter the conclusion that 4 S and 5 S RNA were improper markers for the estimation of poly(A)-segment size (Figure 1) (Burness et at., 1975). One solution to the problem is the replacement of heteropolyribonucleotides with commercially available samples ofpoly(A) standards of known length. In other respects, especially in view of the linear relationship that exists between the mobility of poly(A) segments on 12% polyacrylamide gels (with or without formaldehyde) and

100 ｾ＠

J:

60

ｾ＠

t!)

zw

40

ｾ＠

z

« J: u

20 10

2

4

6

MOBILITY (em)

8

FIGURE 1. Relationship between logarithm of average nucleotide number and mobility of poly(A) on 12% polyacrylamide gels with (0) and without (e) formaldehyde (electrophoresed for 4 hr at 7 rnA/gel or for 2 hr at 4 mA/gel, respectively). The mobilities of 4 S and 5 S RNAs (Krebs ascites tumor cells) in the presence and absence of formaldehyde are also indicated. Reprinted with permission from Burness et al. (1975).

Polyadenylic Acid

7

the logarithm of average nucleotide number (Burness et ai., 1975; Morrison et al., 1973), PAGE appears as an acceptable technique for poly(A) size determination. Comparisons of poly(A) size obtained by different techniques have been made, and provide another example of the necessity for proper standards. Estimates of the weight average molecular weight of mouse myeloma cell cytoplasmic poly(A)-containing RNA, for example, obtained by PAGE or by sucrose-gradient sedimentation, but using rRNA and tRNA as standards, differ by a factor of 2 (Macleod, 1975a). The differences in weight did not stem from either RNA degradation or acid poly(A) doublehelix formation during electrophoresis. If the average poly(A) sequence length is shortened, however, the discrepancy between the two methods decreases. Furthermore, synthetic homopolymeric poly(A) behaved in a manner qualitatively similar to the behavior of poly(A)-containing RNA. Under the conditions of the experiments, poly(A) exists as a stacked singlestranded helix and would be expected to have a larger radius of gyration than rRN A or tRN A of equivalent molecular weight. Comparison of mobilities of poly(A)-containing RNA with RNA markers would therefore result in an underestimation of the sedimentation coefficient and, because of a more effective exclusion from the gel pores, an overestimation of Svedberg values based on apparent electrophoretic mobilities (Macleod, 1975a). In this connection, it should be mentioned that the molecular weight of mouse globin mRNA determined by analytical ultracentrifugation under denaturing conditions (where most secondary structure is lost) is lower than the values determined by sucrose-gradient sedimentation or by analytical gel electrophoresis (Williamson et al., 1971). Although gel electrophoresis has superior resolving power for polynucleotides, DEAE-Sephadex chromatography was found to separate small oligonucleotides (1-6 nucleotides in length) (Burness et al., 1975). This latter technique should, however, be used with extreme caution for estimating poly(A) segment length, since polynucleotides or oligonucleotides eluted from DEAE-Sephadex by the same concentrations of NaCI (even in the presence of 7 M urea) may not be of the same size. Additionally, molecules eluted by different NaCI concentrations are not necessarily of different chain length (Burness et aI., 1975, 1977). Thus, it appears that poly(A) segments are not eluted in strict order of increasing size as the ionic strength is raised, perhaps because of the complex nature of the interaction between poly(A) and the DEAE-Sephadex gel. Relationships between the polarographic behavior of poly(A) and molecular weight have been derived recently (Janik and Sommer, 1972; Brabec and Palecek, 1973). The correlation between the polarographic

8

Timothy P. Karpetsky, Mark S. Boguski, and Carl C. Levy

currents and size of poly(A) was found to be valid for a broad range of poly(A) lengths (25-9600 monomeric units) (Janik and Sommer, 1972). That this technique has not found widespread use probably results from the simplicity and universality of other methods in current use, such as gel electrophoresis. Additionally, although the accuracy and rapidity of directcurrent polarography or pulse polarography are comparable to those of other techniques, these methods (Janik and Sommer, 1972) seem to offer no special advantage. Techniques have been developed for the estimation of 3'-terminal poly(A) segment size that do not require the prior removal of the nonpolyadenylated portion of mRNA. One such method is based on the processive phosphorolysis of mRNA using an excess of E. coli polynucleotide phosphorylase at O°C in the presence of 1 M sodium chloride (Soreq et ai., 1974). Under these conditions, polynucleotide phosphorylase is a specific 3'-exonuclease and will remove the 3'-terminal poly(A) segment without any degradation of the rest of the mRNA molecule. The length of the poly(A) segment may then be determined from the quantity of ADP released as a product of phosphorolysis or by comparison of sizes of the starting polynucleotide and the mRNA stripped of its 3'-terminal poly(A) tract. This method is particularly useful in the determination of sizes of poly(A) segments that contain a small percentage of nucleotides other than adenylic acid (Lingrel et al., 1973; Haff and Keller, 1973). Treatment ofthese types of mRNAs with endonuc1eases (either RNase A or T 1) will cause chain cleavage with a resultant shortening of the length of the poly(A) moiety. Use of polynucleotide phosphorylase circumvents this difficulty and should enable detection and identification of low quantities of heterogeneous nucleotides present in the poly(A) segment. It is interesting to note that phosphorolysis ceases if the tract of poly(A) is protected as part of a ribonucleoprotein complex (Soreq et al., 1974). Similar results would be expected if the poly(A) portion is strongly complexed to another region of the mRNA molecule. Provided the average length of the mRNA is known (Morrison and Lingrel, 1976), the hybridization of [3H]poly(U) with mRNA forms the basis of another technique by which poly(A) segment size may be determined. After completion of binding, the complex is hydrolyzed with pancreatic RNase, and the resulting hybrid products are precipitated with 10% trichloroacetic acid and collected on glass filters for radioactivity measurements. A control reaction of [3H]poly(U) with a known quantity of poly(A) is necessary to determine whether the complex obtained under the particular conditions of ionic strength utilized is the duplex, poly(A) . poly(U), or the triplex, poly(A) . 2 poly(U). The reaction was found to be specific for

Poly adenylic Acid

9

poly(A)-containing RNAs, and no reaction was seen with rRNAs (Morrison and Lingrel, 1976). Although at this time, then, a number of methods exist for the determination of poly(A) segment size or polydispersity, care must be exercised both in the preparation of the mRNA sample for analysis to assure that large portions of poly(A)-containing mRNA are not excluded and in the choice of suitable molecular-weight standards to ensure accurate size information for poly(A) molecules.

3.

3.1.

MESSENGER RNA AND THE 3'-TERMINAL POLY(A) SEQUENCE

Occurrence of Poly(A) in Living Organisms

Although poly(A) as a component of mRNA was first discovered in mammalian cells (for reviews, see Brawerman, 1974, 1976; Greenberg, 1975), it has since proved to be a ubiquitous constituent of other eukaryotic cells as well. Poly(A) sequences are also of widespread occurrence in viruses and in some prokaryotic organisms (Table I). Throughout this review, poly(A) is considered only as a noncoding sequence, i.e., as a polynucleotide accessory to the genetic message with which it is associated. There exists no evidence that the poly(A) sequence of mRNA is translated into a polypeptide product of any kind. A number of studies employing poly(A) as a synthetic message have been carried out (Debov et ai., 1964; Smith, M. A., et ai., 1966; Malpoix, 1967; Tanaka, K., and Teraoka, 1968; Hfulninen and Alanen-Irjala, 1968; Ikemura and Fukutome, 1969; Fabry and Rychlik, 1974), but most ofthese studies were done before the realization that poly(A) exists in the cell as an adjunct to the coding portion of mRNA. These studies will not be discussed further.

3.2.

Poly(A) Sequences in Prokaryotes

Following the discovery ofpoly(A) in mammalian and other eukaryotic cells, it was natural to search for this homopolymeric sequence in prokaryotes. Early studies seemed to indicate that poly(A) was absent from E. coli (Terzi et ai., 1970; Perry et ai., 1972a), and these findings led to speculations ranging from defining a role for poly(A) in terms of a function for eukaryotes not shared by prokaryotes to the evolutionary origin of mitochondria (Perlman et ai., 1973) and chloroplasts (Wheeler and Hartley, 1975). For a long time thereafter, poly(A) was considered to be a distinguishing characteristic of the eukaryotic cell. However, poly(A) sequences

Timothy P. Karpetsky, Mark S. Boguski, and Carl C. Levy

10

Table I Occurrence of Poly(A) in Living Organisms a Organism VIRUSES DNA Viruses Vaccinia virus Herpes simplex virus Adenovirus Simian virus 40 Polyoma virus RNA Tumor Viruses Rous sarcoma virus Avian myeloblastosis virus Feline leukemia virus (Richard) Feline sarcoma virus (Gardner) Rauscher leukemia virus Murine mammary tumor virus (C 3H) Murine sarcoma virus Murine leukemia-sarcoma virus Visna virus Mason-Pfizer agent Baboon virus Woolly monkey sarcoma virus Other RNA Viruses Rhinovirus Cow pea mosaic virus Columbia SK virus Polio virus Vesicular stomatitis virus Sindbis virus Newcastle disease virus Equine encephalitis virus Fowl plague virus Semliki forest virus Foot-and-mouth disease virus Encephalomyocarditis virus Influenza virus Mengo virus Sendai virus Bean pod mottle virus Bovine enterovirus PROKARYOTES Escherichia coli Caulabacter crescentus PLANTS Green Algae Chiarella fusca

References

Kates (1970) Bachenheimer and Roizman (1972) Philipson et al. (1971) Weinberg et al. (1972) Rosenthal (1976) Lai and Duesberg (1972) Green and Cartas (1972), Gillespie et al. (1972) Gillespie et al. (1973) Gillespie et al. (1973) Lai and Duesberg (1972) Gillespie et al. (1973) Green and Cartas (1972) Ross et al. (1972) Gillespie et al. (1973) Gillespie et al. (1972) Bender and Davidson (1976) Bender and Davidson (1976) Nair and Owens (1974) El Manna and Bruening (1973) Johnston and Bose (1972) Armstrong et al. (1972), Yogo and Wimmer (1972) Ehrenfeld and Summers (1972), Soria and Huang (1973) Johnston and Bose (1972) Weiss and Bratt (1974) Armstrong et al. (1972) Ghendon and Blagoveshienskaya (1975) Clegg and Kennedy (1974) ChatteIjee et al. (1976) N. O. Goldstein et al. (1976), Hruby and Roberts (1976) Macnaughton et al. (1975), Glass et al. (1975) R. L. Miller and Plagemann (1972), Marshall and Arlinghaus (1976) Pridgen and Kingsbury (1972) Semancik (1974) Newman and Brown (1976) Nakazato et al. (1975), Srinivasan et al. (1975) Ohta et al. (1975)

Scragg and Thurston (1975)

11

Polyadenylic Acid Table I (Continued) Occurrence of Poly(A) in Living Organisms" References

Organism

Fungi Blastocladia ramosa zoospores Trichoderma viride mitochondria Saccharomyces cerevisiae mitochondria Blastocladiella emerson;; zoospores Dictyostelium discoideum Mosses Funaria hygrometrica Polytrichum commune Seed Plants Germinating cotton seeds Vicia faba meristematic root cells Cultured parsley Cultured sycamore Etiolated Phaseolus vulgaris leaves Phaseolus aureus Cultured rice root Matthiola incana Carrot Soybean Barley aleurone Zea mays seedlings Zea mays chloroplasts Pea seedlings Glycine max

Jaworski and Torzilli (1975) D. Rosen and Edelman (1976) McLaughlin et al. (1973), Hendler et al. (1975) Schimmelpfeng et al. (1976) Firtel et al. (1972) Stegmann and Hahn (1974) Seibert et al. (1976) Hammett and Katterman (1975) Esnault et al. (1975) Ragg et al. (1975) Covey and Grierson (1976) Smith (1976) Higgins et al. (1973) Manahan et al. (1973) Grierson and Hemleben (1977) Key et al. (1972) Key et al. (1972) Ho and Varner (1974) Van de Walle (1973) Haff and Bogorad (1976) Gray and Cashmore (1976) Verma et al. (1974)

ANIMALS Protozoa

Tetrahymena pyriformis

Ron et al. (1976), Rodriguez-Pousada and Hayes (1976)

Arthropoda

Artemia salina (brine shrimp) embryos Pieris brassicae (lepidopteran) imaginal discs Apis mellifera (queen bee) venom gland Rhynchosciara americana salivary glands Chironomas tentans salivary glands Drosophila melanogaster salivary glands Aedes albopictus Bombyx mori (larvae) Anthcraca polyphemus .1.ntheraca pernyi chorion

Susheela and Jayarman (1976) Tarroux (1975) Kindas-Mugge et al. (1976) Lara and Okretic (1975) Edstrom and Tanguay (1974) Hirsch et al. (1974) Hirsch et al. (1974) Lizardi et al. (1975) Voumakis et al. (1975) Voumakis et al. (1975) (Continued)

12

Timothy P. Karpetsky, Mark S. Boguski, and Carl C. Levy

Table I (Continued) Occurrence of Poly(A) in Living Organisms a Organism Echinodermata Tipneustes gratilla Colobocentrotus astratus Strongylocentrotus purpuratus Lytechinus pictus Chordata Osteichthyes Trout testis Amphibia Xenopus laevis Oocytes Liver Embryos Aves Chicken Embryo Oviduct Liver Duck Erythroblasts . Erthrocytes Mammalia Human Liver Spleen Reticulocytes Lymphocytes Leukocytes HeLa cells

HeLa cell mitochondria Cultured fibroblasts Rabbit Brain Uterus Spleen Liver Mammary gland Eggs Retic ulocyte s Mouse Liver

References

Dolecki et al. (1977) Dolecki et al. (1977) Nemer et al. (1975), Wilt (1973) Nemer et at. (1975), Wilt (1973), Slater et al. (1973)

1atrou and Dixon (1977), Gedamu and Dixon (1976)

Rosbash and Ford (1974) RyfIel (1976) Sagata et al. (1976)

Salles et al. (1976), Mondall et al. (1974), Hemminki (1974, 1976) Rhoads (1975), Palacios et al. (1973) Jost and Pehling (1976), Geert et al. (1976), Wetekam et al. (1975) Morel et at. (1973) Pemburton and Baglioni (1973)

Frank and Levy (1976) Hieter et al. (1976) Cann et al. (1974), Maniatis et al. (1976) Berger and Cooper (1975) Kuo et al. (1976) Hirsch and Penman (1973), Molloy and Darnell (1973), Kates (1970), Darnell et al. (1971b), Edmonds et al. (1971) Ojala and Attardi (1974a,b), Perlman et al. (1973) Schneider and Shorr (1975) Mahony et al. (1976) Levey and Daniel (1976), Bullock et al. (1976) Nokin et al. (1976) Avadhani et al. (1975) Houdebine (1976) Schultz (1975) Lim and Canellakis (1970) Lim et al. (1970a,b), Avadhani et al. (1975)

13

PoJyadenyJic Acid

Table I (Continued) Occurrence of Poly(A) in Living Organisms a Organism Mouse (continued) Spleen Brain Kidney Plasmacytoma MP1-11 Plasmacytoma MOPC-104E Reticulocytes Myeloma Fibroblasts Sarcoma SI80 Ehrlich ascites cells Ehrlich ascites cell mitochondria L cells Cultured embryo Cultured neuroblastoma Rat Brain Brain cell mitochondria Liver Novikoff hepatoma Morris hepatoma 7800 Hamster Cultured BHK-21 cells Cultured cells Cultured BHK-21 cell mitochondria Chinese Hamster cultured cells (ovary) Guinea pig mammary gland Dog pancreas Porcine cultured thyroid gland Calf Lens Cultured fetal myoblasts Ewe mammary gland

References Cheng and Kazazian (1976) Bantle and Hahn (1976) Ouellette et al. (1975, 1976) Abraham and Eikhom (1975) Wong-Staal et al. (1975) Morrison et al. (1973), Williamson et al. (1974), Mansbridge et al. (1974) Baglioni et al. (1972), C. H. Faust et al. (1973), MacLeod (l975b) Williams and Penman (1975), Johnson et al. (1975) S. Y. Lee et al. (1971), Mendecki et al. (1972) Comudella et al. (1973) Avadhani et al. (1973) Greenberg and Perry (1972) Getz et al. (1976) Morrison et al. (1977), Prasad et al. (1975) Berthold and Lim (1976a,b), De Larco et al. (1975), Lim et al. (1974) Cupello and Rosadini (1976) Tweedie and Pitot (1974), Hadjivassiliou and Brawerman (1966) Desrosiers et al. (1975) Tweedie and Pitot (1974) Dubin and Taylor (1975) Hirsch et al. (1974) Cleaves et al. (1976) Diez and Brawerman (1974) R. K. Craig et al. (1976) Lomedico and Saunders (1976) Becarevic et al. (1973), Poiree et al. (1973) Lavers et al. (1974), Favre et al. (1974) Buckingham et al. (1976) Houdebine and Gaye (1976)

apoly(A) is considered only as a component of mRNA or "messengerlike" RNA and also, in the case of RNA viruses, as a component of the virion. Heterogeneous nuclear RNA containing poly(A) has not been included. An attempt has been made to be comprehensive, but some sources may have been inadvertently overlooked. The references appearing after each entry do not necessarily represent the first or only workers to have detected poly(A) in these organisms. Poly(A) polymerase has been detected in many organisms in which poly(A) sequences have not necessarily been sought. These organisms have not been included in this list.

14

Timothy P. Karpetsky, Mark S. Boguski, and Carl C. Levy

were found to exist in E. coli (Edmonds and Kopp, 1970; Nakazato et ai., 1975; Srinivasan et ai., 1975) and Cauiobacter crescentus (Ohta et ai., 1975, 1978). These findings were not completely unexpected for it had long been known that a poly(A) polymerase exists in E. coli (see Section 4.1).* The poly(A) sequences in these bacteria are covalently attached at or near the 3' termini of large (4-20 S) RNA molecules. The polydispersity and location in the unstable (pulse-labeled) fraction of RNA suggested that these molecules were mRNA, although evidence for their translation was not sought (Nakazato et al., 1975). Prokaryotic poly(A) sequences differ markedly from those of eukaryotes in both average size and steady-state levels. Whereas eukaryotic poly(A) is well known to consist of approximately 50-200 adenylic acid residues, the poly(A) sequences found in bacteria are only about 15-50 nucleotides in length (Nakazato et aI., 1975; Ohta et al., 1975, 1978). In eukaryotic cells, poly(A) sequences are found in the majority of pulselabeled RNA species. In contrast, poly(A) in bacteria is associated with only 1-15% of the total pulse-labeled RNA, depending on the growth medium used (Srinivasan et aI., 1975). As Nakazato et al. (1975) pointed out, these low levels could account for the failure of Perry et ai. (l972a) to detect poly(A) tracts in E. coli, since these sequences are close to the "noise" level reported for the experiments of Perry and co-workers. Also, the Millipore filter technique the latter authors employed is unable to detect short poly(A) sequences (less than 50 residues). Any view of the biological function(s) of poly(A) would have to account for the considerable differences in size and steady-state levels between the poly(A) of prokaryotes and that of eukaryotes. If poly(A) has something to do with the stability and, hence, longevity of mRNA, as some have suggested, the short sequences present in bacterial messages may account for the very short half-lives (minutes) of these molecules in comparison with the much longer half-lives (hours to days) of eukaryotic mRNA. With respect to the low concentration ofpoly(A) in prokaryotes as compared with eukaryotes, Nakazato et ai. (1975) observed that this is perhaps not surprising, since the coupling of translation with transcription in prokaryotes may not allow the accumulation of many mRN As with completed 3' termini. Although poly(A) exists at the 3' terminus of only a low percentage of bacterial mRNAs, many of these molecules may have 3'-terminal oligo(A) tracts that are too short to form stable complexes with oligo(dT)-cellulose *Recently, Cheung and Newton (1978) purified from C. crescentus a polymerase capable of catalyzing poly(A) synthesis in the absence of template.

PolyadenyJic Acid

15

or other poly(A) affinity media. Such oligo(A) sequences have been detected in E. coli mRNAs synthesized after infection with T7 bacteriophage (Kramer et at., 1974) and bacteriophage

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FIGURE 12. CsCI step gradient in B-XV rotor with and without sample zone of bovine serum albumin (BSA). CsCI density gradient simulated ( - - ) , recovered from rotor without (0 0 0 0) and with (x x x x ) BSA. BSA sample (including dimer) zone profile simulated ( - - ) and experimental ( • • • •). Initial gradient: With BSA, 1344 ml [0.01 M tris-HCI (pH 7.5)J; 33ml [sample containing 44 mg BSNml, 0.01 M tris-HCI (pH 7.5), and 3 ml 0.01 M trisHCI (pH 7.5), CsCI to a density of 1.497 glcm 3 J; 290 ml [0.01 M tris-HCI (pH 7.5), CsCI to a density of 1.497 glcm"J. Without BSA, 1373 ml [0.01 M tris-HCl (pH 7.5)J; 293 ml [0.01 M trisHCI (pH 7.5), CsCI to a density of 1.497 glcm"J. Simulated as: 2.4- to 7.97-cm rotor radius, 0.997 glcm 3 gradient density; 7.97- to 8.01-cm, linear interpolation 0.997-1.043 glcm:l; 8.01- to 8.06-cm, 1.043 glcm"; 8.06- to 8.10-cm, linear interpolation 1.043-1.497 glcm 3 ; 8.10- to 8.89-cm, 1.497 glcm" (sample zone 7.99-8.08 cm) . Rotor velocity: acceleration, 0.32 hr, 1500-25,319 rpm; 9.11 hr at 25,319 rpm; deceleration, 0.34 hr, 25,319-1500 rpm. Reproduced from Sartory et al. (1976).

138

6.

Jens Steensgaard and Niels Peter Hundahl Meller

GENERAL DISCUSSION

The compartmental approach and the analytical approach to numerical simulation of gradient centrifugation as described in Sections 4 and 5 have both reached the level at which they can be used directly for solving problems in connection with separative and analytical procedures. The two approaches are not directly comparable, since the aim in the development of the compartmental approach was to create a simple yet useful simulation model, whereas the analytical approach model was developed as a very versatile model with a very high numerical accuracy. Simulation models for technical procedures have many potential uses. One, namely, optimization of procedures has been mentioned previously. Another is for further studies on the behavior of particles during centrifugation. So far, the models described have been developed under the assumptions that the propagation of particles is not influenced by the particle concentration in the sample zone, that the particles do not interact either mutually or with the gradient material, that the pressure does not affect the particle movements, and that the sample zone is not overloaded. Both of the models described have the potential to be extended to include tests for overloading either following the concepts of density inversion (Svensson et al., 1957; Berman, 1966) or the concepts of hydrodynamic instability of Meuwissen (1973). Theoretical testing for possible overloading may tum out to require use of a simulation model. Finally, the use of these simulation models may be advantageous when the design of new rotor types and centrifuges is being planned. The technique of computer simulation is gradually being used more and more in the broad field of subcellular biochemistry. To illustrate the diversity of problems that currently are attacked by use of computer simulation, a few examples are given below. Starting with the smallest particles, protein folding, unfolding, and fluctuations were studied by Taketomi et al. (1975) using a lattice model. Complex formation in immunological systems was described by Steensgaard et al. (1975, 1977). Computer simulation was used to optimize enzymatic assays (London et al., 1974), and to study factors that cause sigmoidal substrate saturation curves in enzymatic systems that involve enzyme inactivation (Fisher and Keleti, 1975). Protein interactions in the analytical ultracentrifuge were investigated by combining a program for interactions with a program for simulation of analytical ultracentrifugation (Gilbert and Gilbert, 1973). Comparison of the metabolism in brain slices with the metabolism in the intact brain of guinea pig led to a program for simulation of the metabolism of glutamate, glutamine, y-aminobutyrate, and the Krebs cycle (Garfinkel et al.,

Computer Simulation of Density-Gradient Centrifugation

139

1975). An analysis of firefly communication was used to develop a computer model of firefly flash sequences (Carlson and Soucek, 1975). Functional aspects such as the renal handling of urea were investigated with the aid of a computer model of a countercurrent system (Stewart, 1975), and cell-growth kinetics of tumor cells inspired computer modeling (Kim and Woo, 1975). In conclusion, we believe that computer simulation will be used increasingly in the future. Computer simulation is especially likely to find use as an optimizing tool for experimental procedures and as a means of evaluating the consequences of new theories. It would be very helpful if scientists planning new programs could agree on writing programs in simple languages that are easily adaptable to computers other than their own, thus facilitating mutual exchange of programs.

7. REFERENCES Anderson, N. G. (ed.), 1966, The development of zonal centrifuges and ancillary systems for tissue fractionation and analysis, Nat!. Cancer [nst. Monogr. 21. Barber, E. J., 1966, Calculation of density and viscosity of sucrose solutions as a function of concentration and temperature, Nat!. Cancer [nst. Monogr. 21:219-239. Berman, A. S., 1966, Theory of centrifugation: Miscellaneous studies, Natl. Cancer [nst. Monogr. 21:41-76. Bishop, B. S., 1966, Digital computation of sedimentation coefficients in zonal centrifuges, Nat!. Cancer [nst. Monogr. 21:175-188. Carlson, A. D., and Soucek, B., 1975, Computer simulation of firefly flash sequences, J. Theor. Bioi. 55:353-370. Cox, D. J., 1965a, Computer simulation of sedimentation in the ultracentrifuge. I. Diffusion, Arch. Biochem. Biophys. 112:249-258. Cox, D. J., 1965b, Computer simulation of sedimentation in the ultracentrifuge. ll. Concentration-independent sedimentation, Arch. Biochem. Biophys. 112:259-266. Cox, D. J., 1967, Computer simulation of sedimentation in the ultracentrifuge. ill. Concentration-dependent sedimentation, Arch. Biochem. Biophys. 119:230-239. Cox, D. J., 1969, Computer simulation of sedimentation in the ultracentrifuge. IV. Velocity sedimentation of self-associating solutes, Arch. Biochem. Biophys. 129:106-123. Eikenberry, E. F., Bickle, T. A., Traut, R. R., and Price, C. A., 1970, Separation of large quantities of ribosomal subunits by zonal centrifugation, Eur. J. Biochem. 12:113-116. Fisher, E., and Keleti, T., 1975, Sigmoidal substrate saturation curves in Michaelis-Menten mechanism as an artefact, Acta Biochim. Biophys. Acad. Sci. Hung. 10(3):221-227. Funding, L., 1973, Estimation of equivalent sedimentation coefficients with zonal rotors, in: European Symposium of Zonal Centrifugation in Density Gradient, Spectra 2000 (I.-C. Chermann, ed.), Vol. 4, pp. 45-49, Editions Cite Nouvelle, Paris. Garfinkel, D., London, J. W., Dzubow, L., and Nicklas, W. J., 1975, Computer simulation of the metabolism of guinea pig brain slices, and how they differ from the intact brain, Brain Res. 92:207-218.

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Gilbert, L. M., and Gilbert, G. A., 1973, Sedimentation velocity measurement of protein association, in: Methods in Enzymology (S. P. Colowick and N. O. Kaplan, eds.), Vol. XXVII, pp. 273-2%, Academic Press, New York. Hinton, R. H., 1971, Computational approaches in the processing of zonal results, in: Separations with Zonal Rotors (E. Reid, ed.), pp. Z-5.1-Z-5.1O, Wolfson Bioanalytical Centre, University of Surrey, Guildford, England. Jfft, J. B., Voet, D. H., and Vinograd, J., 1961, The determination of density distributions and density gradients in binary solutions at equilibrium in the ultracentrifuge, 1. Phys. Chem. 65:1138-1145. Johns, P., and Stanworth, D. R., 1976, A simple numerical method for the construction of isokinetic sucrose density gradients, and their application to the characterization of immunoglobulin complexes, 1. Immunol. Methods 10:231-252. Kim, M., and Woo, K. B., 1975, Kinetic analysis of cell size and DNA content distributions during tumor cell proliferation: Erlich ascites tumor study, Cell Tissue Kinet. 8:197-218. Leach, J. M., 1971, Data processing of zonal centrifuge experiments, in: Separations with Zonal Rotors (E. Reid, ed.), pp. Z-4.1-Z-4.16, Wolfson Bioanalytical Centre, University of Surrey, Guildford, England. London, J. W., Yarrish, R., Dzubow, L. D., and Garfinkel, D., 1974, Computer simulation and optimization, as exemplified by the enzyme-coupled aminotransferase (transaminase) assays, Clin. Chem. 20(11): 1403-1407. Ludlum, D. B., and Warner, R. C., 1965, Equilibrium centrifugation in cesium sulfate solutions, 1. Bioi. Chem. 240:2961-2965. Martin, R. G., and Ames, B. N., 1%1, A method for determining the sedimentation behaviour of enzymes: Application to protein mixtures, 1. BioI. Chem. 236:1372-1379. Meuwissen, J. A. T. P., 1973, Hydrodynamic instability: An explanation of anomalous zone spreading in density gradient methodology, in: European Symposium of Zonal Centrifugation in Density Gradient, Spectra 2000 (J.-C. Chermann, ed.), Vol. 4, pp. 21-31, Editions Cite Nouvelle, Paris. Noll, H., 1967, Characterization of macromolecules by constant velocity sedimentation, Nature (London) 215:360-363. Norman, M. R., 1971, Simple equations for relating volume to radius in "Boo type zonal rotors, in: Separations with Zonal Rotors (E. Reid, ed.), pp. Z-3.1-Z-3.4, Wolfson Bioanalytical Centre, University of Surrey, Guildford, England. Pollack, M. S., and Price, C. A., 1971, Equivolumetric gradients for zonal rotors: Separation of ribosomes, Anal. Biochem. 42:38-47. Pretlow, T. G., 1971, Estimation of experimental conditions that permit cell separations by velocity sedimentation on isokinetic gradients of Ficoll in tissue culture medium, Anal. Biochem. 41:248-255. Pretlow, T. G., Boone, C. W., Shrager, R. I., and Weiss, G. H., 1969, Rate zonal centrifugation in a Ficon gradient, Anal. Biochem. 29:230-237. Price, C. A., 1973, Equivolumetric gradients: Apparent limits on resolution and capacity imposed by gradient-induced zone narrowing, in: European Symposium of Zonal Centrifugation in Density Gradient, Spectra 2000 (J.-C. Chermann, ed.),Vol. 4, pp. 71-81, Editions Cite Nouvelle, Paris. Rickwood, D., 1976, Metrizamide-A gradient medium for centrifugation studies, Nyegaard & Co., Oslo, Norway. Sartory, W. K., Halsall, H. B., and Breillat, J. P., 1976, Simulation of gradient and band propagation in the centrifuge, Biophys. Chem. 5:107-135. Schumaker, V. N., 1967, Zone centrifugation, in: Advances in Biological and Medical Physics (c. A. Tobias and J. H. Lawrence, eds.), pp. 245-339, Academic Press, New York.

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Spragg, S. P., Morrod, R. S., and Rankin, C. T., Jr., 1969, The optimization of density gradients for zonal centrifugation, Sep. Sci. 4:467-479. Steensgaard, J., 1970, Construction of isokinetic sucrose gradients for rate-zonal centrifugation, Eur. J. Biochem. 16:66-70. Steensgaard, J., and Funding, L., 1974, Computer simulation of rate-zonal centrifugation, in: Methodological Developments in Biochemistry (E. Reid, ed.), Vol. 4, pp. 55-65, Longman, London. Steensgaard, J., and Hill, R., 1970, Separation and analysis of soluble immune complexes by rate-zonal ultracentrifugation, Anal. Biochem. 34:485-493. Steensgaard, J., Funding, L., and Meuwissen, J. A. T. P., 1973, Simulation of rate-zonal centrifugation on a digital computer, Eur. J. Biochem. 39:481-491. Steensgaard, J., Funding, L., and Meuwissen, J. A. T. P., 1974, A FORTRAN program for simulation of zonal centrifugation, in: Methodological Developments in Biochemistry (E. Reid, ed.), Vol. 4, pp. 67-80, Longman, London. N. P. R., 1975, Computer simulation of Steensgaard, J., Johansen, R. K. W., and ｍｾｬ･ｲＬ＠ immunochemical interactions, Immunology 29:571-579. Steensgaard, J., Maw Liu, B., Cline, G. B., and Moller, N. P. R., 1977. The properties of immune complex-forming systems-a new theoretical approach, Immunology 32:445456. Steensgaard, J., Moller, N. P. R., and Funding, L., 1978, Rate zonal centrifugation: Quantitative aspects, in: Centrifugal Separations in Molecular and Cell Biology (G. B. Birnie and D. Rickwood, eds.), pp. 115-168, Butterworths, London. Stewart, J., 1975, Urea handling by the renal countercurrent system: Insights from computer simulation, Pfluegers Arch. 356:133-151. Svedberg, T., and Pedersen, K. 0., 1940, The ultracentrifuge, Oxford University Press, Oxford. Svensson, R., Ragdahl, L., and Lerner, K.-D., 1957, Zone electrophoresis in a density gradient: Stability conditions and separation of serum proteins, Sci. Tools 4:1-10. Taketomi, R., Ueda, Y., and Go, N., 1975, Studies on protein folding, unfolding and fluctuations by computer simulation. 1. The effect of specific amino acid sequence represented by specific inter-unit interactions, Int. J. Peptide Protein Res. 7:445-459. Vinograd, J., and Hearst, J. E., 1962, Equilibrium sedimentation of macromolecules and viruses in a density gradient, Fortschr. Chem. Org. Naturst. 20:372-379. Walsh, G. R., 1975, Methods of Optimization, J. Wiley & Sons, London. Wright, R. R., Pappas, W. S., Carter, J. A., and Weber, C. W., 1966, Preparation and recovery of cesium compounds for density gradient solutions, Natl. Cancer Inst. Monogr. 21:241249.

Chapter 3

Crown-Gall and Agrobacterium tumefaciens: Survey of a Plant-CellTransformation System of Interest to Medicine and Agriculture U. C. Knopf IPRIP University of Neuch:1tel 2001 Neuch:1tel, Switzerland

1.

INTRODUCTION

Crown-gall tumors (Figure 1) on various plants were described in Europe by several naturalists as early as the last century, and were generally ascribed to the action of insects or mechanical injury (Smith et al., 1911). According to Smith et al. (1911), Cavara (1897) in Italy was the first to demonstrate the bacterial nature of the disease around 1897 by means of inoculations from pure cultures. However, his studies, as well as those of other writers of southern Europe on this subject, were generally overlooked. In 1907, Smith and Townsend (1907) submitted a paper to Science in which they reported their findings on the causal agent of crown-gall tumors. Their results also showed that a bacterium was the etiological agent of the neoplasm, and they called it Bacterium tumefaciens. Their finding attracted immediate interest, especially from animal pathologists (Jensen, 1910, Levin, J., and Levine, 1918), since in their eyes it was the first instance in which a neoplasm could be associated with an infectious agent and therefore induced under defined experimental conditions. While some animal pathologists started to search for bacteria in animal neoplasms, it was shown that the causal agents of some animal tumors were filtrable ("viral") and thus not of bacterial origin (Rous, 1911; Rous and Murphy, 143

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1914). As a result, crown-gall disease and its infectious agent, mote recently called Agrobacterium tumefaciens (Conn, 1942), became more and more the subject of studies of plant pathologists. However, with the years, fundamental similarities in the cellular processes of different cells were recognized. Much fundamental biological and especially genetic knowledge accumulated from the study of bacteria and their viruses. Cancer researchers made the finding, among other findings, that different agents such as viruses, chemicals, and irradiation could induce neoplasms, and some started to recognize that the question whether there is a common subcellular and molecular basis of neoplasms might be the fundamental problem to be answered. On the other hand, agricultural scientists, in their search for creating new genetic variability among plants, became interested in the possibility of modifying plant cells at the molecular level. With these prospects, the crown-gall disease, and its infectious agent, Agrobacterium tumefaciens, attracted more and more interest among biologists from different fields in succeeding years.

FIGURE 1. Naturally occurring crown-gall tumor on an oak tree.

145

Crown-GaD and Agrobacterium tumefaciens

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2.

OVERVIEW OF THE PROCESS OF PLANT· CELL TRANSFORMATION BY AGROBACTERIUM TUMEFACIENS

The process of cell transformation by A. tumefaciens can be subdivided into three phases (Figure 2): Conditioning. During this phase, a normal plant cell becomes competent for transformation by the bacterium, or in other words, it becomes a potential tumor cell. Conditioning of the plant cells is achieved without the presence of the bacteria by wounding the plant. Induction. During this phase, potential tumor cells are being transformed by the bacteria into tumor cells. According to a hypothesis of Braun (1947a), the bacteria elaborate a "tumor-inducing principle" (commonly abbreviated TIP) that is responsible for the transformation of conditioned plant cells. Proliferation. Once cellular transformation has been accomplished, the cells continue to grow abnormally and autonomously. The presence of the bacteria in this phase is no longer necessary. In the subsequent presentation, discoveries about and problems of the crown-gall system and Agrobacterium will be described following more or less the scheme outlined above.

3.

CONDITIONS FOR PLANT· CELL TRANSFORMATION BY AGROBACTERIUM TUMEFACIENS

It is quite simple to induce experimental tumors with A. tumefaciens. A few conditions must be observed before tumorigenic bacteria can interact with the plant cells.

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3.1. Dicotyledonous Host Plants or Gymnosperms

Agrobacterium tumefaciens has a wide host range (Tamm, 1954), which, however, seems to be limited to dicotyledons and gymnosperms, since it has not been possible thus far to produce tumors on monocotyledonous plants. A number of systems have been developed to produce tumors experimentally. R. M. Klein (1955) described a method with carrot disks (Figure 3). J. A. Lippincott and Heberlein (1965) showed that crowngall tumors may be initiated on primary leaves of pinto beans and that a general relationship exists between the number of tumors per leaf and the number of bacteria in the inoculum. Kurkdjian et al. (1974) described an experimental system with pea seedlings. Other plants commonly used are plantlets of sunflower, datura, and tobacco. With the use of these methods, plants or bacteria can be scored for tumorigenicity after 5 days to 3 weeks. 3.2.

A Temperature below 30"C

Riker (1926) discovered that cell transformation in this system could not be achieved at temperatures above 30°C. The growth of the bacteria is not inhibited at this temperature, nor is the development of the host plant. Experiments by Braun (1952) showed that conditioning takes place at 25°C as well as at 32°C. Furthermore, once cellular transformation has been accomplished by the bacteria, the tumor cells develop into neoplastic growth at temperatures above 32°C (Braun, 1947b). Since the proliferation phase is also not affected, the inactivating effect oftemperature must act in one way or another on the induction phase, or even on the TIP. 3.3. 3.3.1.

A Wound or Wound Stimulus The Conditioning Effect

That it was not enough to bring a suitable plant into contact with the bacteria was recognized from the beginning of crown-gall research (Smith and Townsend, 1907). However, the essentiality of the wound was initially conceived solely in terms of an entrance site for the invading bacteria (Smith et al., 1911). Smith and his co-workers believed the bacteria to be intracellular, and the wound was thought to be necessary to get the bacteria into ruptured cells. However, evidence accumulated from microscopic and electronmicroscopic studies and other experiments (Riker, 1923; Robinson and Walkden, 1923; Magrou, 1927; Rack, 1953; HoW, 1961; Ryter and Manigault, 1964; Gee et aI., 1967; Schilperoort, 1969; Bogers, 1972) indicating that the bacteria are located in the intercellular spaces during tumor induc-

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tion. Although there is now evidence for tumor-induction sites for the bacteria on the plant cell wall or membrane (Lippincott, J. A., and Lippincott, 1969) (see also Section 4.1), it is possible that wounding a plant does more than just give access to these sites. Supporting evidence for this view can be seen in the conditioning effect first demonstrated by Braun (1952). Braun (1952) found evidence for the conditioning effect by comparing the response of wounded tissue that had been permitted to heal for 2 days prior

FIGURE 3. Crown-gall tumors induced on carrot disks by A. tumefaciens. The disks not showing any tumors were inoculated with a nontumorigenic strain of A. tumefaciens.

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U.C.Knopf

to inoculation with the bacteria with the response obtained when the bacteria were introduced into the plant directly after wounding. In both cases, the bacteria were allowed to act for only 24 hr at 25°C, after which time tumor induction was interrupted by a shift to the nonpermissive temperature. These experiments showed that plants that were wounded 2 days prior to inoculation with the bacteria developed large tumors, while those inoculated at the time of wounding showed no tumorous reaction. In consequence, it could be shown that conditioning takes places gradually, reaching a maximum and declining again as wound healing progresses. At 96-120 hr after the wound stimulus, the cells are no longer susceptible to transformation by the bacteria. 3.3.2. Subcellular Events in Plant Cells after a Wound Stimulus Even though the conditioning effect could be nicely demonstrated in this system, its significance at the subcellular level is still open to speculation. A few hints as to what could be involved might be deduced from the following additional results: After a dicotyledous plant has been wounded, changes in the physiology of essentially resting cells in the wound region occur (Bloch, 1941, 1952; Click and Hackett, 1963; Montoya et al., 1977). These modifications include, among others, changes in the permeability of the cell membranes and respiratory modifications. In addition, new substances are synthesized in the wound, such as wound hormones, new proteins, RNAs, and basic amino acids. That one of these substances of the wound juice could be important for the conditioning effect is supported by the facts that the application of fluid collected from plant wounds to small wounds inoculated with Agrobacterium increases the tumor size (Hildebrand, 1942) and washing wounds prior to bacterial inoculation or in the first 6 hr after inoculation inhibits tumor formation (Klein, R. M., 1965; Kurkdjian and Manigault, 1969). It was also found that the time interval between the wound stimulus and the first cell division is temperaturedependent and ofthe order of 15 hr at 36°C and 35 hr at 25°C on Kalanchoe daigremontiana (Lipetz, 1966). The discovery that sensitivity to transformation of the same plant is optimal at about 27-36 hr (at 25°C) after wounding, i.e., before the first cell divisions take place (Lipetz, 1966), and a study of DNA synthesis during the wound reaction (Kupila-Ahvenniemi and Therman, 1971) led to the suggestion that plant DNA synthesis and with it the derepression of certain gene regions could be the first critical event in the conditioning phase of crown-gall cell transformation. Apart from this, some researchers (Guille et al., 1968; Guille and Quetier, 1970) reported that they were able to detect a newly synthesized, heavy satellite

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DNA * in Solanum Iycopersicum, Scorzonera hispanica, Glycine max, and Datura stramonium 24-72 hr after a wound stimulus. The satellites were reported to have a high guanine-cytosine content. On the basis of molecular hybridization experiments, it was further claimed that they had a high specificity for ribosomal RNA (rRNA). Since this nuclear heavy satellite DNA was found to hybridize to a certain degree with A. tumefaciens DNA, it was proposed (Guille and Quetier, 1970) that these hybridization sites might represent "hot spots" for the adsorption and later integration of bacterial DNA. The significant part of the conditioning phase was therefore seen as being the synthesis and eventual amplification of this nuclear heavy satellite DNA. However, other researchers (Pearson and Ingle, 1972; Broekaert and Van Parijs, 1975) could not find any evidence for wound-stimulus-induced satellite DNAs and suggested that previous results were probably due to bacterial contamination. But is also interesting to mention in this context and with what was said under Section 3.1 that it was shown in a systematic study (Ingle et al., 1973) that satellite DNAs are often observed among dicotyledons, while they are generally absent in monocotyledous plant species. Thus far, there is one exception: Cymbidium, in which a lowdensity satellite DNA was found (Capesius et al., 1975). 4.

4.1.

PROPERTmS AND PRODUCTS OF AGROBACTERIUM TUMEFACIENS

Induction of Crown-Galls

The ability to induce neoplasms on plants is the most striking characteristic of A. tumefaciens. Of course, there are also strains of A. tumefaciens that are unable, or have lost the capacity, to induce neoplasms. The ability of A. tumefaciens to induce or not induce plant tumors can be correlated with, respectively, the presence or the absence of a large plasmid (Zaenen et al., 1974) (for details, see Section 5). Apart from this, there are other prerequisites for successful tumor induction by the bacteria. The necessity for the bacteria to attach to specific tumor-induction sites at the plant cells was shown by cross-inoculations (Lippincott, J. A., and Lippincott, 1967, 1969): if avirulent or UV-killed virulent bacteria were inoculated together, or the avirulent before the virulent bacteria, tumor formation was decreased or inhibited. On the other hand, if avirulent *In this chapter, the tenn "satellite DNAs" refers to nuclear DNA fractions that can be separated from the main band after neutral CsC! equilibrium centrifugation.

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bacteria were inoculated after the virulent bacteria, tumor formation was not inhibited. According to B. B. Lippincott et al. (1977), wound-exposed portions of the host plant cell walls constitute the natural attachment site essential to Agrobacterium tumor induction. The exact nature of the host cell wall components involved in site attachment and the subsequent role of this attachment remain to be determined. Whatley et al. (1976) reported that on the bacterial side, a lipopolysaccharide is involved in site attachment. The observation that mutants of Agrobacterium requiring adenine, methionine, or asparagine are less infectious than their prototrophic parent (Lippincott, J. A., et al., 1965) and induce tumors only at larger wounds in which nutritional requirements may be less stringent (Lippincott, J. A., and Lippincott, 1966) was interpreted as being due to the requirement for bacterial metabolism for tumor induction. 4.2.

General Properties and Classification

Agrobacterium tumefaciens is a gram-negative bacterium classified in Bergey's Manual of Determinative Bacteriology (Bergey, 1974) in the family of Rhizobiaceae. It is a rod-shaped bacterium with dimensions of 0.8 x 1.5-3.0 p,m (Allen and Holding, 1974), motile by peritrichous fiagellae, the most frequent number being 1-4. The doubling time in culture is about 2 hr. Many although not all of the strains of A. tumefaciens and A. radiobacter form 3-ketoglycosides from disaccharides, especially from lactose (Bernaerts and De Ley, 1%3). On the basis of this feature, Bernaerts and De Ley (1963) developed a quick identification test for A. tumefaciens and A. radiobacter. In Bergey's Manual of Determinative Bacteriology, A. tumefaciens is one of the four species of the genus Agrobacterium, the other three species being A. radiobacter (Beijerinck and van Delden) Conn, A. rhizogenes (Riker et al.) Conn, and A. rubi (Hildebrand) Starr and Weiss. The relationship of the genus Agrobacterium to the genus Rhizobium has been examined extensively during the past years in numerous taxonomic studies (Graham, 1964; Mannetje, 1967; Moffett and Colwell, 1968; Skyring et al., 1971; White, L. 0., 1972; Kersters et al., 1973), DNA composition and hybridization studies (De Ley et al., 1966, 1973; Heberlein et al., 1967; Kern, 1968; Gibbins and Gregory, 1972), physiological tests and studies of pathogenicity (Keane et al., 1970; Lippincott, J. A., and Lippincott, 1969; Panagopoulos and Psallidas, 1973; Lippincott, J. A., et al., 1973), and studies of cell envelope composition and isozyme patterns (Graham, 1965; Manasse and Corpe, 1967; Graham and O'Brien, 1%8; Skyring et al., 1971; Clark, 1972; Zevenhuizen, 1973).

151

Crown-Gall and Agrobacterium tumefaciens

It appears from these studies that the Agrobacteria and at least the fast-growing Rhizobia (R.ieguminosarum, R. metiioti, R. phaseoli, and R. trijolii) are closely related (Graham, 1964; Heberlein et ai., 1967; Moffett and Colwell, 1968; White, 1972). There are differences between the two genera in their interactions with the plant cells. Furthermore, they have different viral pathogens (Roslycky et ai., 1963; Boyd et ai., 1970). On the basis of numerical analysis of 92 characters and studies of DNA hybridization and hybrid thermal stability, J. A. Lippincott and Lippincott (1975) showed that most Agrobacteria could be assigned to two clusters. Strains of cluster 1 are characterized as producing 3-ketolactose (see above for the test), growing on salts-plus-carbohydrate media and giving a neutral or alkaline litmus milk reaction. All prototrophic A. radiobacter and most A. tumefaciens strains are in this group, which corresponds to biotype 1 of Keane et af. (1970) and group 1 of L. O. White (1972). Strains of cluster 2 give a negative 3-ketolactose response, weak or no growth on salts-pluscarbohydrate media, and an acid litmus milk reaction. This cluster corresponds approximately to biotype 2 of Keane et ai. (1970) and group 3 of L. O. White (1972) and includes all A. rhizogenes strains and most auxotrophic strains of A. tumefaciens and A. radiobacter (Lippincott, J. A., and Lippincott, 1975). 4.3.

Differential Ability to Use Unusual Amino Acids as Sole Nitrogen Source

Unusual amino acids (see also Section 6.2) such as octopine and nopaline (Figure 4) were isolated from crown-gall tumor tissues of Scorzonera hispanica (Menage and Morel, 1964) and Opuntia vulgaris (Menage and Morel, 1966; Goldmann et al., 1969). Furthermore, Goldmann et af. (1968) showed that tumors induced by A. tumefaciens strain T37 form nopaline, whereas bacteria-free tumors induced by strain B6 form octopine. Petit et ai. (1970) subsequently found that tumors contained either octopine

/ NH2

HN

=

C

HN

'NH - (CH2)3 - CH - CDDH

=

/NH2 C 'NH - (CH2)3 - CH - CDDH

I

I

NH

NH I

I

CH3 - CH - CDDH OCTOPINE

HDDC - (CH2)2 - CH - CDDH NOPALlNE

FIGURE 4. Chemical structures of octopine and nopaline (Menage and Morel, 1964; Goldmann et al .• 1969).

U.C.Knopf

152

or nopaline, depending on the bacterial strain that originated the tumor. This striking difference could then be correlated with the ability of the tumor-inducing bacteria to degrade either one of these amino acids; thus, bacteria that induce octopine-forming tumors were found to degrade octopine but not nopaline and vice versa. Later, J. A. Lippincott et al. (1973) showed that there are exceptions to this generalization: certain strains of A. tumefaciens such as Ag6, TTl33, and P2 can utilize both octopine and nopaline; certain strains such as AT-1 and AT-4, both tumorigenic, are unable to utilize either one ofthese amino acids; and finally, certain strains such as EU 6 and 181, which degrade nopaline, initiate tumors that do not contain nopaline. The enzyme systems responsible for the oxidation of octopine and lysopine in the bacteria are cytochrome-linked membrane-bound oxidases and require no cofactors (Jubier, 1972; Bomhoff, 1974). More about these interesting amino acids and enzyme systems will be written in Sections 6.2 and 7.2. 4.4.

Production of Plant Growth Substances

Experiments by Brown and Gardner (1936), Link et al. (1937), Dame (1938), and Berthelot and Amoureux (1938) suggested that A. tumefaciens can form J3-indoleacetic acid. This was confirmed by Kaper and Veldstra (1958), Rodriguez de Lecea et al. (1972), and Sukanya and Vaidyanathan (1964). There is evidence that the bacteria actually produce this hormone in the host plant (Lippincott, J. A., and Lippincott, 1968). Furthermore, certain strains of A. tumefaciens fail to induce tumors without added auxin (Braun and Laskaris, 1942; Klein, R. M., and Link, 1952), which suggests that bacterial auxin is required in the transformation process. Upper et al. (1970) reported that culture filtrates of A. tumefaciens contain a cytokinin, which is indistinguishable from 6-(3-methyl-2-butenylamino) purine by gas-liquid or thin-layer chromatography, by its UV spectrum, and by its biological activity. Galsky and Lippincott (1967) reported that they could extract a gibberellinlike substance from cells of certain strains of A. tumefaciens, but no correlation between the production of this gibberellinlike substance and the ability to form tumors could be found. 4.5.

Production of Polysaccharides

In 1942, McIntire et al. (1942) reported finding a low-molecular-weight (3600-dalton) polysaccharide in the culture medium of A. tumefaciens. The polysaccharide represented 15-20% of the metabolized sugar and was composed entirely of glucose. Reeves (1944) suggested on the basis of

Crown-Gall and Agrobacterium tumefaciens

153

optical rotation studies in water and cuprammonium solution that the glucose units were linked through the 2 position. Results obtained after methylation (Putnam et ai., 1950) also indiCated that a substantial portion ofthe glucopyranose residues were linked through 1-2 glucosidic linkages. These results were confirmed later by Madsen (1962). The glucan described above is different from the mucopolysaccharide found by Conner et al. (1937) to be produced by A. tumefaciens. The latter was found to contain glucose and small amounts of uronic acid. 4.6.

Production of Vitamins

Agrobacterium tumefaciens produces considerable quantities of biotin, large amounts of riboflavin, and moderate amounts of thiamin and pantothenic acid (McIntire et ai., 1941). 4.7.

Production of Antibiotics

The production of an antibiotic by certain strains of Agrobacterium such as T37 and HIOO was discovered by Stonier (1960). He called the antibiotic agrobacteriocine I. The antibiotic was found to be readily diffusible through 1.1% agar and dialysis bags. An antibiotic that should be mentioned with regard to Section 5.1.2 below is the so-called agrocine 84. This antibiotic, which has been shown to be a 6-N-phosphoramidate of an adenine nucleotide analogue (Roberts et aI., 1977), can be isolated from a nonpathogenic strain of Agrobacterium radiobacter (strain 84). It selectively inhibits the growth strains that have acquired tumorigenicity through the transfer of the TIP plasmid (see Sec-

tion 5.1.2), while most nonpathogenic strains are not affected. 5.

MOLECULAR COMPONENTS, GENETIC SYSTEMS, AND SEARCH FOR THE TUMOR-INDUCING PRINCIPLE (TIP) OF AGROBACTERIUM TUMEFACIENS

5.1. 5.1.1.

DNA and DNA Plasmids DNA

De Ley et al. (1966) obtained a T m value for several A. tumefaciens DNAs in the narrow range of93.9-95.0°C and an average guanine-cytosine content of 59.9-62.8%. For six strains of A. tumefaciens, Heberlein et al. (1967) determined the guanine-cytosine content and found it to range from 60.6 to 62.2%.

154

U. C.Knopf

The buoyant density of A. tumefaciens was reported as 1.718 ± 0.0006 g/cm3 at 20°C (Schilperoort, 1969; Stroun and Anker, 1971). With the use of this value and the equation of Schildkraut et at. (1962), a guanine-cytosine content of 59.2% can be calculated. Indirect evidence for an implication of bacterial DNA in tumor induction was derived by Bopp (1961, 1964, 1965) from his experiments with base analogues such as 5-fluorodeoxyuridine, 5-fluorouracil, 5-bromouracil, and 5-bromodeoxyuridine. He reported that tumor induction could be inhibited when these compounds were introduced into infected plant tissue during the induction period. According to Bopp, there was no inhibition of the wound-healing process (conditioning) and the growth of bacteria by these products. Transfer of virulence with bulk DNA of A. tumefaciens has been reported. D. Klein and Klein (1953, 1956) reported that they were able to transfer virulence from a tumorigenic strain to a nontumorigenic strain of A. tumefaciens. Later, Kern (1965, 1969) reported that he was able to transform R. leguminosarum C into a tumor-producing strain with DNA from A. tumefaciens. Tumor induction with apparently sterile and pure bulk DNA from A. tumefaciens was reported (Kovoor, 1967). Other research (e.g., that of Stroun et al., 1971), although under conditions different from those of Kovoor, was not able to demonstrate plant-cell transformation with pure, noncontaminated bulk DNA from A. tumefaciens. 5.1.2.

DNA Plasmids

In 1969, Schilperoort (1969) reported in his thesis that in an electronmicroscopic study of Agrobacterium DNA, he found small amounts of circular DNA with a contour length varying from 0.4 to 2 /Lm. In 1974, Zaenen et al. (1974) reported that they found large DNA plasmids (54.1/Lm corresponding to a molecular weight of 112 x 106 daltons) in certain strains of A. tumefaciens (Figure 5). According to these authors, only a few copies of this large plasmid are present per bacterial genome. The fact that at this time no nontumorigenic strains were found that carried a large plasmid led these authors to propose the hypothesis that' 'the tumor-inducing principle (Braun, 1947a) in crown-gall-inducing Agrobacterium strains is carried by one or several large plasmids of various lengths." Although it was reported later (Merlo and Nester, 1977) that nontumorigenic Agrobacterium strains also carried large plasmids, more evidence accumulated that in tumorigenic strains, a large plasmid or parts of it are involved in tumorigenesis: On the one hand, a correlation between the loss of a large plasmid and the loss of the tumor-inducing capacity was 100% (Van Larebeke et ai., 1974; Hamilton and Chopan, 1975; Watson et at., 1975). On the other hand, the

Crown-Gall and Agrobacterium tumefaciens

155

FIGURE 5. Electron micrograph of a large DNA plasmid of A. tumefaciens canying genes involved in tumorigenesis. The smaller plasmids visible in the photograph are size markers. Isolation, purification, electron microscopy and other properties of the plasmid will be described elsewhere.

introduction of the virulence-associated plasmid into an avirulent-plasmidless strain always conferred virulence on the recipient strain (Hamilton and Chopan, 1975; Watson et aI., 1975; Van Larebeke et al., 1975). Several Agrobacterium strains have more than one plasmid (Currier and Nester, 1976), which might differ in size (from 25 X 106 to 150 X 106 daltons). However, there is so far no evidence for any natural association of tumorigenicity with a smaller plasmid.

156

U. C.Knopf

A high degree of homology among the large plasmids of different bacterial strains carrying pathogenicity genes would have been expected if many sequences of the plasmid were needed to code for gene product(s) involved in tumorigenesis. However, data elaborated by Currier and Nester (1976) show that homologies among different plasmids range anywhere from 3 to 100%. A pattern among different plasmids was evident to the extent that two genetically distinct groups of plasmids could be identified: the plasmids that are closely related to the plasmid of A. tumefaciens A6, an octopine-utilizing strain (see also Section 4.3), and the plasmids that are closely related to A. tumefaciens C58 , a nopaline-utilizing strain (see also Section 4.3). Few naturally occurring genetic markers have thus far been discovered for the large plasmids known to code for virulence-associated traits. Already in 1972, Petit and Toumeur (1972) observed that in one case, the loss of virulence is accompanied by loss of the ability to degrade octopine. Later, utilization of octopine or nopaline was shown by Lippincott et ai. (1973) to be highly correlated with virulence. Subsequently, it was suggested (Kerr, 1975; Bomhoff et ai., 1976; Montoya et ai., 1977) that octopine and nopaline synthesis in the plant and their breakdown by different strains of Agrobacterium are controlled by genes located at least most of the time in the virulence plasmid. Furthermore, in strains C58 and K27, sensitivity to agrocine 84 (see Section 4.7) is a characteristic property conferred by the virulence-associated plasmid (Chilton et ai., 1974; Watson et ai., 1975; Engler et ai., 1975). However, there are virulent strains that are not sensitive to this bacteriocin (Chilton et ai., 1976). Finally, some bacteriophages were found (Van Larabeke et ai., 1975) that are excluded by plasmid-carrying strains. As briefly mentioned above, the transfer of plasmids among strains of Agrobacterium and eventually other bacteria is possible. Kerr (1969, 1971) discovered and developed an in vivo transfer system: nonpathogenic strains of A. radiobacter became pathogenic when reisolated from tomato crown-gall tumors produced by pathogenic A. tumefaciens in the presence of nontumorigenic A. radiobacter. Later, it could be shown (Van Larebeke et ai., 1975) that in this system, acquisition of the large plasmid parallels acquisition of tumorigenicity. In vitro transfer of the large plasmid and virulence was achieved on the one hand by research workers using the RP4 plasmid for promotion of the transfer (R. A. Levin et ai., 1976; Chilton et ai., 1976). Conjugation was proposed as the transfer mechanism. On the other hand, in vitro transfer of Agrobacterium plasmids without the promotion ofRP4 has been achieved (Kerr et al., 1977; Genetello et al., 1977). These experiments suggested, furthermore, that the rare amino acids such as octopine and nOPaline promote the plasmid transfer and that they could function as derepressors of genes involved in this transfer.

Crown-Gall and Agrobacterium tumefaciens

157

The transfer by means of a conjugative process of an oncogenic plasmid from a virulent strain of A. tumefaciens to a strain of that organism that had been cured ofthe plasmid is thermosensitive (Tempe et al., 1977). Since the thermo sensitive step found in the conjugative process appears similar in every respect to the thermosensitive step that is involved in the transformation of a normal cell to a tumor cell (see Section 3.2) it was suggested (Tempe et al., 1977) that both phenomena have a common basis. 5.2. An RNA Polymerase and Its Components A rifampicin-sensitive RNA polymerase of the tumorigenic strain A. tumefaciens B6806 was extensively purified and analyzed (Knopf, 1974a). The subunit structure of the highly purified enzyme was found to be similar to, although not identical with, the RNA polymerase of E. coli (Figure 6). It can be described as {3', {3, x' and a with molecular weights of 160,000, 150,000, 98,000, and 41,000 ± 10% daltons, respectively. On the basis of experiments with the drug rifampicin, Stroun (1971) postulated that transcription of presumed bacterial DNA within the plant cell takes place by means of a bacterial-DNA-dependeIit RNA polymerase. 5.3.

RNA

While Zaenen et al. (1974) hypothesized that a large DNA plasmid might carry the TIP, another group (Beljanski et al., 1974) reported that they were able to isolate two RNA fractions that were oncogenic from both tumorigenic and nontumorigenic strains of Agrobacterium. One of these RNA fractions was reported to be bound to an RNA-directed DNA polymerase, while the other was associated with the bacterial DNA. From sedimentation in linear sucrose gradients, it was concluded that their size corresponded to molecules sedimenting at about 5-6 S. Since no hypochromicity was found in the presence of ribonuclease A, it was concluded that these tumorigenic RNAs were single-stranded. Somewhat later, Roussaux (1975) reported the isolation of tumorigenic RNA from crown-gall tumors. Although there has been no confirmation of these results published so far, it should be noted that RNA was always thought to be involved in tumorigenesis at one stage or another, since the report of Braun and Wood (1966) that ribonuclease A but not deoxyribonuclease could significantly inhibit tumor inception .. 5.4.

Ribosomes and Their Components

Ribosomes from tumorigenic and nontumorigenic Agrobacterium strains have been isolated and analyzed (Knopf, 1977). The hydrodynamic

158

U. C. Knopf

>....

e:;; z

....c -'

B

A

A r : ATP+H 20 Nuclease ADP+Pi

A

A

+--I A +--I

+-I

A

+--i

A

+---l

Retention and amplification of one fragment, loss of others

BCD

.---; +---l ｾ＠

E

+-i

MtDNA fragments

B

＼ｾｴ｜Ｂｬ＠ ＧＭｾＯｉ＠

......... ｾ＠

/' ｾＧﾫＢＯ＠ "

fl

l

Proteins coded in nucleus and synthesized in cytoplasm:

I

S-----tt---ll___ I

I

t ］ＺＱｾＭｎ /Subunits of: I 1. Oligomycin/ sensit ive ...-:::::::It:=:::::;:;;...J ATPase 2. Cytoch rome-";:::::::jl:::::::::---. oxidase complex 3. Cyt b· c,

mtDNA

/ Inner membrane

{

Mitoch,:,ndrial ribosomal protems

j

l3-0Xidation enzymes TCA cycle enzymes mtDNA polymerase mtRNA polymerase

_____ {outer ":,embrane protems Cyt c Cyt c, Flavoproteins Subunits of: 1. Oligomycinsensitive ATPase 2. Cytochrome oxidase complex \ 3. Cyt b· c, complex Outer membrane

FIGURE 7. (A) Summary of events accompanying ethidium bromide induction of petite mutation. (A-E) Hypothetical gene loci; (EtBr) ethidium bromide. (B) Summary of interactions involved in the assembly of mitochondrial components. (- - -) Pathways that are lost in petite mutants.

217

The Petite Mutation in Yeast

the lack of genes essential to growth, survival, and division associated with the mitochondrial genome, or does this mutation serve some useful purpose for the yeast? In all probability, the former is correct, but it is nevertheless tempting to speculate that the petite mutation may confer some evolutionary advantage. It is known that certain treatments that inhibit growth of Saccharomyces cerevisiae are considerably less effective against petite mutants. Thus, the presence in the growth medium of cobalt (Horn and Wilkie, 1966), 2,3,5-triphenyl-tetrazolium chloride (Bachofen et at., 1972), cycloheximide (Bilinski et at., 1974), or nalidixic acid (Carnevali et at., 1976) strongly favors the growth of petite mutants. The reasons for this selective advantage of petite mutants are not altogether clear, but there is the possibility that from time to time, in the wild, yeast might encounter analogous conditions in which growth of petite mutants would be favored. Clearly, the ability to produce viable petite mutants here would lead to proliferation of petite-positive as opposed to petite-negative species. The possibility would remain at a later time to reacquire a functional mitochondrial genome by sexual means. According to Bulder (1963), petite mutant strains do not seem to be particularly uncommon in nature, which is the situation one would expect to find if the hypothesis set out above were correct. Of 60 or so strains of Saccharomyces cerevisiae held at that time in the Centraalbureau voor Schimmelcultures, 3 proved to be petites. What is more, it looked as though all the strains of Schizosaccharomyces versatilis and Torulopsis lactis-condensi were petites. The petite mutation will undoubtedly continue to be studied even after a fairly complete picture of the mutagenic process and of the mutants themselves has been established. In particular, petite mutants will continue to provide an indispensable tool for mitochondrial gene mapping and will provide large quantities of relatively short segments of mtDN A that should be of considerable value for nucleotide sequencing of the mitochondrial genome. In conclusion, the probable course of petite mutation (induced by ethidium bromide) and the biochemical consequences of the mutation are summarized in Figure 7 and Table V.

Table V

Summary of the Biochemical Consequences of the Petite Mutation 1. 2. 3. 4.

Loss of ability to synthesize mt rRNAs and usually tRNAs and mRNAs as well Consequent loss of mitochondrial protein synthesis Loss of cyt b . c" cytochrome oxidase, and oligomycin-sensitive ATPase subunits. Consequent loss of oxidative phosphorylation capacity

218

Peter A. Whittaker

10. APPENDIX: ABBREVIATIONS AND TERMS

C, cap], RIB /: Alternative designations, used by different research groups, of a mtDNA gene controlling the degree of sensitivity to chloramphenicol. The gene is thought to specify a region of the 23 S mt rRNA. E, ery/, ery2, RIB II , RIBw: Alternative designations, used by different research groups, of mtDNA genes controlling the degree of sensitivity to erythromycin. The ery] and ery2 (RIBII and RIBIII ) genes independently affect erythromycin sensitivity. They specify different regions of the 23 S mt rRNA. Hybridization level: A measure of the total amount of base sequences in a DNA population homologous with a particular RNA (or DNA) molecule. In this review, hybridization levels are given relative to the hybridization of the same RNA (or DNA) with p+ mtDNA. Thus, petites whose mtDNA has a higher hybridization level than 1.0 with a particular tRNA will have a greater number of copies of that tRNA gene per unit length of mtDNA than grande cells. Kinetic complexity: A measure of the total length of unique base sequence in a DNA population, obtained from a study of the renaturation kinetics of denatured DNA. The complexities are given in this review relative to p+ mtDNA, which is taken as 1.0. Petites retaining and amplifying only a very short segment of p+ mtDNA would have low kinetic complexity. mit mutants: Mitochondrial DNA gene mutations that affect the synthesis of components of the oxidative phosphorylation system. pet mutants: Chromosomal gene mutations that affect the synthesis of components of the oxidative phosphorylation system. Suppressiveness: A property of the majority of p- petites. If these are crossed with a wild-type strain, the aberrant mitochondrial genome of the p- strain suppresses the transmission of the mitochondrial genome of the grande parent in a proportion of the haploid progeny. The proportion of petite progeny is referred to as the "degree of suppressivity. " p factor: Extrachromosomal factor whose genetic function is lost or grossly impaired in petite mutants. Now considered to be identical with mtDNA. pO petites: Petite mutants lacking mtDNA. p- petites: Petite mutants with retained mtDNA. w gene: Mitochondrial DNA gene locus determining the polarity of transmission of certain mitochondrial gene markers in a yeast cross. In a cross w+ X W-, genes closely linked to the w+ allele are transmitted to the progeny in preference to those linked to the w- allele.

The Petite Mutation in Yeast

11.

219

REFERENCES

Allen, N. E., and MacQuillan, A. M., 1969, Target analysis of mitochondrial genetic units in yeast, J. Bacteriol 97: 1142-1148. Allmark, B. M., Danks, S. M., and Whittaker, P. A., 1977, Isolation and characterization of respiration-deficient mutants of Kluyveromyces lactis, a petite-negative yeast, Biochem. Soc. Trans. 5:1498-1500. Arakatsu, Y., 1971, Action of acriflavine on growth and mutation in yeast. 1. Nitrogen sources affecting mutation-induction, Mutat. Res. 12:235-248. Arakatsu, Y., 1972, Action of acriflavine on growth and mutation in yeast. II. Kinetic study on the effect of glutamate on respiration-deficient mutation-induction, Mutat. Res. 14:165184. Avers, C. J., 1967a, Heterogeneous length distribution of circular DNA filaments from yeast mitochondria, Proc. Natl. Acad. Sci. U.S.A. 58:620-627. Avers, C. J., 1967b, Distribution of cytochrome c peroxidase activity in wild type and petite cells of bakers' yeast grown aerobically and anaerobically, J. Bacteriol 94: 1225-1235. Avers, C. J., Pfeffer, C. R., and Rancourt, M. W., 1965, Acriflavine induction of different kinds of "petite" mitochondrial populations in Saccharomycyes cerevisiae, J. Bacteriol. 90:481-494. Azzi, A., and Santato, M., 1971, Interaction of ethidium and mitochondrial membrane: Cooperative binding and energy-linked changes, Biochem. Biophys. Res. Commun. 44:211217. Bachofen, V., Schweyen, R. J., Wolf, K., and Kaudewitz, F., 1972, Quantitative selection of respiratory deficient mutants in yeast by triphenyltetrazolium chloride, Z. Naturforsch. 27b:252-256. Bastos, R. N., and Mahler, H. R., 1974, Molecular mechanisms of mitochondrial genetic activity: Effects of ethidium bromide on the deoxyribonucleic acid and energetics of isolated mitochondria, J. Bioi. Chem. 249:6617-6627. Bech-Hansen, N. T., and Rank, G. H., 1973, The bivious suppressiveness of cytoplasmic petites of S. cerevisiae lacking in mitochondrial DNA, Mol. Gen. Genet. 120:115-124. Bechmann, H., Kruger, M., Boker, E., Bandlow, W., Schweyen, R. J., and Kaudewitz, F., 1977, Formation of rho- petites in yeast. 2. Effects of mutation TSM-8 on mitochondrial functions and rho factor stability in Saccharomyces cerevisiae, Mol. Gen. Genet. 155:4151. Benson, R. W., 1972, Characterization of yeast nuclear and mitochondrial DNA-dependent RNA polymerases, Fed. Proc. Fed. Am. Soc. Exp. Bioi. 31:472. Bernardi, G., and Timasheff, G. N., 1970, Optical rotatory dispersion and circular dichroism properties of yeast mitochondrial DNA's, J. Mol. Bioi. 48:43-52. Bernardi, G., Faures, M., Piperno, G., and Slonimski, P. P., 1970, Mitochondrial DNA's from respiratory-sufficient and cytoplasmic respiratory-deficient mutant yeast, J. Mol. Bioi. 48:23-42. Bilinski, T., Jachymczyk, W. J., and Kotylak, Z., 1974, The dependence of cytosole protein biosynthesis-resistance to cycloheximide in yeast on changes in mitochondrial activity, Mol. Gen. Genet. 129:243-248. Boguslawski, G., Vodkin, M. H., Finkelstein, D. B., and Fink, G. R., 1974, Histidyl-tRNAs and histidyl-tRNA synthetases in wild type and cytoplasmic petite mutants of Saccharomyces cerevisiae, Biochemistry 13:4659-4667. Bolotin, M., Coen, D., Deutsch, J., Dujon, B., Netter, P., Petrochilo, E., and Slonimski, P. P., 1971, La recombinaison des mitochondries chez Saccharomyces cerevisiae, Bull. [nst. Pasteur (Paris) 69:215-239.

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Chapter 5

The Role of Lipids in the Structure and Function of Membranes Giorgio Lenaz Institute of Biochemistry, Faculty of Medicine and Surgery University of Ancona Ancona, Italy

1.

INTRODUCTION

The increased knowledge of the properties of membrane lipids (Ansell et ai., 1973) and oflipid-protein interactions (Singer, 1971; Lenaz, 1973, 1977; Vanderkooi, G., 1974) allows a better understanding ofthe role of lipids in membrane structure and functions. Nevertheless, a unifying picture of such a role is lacking, and it is often tacitly assumed that lipids have different roles; this is indeed the main conclusion emerging from analysis of the literature. In fact, lipids in membranes have different functions, affecting enzymic activity positively or negatively, being determinants of permeability properties and transport and being involved in the action of membrane binding sites and receptors. Moreover, they are determinants of membrane phenomena involving fusion processes (e.g., cell movement, pinocytosis, cell division, cell adhesion, secretion). In such functions, lipids may be specific or not. The physical state of a lipid, besides the specific chemical nature of certain groups, appears to be very important in its functions. It seems therefore appropriate to assign to lipids many different roles. Abbreviations used in this chapter: (ANS) l-anilinonaphthalene 8-sulfonate; (BDH) ,B-hydroxybutyrate dehydrogenase; (.cAMP) cyclic 3' ,5' -AMP; (CD) circular dichroism; (cyt) cytochrome; (DML) dimyristoyllecithin; (DOL) dioleyllecithin; (DPL) dipalmitoyllecithin; (DSC) differential scanning calorimetry; (EFA) essential fatty acid(s); (EM) electron microscopy; (ESR) electron spin resonance; (excimer) excited dimer; (GH) growth hormone; (HDL) highdensity lipoproteins; (NMR) nuclear magnetic resonance; (PC) phosphatidy1choline (lecithin); (PE) phosphatidylethanolamine; (PG) prostaglandin; (PS) phosphatidylserine; (TEMPO) 2,2,6,6-tetramethyl piperidine-l-oxyl.

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However, one property of lipids may be a common feature of their different roles. Lipids represent at the same time an asymmetrical barrier separating two different compartments and a medium in which specific functional components of the membrane (the proteins) are dissolved. The solvent function of lipids in biomembranes has received much attention recently, and the properties of such solutions have been examined. There is a fundamental difference between the universal bulk solvent of cells (water) and the lipids in membranes (which are mainly phospholipids). Lipids represent a bidimensional solvent (Vanderkooi, G., 1974) endowing membrane components with vectorial properties. There are, however, additional differences that may account for the several roles of lipids. First, lipids in membranes are heterogeneous, and consist of several species differing in the nature of both the polar head and the hydrophobic tails (Table I); the existence in most natural lipids of two fatty acyl chains gives rise to a large degree of molecular heterogeneity. Second, a phospholipid molecule is very complex, and its properties differ from the polar head to the hydrophobic tails; not only are there obvious differences in polarity, but also there is a fluidity gradient, changing with chain length and unsaturation ofthe chains. The overall properties are furthermore modified by the presence of other lipid molecules such as cholesterol, neutral lipids, and Table I Main Amphipathic Lipids Found in Membranes

Charge Lipid Phosphatidylcholine (lecithin) (PC) Phosphatidylethanolamine (PE) Phosphatidylinositol (PO Phosphatidylinositol phosphates Phosphatidylglycerol (PG) Diphosphatidylglycerol (cardiolipin) (DPG) Phosphatidylserine (PS) Phosphatidic acid (PA) Lysolecithin (LPC) Sphingomyelin Cerebrosides Cerebroside sulfate Gangliosides Cholesterol

Fatty acids"

+ I 0 0 0 0

1 0

1 1

3-5 1

2 2 2

1

1

0 0 0 0

0

2 2 2

2 2 4

2 2

1 1-3 0

o

aPrincipal fatty acids: 16:0; 16: 1; 18 :0; 18: 1; 18 :2; 18:3; 20:4; 22 :6; 24:0; some odd-chain and branchedchain acids, Fatty aldehydes: 16:0; 18:0.

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gangliosides. Since lipid and protein molecules have comparable dimensions, compared to the difference between the sizes of protein and water molecules, the interactions between proteins and lipids will depend on several associations among different parts of the surface of both interacting molecules. This leads to the possibility of very specific interactions, which may be important for function. One of the purposes of this review of the role of lipids is to give experimental support to the idea that lipids are involved in maintaining an optimal conformation for membrane proteins. In much the same way as the polar aqueous environment determines the folding of water-soluble proteins, by hiding hydrophobic groups in the protein interior (Kauzmann, 1959; Tanford, 1962), membrane proteins assume the conformation having the minimal free energy in a hydrophobic medium by optimizing hydrophobic interactions with lipid alkyl chains (Singer, 1971). In membrane proteins, however, there are increased chances of variations in such interactions and in the ensuing conformation, due to the possibility of changing the physical state of the lipids in vivo by affecting both the polar part and the hydrophobic tails through the action of chemical and physical means.

2. 2.1.

PROPERTIES OF THE LIPID BILAYER Lamellar Systems

Amphipathic lipids in membranes have a bilayer arrangement (Danielli and Davson, 1935; Vanderkooi, G., and Green, 1970) (Figure 1). (See p. 403 for a further discussion of lipid bilayers.) Many properties of natural membranes depend on such an arrangement and are mimicked by artificial membranes made up of purified lipids (Luzzati, 1968; Bangham, 1968). Several model lipid systems are available to study phospholipids and their interactions with soluble and membrane proteins; the most used systems are liposomes [either multibilayered lamellae obtained by shaking phospholipids in water (Bangham, 1968, 1972) or single-bilayer vesicles obtained by ultrasonic irradiation (Huang, 1969) or by other means] and the so-called black lipid membranes (bilayers formed in a hole separating two aqueous compartments) (Szabo, 1972; Papahadjopoulos, 1973). While liposomes are best suited to study enzymatic and transport properties of biomembranes by adding the appropriate protein components, black lipid membranes are particularly suitable to the study of electrical properties of membranes. In addition, studies with lipid monolayers at the air-water interface (Cadenhead, 1970; Paphadjopoulos, 1973) have contributed to a great extent to knowledge of membrane structure, and they may indeed be considered as

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POLAR

NONPOlAR POLAR

｛Ｔｾｬｩ＠ [

[I"V'''''''V'\I''V'\'''......ｾ＠

FIGURE 1. Scheme showing the lamellar disposition of a homogeneous phospholipid in water and the thermal transition from the liquid-crystalline phase (left) to the gel phase (right).

half-membranes maintaining the molecular relationships found in bilayer membranes. 2.2 Thermotropic Phase Changes and Phase Separations The most characteristic property of amphipathic lipids is that they undergo a thermotropic phase transition (Chapman, 1969; Luzzati, 1968)(Figure 1). When a pure crystalline lipid is heated, an endothermic transition occurs in which the hydrocarbon chains of the component fatty acids melt and become liquidlike in mobility; such a transition is detected by differential scanning calorimetry (DSC)(Chapman, 1973a): at constant pressure, the free-energy change of melting is zero and the entropy increase (disorder) of melting is accomplished at the expense of an enthalpy change (heat absorption) (Figure 2). The endothermic transitions detected by DSC are sharp for homogeneous lipids; the transition temperature depends on both the polar and the apolar phospholipid moieties. The more unsaturated the lipid, the lower is the transition temperature; similarly, the transition temperature is directly proportional to the length of the fatty acid chain. These effects are a consequence of the importance of Van der Waals attractions among fatty acid chains in assuring lipid adhesion. In addition, different lipid classes, even those having the same fatty acid composition, melt at different temperatures. For example, phosphatidylethanolamine (PE) has a higher transition temperature than the corresponding phosphatidylcholine (PC), due to the bulky properties of the choline head group (Figure 3), which decreases cohesion of the hydrocarbon chains; e.g., dimyristoyl PE melts at 45°C, while dimyristoyl lecithin (DML) melts at 25°C (Reinert and Steim, 1970). In the case of binary phospholipid mixtures, two transitions may become apparent (Chapman et al., 1974); in the temperature region

237

The Role of Lipids in Membranes

ｾｈ＠

t

Endothermic

20

10

30

40

50

'C

60

FIGURE 2. A calorimetric scan for dipalmitoyiiecithin in water (4 : 1).

ｾ＠ 1

j

W 1

1

FIGURE 3. Schematic model of the terminal head groups in phosphatidylcholine and phosphatidylethanoiamine. showing that interchain distance is higher in the case of phosphatidylcholine because of the bulky trimethylammonium group of choline.

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Giorgio Lenaz

between the two endothermic peaks, or also in the case of a broad single peak, solid and fluid phases coexist in the same bilayer, the solid phase containing an excess of the component having the higher transition temperature (Figure 4). This phenomenon is a phase separation and has been particularly investigated by spin-labeling techniques (Shimshick et aI., 1973). The spin labels (Jost et al., 1971; Smith, 1. C. P., 1972; Keith et al., 1973; Seelig, 1976) are nitroxide derivatives in which the N ｾ＠ 0 group contains an unpaired electron that renders the molecule paramagnetic. The spin label absorbs electromagnetic radiation in the presence of an external magnetic field, and the interaction of the unpaired electron with nuclear spin produces a hyperfine splitting of the absorption spectrum with three lines at increasing magnetic field. The shape of the spectrum reflects the orientation and the motion of the label and the polarity of its environment. Water-soluble nitroxides such as 2,2,6,6-tetramethyl piperidine-1-oxyl (TEMPO) have been used to detect phase separations in lipid mixtures (Shimschick et al., 1973). The upper field line of the electron spin resonance (ESR) spectrum has a different position in polar and nonpolar environments, and duplicates when the label is partitioned among both environments. By plotting the ratio of the two peak heights against temperature, one observes, in binary mixtures, two discontinuities, which have been associated with the onset and completion of phase separation. The temperature region between the two discontinuities corresponds to the range in which solid and fluid regions coexist. A similar phase separation may be shown even in the case of pure homogeneous lipids, since there is formation of quasi-crystalline clusters of lipid molecules well above the phase transition (Lee, A. G., et al., 1974); only at very high temperatures do the molecules exist in their true monomeric state.

ｾ＠

nli ｾｵ＠ (J

ｾ＠

-II [ J.

II

ｾ＠

..

r

FIGURE 4. Scheme showing phase separation in a binary mixture of phospholipids having different transition temperatures. (.) Polar heads of the component having higher transition temperature.

239

The Role of Lipids in Membranes

2.3. Lipid Viscosity Spin labels and fluorescent probes have been largely used to investigate the viscosity of the lipid bilayer. Using as spin labels fatty acid or phospholipid derivatives, the shape of the electron paramagnetic resonance (EPR) spectra is very sensitive to probe rotational mobility (Figure 5). The order parameter S describes the state of the lipid chains: S

=

Til - Tl. T zz - 0.5 (Txx + Tyy)

a a'

where TzzI TxxI and Tyy are constant values and represent the distances in gauss between the hyperfine splitting extremes for a nitroxide incorporated into a rigid crystal, with the nitroxide axis parallel to the three principal coordinates of the crystal; Til and T.l are experimental values found by analysis of the spectrum of a nitroxide immersed in a nonoriented and usually not crystalline system, and represent the distances in gauss between given bands in the ESR spectrum, and are sensitive to the viscosity of the system; and a and a I are also distance parameters measured from the spectra and represent the isotropic component of the hyperfine interac-

A H-

(gauss)

FIGURE 5. EPR spectra of 5-doxyl stearic acid in water (A) and in phospholipids (B).

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Giorgio Lenaz

tion in the rigid lattice and in the experimental system, respectively (Jost et ai., 1971; Smith, I. C. P., 1972; Keith et ai., 1973; Lenaz, 1977). The lipid viscosity decreases with temperature, and discontinuities are found in Arrhenius plots ofthe motion parameters (Raison et ai., 1971); in simple lipid mixtures, the breaks appear to be related to the onset and completion of phase separation, but in complex mixtures and in membranes, they may be ascribed to formation of quasi-crystalline clusters or phase separations within fluid bilayers (Davis et ai., 1976) (see Section 4.3.5b). Fatty acid spin labels have been found to have preferential affinity for fluid areas when fluid and solid areas coexist (Oldfield et ai., 1972). Such preferential distribution results in extrusion of the labels from solid to fluid bilayer areas; on the other hand, sterol spin labels do not appear to be segregated and are better suited to study average mobility when phase separation occurs (Butler et ai., 1974). Spin labels have also shown a fluidity gradient in bilayers, with mobility increasing from the polar head groups toward the center of the bilayer (Jost et al., 1971; Hegner et al., 1973) (Table II). Seelig and co-workers (Seelig and Niederberger, 1974) confirmed by deuterium nuclear magnetic resonance (NMR) that a fluidity gradient exists from the surface to the core of the bilayer, but it has a different pattern in comparison with that detected by ESR; the reason may be in the perturbing effect of nitroxides in the lipid bilayer. Also, fluorescent probes (Chapman and Dodd, 1971; Radda and Vanderkooi, 1972; Yguerabide, 1973) have been applied to test lipid fluidity in bilayers. The quantum yield of some probes, including the widely used 1anilinonaphthalene 8-sulfonate (ANS) and N -phenyl naphthyl amine (NPN), is sensitive to the viscosity of their microenvironments (as well as to their polarity) (Trauble and Overath, 1973; Lenaz et ai., 1975a, 1976). Table II Fluidity Gradient in Lipids and in Mitochondrial Membranes u Mobility of spin label b Membrane

5-NS

Lipid vesicles Mitochondrial membranes a Modified b

12-NS

16-NS

6-7

4-5

1.8-2.5

15-20

9-10

3-4

by Lenaz et al. (1974). Mobility was measured by the empirical ratio of the height of the median and high field peaks in the EPR spectrum. The ratio is 1 for a nitroxide freely tumbling in solution and increases with increasing rigidity. For limitations in such expression of fluidity, cf. Lenaz (1977). The spin labels designated as 5-NS, 12-NS, and 16-NS are stearic acid derivatives having nitroxide (doxyl) groups in the 5, 12, and 16 position, respectively.

241

The Role of Lipids in Membranes

ANS is located on the surface ofthe lipid bilayer, while NPN is dissolved in the lipid core; they therefore detect superficial and deep mobility of the lipids, respectively. The quantum yield of the probes increases with decreasing temperature, and therefore is proportional to lipid viscosity in a fluid membrane; however, at the transition temperature, there is a large decrease of fluorescence, due to decreased solubility of the probes in the solid phase. This conspicuous change was used to detect phase transitions in membranes (Tdiuble and Overath, 1973) (Figure 6). Other probes have now become of wide use (Shinitzky and Inbar, 1974; Vanderkooi, J. M., et ai., 1974; Papahadjopoulos et ai., 1973; Cogan et ai., 1973; Faucon and Lussan, 1973) such as perylene and anthracene derivatives, which are located, like NPN, in the interior of the lipid bilayer. The polarization of their fluorescence is directly proportional to the viscosity of the bilayer core, and slope discontinuities in the temperature de pen-

> ｾ＠

II)

z

25

w

ｾ＠

z

'20

w

...J

10

u..

transition temperature

5 0

20

30

40

50

60

70

TEMPERATURE FIGURE 6. Phase transitions in distearoyllecithin dispersions, detected by ANS and NPN fluorescence. Redrawn from Triiuble and Overath (1973).

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Giorgio Lenaz

dence of fluorescence polarization have been used to probe phase transitions. The microviscosity 'Y/ of lipid bilayers can be calculated from· the following equation (Feinstein et al., 1975):

rO KTT -=1+-r

'Y/ VCr)

where rO is the limiting anisotropy and r the anisotropy measured under the experimental conditions, T is the mean fluorescence lifetime, K is the Boltzmann constant, T is absolute temperature, and VCr) is the effective rotational molecular volume. The formation of pyrene-excited dimers (excimers) is also used to investigate lipid viscosity (Galla and Sackmann, 1974; Vanderkooi, J. M., and Callis, 1974), following the reasoning that excimer formation is dependent on the rate of diffusion of the lipid-soluble probe in the fluid bilayer. In liquid-crystalline lipids, several techniques have shown that lateral motion of the individual molecules occurs very· rapidly with diffusion coefficients in the order of 10-8 cm 2/sec (Scandella et ai., 1972; Galla and Sackmann, 1974), whereas the jump of lipid molecules from one to the other side of a bilayer (flip-flop) occurs at extremely low rates, with halftimes ofthe order of up to several hours (Kornberg and McConnell, 1971). 2.4. Summarizing Concepts The combined use of several physical techniques has allowed us to understand the main properties of lipid bilayers, which are summarized below (cf. Lenaz, 1977): 1. Heating pure lipids induces a sharp transition with melting of the fatty acid chains, whereas mixed lipids undergo phase separations. 2. Phase separations are induced not only by temperature changes, but also by changes of pH, ionic strength, and addition of specific metal cations (e.g., Ca2+); these isothermal changes (Trauble and Eibl, 1974) may be of great physiological significance. 3. The temperatures at which phase transitions and phase separations occur depend on the nature of both the fatty acid chains and the polar moieties. 4. In addition, the presence of cholesterol in a bilayer induces very interesting changes (Demel and De Kruyff, 1976; Lenaz, 1977). Cholesterol, up to a 1:1 ratio, with phospholipids forms complexes with the phospho-

The Role of Lipids in Membranes

243

lipid molecules and induces an intermediate degree of fluidity (Chapman, 1973b), so that crystalline lipids are more mobile while fluid lipids become more viscuous. Cholesterol abolishes the phase transition by abolishing the cooperativity of the melting process. 5. Lipids in the liquid-crystalline state are highly mobile and undergo rapid lateral diffusion, whereas the vertical movement from one monolayer to the other in a membrane is usually a very slow process. 6. Lipids are distributed asymmetrically in single-bilayer vesicles (Litman, 1974, 1975; Michaelson et al., 1973, 1974; Yeagle et al., 1976), so that bulky or charged groups and unsaturated fatty acids will preferentially stay in the external monolayer, in order to minimize repulsions and stretching in vesicles having a very low radius of curvature.

3.

LIPID-PROTEIN INTERACTIONS AND LIPID ORGANIZATION IN MEMBRANES

3.1. Lipid-Protein Interactions 3.1.1. Membrane Proteins The properties summarized above concern amphipathic lipids independently of the presence of proteins. Proteins are now known to affect bilayer properties very strongly. The structure of biological membranes is often described by the fluid mosaic model of Singer and Nicolson (1972). Not all membranes, however, appear to be adequately described by this model (Vanderkooi, G., and Green, 1970). Proteins are contained and immersed to different extents in the lipid bilayer, and the membrane may be envisaged as a two-dimensional solution of proteins in the lipid milieu (Vanderkooi, G., 1974). Proteins undergo different degrees of penetration into the bilayer (Singer, 1971; Green, 1972). At one extreme, certain proteins do not penetrate at all and are bound by polar interactions with the membrane surfaces; these are called peripheral or extrinsic proteins. On the other hand, integral or intrinsic proteins penetrate to different extents and with different modalities. Examples of different locations are given by cytochrome (cyt) h5 in endoplasmic reticulum (Spatz and Strittmatter, 1971), glycophorin in erythrocyte membranes (Marchesi, V. T., et al., 1972), and cytochrome oxidase in the inner mitochondrial membranes (Schatz and Mason, 1974). The different situations are depicted schematically in Figure 7.

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Giorgio Lenaz

FIGURE 7. Different possible dispositions of membrane proteins. (a) An extrinsic protein; (b) an intrinsic protein anchored to the bilayer by a hydrophobic segment (e.g., microsomal cyt bs); (c) an intrinsic protein spanning the membrane (e.g., erythrocyte membrane glycophorin); (d) an intrinsic protein complex formed by different subunits (e.g., mitochondrial cytochrome oxidase); (e) an intrinsic protein completely immersed in the bilayer core (e.g., sarcoplasmic reticulum proteolipid). (See p. 411 for a further discussion of biomembrane models.)

3.1.2. Extrinsic and Intrinsic Proteins

The types of interactions between lipids and proteins are related to the extent of penetration (Lenaz, 1977). Extrinsic proteins appear mainly bound to the membrane surfaces by means of polar forces (Marchesi, S. L., et ai., 1970; Hanahan, 1973), and they may accordingly be removed by simple means such as changes of pH, ionic strength, and chelating agents. It is not excluded, however, that extrinsic proteins may also undergo some extent of penetration into the membrane; e.g., cyt c in its reduced form binds more firmly to mixed phospholipids than ferricytochrome c (Ivanevitch et ai., 1974). Letellier and Schechter (1973) found thatferricytochrome c is bound mainly electrostatically, while ferrocytochrome c is bound hydrophobically; as a result, the complex is dissociated by salt only in the case of the oxidized cytochrome. For true intrinsic proteins, hydrophobic interactions become predominant; the amino acid composition of intrinsic proteins, particularly rich in nonpolar residues (Capaldi and Vanderkooi, 1972), is in line with the view that a large extent of the protein surface is hydrophobic and hence can interact with the fatty acyl chains ofthe phospholipids. The extent of polar interactions has not, however, been determined, and may be very large; when intrinsic proteins protrude out of the membrane, as is often the case, there must be large degrees of interactions with the polar heads of the lipids. Polar interactions will be minimal for polypeptides completely immersed in the bilayer, as is the case for certain "proteolipids" [from

The Role of Lipids in Membranes

245

myelin (Folch and Lees, 1951), sarcoplasmic reticulum (MacLennan and Yip, 1973), and mitochondria (e.g., Kadenbach and Hadvary, 1973)]. 3.1.3.

Effect of Proteins on Lipid Fluidity

The interactions of lipids and proteins give rise to mutual changes of the interacting molecules. I will describe the effects of lipids on protein conformation in a later section; in this section, I will describe the effects of proteins on the physical state of membrane lipids. Such effects have been investigated by several techniques, such as DSC, spin-labeling, X-ray diffraction, and freeze-fracture electron mircoscopy (EM). Proteins affect lipids in substantially three ways (Figure 8) (Papahadjopoulos et at., 1975a). Proteins acting in the first way are basic proteins showing strong electrostatic binding; they increase the enthalpy of transition with either an increase or no change in transition temperature. Their behavior is shared by inorganic cations such as Ca2+ (Chapman et at., 1974; Verkleij et at., 1974), and is due to immobilization of the lipid polar heads. A second group of proteins , such as cyt c, also show ionic binding with the lipid bilayer, but decrease both !:::.H and temperature of transition; these proteins partially penetrate the bilayer with deformation of the bilayer itself. The shifted transition is probably related to a reorganization of the polar groups by ionic interaction with the charged groups of the proteins in fixed positions (Lenaz, 1977) leading to less efficient packing of the lipid chains. A third group of proteins [myelin proteolipid, gramicidin A, and certain amino acid copolymers (Bach and Miller, 1976)] induce a decrease of !:::.H but have no effect on the transition temperatures. The interpretation

FIGURE 8. Different interactions of proteins with the lipid bilayer. (A) Electrostatic binding (I1H increased, T t increased); (B) polar binding with partial penetration (I1H decreased, Tt decreased); (C) hydrophobic penetration (I1H decreased, Tt unchanged).

Giorgio Lenaz

246

of this behavior is that these proteins undergo complete penetration with hydrophobic binding to a limited number of lipid molecules, leaving the rest of the bilayer unperturbed. Only the behavior of the unperturbed bilayer will be detected, while a fraction ofthe lipids, directly bound to the protein, is masked in its thermodynamic behavior, probably because it undergoes an extremely broad transition, as in the case of cholesterol (Table III). 3.1.4.

Boundary Lipids

The properties of the lipids directly in contact with intrinsic proteins have been the subject of much recent study. Different techniques have shown that 20-30% of the lipids in several membranes behave differently from the remaining lipids and do not share the properties of lipid bilayer (Figure 3) (Blazyk and Steim, 1972; Trauble and Overath, 1973; Metcalfe et al., 1972; Shechter et al., 1974). Jost et al. (1973a,b) found by spin-label studies of cytochrome oxidase at increasing lipid contents that highly immobilized lipids persisted up to 0.2 mg lipidlmg protein; only above that value was a progressive increase of motional freedom found. The results indicated that on lipidation of the lipid-poor oxidase complex, the amount of bound lipids remained constant and a new class of lipids, essentially having the properties of free bilayer, appeared. Jost et al. (1973a) calculated that only one layer around the protein is highly immobilized; these immobilized lipids were called boundary lipids and are variously described as the lipid annulus or halo. The presence of boundary lipids has been confirmed for several other proteins, such as cyt b 5 (Dehlinger et al., 1974), myelin proteolipid (Papahadjopoulos et al., 1975a,b; Boggs et al., 1976), sarcoplasmic reticulum proteolipid (Laggner and Barratt, 1975), and sarcoplasmic reticulum ATPase (Warren et al., 1974b), and

Table III Effects of Different Proteins on Phospholipid Bilayers a Group

2

3

Protein Ribonuclease Polylysine Al myelin protein Cyt c Myelin proteolipid Gramicidin A (Lys-Phe)n

Penneability Small increase Small increase Large increase Large increase Large increase Large increase Large increase

"Freely after Papahadjopouios et al. (1975a) and Bach and Miller (1976).

aH

aT

Increase Increase Decrease Decrease Decrease Decrease Decrease

No effect Increase Decrease Decrease No effect No effect No effect

The Role of Lipids in Membranes

247

FIGURE 9. Boundary lipids. The scheme shows that the first layer of lipids surrounding an intrinsic protein is in a viscous condition but does not undergo a normal thermal transition.

is also supported by other techniques besides spin labeling, such as DSC, fluorescence, and X-ray diffraction (Table IV). In the case of Sindbis virus membrane (Sefton and Gaffney, 1974), the observed rigidity was also ascribed in part to interaction with the ribonucleoprotein core. The exchange of lipids from the boundary layer to the free bilayer is several orders of magnitude slower than lateral mobility within the bilayer (Warren and Metcalfe, 1976; Jost et at., 1977): for this reason, the boundary layer forms a unique body with the intrinsic protein and follows it in its lateral and rotational movements (see later). The modification of the lipid structure does not appear, however, to be confined to the lipid layer, one molecule thick, comprising the annulus; theoretical calculations (Marcelja, 1976) show that the modification of the lipid extends two or three layers beyond the annulus. The ordering oflipids

Table IV Evidence for Boundary Lipids DSC

ESR Fluorescence X-ray Enzymatic activity

A fraction of lipids does not show thermal transition (Papahadjopoulos et ai., 1975a). Lipids are immobilized near the protein (Jost et ai., 1973a,b). A fraction of lipids does not show thermal transition (Trauble and Overath, 1973). A fraction of lipids does not show thermal transition (Shechter et ai., 1974). Some 20-30 molecules of lipids per molecule of enzyme are necessary to give maximal activity (Jost et ai., 1973a,b). There is specificity in the lipid molecules that directly surround the enzyme (Warren et ai., 1975a).

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Giorgio Lenaz

within the annulus has a modified temperature dependence and becomes a continuous function of temperature for low lipid/protein ratios. The extent of lateral influence of the protein may be quite large and is highly temperature-dependent (Kleemann and McConnell, 1976). These properties must be considered in discussing aggregation of intrinsic proteins accompanying temperature-induced phase separations and the anomalous temperature dependence of membrane-bound enzymes. 3.2. Asymmetry of Membrane Components It must be stressed here, although the reader is referred to other reviews (Lenaz, 1974, 1977; Nicolson, 1976; De Pierre and Dallner, 1975; Vanderkooi, G., 1974), that each natural membrane, although it is a mosaic of lipids and proteins, must be considered as a special case, and is highly specific in its molecular relationships.

3.2.1. Asymmetry Membrane proteins are asymmetrically distributed (Carraway, 1975; Lenaz and Sechi, 1976). This is true not only for extrinsic proteins, which are bound to only one side ofthe membrane, but also for intrinsic proteins, which are immersed asymmetrically, and available to different extents at the two sides of the membrane. Nonpenetrating reagents (Carraway, 1975) have allowed workers to detect membrane protein asymmetry and to recognize that certain proteins are immersed more in one half of the bilayer than in the other half, whereas other proteins span the entire membrane thickness. The latter is the case for glycophorin, which was recognized to tranverse the erythrocyte membrane asymmetrically (Steck, 1974; Juliano, 1973), with its amino-terminal end outside, bound to carbohydrate units, and its carboxyl-terminal end on the cytoplasmic side. The two extremes are linked by an extremely hydrophobic segment that spans the bilayer (Segrest and Kohn, 1973). This segment is responsible for the appearance of the intramembrane particles observed by freeze-fracture EM, as demonstrated by Segrest et al. (1974b) by reconstitution studies using the isolated fragment and lecithin. Membrane lipids are also asymmetrical (Lenaz and Sechi, 1976); this is the case not only in artificial lipid vesicles having a low radius of curvature, but also in several natural membranes. Studies employing phospholipases, chemical probes, and immunological methods (Casu et al., 1969; Verkleij et at., 1973; Gordesky et at., 1975; Zwaal et at., 1975; Barsukov et at., 1976) have shown that the erythrocyte membrane is highly asymmetrical in its lipid distribution, having most choline phospholipids and cholesterol

The Role of Lipids in Membranes

249

outside and PE and phosphatidylserine (PS) inside. Asymmetry of lipids has also been detected in other natural membranes, and is believed to be a cause of different fluidity of the two membrane halves (Fisher, 1976). According to Sheetz and Singer (1974), membranes whose proteins and lipids are asymmetrical can act as bilayer couples; i.e., the two halves can respond differently to a perturbation. This would lead to various functional consequences, including changes in shape in the intact cells. 3.2.2.

Reasons for Asymmetry

The asymmetry oflipids is well documented both in pure lipid vesicles and in natural membranes. The reason for asymmetry may be the necessity to accommodate bulky or charged phospholipids outside when a membrane has a low radius of curvature (Litman, 1974, 1975); in other instances, there may be specific biosynthetic and metabolic reasons. In erythrocytes, lecithin is exchanged into the external membrane monolayer from serum lipoproteins (Renooj et at., 1976) in the presence of the enzyme lecithin: cholesterol acyltransferase (Reed, C. F., 1968; Glomset and Norum, 1973). In all membranes, the asymmetry may be due to the properties of the phospholipid exchange proteins (Wirtz, 1974; Johnson and Zilversmit, 1975), required to distribute phospholipids among cell membranes after their initial biosynthetic localization. The extremely slow exchange rate of phospholipids between the two membrane monolayers allows the maintenance of the established asymmetry (Rothman et at., 1976; Renooj et at., 1976). In the influenza virus, membrane phospholipid asymmetry probably reflects a similar asymmetrical distribution in the host cell surface membrane (Rothman et ai., 1976). In most cases, proteins are distributed asymmetrically in natural membranes, but their distribution becomes more homogenous after different treatments, including solubilization or delipidation followed by reconstitution of the membrane, as observed for sarcoplasmic reticulum (Packer et ai., 1974) (Table V). In other cases, intrinsic proteins appear to be asymmetrically distributed as a consequence of specific binding with asymmetrical extrinsic proteins. There is often present a chain of events in which it is difficult and perhaps not significant to find a starting point. 3.2.3.

Asymmetry of Lipids and Distribution of Proteins

Although it has not been clearly demonstrated in experimental studies to be the case, it is reasonable to assume that the presence of asymmetrical lipids could affect protein distribution in two ways. First, there may be a specific preference of a given protein for a specific type of lipid. For

Giorgio Lenaz

250

Table V Membrane Particles in Freeze-Fracture Faces of Sarcoplasmic Reticulum a Preparation Control vesicles Reconstituted vesicles

Fracture face

Particles!/LID2

Concave Convex Concave Convex

5625 650 4680 4471

aFrom Packer et al. (1974).

example, ,B-hydroxybutyrate dehydrogenase (BDH) appears to select lecithins in its environment (Houslay et al., 1975), and sarcoplasmic reticulum ATPase excludes cholesterol and acidic lipids (Warren et al., 1975b), while myelin proteolipid appears to have high affinity for cholesterol (London et al., 1974). If the two bilayer halves contain different lipids, proteins will become asymmetrically distributed in order to fulfill this preference. Another possibility is a different affinity of protein for a different physical state assumed by lipids in the two membrane halves as a consequence of chemical asymmetry (Rottem, 1975; Wisniesky et al., 1974; Haest and Deuticke, 1976). It has been demonstrated that in the case of lipid bilayers undergoing a phase separation, proteins are squeezed out of the regions that are becoming solid into the more fluid areas (Haest et al., 1974; James and Branton, 1973; Kleemann and McConnell, 1976). Such a preference has also been proved for cholesterol: when two lipids having different melting points are present in the same bilayer, and cholesterol is added, it is preferentially associated with the more fluid component (De Kruyff et al., 1974). It is therefore plausible that in an asymmetrical bilayer, proteins are preferentially shifted to the more fluid monolayer. 3.2.4. Function of Extrinsic Proteins Asymmetrically distributed extrinsic proteins, together with cytoskeletal elements (microfilaments and microtubules), represent an important anchoring support for intrinsic proteins (Nicolson, 1976). Spectrin is recognized to form a meshwork on the inner surface of the erythrocyte membrane and is in contact with the C-terminal end of glycophorin (Figure 10). The presence of spectrin has been considered a determinant for the limitation of motion of intramembrane particles (glycophorin) in the bilayer (Elgsaeter et al., 1976); such limitation prevents rapid protein diffusion in the membrane. A similar suggestion was advanced by Lenaz (1977) in the inner mitochondrial membrane, in view of the greatly increased amount of

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251

FIGURE 10. Scheme showing how spectrin prevents the lateral mobility of the erythrocyte membrane glycoproteins. Integral glycoproteins are the vertical molecules spanning the bilayer, while spectrin is represented by the horizontally placed molecules at the bottom.

lipids that can be bound to membranes devoid of extrinsic proteins (Lenaz et ai., 1970b).

3.2.5. Protein Complexes Membrane proteins are often arranged as complexes. The best-known examples are those of the inner mitochondrial membrane, especially cytochrome oxidase (Vanderkooi, G., 1974) and ATPase (Senior, 1973; Tzagoloff et ai., 1973). The cytochrome oxidase complex consists of six or seven proteins (depending on the species of origin), four of which are considered extrinsic according to the criteria of water solubility and reactivity to nonpenetrating, membrane-impermeable reagents (Poyton and Schatz, 1975a,b; Eytan et ai., 1975). The other components are very hydrophobic and are believed to be located in the inner core of the membrane. Since other lines of evidence have indicated that cytochrome oxidase is accessible to both sides of the inner membrane (Schneider, D. L., et at., 1972), the complex may be envisaged as a series of polypeptides arranged in such a way as to span the membrane as a whole. Similar considerations apply for the ATPase complex (Pedersen, 1975), in which an extrinsic component (P1) protruding into the matrix space is linked through a small intermediate protein to a "membrane factor" composed of four intrinsic subunits. The intrinsic and extrinsic subunits also appear to be separate entities by considerations deriving from biogenetic studies of mitochondrial protein complexes. In fact, the intrinsic components are synthesized by the intramitochondrial protein-synthesizing

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genetic apparatus, while the extrinsic proteins are synthesized via information from nuclear genes on cytoplasmic ribosomes (Tzagoloff et al., 1973). 3.3. Protein Mobility

Proteins are able to diffuse and to rotate in the fluid lipid medium (Cherry, 1975) (Table VI). 3.3.1. Aggregation of Intramembrane Particles

Qualitative demonstrations of protein lateral movements come from the finding that different agents induce protein aggregation in membranes (Haest et al., 1974; Elgsaeter et al., 1976; McIntyre et al., 1974; Pinto da Silva and Branton, 1972). Aggregation has been detected by means of freeze-fracture EM. With this technique, intramembrane particles usually appear distributed in a statistically homogeneous fashion; however, several treatments may induce aggregation of the particles, leaving patches of protein-free lipid bilayer. One such treatment is the cooling of the membrane below the thermotropic phase transition (Figure 11); the aggregation phenomenon is rather complex, however, and is linked to the phenomenon of phase separation. According to Marcelja (1976), the change in order of the lipid molecules surrounding a protein leads to an indirect, lipidmediated interaction among protein molecules, which may give rise to protein aggregation. Each protein molecule perturbs the structure of the surrounding lipids; however, the total perturbation of the lipid structure is lower than the individual contributions, causing a net attractive force between the proteins. The presence of protein aggregation is the result of the thermotropic behavior oflipids, but transitions do not always cause protein aggregation.

Table VI Demonstration of Protein Movements in Membranes Lateral diffusion (10-9 _10- 12 cm2/sec)

Rotation

Aggregation of intramembrane particles: cooling, pH changes, lectins Diffusion of superficial antigens Diffusion of fluorescent-labeled proteins Polarization of light absorption by protein chromophores induced by protein cross-linking Enzymatic studies Lack of light polarization by protein chromophores Light polarization of "slow" chromophores

FIGURE 11. Effect of temperature on the aggregation of intramembrane particles as detected

by freeze-fracture EM. (A) Escherichia coli membrane vesicles isolated from cells grown in the presence of oleic acid, frozen from 45"C; (B) E. coli membrane vesicles isolated from cells grown in the presence of elaidic acid, frozen from O°C. Reproduced from Shechter et al. (1974) with the kind permission of Springer-Verlag.

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Giorgio Lenaz

For example, when branched-chain fatty acids are present, as in Staphylococcus aureus membranes, no particle aggregation is observed, even well below the transitions as determined by DSC (Haest et al., 1974). The presence of cholesterol in a membrane prevents particle aggregation, in accord with its abolishing lipid-phase separation (Rottem et al., 1973; Duppel and Dahl, 1976). Other treatments, not directly related to lipids, induce protein aggregation; one such treatment is related to the trans membrane control (Nicolson, 1973b, 1976), such as that exerted by spectrin on the erythrocyte membrane integral proteins. Spectrin is an extrinsic component located on the inner side of the erythrocyte membrane (Juliano, 1973); extracted spectrin and actin have very low solubility near their isoelectric points (pH 4.8) and are precipitated by Ca2 +, Mg2+' polylysine, or basic proteins. All these conditions also induce intramembrane particle aggregation in spectrin-deficient ghosts, as a consequence of the aggregation of the residual spectrin molecules (Elgsaeter et al., 1976). When fresh ghosts (not deficient in spectrin) are exposed to the same conditions, only limited aggregation occurs; the contraction of the spectrin meshwork compresses the bilayer, causing it to extrude protein-free lipid vesicles. 3.3.2. Diffusion of Superficial Proteins Protein aggregation implies lateral displacement in the lipid phase. Such displacement has been proved qualitatively by other techniques. Superficial antigenic sites and receptors in plasma membranes are associated with the intramembrane particles; the demonstration that these sites may diffuse in the membrane, and collect under certain conditions to form a "cap" in a limited region of the cell surface, has allowed a different approach to the investigation of protein movements in membranes (cf. Nicolson, 1973a). Frye and Edidin (1970) visualized surface antigens of two different cell lines using immunofluorescence techniques and fused the cells with inactivated Sendai virus; after fusion, the antigens rapidly became intermixed, allowing these workers to calculate a lateral diffusion constant of 10-10 cm2/ sec. The mixing process is strongly temperature-dependent (Petit and Edidin, 1974). In another experiment, Edidin and Fambrough (1973) observed the spreading of a fluorescent antibody on the surface of a cultured muscle fiber and calculated a diffusion coefficient of 1-3 x 10-9 cm2/sec. Peters et al. (1974) observed in a fluorescence microscope a single erythrocyte ghost labeled with fluorescein and bleached on one half only; no diffusion of fluorescein into the bleached half could be detected in a period of 20 min, in accordance with motion restriction for erythrocyte

The Role of Lipids in Membranes

255

membrane proteins (Verma and Wallach, 1975). On the other hand, in cultured mouse fibroblasts labeled with fluorescein isothiocyanate, a recovery of fluorescence was found, with a calculated diffusion coefficient of 2.6 x 10-10 cm2/sec (Edidin et ai., 1976). The lateral diffusion of rhodopsin in frog retina was also observed using a microspectrophotometric technique (Liebman and Entine, 1974) with a diffusion coefficient of 5.5 x 10-9 cm 2/ sec. Similar values were found by Poo and Cone (1974), who observed that when the rhodopsin is bleached on one side of the membrane, the rhodopsin on the other side diffuses rapidly until uniform distribution is achieved on both sides. 3.3.3. Physicochemical Demonstration of Protein Movements Physicochemical approaches have also been attempted to show protein diffusion and rotation in membranes (Cherry, 1975, 1976), but until now they have not been without ambiguities. In retinal rod membranes, the rate of decay of dichroism ofrhodopsin after flash photolysis gave a rotational relaxation time of about 20 p.,sec at 20°C (Cone, 1972), whereas in bacteriorhodopsin from Haiobacterium halobium, the relaxation time was calculated to be slower than 20 msec (Razi-Naqvi et al., 1973). In retinal rod membranes, P. K. Brown (1972) showed that a persistent dichroism in rhodopsin absorption could be induced by previous treatment with glutaraldehyde, which is a cross-linking agent for proteins. This was taken as demonstration that proteins are free to approach each other for a sufficient fraction of time to become cross-linked, thereby implying freedom of diffusion. In cytochrome oxidase, Junge and De Vault (1975) found polarization of the 445 nm absorption of the CO complex in mitochondria, suggesting that either the enzyme rotates around a single axis parallel to the symmetry axis of the a3 heme or that it is completely immobilized. Also, EM (Vanderkooi, G., 1974) suggests a rigid lattice structure for cytochrome oxidase. The fact that in the inner mitochondrial membrane, lipid extraction does not alter the microscopic membranous appearance (Fleischer, S., et ai., 1967) and that the amount of lipids can be increased above the natural range only when extrinsic proteins are removed (Lenaz, 1977)(cf. Section 3.2.4) suggest a fixed structure for the intrinsic proteins in the intact membrane. These observations suggesting that proteins are restricted in motion in the mitochondrial membrane are in contrast to the cold-induced aggregation of intramembrane particles in the same membrane, suggesting that proteins are in a very mobile situation above the phase transition of the lipids (between -4° and -12°C) (Hochli and Hackenbrock, 1976).

256

Giorgio LeDaz

The use of "slow" probes for measuring protein mobility in the microsecond and the millisecond range rather than in the nanosecond range, as with fluorescent probes, was explored in Chapman's laboratory (Razi-Naqvi et al., 1973; Cherry, 1975); these probes involve the signals arising from triplet-triplet absorption of bound suitable probes such as eosin. 3.3.4.

Enzymatic Demonstration of Protein Diffusion

Enzymatic evidence for protein diffusion was obtained for NADH-cyt hs reductase (Rogers and Strittmatter, 1974a,b), in which the reductase flavoprotein and cyt hs are proteins partly enbedded in the microsomal membrane. The kinetics of cyt hs reduction suggest that the two proteins

meet via lateral diffusion in the plane of the membrane. According to Archakov et al. (1975), certain membrane proteins are specialized in lateral diffusion, because this process could be more rapid and efficient than bulk diffusion in the cytoplasm and could lead to very efficient equilibration between redox equivalents or between substrates in the same membrane or also in different membranes. These authors found that NADH-cyt hs reductase can also carry out electron transport between different microsomal vesicles.

4.

4.1.

EFFECTS OF LIPIDS AND THEIR PHYSICAL STATE ON THE PROPERTIES OF BIOMEMBRANES Means Employed to Investigate the Effects of Lipids in Membrane Functions

Before the role of lipids in catalytic membrane functions is approached more directly, it is convenient to discuss how the information has been obtained. Several criteria have been applied, and all have been useful in some way to obtain partial information. The initial ways of investigating lipid dependence of membrane functions were to extract the natural lipids by solvents or phospholipase digestion and to add back natural or synthetic lipid mixtures or purified single phospholipid species in order to observe the specificity in the restoration of the lost activity (Fleischer, S., et at., 1962; Fleischer, S., and Fleischer, 1967; Lenaz et at., 1970a; for a review, cf. Lenaz, 1974). A method that has certain advantages and was described more recently by Warren et al. (1974b) is that oflipid substitution (Figure 12); the natural membrane is dissolved with a detergent and dialyzed against an

o

Ａ＼Ｎｾ＠

\::::-(:0. ｾ＠

. if.

Q'

ｾ･＠

Ｎｴ＾ｾｶ＠

ｏｯＬｾ＠

• 0, 0,'

ｾＦｗ＠

re

eo,o,

. n'v

' ",0,'

'l;+-V ... 0'" , . •

•

CIOI)

ｾ＠

FIGURE 12. Scheme showing the lipid-substitution method. The membrane is solubilized in the presence of detergent, and the individual proteins are solubilized together with mixed phospholipid-detergent micelles. Dialysis in the presence of excess phospholipids (of the original or different composition) will re-form a membrane in which the original disposition of lipids and proteins is largely m&intained.

Detergent

＼＿ｊｾ＠

Vb;l;

SO/,

ｾ＠

.....

f

5'

f'"

ｾ＠

ｾ＠

t

258

Giorgio Lenaz

excess ofthe phospholipid to test. If the process is accomplished twice with a lO-fold excess of the phospholipid to test over the natural components, the result will be a 99% substitution. If one desires to change the lipid physical state without changing lipid composition, besides carrying out the investigation at different temperatures, one may take advantage of perturbing agents such as organic solvents or anesthetics (Seeman, 1972; Lenaz et at., 1975a) or other agents that affect membrane fluidity such as adamantane (Eletr et at., 1974); these compounds dissolve in the membrane, inducing fluidization. The fluidization induced by solvents is analogous to the depression of freezing point induced by different solutes in water (Hill, 1974):

where eland c 2 are the concentrations of the solute in the liquid and in the solid, respectively, and Q is the transition enthalpy. This is the basis for the entropic theory of anesthesia (Hill, 1974). Anesthesia is the result of any treatment that increases membrane disorder, and will be produced when the free energy of a nonaqueous phase has been changed by a critical amount, independent of the method used to induce this change. Another interesting approach for testing the effect of lipids is to change the lipid composition in vivo. This can be easily accomplished in microorganisms by selection of mutants incapable of synthesizing fatty acids, and adding selected fatty acids in the culture medium (Razin et at., 1966; Esfahani et at., 1971). In the case of higher animals, changes in lipid composition of various tissues may result from dietary changes [diets deficient in essential fatty acids (EFA) or supplemented with oils of known fatty acid composition (e.g., Farias et at., 1975)]. The results may be quantitatively significant if the lipid and fatty acid composition of the membranes is studied along with the physical state of the lipids and the functional parameters one wishes to investigate, in order to establish as precise correlations as possible. 4.2.

Permeability and Transport

4.2.1. Effect of Lipids on Permeability

The passive diffusion of nonelectrolytes through lipid bilayers, investigated either by release of trapped solutes from liposomes or by the osmotic behavior of liposomes following the addition of the compound to be tested,

The Role of Lipids in Membranes

259

is very much dependent on the lipid compositon of the bilayer (Wright and Diamond, 1969). The degree of unsaturation of the fatty acid chains is an important factor in determining the permeability of liposomes (De Gier et at., 1968; Chen et at., 1971); an exceptionally high degree of unsaturation of the retinal rod membrane [with up to 45% docosahexaenoic acid 22: 6 (Daemen, 1973; Hendriks et at., 1976)] may be responsible for the extremely high permeability to Na+ of liposomes derived from rod outer segment lipids, even when compared with total retinalliposomes. Another important factor in permeability is the presence of cholesterol; increasing cholesterol in liposomes decreases the permeability for glycerol (De Gier et at., 1968; Demel et at., 1972), ascorbate (Schreier-Muccillo et at., 1976), and monovalent cations (Scarpa and De Gier, 1971; Papahadjopoulos et at., 1971; Szabo, 1974). The effect of cholesterol of reducing solute permeability is also found in natural membranes (Grunze and Deuticke, 1974; Deuticke and Ruska, 1976; De Kruyff et at., 1972, 1973). All these effects are clearly related to lipid fluidity, which is increased by the presence of polyunsaturated fatty acids and decreased by cholesterol. Cholesterol induces a permeability decrease through formation of a 1 : 1 complex with the phospholipids (Demel and De Kruyff, 1976); there is a specific reduction of permeability by cholesterol up to this value. In erythrocyte membranes, the value is lower (ratio cholesterol/polar lipids of 0.6), and this is due in part to the presence of proteins (Deuticke and Ruska, 1976), with their property of sequestering part of the lipids (boundary lipids). Indeed, Warren et at. (1975b) found that cholesterol is excluded from the lipid annulus surrounding sarcoplasmic reticulum ATPase; this may be a property for most intrinsic membrane proteins. It has been found that an enhanced release of trapped solutes from liposomes ensues near the phase-transition temperature of lipids (Haest et at., 1972; Papahadjopoulos et at., 1973; Inoue, 1974; Nicholls and Miller, 1974; Blok et at., 1975). Also, ANS transport into liposomes is maximal at the lipid-phase transition (Tsong, 1975). The enhanced permeability near transition exhibits strong selectivity with respect to the molecular size of the permeating compound, and the extent of permeability depends strongly on the length of the paraffin chain in a saturated lecithin (Blok et at., 1975). The finding suggests that the increase in permeability is related to phase separation when solid and fluid lipids coexist in the same membrane. Perturbation of the lipid-phase equilibriuUl by temperature variations results in increased release of trapped solutes (Blok et at., 1976), supportat the boundaries of two ing the concept that a dynamic ｾｱｵｩｬ｢ｲｭ＠ coexisting phases is important for the formation of statistical pores through which the solutes would permeate.

Giorgio Lenaz

260

4.2.2. Effect of Proteins on Permeability

Protein interaction with lipids increases the permeability to various solutes and ions, as shown by experiments with liposomes and by the electric behavior of black lipid membranes (Haydon and Hladky, 1972; Kimelberg and Papahadjopoulos, 1971a,b; Juliano et ai., 1971; Rothstein et ai., 1975). This effect is not necessarily due to a real carrier property of the proteins, and the effect is often nonspecific; it is, however, the result only of penetrating proteins, suggesting that protein penetration perturbs the lipid bilayer creating nonspecific holes or cavities in the regions between proteins and lipids where solutes may permeate. The presence of the annulus of strongly bound immobilized lipids tends to combat this tendency and limits the increase of passive permeation resulting from irregularity in the shape of the protein molecule (Warren et ai., 1975a). The increase of permeability in the presence of proteins may be due to the protein itself, when it is composed of different subunits; this also may not yet represent a specific carrier mechanism. Pinto da Silva (1973) found that there is an enhanced permeation of water through the intramembrane particles visible by freeze-fracture EM, and suggested that this effect is due to cavities between polypeptide subunits (glycophorin). Moreover, there is an increased permeability to water and K+ in liposomes in which the glycophorin hydrophobic segment has been included, and a decreased resistance of black lipid membranes (Lea et al., 1975), suggesting that the peptide, by penetration, induces local disordering of the bilayer (Table VII).

4.2.3.

Carrier-Mediated Transport

A real carrier mechanism is due to formation of specific saturable "pores" through a bilayer membrane. Different carrier mechanisms have Table VII Effect of Glycophorin Hydrophobic Segment Tos) on Permeability Properties a K+ leakage from liposomes (% in 35 min)

Resistance of BLMb (f!/ cm2 )

Water permeability (/Lm . sec-I)

0.62 8.6

7.8 x lOS 6.3 x 10"

30.1 41.4

-Tus) +T us) "Prom Lea et al. (1975).

b

(BLM) Black lipid membranes.

The Role of Lipids in Membranes

261

been envisaged (Grell and Funck, 1973; Hladky et ai., 1974; Stein, 1972; Urry, 1975) (Figure 13). The mobile-carrier mechanism is exemplified by valinomycin (Ohnishi and Urry, 1970); this antibiotic coordinates K+ in a polar inner shell, while the exterior of the molecule is hydrophobic and lipid-soluble. By virtue of its lipid solubility, the complex can move back and forth across the membrane, discharging the K+ on the side where it is less concentrated (unless the process is coupled to an energy source, as in mitochondria). This type of mechanism is strongly temperature-dependent, and sharp discontinuities are present in Arrhenius plots of the transport at the transition temperature ofthe lipids composing the bilayer (Krasne et ai., 1971). Although documented for Valinomycin and other antibiotics, such as enniatins A, B, and C (Ovchinnikov, 1974), the actins (Ciani et ai., 1969; Eisenman et at., 1973), and nigericin (Lutz et at., 1970), and also for carriers to divalent cations, such as carboxylic acid A23187 (Reed, P. W., and Lardy, 1972; Pfeiffer and Lardy, 1976), there is no evidence for this type of transport in natural membranes of higher organisms (cf. Read and McElhaney, 1976), although breaks in Arrhenius plots for many transport systems (Esfah&ni et at., 1971; Fox and Tsukagoshi, 1972; for a review, cf. Lenaz, 1977), including H+ transport in chloroplasts (Yamamoto and Nishimura, 1976), have been described. Indeed, there is much evidence against this mechanism (Read and McElhaney, 1976), and the highest credit is given to a fixed-channel mechanism (Naftalin, 1970) exemplified by another antibiotic, gramicidin A

FIGURE 13. Different carrier mechanisms for transport of solutes across membranes. (I) A mobile carrier accepts a solute in the hydrophobic interior and discharges it to the other side. (II) Channel mechanisms. Left: a channel formed by the secondary structure of a peptide molecule (e.g .• gramicidin A). Right: a channel formed by the interstices between subunits of a multiprotein complex. (III) A conformational mechanism, whereby a solute binds to a subunit in a complex and is discharged on the other side by conformational changes involving rotation of individual subunits.

262

Giorgio Lenaz

(Hladky et ai., 1974). Gramicidin A forms an unusual type of helix, the 1T(L,D) helix, in which alternating Land D residues (Urry, 1971, 1972) are coiled in such a way as to leave a larger space than an a helix. The gramicidin channel is formed by two molecules linked head-to-head by hydrogen bonds, and is highly hydrophobic outside in contact with the lipid bilayer, which is strongly immobilized (Chapman et ai., 1974); the inside of the helix is polar and forms a water-filled channel (Veatch et aI., 1975) in which K+ moves through linkage to successive CO groups of the helix. Accordingly, gramicidin A increases the conductance of black lipid membranes (Urry et ai., 1971a). Other molecules such as alamethicin (Mueller and Rudin, 1%8; Hladky et ai., 1974; Gordon, L. G. M., and Haydon, 1976) form channels in lipid bilayers. The K+ transport through the gramicidin channel is not temperature-dependent (Krasne et ai., 1971), in accord with the fixed character of the peptide dimer in the bilayer. Peptides having the properties of channels crossing lipid bilayers have been built according to the general formula of gramicidin, with glycine substituting for D residues (Goodall and Urry, 1973). Peptides such as Nformyl-(Ala-Ala-GlY)4' COOMe form channels across bilayers with discrete increments of conductance of the order of 10-10 ohm- 1 (Goodall and Urry, 1973; Goodall, 1973). 4.2.4. Natural Carriers Several ionophoric materials of (poly)peptide nature have been isolated from natural membranes (Table VIII). Such ionophores have been tested particularly in black lipid membranes and have been found to increase the conductance of the bilayers by several orders of magnitude (Redwood et ai., 1973; Shamoo and MacLennan, 1974; Lea et ai., 1975; Shamoo and Albers, 1973; Shamoo et ai., 1974, 1976; Blumenthal and Shamoo, 1974; Changeux et ai., 1970; Romine et ai., 1974; Reader and De Robertis, 1974; Sachs et ai., 1974). The conductance increases are often evoked by specific ions, suggesting that the physiological carriers have been isolated. The electrophysiological characteristics of stepwise increases of conductance by the aforenamed ionophores suggest a channel mechanism (Katz and Miledi, 1971). Many natural transport mechanisms are strongly temperature-dependent, with breaks or discontinuities occurring at temperatures related to phase transition of the lipids (Esfahani et ai., 1971; Fox and Tsukagoshi, 1972). The existence of discontinuities at the phase transition is not incompatible with a fixed-channel mechanism; in fact, the transport process is complicated by the recognition sites and enzymatic mechanisms associated

263

The Role of Lipids in Membranes

Table VIII Some Natural Proteins with Ionophoric Activity Protein A peptide fraction from sarcoplasmic reticulum ATPase A peptide fraction from (Na+ -K+)-ATPase Acetylcholine receptor from Torpedo

ATPase from Streptococcus faecalis Mitochondrial glycoprotein + Ca2+ Glycophorin hydrophobic segment Bacteriorhodopsin A strongly bound protein fraction from erythrocyte membranes Ionophoric material from cell membrane preparations

References Shamoo and MacLennan (1974, 1975) Shamoo and Albers (1973), Shamoo and Myers (1974) Shamoo and Eldefrawi (1975), Romine et al. (1974), Jain (1974), Goodall et al. (1974) Redwood et al. (1973) Prestipino et al. (1974) Lea et al. (1975) Racker and Hinkle (1974) Lossen et al. (1973) Shamoo et al. (1974)

with the "channel" proper, and the whole conformation ofthe protein may be extremely sensitive to the state of the lipid (Lee, A. G., 1976). The significance of discontinuities in Arrhenius plots will be discussed in Section 4.3.5. A third mechanism described by Stein (1972) on the basis of ｫｩｮ･ｴｾ｣＠ properties of natural transport processes indicates that transport occurs through cooperative conformational changes of polypeptide subunits of larger protein complexes. The conformational changes should be such as to release the bound solute on the other side of the membrane. This possibility is considered difficult on thermodynamic grounds, since it involves rotation of protein subunits around the plane ofthe membrane; such rotation should be limited by the polarity of the membrane surface and the hydrophobicity of the membrane core, which favors a fixed position of the protein in the axis traversing the membrane and would allow only rotation around the latter axis or lateral diffusion. Indeed, Dutton e/ al. (1976) eliminated the possibility of a rotating carrier for Ca2+ transport in sarcoplasmic reticulum by showing that antibodies against a covalent label to (Ca2 +)-ATPase had no effect on Ca2 + transport. They also suggested a model in which a subunit aggregate spans the membrane, leaving a water-filled channel in which the ion can move. The characteristics of natural channels, however, so greatly resemble those of peptide antibiotics, that one can postulate that the mechanisms of

264

Giorgio Lenaz

channel opening and gating are similar and due to secondary structure changes rather than to gross protein conformational changes or quaternary structure changes. 4.3.

Lipids and Enzyme Activity

4.3.1. Lipid-Dependent Enzymes It has long been known that lipids are required for activity of many membrane-bound enzymes. Lists of enzymes having a lipid requirement are available in the literature (Triggle, 1970; Lenaz, 1974, 1977; Fourcans and Jain, 1974; Rothfield and Romeo, 1971). In general, it is found that lipids are required for the activity of membrane enzymes and increase enzymic activity in lipid-depleted membranes. The lipid-requiring enzymes are represented at least in part by intrinsic proteins such as the complexes of the mitochondrial respiratory chain (Brierley et at., 1962), mitochondrial ATPase (Dabbeni-Sala et at., 1974), plasma membrane (Na+-K+)-ATPase (Tanaka et at., 1971), sarcoplasmic reticulum (Ca2+)-ATPase (Martonosi, ｾＹＶＩＬ＠ and microsomal NADH-cyt b5 reductase (Rogers and Strittmatter, 1973); in other cases, the lipid-requiring enzymes are isolated with greater ease from the membranes, and their binding to the lipids in vivo may not be a very strong hydrophobic interaction. In other instances, lipids affect (but sometimes in a negative fashion) the activity of enzymes that are watersoluble and are usually considered not to be membrane-associated in vivo. This is the case with glutamate dehydrogenase (Dodd, 1973), pyruvate oxidase (Cunningham and Hager, 1971a,b), hexokinase (Knuell et al., 1973), and others. It may be considered that for these enzumes, binding to lipids represents an important regulatory mechanism (see later).

4.3.2. Specificity of Lipids a. Absolute Specificity. Certain enzymes have an absolute requirement for one type of phospholipid, or at least a preference for certain phospholipid classes. Mitochondrial BDH has an absolute requirement for lecithin (Jurtshuck et at., 1961; Gotterer, 1967; Gazzotti et at., 1975); the specificity is absolute for the choline head group, although unsaturation of the lecithin is also required. However, saturated lecithins may restore activity in the presence of unsaturated PE, while PE itself is inactive (Wilschut et al., 1976). Clearly, the need for the choline head group and that for unsaturation represent two different phenomena. The minimal requirements for BDH activity were studied by Gazzotti et al. (1975) and

265

The Role of Lipids in Membranes

Table IX Lipid Specificity for ,B-Hydroxybutyrate Dehydrogenase a

Compound

L-a-di C 4:0 PC L-a-di C 6:0 PC L-a-di C8:0 PC L-'a-di C lO :0 PC L-a-di C'2:0 PC L-a-di C'4:0 PC L-a-di C'6:0 PC L-a-di C,s:o PC L-a-1-C'8:o' 2-C'8:' PC L-a-di C,s:, PC L-a-di C'8:2 PC L-a-C'2:0 LPC Stearylphosphorylcholine

Maximal relative activity

28 95 77 72 33 44

Concentration for 50% activation (f.LM)

13

X

103

740 41

2.4 0.4 2.3

76

26

75

46 4.7 1.4 0.7 150 2.1

100

49 32

25 128

aFrom Table I of Grover et ai, (1975),

by Grover et al. (1975) (Table IX). Lecithins, lysolecithins, and stearylphosphorylcholine form active complexes with the enzyme; the L-a-di C4 :0 is the smallest lecithin forming an active complex, and L-a-C 12 : 0 is the smallest lysolecithin. A hydrophobic chain followed by a negative and a positive charge is the minimal structural requirement of an activator; however, complexes of appreciable stability are formed only with those phospholipids that exist in bilayer-membrane-like structure. The lecithins having saturated fatty acids activate the enzyme but form less stable complexes. In contrast to BDH, PE, but not lecithin, reactivates glucose-6-phosphatase activity lost on phospholipase C digestion of microsomes (Duttera et al., 1968). b. Partial Specificity. Many enzymes are restored only or optimally by negatively charged phospholipids. (Na+ -K+)-ATPase appears to require PS or other negatively charged phospholipids (Tanaka and Sakamoto, 1969; Goldman, S. S., and Albers, 1973; Walker and Wheeler, 1975); this is a rather strict requirement. The specificity is much less in restoration of respiratory activity in lipid-depleted mitochondria or in lipid-depleted respiratory complexes (Fleischer, S., et al., 1962); in these instances, small amounts of negatively charged phospholipids such as cardiolipin appear to

266

Giorgio Lenaz

optimize the ratios of the neutral phospholipids PC and PE (Racker and Kandrach, 1973). Respiratory-chain activity also depends on the presence of phospholipids containing unsaturated fatty acids. For example, ATP_32Pj exchange in vesicles free of electron-transfer complexes (Kagawa and Racker, 1971) or oxidative phosphorylation linked to the NADH-CoQ reductase segment of the respiratory chain (Ragan and Racker, 1973a,b) requires an optimal PE/PC ratio of 4: 1; however, in the presence of low amounts of cardiolipin (0.05-1.5%), the optimal ratio changes to 1: 1 PE/PC. This kind of effect has been studied in detail for mitochondrial ATPase; stimulation of the enzyme by PC was investigated in a lipid-free preparation obtained by cholate extraction of mitochondria (Dabbeni-Sala et ai., 1974). The low stimulatory activity of PC was increased by either introduction of negative amphipathic substances into the zwitterionic liposomes or addition of Cl- to the incubation system. Activation by PC was strongly dependent on the acyl-chain composition of the phospholipid, while activation by negatively charged liposomes was independent (Bruni et ai., 1975). Liposomes of acidic phospholipids or PC containing anionic compounds prevented the (ATP + Mg2+)-induced decrease of ATPase activity in submitochondrial particles containing the ATPase inhibitor subunit; in addition, acidic phospholipids prevented the inhibition of isolated ATPase by purified ATPase inhibitor. In the cases described above, a negative zeta potential seems the minimal requirement for an optimal activity of the membrane-bound enzyme. 4.3.3.

Kinetics of Membrane-Bound Enzymes

Membrane-bound enzymes in the presence of lipids have several kinetic differences in comparison with soluble enzymes or with the same membrane enzymes after delipidation. Moreover, kinetic changes are also shown by varying the lipid composition, when several lipid types can reactivate the enzyme. The literature on this subject is scattered, but there are several observations pointing to certain types of kinetic changes occurring after solubilization or delipidation of a membrane enzyme (Fourcans and Jain, 1974; Lenaz et ai., 1975a; Lenaz, 1977). In many instances, solubilization or delipidation results in complete inactivation of an enzyme; when loss of activity is only partial, or when delipidation may be accomplished in a stepwise fashion, changes in V max and Km may be investigated. In most cases described, solubilization or delipidation of an enzyme, or addition of solvent to perturb lipid-protein interactions, is accompanied by a decrease of both V max and Km for the substrate. In some cases, the

The Role of Lipids in Membranes

267

opposite is true, and this appears to be the case with many loosely bound enzymes; in still other cases, there is a decrease of V max but no change in Km. A list of some of the observed changes is presented in Table X. An analysis of such phenomena has not been attempted for most of the observations. A very interesting study to this purpose is that of Hegyvary (1973) on the effect of organic solvents on (Na+ -K+)-ATPase. Alcohols were found to inhibit the enzyme noncompetitively with respect to ATP, i.e., with a decrease of both Vmax and Km for ATP. In the presence of alcohols, the apparent affinity of the enzyme increased for Na+ and ATP and decreased for K+. By pulse-labeling, it was shown that the solvents enhanced the interaction of ATP with the enzyme, but decreased the net dephosphorylation of phosphoenzyme in presence of K+. In other words, for the reaction

organic solvents enhance the stability of E-P by decreasing both Km [approximately k2/ kl or (k2 + k3)/ kl if k3 is large] and k3 (which is proportional to V max). Considering that alcohols break lipid-protein interactions in the membrane (Lenaz et ai., 1976) and that their addition is equivalent to partial delipidation (Lenaz et ai., 1975b), the effect of phospholipids on this enzyme would be to keep a high turnover of the enzyme-substrate complex by favoring both its dissociation and product formation. In the case of Ca2 +-ATPase from sarcoplasmic reticulum, phospholipids have been found not to be required for formation of phosphoenzyme intermediate, but to be required for the subsequent hydrolysis of the phosphoenzyme (Martonosi, 1969; Meissner and Fleischer, 1972; Hidalgo et ai., 1976). A decrease of both V max and Km for ATP in mitochondrial ATPase is observed after addition of several solvents and anesthetics to mitochondria or submitochondrial particles, as well as by phospholipase A2 hydrolysis of the mitochondrial membrane (Landi et ai., 1976) (Figure 14). This comparison appears to substantiate the proposal that solvents affect the enzymic protein indirectly through alterations of the lipids (Lenaz et ai., 1975a,b). In contrast to this study, Swanljung et ai. (1973) had found that a lipid-poor ATPase isolated by cholate treatment has a higher Km for ATP than the phospholipid-reconstituted enzyme. The effect of solvents and anesthetics was shown in another study (Lenaz et ai., 1976, 1978) to consist not only in fluidization of the lipids, but also largely in breakage of lipid-protein interactions, since the disordering effect, shown by spin-label techniques, is more pronounced in membranes

268

Giorgio Lenaz

Table X Kinetics of Membrane-Bound Enzymes a Enzyme

Treatment

Vrnax

Km

References

Decreased lipid content Delipidation

Decrease

Decrease

Gotterer (1%7)

Increase

Increase

Glucose-6-phosphatase

Phospholipase A Detergent

Decrease Decrease

Decrease Decrease

Succinate-cyt c reductase (Na+ - K+)-ATPase Beef brain Electrophorus

Delipidation

Decrease

Decrease

Cunningham and Hager (l971a,b) Zakim (1970). Soodsma and Nordlie (1%9) Yu et al. (1973)

Delipidation Phospholipase C

Decrease Decrease

No change Decrease

Cytochrome oxidase

Delipidation

Decrease

Decrease

ATPase (beef heart mitochondria)

Phospholipase A2

Decrease

Decrease

Tanaka et al. (1971) S. S. Goldman and Albers (1973) W. L. Zahler and Fleischer (1971) Landi et al. (1976)

Butanol Alcohols Delipidation

Decrease Decrease Decrease

Decrease Decrease Increase

Landi et al. (\976) Hegyvary (1973) Swanljung et al. (1973)

BDH Pyruvate oxidase

(Na+ - K+)-ATPase ATPase (mitochondrial, isolated)

than in isolated lipids, and involves a disappearance of the immobilization induced by intrinsic proteins on membrane lipids. The kinetics of membrane-bound enzymes may also be affected by changing lipid composition in vivo_ The activity of (Mg2+ -Na+ -K+)ATPase ofliver plasma membranes was higher in EPA-deficient rats; there was an increase of both V max and Km for ATP in comparison with EPAsupplemented membranes (Brivio-Haugland et at, 1976). An increase of (Na+ -K+)-ATPase specific activity was also observed in brain homogenates and synaptosomal membranes from EPA-deficient mice (Sun and Sun, 1974). On the other hand, in cholesterol-enriched human erythrocyte membranes, there was a marked inhibition of a cotransport of Na+ and K+ sensitive to the diuretic furosemide, but not to the classic inhibitor of (Na+ -K+)-ATPase, ouabain, accompanied by a significant decrease of Km for both Na+ and K+ (Wiley and Cooper, 1975). In reconstitution experiments in which different types of lipids are involved, few studies have examined the affinity of enzymes for their substrates, and usually only the rates of enzymic activity were measured. When Km was measured, it was found to be unaffected (Kimelberg, 1975) or also modified (Swanljung et al., 1973). In a model study (Cho and Swainsgood, 1974), rabbit muscle lactate

269

The Role of Lipids in Membranes

dehydrogenase was covalently bound to glass beads. The bound enzyme exhibited an increased K m for N ADH and a decrease of K m for pyruvate. 4.3.4.

Allosteric Behavior

Changes in the allosteric behavior of membrane-bound enzymes have been described as a result of changing lipid composition in experimental animals fed different diets (Farias et ai., 1975) (Table XI). A ｾ＠ V 100

070

040

1

o

B

1 232

5.85

1163

ATpmM-1

2325

1 V

• Butanol

005

Control

D

003

.001

-"""'--------1-------,-------....10

ａｾｰｭｍＭＧ＠

FIGURE 14. Effect of phospholipase A2 and of n-butanol on the Lineweaver-Burk plots of mitochondrial ATPase. (A) Phospholipase A, treatment; (B) n-butanol treatment.

270

Giorgio Lenaz

Table XI Allosteric Behavior of Some Membrane-Bound Enzymes Parias et al. (1975) Enzyme (Na+ -K+)-ATPase

Acety \cholinesterase (Mg'+ -CaH)-ATPase Acetylcholinesterase - Cholesterol + Cholesterol

Source

Effector

Erythrocyte

Na+ K+ PPP-

Erythrocyte Heart Kidney Brain Erythrocyte Erythrocyte Erythrocyte

n + EPA a

-

ppPMg'+ P-

2.9 2.1 2.8 2.1 1.9 1.9 1.6 1.6 2.0

Pat-free

-

2.0 1.4 1.6 1.4 1.0 1.0 1.8 1.0 1.3

- 1.5 - 1.0 Landi et al. (1976)

Enzyme

Effector

Mitochondrial ATPase

Oligomycin

Treatment

Phospholipase A2 Butanol 0.1 M

na

-2 -1 -1

"(n) Hill coefficient indicative of cooperativity; (EFA) Essentiru fatty acids.

In rats fed a fat-free diet, the allosteric kinetics for fluoride inhibition of ATPases from different tissues changed from Hill coefficients of - 2 to - 1, in comparison with corn-oil-supplemented rats (Farias et al., 1970), and a relationship was found with a change of the double bond index/saturation ratio (the ratio between the total number of double bonds and the number of saturated fatty acids as a percentage of total fatty acids), indicative of fluidity (Bloj et al., 1973a,b). (Ca2+)-ATPase from E. coli presented similar behavior in relation to membrane fluidity. The activation by Mg2+ of erythrocyte (Mg2+ -Ca2+)-ATPase also shows a sigmoidal character, though only in the presence of 0.2 mM Ca2+, but the Hill coefficients for activation range from 2.0 in rats fed corn oil down to 1.13 in rats fed hydrogenated fat (Galo et al., 1975); there is a strict correlation between the absolute values for n and the ratio between the double bond index and saturated fatty acids in erythrocyte lipids. Also, the effects of loading membranes with cholesterol in vitro are consistent with there being a direct relationship between membrane fluidity and allosteric behavior of certain enzymes (Bloj et al., 1973a).

The Role of Lipids in Membranes

271

Partial lipid removal or n-butanol addition to submitochondrial particles changes the oligomycin inhibition curve of ATPase from sigmoidal to hyperbolic, with n changing from about -2 to -1 (Landi et al., 1976). On the other hand, solubilization of E. coli ATPase induces a change from noncooperativity in the bound state to positive cooperativity in the soluble enzyme (Carreira and Munoz, 1975). According to Farias et al. (1975), the allosteric behavior, shown as a change in cooperativity, is the most sensitive index of membrane lipid fluidity. 4.3.5.

Arrhenius Plots of Membrane-Bound Enzymes

A kinetic parameter that has been widely studied in recent years as a function of the physical state of membrane lipids is the temperature dependence of enzymic activity, showing large deviations from the linear pattern of most soluble enzymes (Raison, 1973; Lenaz, 1977) predicted by the equation of Arrhenius: E == 2 303 R'P d log k A· dT

where EA is the activation energy and k is the rate constant. EA is related to the enthalpy difference tlHt. between the transition state and the reactant by

a. Breaks in Arrhenius Plots. In contrast to Arrhenius plots of most soluble enzymes showing straight lines with constant activation energies over a wide range of temperatures, those of most membrane-bound enzymes show sharp discontinuities with sudden increases of EA below the discontinuities (usually 2- to 4-fold increases) (Figure 15). Lists of membrane-bound enzymes showing discontinuities in Arrhenius plots have been presented in other reviews (Lenaz, 1977; Sechi et al., 1973). The number of such enzymes known is increasing, and the discontinuities have been rigorously confirmed in many cases after account was also taken of possible artifacts arising from Km changes with temperature (so that assays may not be constantly under V max conditions) (Sullivan et al., 1974) and of temperature-dependent pH changes (Kimelberg, 1975). Breaks in Arrhenius plots are not confined to such membrane activities as membrane-bound enzymes, but are also found in permeability properties (Nobel, 1974), in transport systems (see above), in hormone binding to membranes (Bashford et al., 1975), in hormone-stimulated adenylate

272

Giorgio Lenaz

4

-----

0-0

CI

E c

E

-:::,. Cl.

'0 E

control Triton-X-l00 n- butanol

3

ｾ＠

::>

...u> "u ::u

a. 2

Gl

III

CI

0 -'

32 Ｓｾ＠

3A

3S Ｓｾ＠

FIGURE 15. Discontinuities in Arrhenius plots of mitochondrial ATPase.

cyclase (Houslay et ai., 1976), and in physiological activities that are to be considered as resulting from complex biochemical reactions (Raison, 1973). The correlations are usually complex; e.g., the ouabain-sensitive K+ influx in erythrocytes exhibits far less temperature sensitivity than the (Na+ -K+)-ATPase prepared from the same cells. The physiological significance of such breaks is substantiated by the adaptation of many organisms so as to grow at low temperature by increasing lipid unsaturation and therefore fluidity. In such organisms, breaks in Arrhenius plots of membrane enzymes occur at lower temperatures or do not occur at all (Lyons and Raison, 1970; Raison and Lyons, 1970; Lyons, 1973; Raison, 1973; Sinensky, 1971; Tanaka and Teruya, 1973; McMurchie et ai., 1973; Smith, C. L., 1973; Keith et ai., 1975; Wodtke, 1976).

The Role of Lipids in Membranes

273

Sometimes the Arrhenius plots may be modified by assay conditions; comparison of temperature dependence of an enzyme with lipid properties or comparison of different activities in the same membrane should also consider the assay conditions (addition of extrinsic proteins, metal cations, pH differences). For example, in our hands, cytochrome oxidase in mitochondria showed no break when assayed using reduced cyt c as substrate, but a break at 24°C in a manometric assay with ascorbate as substrate (Sechi et ai., 1973). However, even using a similar spectrophotometric assay, M. P. Lee and Gear (1974) found no break in cytochrome oxidase activity, whereas Wilschut and Scherphof(1974) found two breaks at about 17° and 25°C. In fact, assay conditions can modify the fluidity of the membrane, and there is no reason to doubt that the breaks usually represent true indications of phase changes of the membrane lipids. Lipid perturbation with solvents and anesthetics (Lenaz et ai., 1975a) or other perturbers such as adamantane (Eletr et ai., 1974) affects the break temperature and the activation energies of membrane-bound enzymes.· Phospholipase A2 hydrolysis of mitochondrial membranes enhances the break temperature of ATPase (Landi et al., 1976) and of succinate cyt c reductase and cyt c oxidase (Wilschut and Scherphof, 1974); the activation energies of ATPase are increased (Landi et al., 1976) and those ofrespiratory activities are decreased (Wilschut and Scherphof, 1974). Also, the breaks in Arrhenius plots of microsomal enzymes, glucose 6-phosphatase, and UDP-glucuronyltransferase were abolished by phospholipase A treatment (Eletr et al., 1973). Furthermore, detergents may abolish the discontinuities (Lenaz et al., 1972). Besides the effects of medium composition on the physical state of lipid dispersions (and hence on Arrhenius plots of enzymes), the presence oftransportable substrates or the metabolic state of the cell was also found to affect the membrane lipid transition; Zimmer et al. (1975) found opposite changes induced by glucose and sorbose on the transition of erythrocyte membranes. The state of the glycolytic metabolism (i.e., the number of glycolyzing and nonglycolyzing cells) and the ATP level in erythrocytes were found to affect lipid-protein interactions and membrane organization (Gazitt et al., 1975; Haest and Deuticke, 1975). b. Correlations of Breaks and Lipid Physical State. In many investigations, attempts have been made to correlate the breaks in Arrhenius plots with critical temperatures associated with phase transitions of the membrane lipids. Raison et ·al. (1971) found a break in mitochondrial enzymic activities correlated with a break in the temperature dependence of the mobility of stearic acid spin labels in the membrane. A similar correlation was found for (Na+ -K+)-ATPase (Grisham and Barnett, 1973)

274

Giorgio Lenaz

and microsomal enzymes (Eletr et ai., 1973). Studies of reconstitution in vitro with different phospholipids and purified enzymes have shown correlations (but not identities!) between breaks in Arrhenius plots of enzymic activity and the fluidity, or the transition temperatures, of the added phospholipids (Kimelberg and Papahadjopoulos, 1974; Charnock and Bashford, 1975); also, changes in lipid composition affected by medium changes (for microorganisms) or dietary changes (for higher animals) have shown such correlations (Esfahani et al., 1971; Fox and Tsukagoshi, 1972; Solomonson et al., 1976). In a more recent investigation, Raison and McMurchie (1974) found two breaks in Arrhenius plots of succinoxidase. In rat liver mitochondria, the discontinuities occurred at 24° and goC; in sheep liver mitochondria, they occurred at 29° and 16°C. The two breaks were correlated with the onset and completion of "lateral phase separation" of membrane lipids, as the result of the abrupt changes in molecular ordering observed on spinlabeled membrane lipids at the same temperatures at which the breaks occurred. Similar results and correlations were obtained for other enzymic activities by Kimelberg (1975), Linden et al. (1973), Wisnieski et al. (1974), and Gruener and Avi-Dor (1966). In some studies, different activities from the same membrane exhibit breaks at different temperatures (Lenaz et al., 1972; Esfahani et al., 1971; Mavis and Vagelos, 1972; Lee, M. P., and Gear, 1974). Table XII shows examples for the mitochondrial membrane enzymes. There may be a number of reasons for such findings. Besides differences in assay conditions (see above), one may envisage a specific association of lipids having different fluidities. In addition to this possibility of a microcompartmentation in the plane of the membrane, it is possible that the different fluidities depend on a more or less deep situation ofthe enzyme in the membrane (for the fluidity gradient of the bilayer), or its situation in one or the other monolayer comprising the lipid bilayer. Occurrence at different temperatures of multiple breaks associated with lipid physical changes was described for lipid order parameters (cf. Section 2.3) obtained by stearoyl nitroxide spin-label ESR techniques (Morrisett et al., 1975). The question of the correspondence of the break of an Arrhenius plot of an enzymic activity to a physical change is very important, and the literature on this subject appears confusing. One reason is that different physical techniques measure different physical changes (Lenaz et ai., 1975a). Morrisett et al. (1975) investigated the relationship between organization of lipid components of E. coli membranes, studied by spin-labeled fatty acids and pyrene excimer fluorescence, and the activities of two

ATPase BDH 4. Succinate oxidase

Cyt c oxidase 3. Succinate oxidase Succinate-cyt c reductase Cyt c oxidase

Succinate Malate-pyruvate State 3 respiration State 4 respiration C l-CCP respiration DNP-ATPase ATP-supported Ca2 + uptake Respiration-supported Ca2 + uptake Valinomycin-K+ uptake Energy -swelling Succinate-cyt c reductase NADH-cyt c reductase Cyt c oxidase ATPase in sonicated particles 2. Succinate-cyt c reductase

I. ADP phosphorylation

Enzyme or enzyme system

16.0 18.5 19.0 23-24 12 8-24 17.9

12-23 21-31 18-27 26.0 20.0 26.0

16.9-28.0 18.0-30.0 17.5-27.5 16.0-30.0 17.0-28.0 15.7-25.3 19.0-27.5 12.5-26.5 12.0-28.5 13.0-27.5 27.5 27.5

Break tempts) (OC)

Rat liver mitochondria in state 3 Trout mitochondria

Keilin-Hartree particles Rat liver mitochondria Chilling-sensitive plant

(Assayed manometrically) (Assayed spectrophotometically)

Beef heart mitochondria

(+ phospholipase A)

Source or (comments)

Table XII Breaks in Arrhenius Plots of Mitochondrial Enzymes

Zeylemaker et al. (1971) Kumamoto et al. (1971) Kumamoto et al. (1971) Raison and McMurchie (1974) Kemp et al. (1968) Lyons and Raison (1970)

Sechi et al. (1973)

Wilschut and Scherphof (1974)

M. P. Lee and Gear (1974)

References

ｾ＠

....

N

'=' § Dehydrogenase Atebrin

Indophenol

I

e

>b

\ e-

(

i

>X

Azide Triiodothyronine

FIGURE 4. Scheme for a sequence of redox carriers in the plasma membrane arranged to account for general inhibition by atebrin and partial inhibition of dehydrogenase, but stimulation of oxidase, by triiodothyronine and azide.

Frederick L. Crane et al.

368

Table XI Values Reported for NADPH-Cytochrome c Reductase Activity in Plasma Membrane Preparations

Source

NADPH-cyt c reductase activity (nmoVmin per mg protein)

Rat liver

Rabbit liver

Chick fibroblast

Chick synaptosome Rat synaptosome

66-117 243 120 10 8 1-10 Trace, 0 0 0 9 9 52 0 H)

Chick myoblast Rabbit leucocyte

4

< 0.8

Reference B. Fleischer and Fleischer (1970) Wisher and Evans (1975) Brown et al. (1976) Berman et al. (1969) Colbeau et al. (1971) Aronson and Touster (1974) Crane (unpublished) Ichikawa and Mason (1973) Ichikawa and Yamano (1970) Bingham and Burke (1972) Schimmel et al. (1977) Schimmel et al. (1973) Babitch et al. (1976) Morgan et ai. (1971) Gurd et al. (1974) Vandenburgh (1977) Woodin and Wieneke (1966)

Squid retinal axon Rabbit neuron Guinea pig pancreas KB cells LI210 mouse leukemia cells Rat adipocyte Rat intestinal epithelium (basal-lateral) Dictyostelium discoideum Acanthamoeba castellanii Oat roots Phaseolus aureus hypocotyl Human erythrocyte Human intestine brush border Mouse liver Bovine adrenal chromaffin granule

0 2 0.7 50

Fischer et al. (1970) Henn et al. (1972) Meldolesi et al. (1971) Charalampous et al. (1973)

1.9 180 85

Tsai et al. (1975) Giacobino and Chmelar (1975) Crane (unpublished)

0.2 25 4-8 0 14

Douglas et al. (1972) A. A. Green and Newell (1974) Gilkes and Weeks (1977) illsamer et al. (1971) Hodges and Leonard (1974)

41 (?) 0 Trace 6.5 0

Bowles and Kauss (1976) Zamudio and Canes sa (1966) Schmitz et at. (1973) Goldenberg et al. (1978) Flatmark et al. (1971a)

Debydrogenases oftbe Plasma Membrane

369

drogenase in plasma membrane should be considered carefully. Very few studies have been done using alternative acceptors or selective inhibitors. MukheIjee and Lynn (1977a) reported an NADPH oxidase in adipocyte plasma membranes and reported that it is strongly stimulated by insulin. This activity is also stimulated by tbiol reagents such as p-hydroxymercuribenzoate or tosyl-L-Iysyl chloromethyl ketone. Hydrogen peroxide production parallels the oxidation of NADPH, which suggests the autooxidation of a flavoprotein dehydrogenase. Vas siletz et ai. (1967) also reported NADPH oxidase in liver plasma membrane at 1.5 times greater activity than NADH oxidase. They found NADH oxidase and NADPH oxidase in plasma membrane to be about one third the respective activity in microsomes. Indirect evidence was presented for NADPH oxidase in leucocyte membranes during phagocytosis (Takanaka and O'Brien, 1975), but the majority of the induced, neutrophil, cyanide-insensitive NADPH oxidase appears to be associated with a flavoprotein in internal membranes (Babior and Kipnes, 1977), though it is apparently not derived from the activity of the soluble myeloperoxidase (Kakinuma and Chance, 1977; Murphy, 1976). Woodin and Wieneke (1966) found no NADPH oxidase in rabbit leucocyte membranes. The importance of a membrane-based NADPH oxidase as a source of H 20 2 in leucocytes is evident from the fact that chick leucocytes that are deficient in myeloperoxidase still produce H2 0 2 during phagocytosis (Bellavite et ai., 1977). Emmelot and Benedetti (1967) found no NADPH oxidase in rat liver plasma membranes in contrast to rates of 5-10 nmol/min per mg protein for endoplasmic reticulum. We have also observed a similar lack of activity for rat liver membrane when assays are carried out under similar conditions. Endoplasmic reticulum can also oxidize NADPH either through the cyt P450 or directly by autooxidation of the NADPH dehydrogenase. The latter reaction will generate H2 0 2 and is stimulated by catechols (Augusto et ai., 1973; Hildebrandt and Roots, 1975). Oxidation through P450 is stimulated by a suitable substrate, such as phenobarbitol, for the mixedfunction oxidase and does not lead to peroxide formation. A mixedfunction NADPH oxidase of this type, which hydroxylates dehydroepiandrosterone, was reported in calf lens fiber plasma membrane (Vermorken et ai., 1977). 3.4.

Xanthine Oxidase

Xanthine oxidase in milk has been known to be associated with membranes in the milk fat since Morton's studies in 1954 (Morton, 1954). It has usually been assumed that these membranes were derived from endo-

370

Frederick L. Crane et aI.

plasmic reticulum, but more recent studies have identified them as specialized apical plasma membrane of the milk-secreting cells (Keenan et al., 1970). There is also a certain amount of the enzyme in the cytosol. Xanthine oxidase is found in other tissues such as liver and intestine, but the location in these tissues has had almost no study (Bray, 1975). Activity has recently been found in rat liver and adipocyte plasma membranes, but the proportion of soluble to membrane-bound activity remains to be determined (Table XII). Jarasch et al. (1977) compared the activity in milk fat globule membranes with the activity in endoplasmic reticulum from the same tissue, and found that the xanthine oxidase is clearly concentrated in the plasma membrane. A similar difference is seen between liver plasma membrane and endoplasmic reticulum (Crane and Mom!, 1977). Both Briley and Eisenthal (1975) and Jarasch et al. (1977) found that the xanthine oxidase in the membrane fraction is tightly bound and is only partially extracted with salt solutions. From relative activity comparision with the purified enzyme, it can be estimated that xanthine oxidase is up to 10% ofthe protein in milk fat globule membrane. The enzyme appears to be on the cytoplasmic side of the membrane (Nielsen, C. S., and Bjerrum, 1977). Briley and Eisenthal (1975) pointed out that the soluble purified xanthine oxidase has a ratio of xanthine oxidase activity to NADH oxidase activity (X: N ratio) of 110, whereas the membrane-bound enzyme has an X:N ratio of 50. By adding inactivated membrane material to the purified enzyme, they were able to lower the X:N ratio. The same effect could be induced by solvents in the assay mixture. They concluded that the association with membrane alters the catalytic properties of the enzyme. Table XII Xanthine Oxidase or Dehydrogenase Activity in Plasma Membrane Preparations Xanthine dehydrogenase activity (nmol/min per mg protein) Source

Acceptor

Plasma membrane

Endoplasmic reticulum

Bovine mammary gland a Rat mammary gland a Rat liver

O2 O2 MBb

180 10 17

3 2

Rat adipocyte

Cyt c

o

Reference Jarasch et al. (1977) Jarasch et al. (1977) Crane and ｍｯｲｮｾ＠ (1977)

aMilk fat globule membranes. "Methylene blue.

8

Crane and Low (unpublished)

Dehydrogenases of the Plasma Membrane

371

Aside from a pathway of purine catabolism, there is no major function for xanthine oxidase, and individuals genetically deficient in the enzyme do not appear to suffer serious consequences (Bray, 1975). It should be noted, however, that there have occasionally been suggestions of more than one enzyme (Tuttle and Krenitsky, 1977; Bray, 1975). It has also been common to assay soluble xanthine oxidase activity and not membrane-bound activity. Both insulin (Wu et al., 1977) and vitamin E deficiency (Catignani et al., 1974) increase the soluble xanthine oxidase in liver, but their effect on membrane-bound enzyme is not known. Xanthine oxidase acts as a superoxide generator when substrates are oxidized with molecular oxygen (Fridovich, 1970; Kellogg and Fridovich, 1975). The close association of the enzyme with unsaturated lipids in a membrane suggests an influence on membrane composition. Oxidation of cathecholamines, such as epinephrine, in conjunction with xanthine oxidase also involves superoxide (Bray, 1975). 3.5.

Other Dehydrogenases

Substrates other than the pyridine nucleotides and xanthine may be oxidized by enzymes in the plasma membrane. Specific examples in which the plasma membrane may be implicated are a-tocopherol, glutathione, and retinol. a-Tocopherol has long been considered as a possible antioxidant for plasma membrane lipids. J. Green (1972) proposed that specific oxidation reactions in the plasma membrane may account for the good antioxidant activity of tocopherol in the membrane in contrast to its poor activity in solution. Specific unknown products of a-tocopherol oxidation were recognized in erythrocyte membranes (Shimasaki and Privett, 1975). A specific effect of a-tocopherol, in contrast to artificial antioxidants, is preventing ascorbate inhibition of ACTH stimulation of adenylate cyclase in adrenal cells. This suggested an enzymatically defined role for tocopherol (Nathans and Kitabchi, 1975). Evidence was also presented that tocopherol deficiency induces an increase in phosphodiesterase activity and that tocopherol analogues can inhibit the enzyme (Schroeder, 1974). Oxidation of glutathione with milk fat globule membranes (Kitchen, 1974) as well as with several membrane fractions from liver (Isaacs and Binkley, 1977) has been reported, and would most likely be catalyzed by membrane-bound glutathione peroxidase. Thiol transferase, which leads to mixed disulfide formation between glutathione and proteins, is also found in membrane fractions, some of which may contain plasma membrane (Varandani, 1973; Isaacs and Binkley, 1977). Glutathione was shown to activate the adenylate cyclase in sea urchin membrane fractions (Gentleman and Mansour, 1974). The many effects of glutathione as a redox agent on

Frederick L. Crane et al.

372

plasma-membrane-controlled functions such as ATPase led Dikstein (1971) to propose a regulatory function at the cell membrane. A tightly bound NADPH-retinal reductase was found in retinal rod outer segment membranes (De Pont et ai., 1970; Futterman, 1963). This enzyme is different from the soluble retinol dehydrogenase of whole retina, which uses both NADH and NADPH. Since the membranes of the outer segment are derived from plasma membrane (Daemen, 1973) and since retinol is found in rod outer segment plasma membrane (Basinger et ai., 1976) as well as in liver, kidney, and erythrocyte plasma membranes (Mack et ai., 1972), the general distribution of retinal reductase should be considered. Ascorbic acid oxidase activity was reported in some preparations that contain plasma membrane (Honda, 1957; Forti et ai., 1959; McConnell, 1965).

4.

4.1.

RELATIONSHIP OF DEHYDROGENASES TO MEMBRANE FUNCTION Energy-Linked Transport

Evidence for plasma membrane transport function coupled to electron flow has been presented. The generation of a proton gradient by directed electron flow across a membrane is the most widely accepted energycoupling theory (Mitchell, 1976, 1977). If there is an electron-transport system in the plasma membrane, then it may be used for direct energization of metabolite transport without mediation by ATP, as in the calcium ion and metabolite transport systems of mitochondria and bacterial plasma membrane (Moyle and Mitchell, 1977; Mitchell, 1972; Kaback, 1972). Direct coupling of membrane oxidoreduction carriers and ion transport was proposed for plants (Lundegardh, 1945; Robertson, 1968) and for animals (Conway, 1951). The discovery of the Na+,K+-ATPase as the basis for Na+ and K+ transport (Skou, 1965) and the lack of redox elements in plasma membranes has discouraged interest in direct coupling. Stimulation of respiration in cells by added salts is generally interpreted as an indirect effect on coupled mitochondrial oxidation. The ADP produced by the hydrolysis of ATP during transport returns to the mitochondria and stimulates respiration. A precise definition of the relationship among salt-induced respiration, ADP levels, and mitochondrial stimulation remains to be made (Hersey, 1969). For example, Gubitz et al. (1977) reported that ouabain inhibits Na+,K+-stimulated respiration in brain, even in the presence of the uncoupler 2,4-dinitrophenol, which eliminates the requirement for ADP for

Dehydrogenases of the Plasma Membrane

373

respiration. There are also unexplained differences in the effects of respiratory inhibitors on plant membrane potentials, although the overall ion transport correlates well with ATP levels (Anderson, W. P., et al., 1974). However, active extrusion of CaH by Paramecium is not correlated with ATP supply (Browning and Nelson, 1976), and Wins and Schoffeniels (1968) reported inhibition of the MgH ,CaH-stimulated ATPase of erythrocyte membranes by redox agents and inhibitors such as indophenol and atebrin. As reviewed by Hersey (1974), proton secretion by gastric mucosa has been proposed to be driven by electron transport or by ATP. A protonstimulated ATPase was found in gastric mucosa (Chang et al., 1977; Schackmann et al., 1977), but its location on plasma membrane was questioned (Van Amelsvoort et al., 1977). Hersey (1977) also noted unusual amytal effects on HC1 secretion, which are not reversed by agents that bypass mitochondrial amy tal inhibition. The mechanism of plasma membrane proton transport remains to be determined. Amino acid transport in bacteria is coupled directly to plasma membrane electron transport (Kaback, 1972). In animals, the most favored mechanism has involved cotransport with Na+ and removal of Na+ by the ATPase. A direct redox-driven amino acid transport system was described in isolated plasma membranes by Garcia-Sancho et al. (1977), and there is evidence that this system may be specifically inhibited by wheat germ agglutinin, which does not inhibit the Na+,K+-ATPase (Li and Kornfeld, 1977). Inhibition by atebrin is similar for the amino acid transport and the NADH oxidase of plasma membranes. The unexplained effects of cyanate and triiodothyronine on a-aminoisobutyrate transport (Lea et al., 1977; Goldfine et at., 1975) may also be caused by an inhibition or stimulation of the plasma membrane redox system, since they do not seem to directly affect ATP levels in the cell. Lever (1977) also proposed an electricalgradient-driven amino acid transport in fibroblasts, and he showed that plasma membrane retains 2.6% of the total NADH oxidase in a homogenate, but only 0.1% of the succinic dehydrogenase. Amino acid transport and glucose transport through plasma membrane have been reported to be stimulated by triiodothyronine. Furthermore, triiodothyronine binding sites have been found on liver plasma membranes (Pliam and Goldfine, 1977), so it is possible that triiodothyronine acts directly on a plasma membrane transport system. The affinity of the plasma membrane site for triiodothyronine and the specificity for triiodothyronine compared with thyroxine are remarkably similar to the affinity and selectivity for triiodothyronine observed in the stimulation of the plasma membrane NADH oxidase (Gayda et al., 1977). It is suggested that a plasma membrane binding site for triiodothyronine [dissociation constant (K d) =

374

Frederick L. Crane et al.

220 nM] may be the site through which triiodothyronine affects the plasma membrane NADH dehydrogenase. Active transport of carnitine through muscle cell membranes is inhibited by azide or lack of oxygen (Rebouche, 1977) or by fluoride, but not by iodoacetate or cyanide (M0lstad et ai., 1977). This suggests a relationship to the azide- and fluoride-sensitive NADH dehydrogenase in plasma membrane (see Table X), rather than dependence on ATP generation. ATP production through either glycolysis or oxidative phosphorylation would be inhibited by iodoacetate or cyanide. Sugar transport through plasma membranes is usually by facilitated diffusion and metabolic conversion inside the cell (Andrews and Lin, 1976). It would not be expected that redox function would be needed to energize transport (Carter et ai., 1972). There are, however, several studies that emphasize redox control of transport in the plasma membrane (Czech, 1977), and these studies will be discussed later. In Saccharomyces fragilis, a system for active transport of sorbose or 2-deoxY-D-galactose, but not glucose, was proposed. The process requires oxygen, it is associated with development of a pH gradient, and a chemiosmotic coupling mechanism was proposed (Jaspers and Van Steveninck, 1977). Most studies of pinocytosis and phagocytosis support the concept that ATP is used to drive the membrane movement by means of the contractile proteins in the cytoplasm. However, there are discrepancies in comparative effects of glycolytic and respiratory inhibitors that remain to be explained (Silverstein et ai., 1977). For example, the selective inhibition of pinocytosis in fibroblasts by 10-3 M azide compared with almost no inhibition of respiration (Steinman et ai., 1974) should be considered in the light of azide effects on the plasma membrane NADH dehydrogenase. Extrinsic dehydrogenases attached to the plasma membrane would not be expected to participate directly in the development of a proton gradient, since they are not oriented to act as transmembrane porters. As pointed out by Schrier (1966) and Fossel and Solomon (1977), ATP generated in a confined region next to the membrane by glyceraldehyde-3-phosphate dehydrogenase could be more readily available for the membrane-bound Na+,K+-ATPase than could ATP from mitochondria. Therefore, enzyme functions could be facilitated by the proximity of other attached enzymes. Detachment of the enzyme in response to metabolic changes in the cell could decrease ATP, or expose the membrane site to cytoplasmic sources of ATP. A similar local supply strategy was proposed for lignin formation in plant cells (Gross, 1977). The malate dehydrogenase associated with the cell wall would produce NADH, which would act as an electron donor for the formation of H 20 2 by a peroxidase in the cell wall. The H20 2 would

Dehydrogenases of the Plasma Membrane

375

then be used for lignin formation. Whether the malate dehydrogenase is attached to membrane fragments in the wall fraction, or is actually in the wall, remains to be determined. 4.2.

Metabolic Conversions

The number of metabolic conversions attributed to the plasma membrane is limited. The elongation of fatty acids by an oxidative process analogous to that known for endoplasmic reticulum was proposed in adipocytes (Giacobino and Chmelar, 1977; Chmelar and Giacobino, 1976). In the endoplasmic reticulum, the process requires the combined action of a fatty acid desaturase, cyt b 5 , and NADH-cyt b 5 reductase. A mixed-function oxidase of unknown type involving NADPH that hydroxylates dehydroepiandrosterone has been found in the lens fiber plasma membrane. It was suggested that this enzyme may be involved in regulation of the lenticular glucose-6-phosphate dehydrogenase (Vermorken et ai., 1977). The nucleotide cyclase enzymes for synthesis of cyclic AMP (cAMP) and cyclic GMP (cGMP) are associated with plasma membranes (Solyom and Trans, 1972), but can also be found in a soluble state (Neer, 1974; Braun and Dods, 1975). Evidence for location in other membranes was reported in yeast (Wheeler et ai., 1974), in the adenohypophysis (McKeel and Jarett, 1974), and in liver (Morre et ai., 1974b; Cheng and Farquhar, 1976a,b; Yunghans and Morre, 1978). The different functions of retinol and retinal in glycolipid synthesis or membrane photoresponse appear to be unrelated in plasma membranes from different tissues. The influence of the redox state of retinol on glycolipid synthesis, however, has not been considered (De Luca, 1977). 4.3.

Peroxide or Superoxide Generation

The generation of superoxide by an NADH oxidase in the plasma membrane may impinge on cell function from several directions. The bestdefined area is that of defense against, or attack on, other cells. Another possible functional significance is the oxidative conversion of metabolites, such as the synthesis of prostaglandins. A further consideration that has not been explored is directed ionic changes or charge transfer in the membrane as the result of generation of the anionic superoxide or as a result of proton shifts involved in H 20 2 formation from superoxide (Fridovich, 1977). Finally, one must consider the possible peroxidation of membrane lipids by the superoxide and its detrimental consequences, or also the reduction of higher-potential compounds such as cytochromes in the plasma membrane.

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Superoxide generation is clearly to be expected in those plasma membrane types that contain xanthine oxidase. The milk fat globule membrane is an excellent example. Small amounts of xanthine dehydrogenase have also been reported in liver and adipocyte membranes, but the generality of the association of xanthine oxidase with plasma membranes or its association with specialized regions of plasma membrane remains to be explored. Generation of superoxide by NADH oxidation in the plasma membrane of leucocytes has been proposed (Briggs et aI., 1975). There is evidence for a special NADH dehydrogenase in the plasma membranes of neutrophils, but the reaction with oxygen has not been studied. It has been reported, however, that this plasma membrane enzyme is missing in chronic granulomatosis, a condition in which the neutrophils lose the ability to kill phagocytosed bacteria (Segal and Peters, 1976). Certainly the major superoxide generation in leucocytes is by means of an NADPH oxidase of uncertain origin located in a granule fraction. The contribution of an N ADH oxidase remains to be established (Iverson et al., 1977). There is evidence that some superoxide production may occur at the cell surface (Goldstein et aI., 1977). Eosinophils have been proposed to attack other cells by surface superoxide generation without accompanying phagocytosis (DeChatelet et al., 1977). Superoxide generation in response to inflamatory agents may also account for increased prostaglandin synthesis under these conditions (Humes et al., 1977; Van Der Ouderaa et al., 1977). The formation of prostaglandins from unsaturated fatty acids requires hydrogen peroxide for the cyclooxygenase step (Morse et al., 1977). The cylooxygenase is located in the microsomal fraction, which is mostly endoplasmic reticulum. Its presence or absence from plasma membrane has not been determined. In the endoplasmic reticulum, the NADPH dehydrogenase has been demonstrated to be a superoxide generator, but the NADH-cyt h5 reductase forms very little superoxide, since it is not a readily oxidizable flavoprotein. Peroxide has also been shown to stimulate glucose oxidation by fat cells and to thus mimic the effect of insulin (Czech et aI., 1974; Czech, 1977). This mimic effect has been related to control of glucose transport by the redox state of membrane thiol groups. The influence of ascorbic acid on cAMP formation and on response to adrenocorticotropin by adrenal cells from tocopherol-deficient rats further suggests an influence of peroxide on cAMP formation (Nathans and Kitabchi, 1975). 4.4.

Redox Control of Plasma Membrane Functions

Considerable evidence has been presented that the redox state of components in the plasma membrane can control transport functions and

377

Dehydrogenases of the Plasma Membrane

the production or breakdown of cyclic nucleotides. The rate of redox exchange for control of function could, of course, be much less than the rate of oxidation required to provide energy for transport or metabolic conversion. For control of function, a single oxidoreduction in a membrane protein could be sufficient, whereas transport or conversion would require stoichiometric redox change. Oxidation or reduction of sulfhydryl groups on the membrane has been reported to inhibit or stimulate transport of both hexoses and amino acids. (Table XIII). Both Czech (1976, 1977) and Kwock et al. (1976) reported conditions under which thiol reagents or oxidants stimulate transport of sugars or amino acids. It has been proposed that an oxidation of sulfhydryl to disulfide activates the transport system. Stimulation was reported with oxidizing agents such as vitamin K5 or HzO z, which appear to mimic the effect of insulin (Czech, 1976). In plasma membrane, insulin was also reported to stimulate an NADPH oxidase that could generate HzO z to induce the disulfide formation (Mukherjee and Lynn, 1977b). The oxidizing agents would form a disulfide to convert the transport system to a high activity state (Czech, 1977). It has been suggested that insulin exerts its effect by activating, in the membrane, a redox enzyme that catalyzes the SH-to-SS conversion, and this conversion could be related to the activity described by Sutherland and Pihl (1967, 1968) (Kwock et aI., 1976) or to the thioredoxin reductase-thioredoxin system (Holmgren, 1977). On the other hand, there have been studies that indicate that oxidizing agents and reagents that react with thiol groups can inhibit transport, especially at high concentrations (Table XIII). Dithiothreitol or mercaptoethanol has also been found to reduce the sensitivity of electrogenic cells of the electric eel and to change acetylcholine binding of isolated membranes. Since no effect on ion transport is Table XIII Effect of Redox Agents and Sulfhydryl Reagents on Membrane Transport Cells

Transport

Redox effect

Reference Czech (1976) Amos et al. (1976) Smith and Ellman (1973) Korbl et al. (1977) Kwock et al. (1976) Pillion et al. (1976)

Fat Fibroblast

Hexose Hexose

Erythrocyte

Hexose

SH ｾ＠ SS stimulates transport. Methylene blue and dehydroascorbate inhibit transport. SH reagents inhibit transport.

Muscle Thymocyte

Xylose Amino acid

Anoxia stimulates transport. SH ｾ＠ SS stimulates transport.

Kidney

Amino acid and hexose

Diamide inhibits transport.

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observed, the reductant appears to directly affect the acetylcholine receptor (Schiebler et al., 1977). Oxidation of methionine in a major plasma membrane sialoglycoprotein (PAS-I) of erythrocyte membranes changes the electrophoretic mobility of the protein. It was suggested that peroxide may account for the formation of the glycoprotein referred to as PAS-4 (Silverberg et al., 1977). It is well recognized that the redox state of a cell can control its metabolic function and development (Warburg, 1956; Schwartz et al., 1974; Szent-Gyorgyi, 1965; Szent-Gyorgyi et al., 1967; Jakob and Diem, 1974; Seglen, 1974; Nath and Rebhun, 1976; Quimby and Kay, 1977). There is also considerable evidence that development and other aspects of cellular function are controlled by a balance of levels of cAMP and cGMP (Goldberg et al., 1973; Leuschen and Amato, 1976; Medoff et al., 1976; Rickenberg and Rahmsdorf, 1975; Shima et ai., 1976; Barber, 1976; Murad and Kimura, 1974; L({)vtrup-Rein and L({)vtrup, 1975; Goldbeter and Segel, 1977; Da Silveira etai., 1977; Costa etai., 1976). Therefore, the oxidationreduction level in the cell may exert its effect by influencing the levels of cyclic nucleotides, in addition to any effects it may have on energy production (Atkinson, 1968). W. B. Anderson and Pastan (1975) showed that NADH levels increase and cAMP levels decrease in virus-transformed cells and that cAMP can reverse the virus-induced morphological changes. Catabolite repression in bacteria and yeast is likewise accompanied by decreased levels of cAMP (Pastan and Perlman, 1970; Fang and Butow, 1970), and is reversed by addition of cAMP (Jacquet and Kepes, 1969). Low oxygen levels were shown to decrease cAMP levels and increase cGMP levels in both nerves (Steiner et ai., 1972) and retina (Orr et ai., 1976). Hypoxic shock increases both cAMP and cGMP in Tetrahymena and induces changes in cyclase and phosphodiesterase activity (Gray et al., 1977). Both ethanol and lactate were shown to inhibit the glucagon-induced formation of cAMP in liver cells when these cells were incubated in the" presence of 3-isobutyl-l-methylxanthine to inhibit hydrolysis of cAMP by phosphodiesterase (Zederman et al., 1977). The general pattern appears to be that any treatment that lowers the redox potential in the cell will decrease the cAMP level. The redox control could, of course, be applied either to the synthesis of cAMP by adenylate cyclase or to the breakdown of cAMP by phosphodiesterases. Possible effects at each of these sites have been reported, and it remains to be determined how each effect relates to cellular cAMP levels. In Escherichia coli, L. D. Nielsen et al. (1973) found that the phosphodiesterase has an absolute requirement for thiol, such as glutathione, and in crude extracts can be activated by NADH or NADPH. Activation of this enzyme would tend to decrease cAMP concentration. Inhibition of adenylate cyclase by glucose was proposed to be linked to the

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379

glucose-transport system (Peterkofsky and Gazdar, 1975). The cyclase is phosphorylated to the active form with phosphoenolpyruvate as phosphate donor. When glucose transport increases, the phosphoenolpyruvate is used to phosphorylate glucose, and consequently the adenylate cyclase becomes dephosphorylated and deactivated. In yeast, a direct inhibition of adenylate cyclase in a membrane preparation by relatively high concentrations of glucose and other sugars was reported (Wheeler et al., 1974). Low and Werner (1976) showed inhibition of adenylate cyclase in adipocyte membranes by NADH, but not by NADPH or NAD. Because of the specificity for NADH, the inhibition may be mediated by an NADH dehydrogenase in the plasma membrane. This may be the same NADH dehydrogenase that is stimulated by catabolic hormones and inhibited by insulin (Low and Crane, 1976; Crane and Low, 1976). A more indirect inhibition of adenylate cyclase by H2 0 2 generated through an NADPH oxidase in adipocyte membranes was suggested by Mukherjee and Lynn (1977a,b). On the other hand, the studies of Clark et al. (1976, 1977) appear to indicate that increased redox potential in the cytoplasm will inhibit cAMP accumulation. For example, addition of pyruvate to liver cells causes a decrease in cAMP, whereas lactate has little effect. Since these studies were done without a phosphodiesterase inhibitor, they may represent combined effects on both adenylate cyclase and phosphodiesterase. The lower level of cAMP in pyruvate-fed cells could indicate a strong stimulation of the phosphodiesterase in the plasma membrane by oxidation of a membrane component, in contrast to the inhibition of cyclase by reduction. In other words, control of both cyclase and the membrane-bound portion of the phosphodiesterase by membrane dehydrogenases needs to be considered. Oxidation has also been reported to induce changes in guanylate cyclase activity from rat uterus. The enzyme can be converted to an activated form by incubation in air. Further oxidation causes inactivation, which can be reversed by dithiothreitol (Kraska et al., 1977). Haddox et al. (1976) showed that the oxidant periodate caused increased proliferation of lymphocytes and an increase in cGMP. The effect of the periodate is reversed by reducing agents such as cysteine to suggest redox control of guanylate cyclase. Light may also influence membrane function by photoreduction of a membrane component. Such photoreduction may be the basis for inhibition of adenylate cyclase in retinal disk membranes (Bitensky et al., 1972; Hendriks et al., 1973) or for the light activation of a cGMP-specific phosphodiesterase (Manthorpe and McConnell, 1975). In smooth muscle, the action of vasodilators such as nitroprusside or glyceryl trinitrate is suggested to involve oxidation of groups on the recep-

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tors (Smith and Kruszyna, 1976). Hypoxia decreases the response of smooth muscle to vasopressin even with glucose present, which indicates a change in sensitivity not based on energy supply (Altura and Altura, 1977). Hypoxia also decreases heart muscle response to epinephrine (Crass, 1977). An oxidase in the plasma membrane that regulates response to hormones could be the basis for effects of this type. The basis for redox control of the nucleotide cyclases remains to be determined. The obvious possibilities have been mentioned. Since the receptor is separate from the adenylate cyclase and may be connected to it by an unknown transducer in the membrane, the redox effects may be at more than one site (Kahn, 1976; Perkins, 1973; Newmark, 1977). The dehydrogenase may reduce or oxidize a sulfhydryl site; it may generate superoxide or peroxide, causing oxidation of a site on the enzyme or even producing a prostaglandin epoxide that may inhibit the cyclase (Dalton et al., 1974; Gorman et al., 1976). A redox reaction in the membrane may also generate a proton that could be used to establish a gradient across the membrane or to control catalytic function of the cyclase, comparable to the proton control of mitochondrial ATPase (Mitchell, 1974). A similar proton flux could be related to the inhibition of cyclase by light in retinal disks, since photogeneration of proton gradients is also known in membranes of photoreceptors (Ostroy, 1977). Intramembranal proton accumulation in a binary membrane (Gould and Cramer, 1977; Crane et al., 1971) could lead to conformational changes or dissociation of receptor sites and cyclase. Evidence for redox-induced shielded pH changes in mitochondria membranes was observed in atebrin fluorescence studies (Huang et at., 1977). Proton secretion by plant cells in response to hormones was correlated with cell extension (Mentze et at., 1977; Cleland, 1971). It is possible that external acidification could be brought about by a proton gradient generated by a plasma membrane redox system. For example, Eldan and Mayer (1972) reported evidence for activation of a NADH-cyt c reductase when lettuce seeds germinate. The reductase could be the mechanism for protongradient formation. The role of flavin and cytochrome in phototropism could also be related to proton-gradient generation in the plasma membrane (poff and Butler, 1974; Brain et at., 1977; Schmidt et at., 1977). Another reflection of this plasma membrane flavoprotein may be seen in light effects on membrane potential in onion guard cells (Zeiger et at., 1977). While it is apparent that cellular membranes are dynamic structures capable of extension, migration, and other forms of movement, we are completely ignorant of the driving forces or transducing mechanisms through which chemical potential is converted into a vectorial displacement of membrane constituents or vesicles. The complement of electron-transport-chain components present in endoplasmic reticulum, Golgi apparatus,

Dehydrogenases of the Plasma Membrane

381

and other endomembranes, and also present in membranes of the cell surface, may provide important components in the energy-transducing mechanisms of membrane dynamics. Their involvement in dynamic processes involving the selective movement or flow of membranes, where some membrane constituents are transferred while others are not, has already been considered (Morn!, 1977a,b).

5.

CONCLUSIONS

It is clear that dehydrogenases are associated with the plasma membrane. Some are easily detached from the membrane and can be described as extrinsic proteins, whereas others are more tightly bound and belong in the category of intrinsic proteins. All evidence at this time indicates that the dehydrogenases are exposed on the inside of the membrane. A possible exception is malic dehydrogenase in plant cell wall fractions. The association with the membrane is specific for certain dehydrogenases or isozymes and is not a random adsorption to the membrane. The relationship between extrinsic dehydrogenases and the membrane has been considered mostly from the standpoint that association-dissociation from the membrane can control the catalytic capacity of the enzyme. There have been suggestions, however, that the relationship may also be

OUTSIDE

t

ｾ＠

w

z

TRANSPORT SYSTEM

In

::iE

w

::iE

------1

+ INSIDE ATP CAMP

FIGURE 5. Schematic representation of the plasma membrane NADH oxidoreductase complex. Possible coupling to transmembrane transport and transmembrane signaling are indicated. Other suggested functions include roles in membrane movements, secretion, and endocytosis. An external site for ferricyanide reduction by erythrocytes has been indicated by Mishra and Passow (1969).

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Frederick L. Crane et al.

meaningful to the membrane in terms of logistical advantage and of restricted or oriented supply of products. It is beginning to appear that the intrinsic dehydrogenases of the plasma membrane can both have a role in energizing certain specialized transport functions and be involved in control of stimulus-response of the plasma membrane, as seen in redox control of cyclic nucleotide formation or breakdown (Figure 5). The relationship at this point is coincidental in that dehydrogenase and cyclase are both. stimulated or inhibited by the same hormones or chemicals. The exact nature of the reduced pyridine nucleotide dehydrogenases, their origin, and their prosthetic groups remain to be determined. The role of these enzymes and xanthine dehydrogenase in the possible production of superoxide or peroxide at the plasma membrane also remains to be examined both from the detrimental and the constructive standpoint. ACKNOWLEDGMENTS

H. Goldenberg received support from the Max Kade Foundation and F. L. Crane from a research career award from the National Institute of General Medical Science. Previously unpublished research was supported by the National Science Foundation, National Institutes of Health, American Heart Association, Indiana Elks, and Swedish Medical Research Council. Helen Crane and Jan Vanderbilt assisted in the preparation' of the manuscript. 6.

REFERENCES

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Chapter 7

Transport Processes in Membranes: A Consideration of Membrane Potential across Thick and Thin Membranes N. Lakshminarayanaiah Department of Pharmacology Thomas Jefferson University Philadelphia, Pennsylvania 19107

1.

INTRODUCTION

Any phase that acts as a barrier preventing mass movement but allowing restricted or regulated passage of one or several species through it may be defined as a membrane. This could be a solid or liquid or even a gas (see Buck, 1976) containing ionized or ionizable groups, or it may be completely unionized. All membranes are active in an operational sense when used as barriers to separate two solutions or phases unless they are too fragile or too porous. Membranes may be broadly classified into natural and artificial or man-made. Natural membranes are thin «100 A), whereas artificial polymeric membranes that have proved their usefulness in several successful unit processes are thick (more than a few micrometers), even though thin Ｈｾ＠ 50 A) membranes of parlodion have been prepared (Lakshminarayanaiah and Shanes, 1963) and characterized (Lakshminarayanaiah, 1965a; Lakshminarayanaiah and Shanes, 1965). In addition, lipid bilayer memA), since the time they were first generated in branes that are also thin ＨｾＵＰ＠ 1962 by Mueller et at. (1962a,b; 1963), have assumed great interest and importance. This is probably due to their close resemblance to natural membranes of living systems. Other model membrane systems of interest The mathematical and electrochemical terms and symbols used in this chapter are defined in the Appendix (Section 5).

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and significance are the so-called "liposomes," which are also called "Bangosomes" (DeGier et aZ., 1968) after their discoverer, Bangham (Bangham et aZ., 1965). Several properties of phospholipid liposomes or vesicles or both were reviewed by Bangham (1968). A rough classification of several types of membranes according to their physical dimensions and chemical structure is shown in Figure 1. A membrane separating two aqueous solutions can be subject to different driving forces, the commonly employed ones, with the exception of gravitational and magnetic forces, being gradients of chemical potential, electrochemical potential, pressure, and temperature. When these driving forces operate singly or in combination, several transport processes occur across the membrane. These processes are outlined in Table I. Some of the transport processes, such as dialysis, electrodialysis, and reverse osmosis, have been investigated extensively and employed industrially to bring about separation of the constituents of aqueous or nonaqueous solutions, or both. All the transport processes indicated in Table I and occurring in several types of membrane system have been covered in books and review articles too numerous to mention all. However, a few books (Helfferich, 1962; Merten, 1966; Spiegler, 1966; Lakshminarayanaiah, 1969a, 1976a; Sourirajan, 1970, 1976; Flinn, 1970; Bier, 1971; Kesting, 1971; Rogers, 1971; Lonsdale and Podall, 1972; Hwang and Kammermeyer, 1975; Lih, 1975; Meares, 1976; Passino, 1976) and review articles (Lakshminarayanaiah, 1965b, 1972, 1974, 1975a, 1978; Buck, 1976) may be personally recommended. Similarly, there are numerous excellent books and review papers dealing exclusively with transport processes in biological membrane systems. The books by Hodgkin (1964), Katz (1966), Stein (1967), Cole (1968), Tasaki (1968), Cereijido and Rotunno (1970), Adelman (1971), Hope (1971), Harris (1972), and Kotyk and Janacek (1975) should prove helpful to those readers who are not familiar with the field. The review articles are too numerous to mention. Annual reviews or other review journals and serials such as Biomembranes (9 volumes), edited by Manson (Plenum Press, New York); Membranes and Ion Transport (3 volumes), edited by Bittar (Wiley-Interscience, New York); and Current Topics in Membranes and Transport (10 volumes), edited by Bronner and Kleinzeller (Academic Press, New York), may be consulted to find a review paper related to any specific area of transport. The literature on bilayer membranes is also extensive. The books by Jain (1972) and Tien (1974) are devoted to descriptions of studies pertaining to lipid bilayers. The series under the general title Membranes edited by Eisenman (1972, 1973, 1975) relates to macroscopic systems and models (Vol. 1), lipid bilayers and antibiotics (Vol. 2), and the dynamic properties oflipid bilayers and biological membranes (Vol. 3). (The molecular organi-

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403

zation of membranes is also discussed extensively in Chapter 5 of the present volume.) This chapter begins with a general description of the properties of bilayers and biomembranes and outlines some transport processes, in particular those processes that result in the development of concentration and biionic electrical potentials across thin (bilayer and biological) membranes. In these considerations, both theoretical and experimental work on membrane potential related to thick ion-exchange membranes has been introduced. Until the time of generation of lipid bilayers in 1962, these membranes served as models for biological membranes. Some key references are given, but no attempt has been made to be exhaustive either in the treatment of the subject or in the references cited.

2.

2.1.

BIOLOGICAL AND LIPID BILAYER MEMBRANES

Chemical Constituents and Physical Structure

In almost every biological cell, water constitutes nearly 80% of the cell. The interior of the cell contains mostly potassium ions, whereas the exterior contains mostly sodium ions. The abundant anion in the extracellular fluid is the chloride ion, whereas the chief intracellular anions are organic ions with a small amount of chloride ions. Most cellular phenomena are controlled by biological membranes (biomembranes). The cell function depends on the exchanges that occur across the cell membrane, which is freely permeable to water and some small ions (e.g., K+, Cl-) and impermeable to other ions (e.g., Ca2+, ｓｏｾＭＩ＠ and colloids. The life of the cell depends on the state of the membrane. Chemical substances can act on the membrane and thus regulate the structure and function of the cell. Our understanding of molecular organization of membranes is still rudimentary despite an impressive amount of theoretical and experimental work from which has emerged some understanding of the gross organization of lipids and proteins in the membrane. Biochemical methods of analysis applied to cell membranes have shown that the biomembrane is composed of lipids and proteins with some oligosaccharides. Lipids form 20-50% of the dry weight of the membrane preparation. Free cholesterol accounts for 20-30% of lipids except in mitochondrial preparation, which has little cholesterol. The rest of the lipids are mostly phospholipids, which are known to interact with cholesterol as experimentally recorded in the evaluation of area per molecule of mixed monolayers of cholesterol and phospholipids [i.e., the area per molecule of mixture is less than the area per molecule of cholesterol plus

MEMBRANES

,

I

I AMPHIPATHIC MOLECULES

I

(e.g. Paraffin, Quartz)

I

LIQUID (e.g. Benzene, Hydrocarbon oil)

I UNCHARGED .-SOLID

I

A Inexcitoble

THICK

"Doped bilayer membranes", Excitable e. g. A lamethicin (Mueller and Rudin, 1968 a) ; ElM (Mueller and Rudin, 1968 b)

( Lakshminarayanaiah e.g. Phosphol ipids, Oxidised and Shanes, 1965) cholestero I "Pure bilayer membranes", Inexcitable (Jain, 1972; Tien, 1974)

Inexcitable

PARLODION

(e.g. Ion exchangers)

I CHARGED

I

-,

Roughly> 100

(Hodgkin, 1964 i Katz, 1966)

e.g. Nerve and Muscle Cells

(Biological Cells) Excitable

NATURAL OR BIO-

I

Roughly < 100 A-

.-----------THIN

ｾ＠

ｾ＠

cr

i·

Ii'...

I

ｾ＠

I

SOLID (Inorganic) e.g. Loosely compacted minerals

I SOLlD(Organic) e.g. Highly cross-linked resins (Wyllie and Patnode, 1950) Dried Collodion (Ling, 1967) Oil impregnated porous filters (I1ani, 1963 j 1966)

FIGURE 1. Classification of thick and thin membranes.

SOLID (Organic) e g Cross -linked ion exchangers (Lakshminarayanaiah, 1969 0)

e.g. Highly compacted minerals

SOLID (Inorganic)

I

ASSOCIATED (Membranes with narrow pores)

DISSOCIATED

Solvents of high dielectric constant with strong acid or bose groups (8eutner , 1933; Osterhout, 1940)

DISSOCIATED

ｾ＠

"'"

f'"

9

ｾ＠

:r'"

r6

l

I I

;:;.

e.g. e.g. Solvents of low dielectric constant (Gemant, 1962 ; Sollner and Shean, 1964; Shean and Sollner, 1966)

ASSOCIATED

I

§

.g

..,

(Liquids)

I

CHARGES, MOBILE

(Membrones with wide pores)

(Solids)

CHARGES, FIXED

I

Ultrafilter or solution diffusion Ion-exchange Ultrafilter Ultrafilter Ion-exchange

Solution diffusion Ion-exchange Polymers

Ion-exchange

Ion-exchange Ion-exchange Ultrafilter or ionexchange

Osmosis

Membrane potential Dialysis

Diasolysis

Osmoionosis

Dufour effect Electrical conduction Piezoelectricity

Transport number of species

Electroosmosis Electrodialysis

Electrophoresis

Electrical field (t:.E)

Ultrafilter or solution diffusion

Mixing or diffusion

Chemical or electrochemical potential (t:..p. or t:./lJ

Membrane type

Transport process

Driving force

Thermal Current Residual polarization and storage of energy Fraction of current carried by the species Solvent Ionic solute and/or solvent transfer Ionic solute

More mobile component Ionic solute

Ionic solute Solute

Solvent

Chemical components

Primary flow

Table I Transport Processes in Membranes a

Separation of large molecules

Solvent transfer Solute removal

Evaluation of membrane permselectivity

Solvent enters concentrated solution (osmometry) Source of emf; negligible solvent flow Solute leaves concentrated solution (hemodialysis) Selective transport of more mobile species Three streams of different concentration generate driving force. Similar to electrodialysis without application of external electric field Gives rise to t:.T Evaluation of membrane resistance Production of electrets

Establishment of chemical equilibrium

Comments

t"'

1:1'

I

'1

I-

e-

:z

ｾ＠

a Based

(tJ.T)

Forced-flow electrophoresis Electrodecantation

Soret effect

Thermo-emf

on Friedlander (1968).

Electrical field (tJ.E) + pressure (tJ.P) Electrical field (tJ.E) + temperature

Temperature (tJ.T)

Thermo-osmosis

Heat conduction

Vacuum

Filtration Hydraulic or mechanical permeability Pressure permeation

Ultrafiltration or reverse osmosis Piezodialysis Streaming potential Streaming current Pervaporation

Pressure (tJ.P)

Transport depletion

Ion-exchange, ultrafilter, or solution diffusion Ion-exchange, ultrafilter, or solution diffusion Ion-exchange, ultrafilter, or solution diffusion Ion-exchange, ultrafilter, or solution diffusion Ion-exchange or ultrafilter Ion-exchange (cation) and ultrafilter

Mosaic ion-exchange Ion-exchange Ion-exchange Solution diffusion

Ultrafilter

Usually cation exchange alternates with ultrafilter in stacks. Ultrafilter (microporous) Ultrafilter or solution diffusion Solution diffusion

Ionic solute and solvent Ionic solute and solvent

Ionic solute

Ionic solute

Solvent

Ionic solute Solvent Ionic solute Selective transport of most mobile component Heat

Selective transport of most mobile component Solvent

Solvent Solvent

Ionic solute

Purification of blood, sewage; still in experimental stage tJ.T supplied by electrical heating and/or cooling; ionic matter concentrates downward and solvent concentrates upward.

Gives rise to tJ.p,; difficult to measure

Solvent may move from hot to cold side or vice versa Source of emf; very few studies

Thermal conductance

Solvent leaves and solution concentrated Product of reduced salinity Generation of emf Current very small; studies rare Separation of liquids and/or gases

Particulate matter retained by sieving Relates to space available for laminar and/or diffusional flow Separation of liquids and/or gases

Simplified high current density electrodialysis

=

i3

ｾ＠ .....

'"

to

=

ｾ＠

...10

ｾ＠

ｾ＠

'" [(i '" 5·

to

ｾ＠

'" 'g :l

003 ｾ＠

408

N. Lakshminarayanaiah

the area per molecule of phosopholipid (Lakshminarayanaiah, 1969a, pp 419-425)]. The lipids contain a variety of fatty acid residues ranging in chain length from 16to 22 carbon atoms and in degree of un saturation from 0 to 4 double bonds (Dowben, 1969; Lee, 1975). The ratio by weight of proteins to lipids ranges from about 1.5 to 4 (Singer and Nicolson, 1972). A good fraction of the protein is involved in the organization of the biomembrane. Membrane proteins have been roughly categorized into peripheral and integral proteins (Singer, 1971). The peripheral proteins are not strongly associated with membrane lipids, and so are easily removed from the membrane by mild treatment. On the other hand, integral proteins are strongly attached to the membrane, and so require drastic treatment to remove them from the membrane. It is assumed that integral proteins confer structural integrity to the biomembranes. The integral proteins are heterogeneous, and probably no one protein is involved in maintaining membrane stability. They are mainly globular in shape, exhibiting considerable amount of a-helical conformation (Leonard and Singer, 1966; Wallach and Zahler, 1966). The membrane lipids and proteins are amphipathic molecules, and so orient themselves at an oil-water interface in such a way that the polar groups are in the aqueous phase and the nonpolar groups in the oil phase. Consequently, for a stable membrane to be formed of lipids and proteins in an aqueous environment, the hydrophobic and hydrophilic interactions should be maximized so that the resulting structure, i.e., the membrane, would exist in a state of lowest free energy. Such structures, bilayer membranes (see Figure 2), have been formed from oxidized cholesterol and phospholipids (Jain, 1972). Experimental incorporation of protein into such a bilayer structure so as to form an independently stable lipid-protein membrane has not been successful (Hanai et ai., 1965), although supported protein-lipid-protein membrane has been constructed (Tsofina et ai., 1966). Several models for the biomembrane have been proposed over the years. The unit membrane structure proposed by Danielli and Davson (1935) contains two monolayers of protein sandwiching the lipid bilayer. This type of organization is considered thermodynamically unstable (Singer and Nicolson, 1972) due to: (1) exposure of nonpolar amino acid residues of the membrane proteins to water and (2) prevention of exposure of polar groups of lipids to water by the layer of protein. Consequently, other modifications have been proposed, some of which are shown in Figures 3 and 4. A modified model of the three-layer structure proposed by Robertson (1964) is shown in Figure 3A. According to this model, the hydrophobic core acts as the barrier to material flow and the polar parts of lipid and

409

Transport Processes in Membranes ｰ｡ｲｴｩｮｾ＠

ｩﾥ｢ｾ＠

Annulus ｾ＠

ｾ＠

85

0--0-" l)::::g 0- "--0

Plateau border

0-0-....-

ｾ｣ｴｧ＠

ｾ＠

ｾ＠

ｾＯ＠ ｾＭｯ＠

0--0

ｾＭｯ＠

--0 --0 0--0

0--

-0

ｾ｣ｯｬｲｳ＠

--0 --0

Interference

ｾ＠

ｾＱ｜ｷｨｩｴ･＠

0--0 0-.-0

0--0

ｾ＠

§-CK ｲｾ＠ ＼Ｗｍｾ＠

Ii ght

0.- 0---0 0---- 0---- 0---

O:-;;.P a

Thinning Membrane

ｾｂｬ｡｣ｫ＠

0----0/ 0---0

g:::::g

o--o;:g ｾＭｑ＠

ｾ＠

ｾ＠

0- ｾ＠ ' " White g::;;.=g light ｾＭｯ＠

ｾｬ＠

ｾ＠ -Ai-=8

?=b- 0....:-0 ｏＭｯｾ＠

ｾ＠

ｾ［ＺＧ＠

L.-1l

0-0

Bi layer Lipid Membrane

FIGURE 2. Diagram illustrating the different stages during thinning of a lipid membrane in aqueous media and indicating the patterns of reflected light. Reproduced from Jain (1972) courtesy of Van Nostrand Reinhold Company.

protein contain sites or specific enzymes or both responsible for the generation of several membrane phenomena. The model suggested by Vanderkooi and Green (1970) is shown in Figure 3B. The essential feature of this model is that two layers of globular protein molecules are in contact with each other, the interspaces between molecules being occupied by phospholipids. The polar groups of the phospholipids exist on the membrane surface, and the fatty acid chains reside in the interspaces. A highly ordered structure was visualized in the original Danielli-Davson model, but other data (Chapman and Wallach, 1968; Chapman et al., 1968; Steim, 1968) point to the existence of a state of disorder, conferring on the core layer properties typical of liquid hydrocarbons. The third model, described by Stoeckenius and Engelman (1969), is shown in Figure 3C. The fluid core is bounded by hydrophilic groups that are not closely packed. The hydrophobic surface of the lipid bilayer is partially covered with proteins that exist partly in a-helical conformation. The proteins also interdigitate with the lipids of the membrane, whose integrity is established and maintained by hydrophobic interactions between the chains of proteins and lipids. The recent fluid mosaic model proposed by Singer and Nicolson (1972)

410

N. Lakshminarayanaiah A

: ｾ＠ Protein

B

c

o--c: Lipid

FIGURE 3. Models for biomembranes. (A) Model proposed by Robertson (1964). (B) Model according to Vanderkooi and Green (1970). (C) Model described by Stoeckenius and Engelman (1969). For details, see the text. Reproduced from Lakshminarayanaiah and Bianchi (1977).

is shown in Figure 4. Figure 4A shows a schematic cross-sectional view of the membrane model. The phospholipids are arranged as a discontinuous bilayer with their polar groups in contact with water. The integral proteins shown with heavy lines represent folded peptide chains, partially embedded in and partly projecting from the membrane. The projecting parts have the polar residues on their surfaces, and the nonpolar parts are embedded in the lipid matrix. Figure 4B is a three-dimensional representation of the lipid-protein (globular) fluid mosaic model. It is considered that the lipid is the matrix in which the integral proteins are incorporated randomly in the plane of the membrane. The extent to which the integral proteins span the width of the membrane is dependent on the size and structure of the integral protein. 2.2.

Properties of "Undoped" Bilayer Membranes and Biomembranes

In view of some similarity in the organization of lipids in bilayer membranes and biomembranes, they might be expected to show some similarities in their physical and other properties. Some of the intrinsic similarities are given in Table II together with a few properties of thin parlodion membranes. .

Transport Processes in Membranes

411

FIGURE 4. Fluid mosaic model for biomembranes (schematic). (A) Cross-sectional view. (B) Three-dimensional lipid-protein (globular) fluid mosaic model. The heavy lines represent globular integral proteins randomly distributed at long range in the plane of the membrane, and at short range, some may form aggregates. Reproduced from Singer and Nicolson (1972) Courtesy of © 1972 holder, American Association for the Advancement of Science. (See Chapter 5 of the present volume for further discussion of membrane models.)

412

N. Lakshminarayanaiah

Table II Comparison of Some Physical Properties of Thin Artificial and Natural Membranes Property

Parlodion a

Membranes lipid bilayer

Natural

Thickness (A) Electron-microscopic picture Surface tension (dyne/ cm) Dielectric breakdown strength(V/cm) Electrical capacitance (j.tFlcm") Electrical resistance (ohm· cm2 ) Water permeability (diffusional)

50-1oob

40--1OO c

40-1oo d

Triple layer C

Triple layer e

0-6 f

0.1-2.0·

=106e

=106e

0.4-1.3
E

E

••••••••••••••••• "•.

413

ENa

0

UJ

-50

••••••••••••. EK .• ｾＭ •• ｾ＠ •••••••••••••••••••••••••••••

3

2

4

Time (msec)

FIGURE 5. Nerve action potential (Em) Membrane potential; (Er) resting potential; (E K and EN.) potassium and sodium equilibrium potentials.

N. Lakshminarayanaiah

414

would generate an equilibrium potential (EeJ that is given by the Nemst equation Eeq

=

(RT/F) In (ao/a;)

(1)

where a o and a; are the ion activities outside (0) and inside (i) the cell and R, T, and F are gas constant, absolute temperature, and Faraday constant, respectively. Since the cell membrane (e .g., nerve cell) at rest is primarily permeable to potassium ion, the equilibrium potential EK is given by (Hodgkin, 1964): EK = 58 log (10 x 0.9/400 x 0.666) = -85.3 mV

where 10 mM and 400 mM are the extra- and intracellular concentrations respectively of the potassium ion and 0.9 and 0.666 are the respective values of activity coefficients. Nerve fibers are easily excited by an electrical stimulus, which acts to discharge the surface membrane of the cell and thus reduce the prevailing resting membrane potential to a lower (depolarization) and unstable level at which excitation occurs. In the region of the cathode, electrical stimulation reverses the existing potential. If the stimulus is strong enough, the local electrical effect is propagated without decrement and with nearly constant velocity as an action potential along the fiber. This propagated action potential or impulse in each axon is an allor-none event arising from a potential change across the cell membrane. Several hypotheses have been put forward to explain the reversal of membrane potential during activity. Of these, the ionic hypothesis formulated by Hodgkin, Huxley, and Katz (Hodgkin and Katz, 1949; Hodgkin et al., 1952; Hodgkin and Huxley, 1952a-d; Hodgkin, 1957, 1964) has been the most popular. According to this, the cell on excitation becomes highly permeable to Na ions and the membrane potential temporarily approaches the equilibrium potential for Na ion (see Figure 5): ENa

= (RT/F) In (NaJNa;)

(2)

The ENa agreed approximately with the value of membrane potential (Ea) in the active state (see Table III). Accordingly, Hodgkin and Katz (1949) demonstrated that with the decrease in the concentration of Na in the outside solution, the amplitude of the action potential decreased. The increase in permeability to Na underlying the action potential was also reflected in a transient increase in the electrical conductance of the membrane during an impulse. Cole and Curtis (1939) showed that during an impulse, the membrane resistance fell from its resting value of 1000 to about 25 ohm . cm2 • Also, it was found that neither the electrical capaci-

415

Transport Processes in Membranes

Table ill Resting, Active, and Equilibrium Membrane Potentials a Membrane potential (mV) Equilibrium

Resting

Active

Tissue

(Er)

(Ea)

EN.

EK

Eel

Loligo axon Sepia axon Carcinus axon

- 61 - 62 - 82

+ 35 + 60 + 52

+49 + 52

- 91 - 89 - 85

- 54

a From

Floyd (1954).

tance of the membrane nor the resistance of the axoplasm changed in any appreciable manner. Further, it has been shown using isotopes that the entry of Na into the cell and exit of K from the cell during an impulse were equal, about 4 pmol/cm2 per impulse (Keynes and Lewis, 1951; Hodgkin, 1964). The electrical events taking place during an action potential, which is an explosive reaction over which the experimenter has no control, were examined by Hodgkin and Huxley (1952a-d) employing the voltage clamp technique (Cole, 1949, 1968). The current responses realized when voltage steps in sequence are applied across the membrane are shown in Figure 6A. The membrane currents are made up of two components-an early transient component due to flow ofNa ions and a late component that levels off to a steady state due to flow of K ions. The maximum values of the early transient current and the steady-state values of the late current are shown in Figure 6B as a function of voltage. The current-voltage curve for the transient peak currents shows a negative electrical resistance in the voltage region in which the threshold for the generation of action potential lies. The kinetics of the conductance change following changes in membrane potential were described by Hodgkin and Huxley (1952d) using a set of empirical equations. The total current I across unit area of membrane is made up of the capacitative current C dE/dt and the ionic current h where C is the membrane capacitance. Thus I

=

C (dE/dt) + Ii

(3)

and Ii = IK + INa + h, where h is an ohmic current usually referred to as the leakage current. The early transient INa is described by

(4)

416

A Ｍ

J

N. Lakshminarayanaiah

B ｾ＠

Current (mAlcm 2)

Ｑｾ･｣＠

ｾ＠ "".,./

t/······.........

5

11 mA/cm 2

4

3 2

E = 60

Voltage (mV)

80

100

\ ................. ｾ＠ \ /.....

f =-20 -3

FIGURE 6. Membrane currents in a squid axon under voltage clamp. (A) Transient current responses following steps of potential to the values of E shown. ( . ... ) Early and late (or delayed) components of membrane current. (B) Peak values of the early transient current (e) and steady-state values of the delayed current (-) derived from (A) plotted as a function of voltage . After Ehrenstein and Lecar (1972).

where gNa is the maximum early conductance and m and h are parameters that describe the kinetics of early conductance change. These parameters follow the equations dm

m oo(E) - m

dt

Tm(E)

dh

h oo(E) - h

dt

Th(E)

(5)

(6)

Similarly, the late current I K is described by

IK

=

gKn 4(E

- E K)

dn

noo(E) - n

dt

Tn(E)

(7) (8)

The functions moo (E), hoo(E), noo(E), Tm(E), Th(E), and Tn (E) were empirically derived from the voltage clamp data and are shown in Figure 7.

417

Transport Processes in Membranes

The membrane currents of Figure 6A may be obtained by solving Eqs. (4)-(8) for I as a function of time for several steps of E. Action potential responses also can be obtained by fixing the value of I and solving the equations for E as a function of time (Hodgkin and Huxley, 1952d). Several experiments show that the early and late currents go through pathways (channels or pores) that are separate entities. The evidence to indicate the existence of independent pathways comes from experiments with selective pharmacological agents that block one component of the membrane current without affecting the other (Hille, 1970; Narahashi, 1973). The drug tetrodotoxin (TTX) blocks the early current without affecting the late component of the membrane current (Narahashi et at., 1964; Kao, 1966). The drug affects the channel itself without being dependent on the nature of the ion going through the channel, since it is found that both the early outward current due to potassium flow (Rojas and Atwater, 1967) and the early inward current due to flow of ions other than sodium (Moore, J. W., et at., 1967; Hille, 1968a, 1970) are blocked by TTX. Tetraethyl ammonium ion (TEA) eliminates the current through the late channel without altering the early current (Armstrong and Binstock, 1965; Hille, 1967). A similar effect is exerted by Cs and Rb ions when they are applied internally (Chandler and Meves, 1965; Adelman and Senft, 1966). The separateness of the early and the late channels is well illustrated by the ammonium ion, which can easily pass through both channels (Binstock and Lecar, 1969). The early component of the current due to NHt ion is

B

A

c

10

msec

/( -50 E(mV)

0

50

50

FIGURE 7. Several parameters of the Hodgkin-Huxley equations. The conductance parameters noo and moo and inactivation parameter hoo are normalized. The time constants Tn, T m, and Th and noo , moo, and hoo are shown as functions of voltage for squid axon membrane whose representative conductances are: gK = 36 mmho/cm 2 and gNa = 120 mmho/cm2 • After Ehrenstein and Lecar (1972).

418

N. Lakshminarayanaiah

blocked by TTX, and the late component, again due to the same ion, is blocked by TEA. The early channel kinetics are governed by two conductance parameters-the activation parameter rn 3 and the inactivation parameter h. Destruction of inactivation by internal application of pronase (Rojas and Armstrong, 1971; Armstrong et al., 1973) shows that, like activation governed by a physical entity rn, inactivation is a physical entity controlled by

h. In summary, nerve action potential or impulse is brought about by the flow of ions (current) through sparsely distributed channels or pores that can be inactivated by pharmacological agents. In the case of some agents (e.g., TEA), kinetic data suggest that these agents block pores through the membrane (Armstrong and Binstock, 1965; Armstrong, 1971). 2.3.

Properties of "Doped" Bilayer Membranes and Biomembranes

In the last decade or so, it was found that several compounds (ionophores), when added in small quantities to one or both of the solutions bounding the bilayer membrane, decreased the high electrical resistance of the membrane. These compounds when they are in the membrane can increase membrane conductance in a number of cases either by acting as carriers of charge across the membrane or creating channels or pores in the membrane through which ions, charged species, or complexes can move down their electrochemical gradients. The literature related to several aspects of these processes is extensive, and excellent reviews exist. Consequently, no attempt is made here to review the field; however, an outline in the form of tables is given with some relevant references. Many of the compounds that have been used by several investigators to study their effects on lipid bilayer membranes may be broadly classified into three groups. Compounds of the first group, which change the high resistance of the membrane (mechanism doubtful), are given in Table IV. In Table V are given those compounds that act on the membrane and facilitate movement of charge either by their own movement or by acting as carriers. In Table VI are given those compounds that act on the membrane to create channels or pores through which charges move. Several aspects of the actions of the compounds given in Tables V and VI have been reviewed elsewhere (Mueller and Rudin, 1969; Haydon and Hladky, 1972; Eisenman et al., 1973a,b; Hladky et al., 1974; McLaughlin and Eisenberg, 1975). In this connection, reference must be made to the work ofYafuso et al. (1974), who found that undoped bilayers formed from oxidized cholesterol exhibit increase in conductance and stepwise change in conductivity, thus pointing to the spontaneous formation of channels in the membrane. This was attributed to formation of membranes from aged lipid solution,

Transport Processes in Membranes

419

Table IV Compounds That Modify Bilayer Membrane Conductance: Some Notes and Comments Aliphatic alcohols: Chain length < Cs increased membrane conductance and > C8 decreased membrane conductance (Cherry et al., 1970). Conductance change due to change in fluidity of the membrane (Paterson et al., 1972). Heptanol: Cation conductance increase, especially of potassium (Gutknecht and Tosteson, 1970). Local anesthetics (nupercaine, tetracaine, cocaine and procaine): Membrane conductance dependent on concentration (Ohki, 1970). N,N'-bisdichioroacetyl-l,12-diaminododecane: Membrane conductance increase (Alkaitis et al., 1972).

ATP + (Na+, K+)-ATPase: Membrane conductance increase dependent on concentration and inhibited by ouabain (Jain et al., 1969, 1972). ATPase extract from Streptococcus fecalis: Membrane conductance increase (Redwood et aI., 1973). Prymnesin: Membrane conductance increase (Moran and Ilani, 1974). Polylysine: Membrane conductance increase and other effects (MontaI, 1972). Protein fraction (Lossen et al., 1973) and glycoprotein (Tosteson et al., 1973) from human red cell membrane: Membrane conductance increase. Polypeptides (copolymers of L-Iysine-L-phenylalanine and L-Iysine-L-serine): Former gave higher membrane conductance increase and the latter relatively small increase (Bach and Miller, 1973); also, former gave small membrane conductance increase in phosphatidyJinositol membrane with effect on its stability (Bach, 1973). Hydrogen peroxide: Membrane resistance and stability decreased with increase in concentration (Van Zutphen and Cornwell, 1973). Detergents: Membrane conductance increase (Seufert, 1965). Triton X-l00: SUblytic concentrations increased membrane conductance (Van Zutphen et al., 1972). Endotoxin: Membrane rupture (Schuster et al., 1970). Lysolecithins: Membrane rupture; shorter chain length and increase in the degree ofunsaturation resulted in loss of lytic effect (Reman et aI., 1969). Filipin complex, filipin II, filipin III, etruscomycin, pimaricin: Membrane disruption provided cholesterol present (Van Zutphen et al., 1971).

since stepwise conductance increase was not observed in membranes formed from fresh lipid solutions. • The reviews by Mueller (1975a,b) and Ehrenstein and Lecar (1977) deal exclusively with compounds that generate voltage-dependent ion conductances whose kinetics are similar to those ofbiomembranes. The kinetic characteristics described by Mueller (1975b) and the discussion by Ehrenstein and Lecar (1977) indicate that the mechanism by which the membrane opens and closes for the movement of ions is probably similar in both bilayer membranes and biomembranes, although the translocator in biomembrane remains unidentified at present. "Gating" is the word that has been used in recent years (see Hille,

420

N. Lakshminarayanaiah

Table V Compounds That Increase Bilayer Membrane Conductance by Carrier or Other Mechanisms: Some Notes and Comments Lipid-Soluble Ions Tetraphenylborate (-): High membrane conductance; limited by diffusion in the boundary layer; at high concentration, anion flux independent of concentration (LeBlanc, 1969; Liberman and Topaly, 1969). Mechanism of transport (Ketterer et ai., 1971; Gavach and Sandeaux, 1975; Benz et ai., 1976a). Impedance characteristics (De Levie et ai., 1974). Current transients (Anderson, O. S., and Fuchs, 1975). Dipicrylamine (-): Membrane conductance increase (Mueller and Rudin, 1969). Mechanism oftransport (Ketterer et aI., 1971; Bruner, 1975; Gavach and Sandeaux, 1975; Benz et aI., 1976a). Comparative study of membrane properties by two methods of measurement (Wulf et ai., 1977). Phenyldicarba-undecaborane (-): Membrane conductance increase (Liberman et ai., 1970a). Salicylate (-): Adsorption caused membrane conductance increase to cations and decrease to anions (McLaughlin, 1973). Picrate (-): Mechanism of transport (Gavach and Sandeaux, 1975). Phloretin (-): Adsorption caused membrane conductance increase to cations and decrease to anions (Anderson, O. S., et ai., 1976; Melnik et ai., 1977). Tetraphenylarsonium (+): Membrane conductance increase small (LeBlanc, 1970). Mechanism of transport (Gavach and Sandeaux, 1975). Tetraphenylphosphonium (+): Small membrane conductance increase (LeBlanc, 1970). Triphenylmethylphosphonium (+ ), dimethyldibenzylammonium (+ ), and trimethylphenylammonium (+): Membrane conductance increase small compared to that due to fat-soluble anions (Liberman and Topaly, 1969). Hexadecyltrimethylammonium (+): Lowered interfacial tension and brought about membrane disruption (TerMinassian-Saraga and Wietzerbian, 1970). Iodine, trinitrobenzene: Membrane conductance increase (Rosenberg and Bhowmik, 1969; Rosenberg and Pant, 1970). Iodide: Membrane conductance increase (Lauger et ai., 1967a; Pashayev and Tsofina, 1968). Iodine + iodide solution: Membrane conductance increase probably electronic (Liiuger et al., 1967a,b), due to Is (Finkelstein and Cass, 1968), due to donor-acceptor complex (Rosenberg and Jendrasiak, 1968), due to I-a (Jendrasiak, 1970). Other mechanisms of conduction (Liberman et al., 1969; Jain et al., 1970; Boguslavskii et al., 1971). Uncouplers of Oxidative Phosphorylation 2,4-Dinitrophenol: Membrane conductance increase due to protons (Bielawski et al., 1966; Rosenberg and Bhowmik, 1969; Rosenberg and Pant, 1970). pH dependence of conductance (Liberman et al., 1970a,b). Frequency dependence of conductance (Lebedev and Boguslavskii, 1971). Conductance increase due to negative complex (McLaughlin, 1972; Foster and McLaughlin, 1974). Pentachlorophenol: Frequency dependence of conductance (Lebedev and Boguslavskii, 1971; Pickar and Amos, 1976). Conductance due to formation of dimers (Smejtek et al., 1976).

Transport Processes in Membranes

421

Table V (Continued) Compounds That Increase Bilayer Membrane Conductance by Carrier or Other Mechanisms: Some Notes and Comments Picric acid: Membrane conductance increase (Rosenberg and Bhowmik, 1969; Rosenberg and Pant, 1970) and dependence on pH (Liberman et ai., 1970a,b). Tetrachlorotrifiuoromethylbenzimidawle: Conductance increase due to protons (Babakov et ai., 1968). Frequency and ionic strength dependence of membrane conductance (Nikol'skaya et ai., 1972). Uncouplers listed above and other uncouplers (salicylic acid, acetoacetic ester, p-trifiuoromethylcarbonylcyanide phenylhydrazone): Conductance increase; membrane potential pHdependent in a nonlinear way (Lea and Croghan, 1969; Liberman, 1970; Liberman et aI., 1970c). (+), (-), or (0) charge membrane conductance increase (Hopfer et al., 1970). Membrane conductance increase due to a dimer (Finkelstein, 1970). Mathematical theory of pH dependence of conductance (Markin et aI., 1969). Carbonylcyanide m-chlorophenylhydrawne: Membrane conductance increase and role of boundary layers (LeBlanc, 1971). Phenylbarene: Membrane conductance increase due to protons (Liberman et ai., 1971). Bis-(2-hydroxy-3,5,6,-trichlorophenyl)sulfoxide: Initiated proton transfer through bilayer membrane (Shkf('b and Mel'nik, 1972). Neutral Carriers Valinomycin: Increased membrane conductance to potassium ions (Mueller and Rudin, 1967; Andreoli et al., 1976a; Shemyakin et ai., 1969). Carrier-mediated transport-theory and experiments (Liiuger and Stark, 1970; Ketterer et al., 1971; Stark and Benz, 1971; Stark et al., 1971; Huang, 1971a;Liiuger, 1972; Benz et al., 1973; Gamble et al., 1973; Stark, 1973; Feldberg and Kissel, 1975; Knoll and Stark, 1975; Benz and Liiuger, 1976; Feldberg and Nakadomari, 1977). Effects of ionic strength (Shkrob et al., 1973), unstirred boundary layers (Ciani et aI., 1975), hydrocarbon chain length and temperature (Benz et al., 1973), and membrane structure (Benz et al., 1977) on ion-mediated transport. Valinomycin analogue also mediates transport (Benz et ai., 1976b). Transport of di- and trinitrophenoiate ions facilitated (Ginsburg, H., and Stark, 1976). Activation enthalpies for conductance (Ginsburg, S., and Noble, 1974). As probe for membrane structure (Szabo et ai., 1972; Stark et ai., 1972). K-conductance increase affected by DDT analogues (Hilton et al., 1973). K-conductance increase blocked by tetrachloro-2-trifiuoromethylbenzimidazole (Kuo and Bruner, 1973) and substituted benzimidazoles (Kuo et aI., 1976) and modified by 4,5,6,7-tetrachloro-2-methylbenzimidazole (Kuo and Bruner, 1976). Probe of Rb-transport by temperature-jump technique (Knoll and Stark, 1977). Nonactin: Membrane conductance increase (Eisenman et ai., 1969; Szabo et aI., 1969; McLaughlin et ai., 1970,1971, 1975; Hladky and Haydon, 1973; Haydon and Myers, 1973; Hladky, 1974a) and role of boundary layers (Hladky, 1973). Kinetic analysis of carrier model (Hladky, 1975a,b; Feldberg and Kissel, 1975; Benz and Stark, 1975) and of relaxation current (Sandblom et al., 1975). Facilitation of transport of di- and trinitrophenolate ions (Ginsburg, H. and Stark, 1976). Monactin: Membrane conductance increase (Eisenman et al., 1969; Szabo et ai., 1969). Analysis of mediated transport (Feldberg and Kissel, 1975; Benz and Stark, 1975). (Continued)

422

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Table V (Continued) Compounds That Increase Bilayer Membrane Conductance by Carrier or Other Mechanisms: Some Notes and Comments Dinactin: Membrane conductance increase (Eisenman et al., 1%9; Szabo et aI., 1%9). Analysis of mediated transport (Feldberg and Kissel, 1975; Benz and Stark, 1975). Trinactin: Membrane conductance increase (Eisenman et al., 1%9; Szabo et al., 1%9) and effect of unstirred layers (Ciani et al., 1975). Kinetic analysis of carrier model for ion transport (Hladky, 1975a,b; Feldberg and Kissel, 1975; Benz and Stark, 1975). Tetranactin: Membrane conductance increase (Krasne and Esienman, 1976). Cyclic polyethers: Membrane conductance increase (Eisenman et al., 1968; McLaughlin et aI., 1970, 1972). Activation enthalpies for ion conduction (Ginsburg, S., and Noble, 1974). Enniatin B: Membrane conductance increase (Dobler et al., 1969; Henderson et al., 1%9; Liiuger, 1972).

Charged Carriers Theory of mediated transport (Huang, 1971b). Nigericin, dianemycin, monensin: Induced proton movement, but not in presence ofuncoupiing agents (Henderson et al., 1%9). X-537A: Mediated bivalent ion transport (Pressman, 1973). A23187: Facilitated calcium movement (Wulf and Pohl, 1977).

1970) to indicate the process by which ion pathways are activated by a stimulus such as a change in membrane potential. It is considered (Ehrenstein and Lecar, 1977) that excitability-inducing material (ElM), alamethicin, monazomycin, hemocyanin, and suzukacillin formed gated channels in bilayer membranes, whereas gramicidin A, amphotericin B, nystatin, and black widow venom formed nongated channels. The existence of discrete channels in the bilayer membrane has been demonstrated by measurements of single-channel conductance and relaxation time for the conductance transition for both gating and nongating molecules. The unit channel conductance and relaxation time of conductance transition are in the range 10- 12_10- 9 mho and - to- 3-10 sec, respectively (Ehrenstein and Lecar, 1977). If the channels are regarded as water-filled pores, the channel diame-

ters may be estimated from their conductances. These are estimated to range from about 1 to 30 A (Ehrenstein and Lecar, 1977). These estimates are to be considered as equivalent diameters without necessarily having any physical meaning, since ionic flow through channels may not be equivalent to that in a bulk electrolyte solution. In agreement with the foregoing, it has been estimated (Ehrenstein and Lecar, 1972; Armstrong, 1974) that the squid axon membrane has a value of about 10-10 mho for the elementary unit of conductance. In other biomembranes, noise measure-

Transport Processes in Membranes

423

Table VI Compounds That Increase Bilayer Membrane Conductance by Creating Channels or Pores: Some Notes and Comments Tyrocidine B: Bilayer conductance increase autocatalytic and not carrier-mediated (Goodall, 1970a,b). Monamycin: Membrane conductance ion-dependent (Goodall, 197Oc). Gramicidin A: Conductance characteristics (Goodall, 1970d, 1971) and fluctuations in steplike fashion (Hladky and Haydon, 1970) discussed. Kinetics of channel formation (Bamberg and Liiuger, 1973; Bamberg and Benz, 1976; Bamberg et al., 1976a; Bamberg and Janko, 1977) and autocorrelation (Zingsheim and Neher, 1974) and noise analysis (Kolb et al., 1975). Single-channel conductance (1.7 x 10-11 mho), duration (0.4-2.2 sec) (Hladky, 1974b), water permeability (Finkelstein, 1974), and other studies (Kolb and Bamberg, 1977; Tredgold et al., 1977). Head-to-head-aggregation-created channels (Urry et al., 1971) and their structure (Veatch et al., 1975; Veatch, 1976) considered. Activation energy for channel closure, 19 kcaVmole (Hladky and Haydon, 1972); for dimer formation, 20 kcal/mole, and its dissociation, 17 kcal/mole (Bamberg and Liiuger, 1974); and enthalpy for ion conduction, 9.3 kcaVmole (Ginsburg, S., and Noble, 1974). Consideration of a model for the channel (Sandblom et al., 1977). Rate theory applied to ion transport. Jump rates of K and Na in the pore, IOB-109/ sec (Liiuger, 1973). Thallous ion interacts with the channel (Neher, 1975). Unit conductance step increase from phosphatidy1choline through phosphatidylethanolamine analogue with decrease in channel lifetime, 1.4 to 0.17 sec (Neher and Eibel, 1977). Effects of synthetic and natural compounds (Bamberg et al., 1976b; Tredgold et al., 1977) and of an analogue of gramicidin A (Apell et al., 1977). Alamethicin: Voltage-dependent conductance change (Mueller and Rudin, 1969; Chapman et aI., 1969; Goodall, 197Oc; Mauro et al., 1972; Gordon and Haydon, 1975). Increased cation conductance by creation of channels (Petkau and Chelack, 1972). Voltage-dependent and -independent conductances (Cherry et al., 1972; Roy, 1975). Exponential conductance increase with five levels (Eisenberg et al., 1973; Hall, 1975). Unit conductance channel properties (Gordon and Haydon, 1972). Activation enthalpy for ion conduction (Ginsburg, S., and Noble, 1974). Conductance of channels in terms of transition-state theory (Gordon, 1973a), thermodynamics (Gordon, 1973b), and models (Gordon, 1974). Kinetics of conducting channel (Gordon and Haydon, 1976). Statistics of pore formation (Boheim, 1974; Boheim and Hall, 1975). Monazomycin: Voltage-dependent conductance change (Mueller and Rudin, 1969; Muller and Finkelstein, 1972a; Mauro et al., 1972; Baumann and Mueller, 1974). Inactivation of voltage-dependent conductance (Heyer et al., 1976b) and production by quaternary ammonium ions (Heyer et al., 1976a). Voltage independence of single channel (Bamberg and Janko, 1976). Fluctuation and relaxation analysis of conductance (Moore, L. E., and Neher, 1976). Channel noise analysis indicates only a fraction of channel conductance fluctuating (Wanke and Prestipino, 1976). Excitability-inducing material (ElM): Dynamic voltage-dependent conductance change (Mueller and Rudin, 1969; Bean et al., 1969). (Continued)

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N. Lakshminarayanaiah

Table VI (Continued) Compounds That Increase Bilayer Membrane Conductance by Creating Channels or Pores: Some Notes and Comments ElM (Continued) Discrete conductance fluctuations due to opening and closing of channels (Ehrenstein et ai., 1970; Gregor'ev and Ermishkin, 1970). ElM-activated areas cation-selective (Kalkwarf et ai., 1972) and cation-conductancevoltage-dependent (Latorre et ai., 1972). Channel characteristics (Bean, 1972) and kinetics (Ehrenstein et ai., 1974; Alvarez et al., 1975a), and single-channel conductance, 3 x 10-11 mho (Hoffman et aI., 1976). Mechanism of gating (Lecar et al., 1975; Ehrenstein and Lecar, 1977). Temperature effect on channel conductance (Latorre et al., 1974). Hemocyanin: Voltage-dependent conductance change (Pant and Conran, 1972; Alvarez et al., 1975b). Relaxation of discrete conductance change order of seconds and that of continuous conductance change order of 10-4 sec (Latorre et al., 1975). Suzukacillin: Discrete conductance fluctuations-kinetics similar to those of alamethicin (Jung et al., 1976; Boheim et al., 1976). Black widow spider venom: Creates channels-single-channel conductance, 3.6 x 10- 10 mho (Finkelstein et al., 1976). DJ-400B: Voltage-dependent conductance change (Mueller, 1975a,b). ATPase extract from StreptococcusJecalis: Voltage-dependent jumps in membrane conductance-unit conductance, 10- 10 mho (Redwood et al., 1973). Paramecium mitochondria: Voltage-dependent anion-selective channel (Schein et aI., 1976). Amphotericin B: Created pores but cholesterol required (Finkelstein and Cass, 1968; Andreoli and Monahan, 1968; Marty and Finkelstein, 1975). Increased water (Andreoli et al., 1969; Dennis et ai., 1970; Holz and Finkelstein, 1970; Cass et ai., 1970) and halide (Kasumov and Liberman, 1972) permeabilities. Discrete change in conductance due to single channel (Ermishkin et ai., 1976). Nystatin: Membrane conductance increase due to formation of pores (Finkelstein and Cass, 1968; Andreoli and Monahan, 1968; Marty and Finkelstein, 1975). Increased halide permeability (Kasumov and Liberman, 1972). Water permeability compared with that in gramicidin pore (Finkelstein, 1974). Discrete change in conductance due to channels (Ermishkin et al., 1976); however, see Romine et al. (1977) to the contrary.

ments (Anderson, C. R., and Stevens, 1973; Sachs and Lecar, 1973; Conti and Wanke, 1975; Conti et ai., 1975, 1977; Begenisich and Stevens, 1975; Neher and Sakmann, 1976; Stevens, 1977) show that the single-channel conductance ranged from 4 to 39 (x 10- 12) mho. In the squid axon membrane, it has been considered (Armstrong, 1974) that the sodium channel conductance is 0.5 x 10- 11 mho and that of potassium channel, 10- 11 mho. The study of the permeability of the Na and K channels to several inorganic and organic ions of differing shape and size has enabled estimation of channel sizes. Hille (1973) determined the relative permeabilities of K, TI, Rb, and NH4 ions in the K channel of the myelinated nerve to be

425

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1: 2.3: 0.91: 0.13. Other ions (Li, Na, Cs, methylamine, guanidine, hydrazine, and hydroxylamine) were relatively impermeable. These relative permeability data were correlated with the minimum pore diameter required to let the ions pass. The values given in Table VII show that the K pore diameter should correspond to 3.0-3.3 A. Similar kinds of ionpermeability measurements presented by Bezanilla and Armstrong (1972) show that ions with crystal diameters from 2.66 A (K+) to 2.96 A (Rb+, NHt) passed through the K pore, while ions with smaller (Na+ = 1.9, Li+ = 1.36 A) and larger (Cs = 3.3, TEA = 8 A) diameters did not. It is considered (Armstrong, 1974) that the K channel has a narrow pore (range of diameter 2.6-3 A) with a mouth diameter of 8 A to just admit the TEA ion. Ions larger in diameter than 3 A are excluded sterically from the pore, whereas ions smaller than 2.6 A are excluded because of unfavorable interaction with the oxygen-lined wall of the pore. An explicit picture of the Na channel was presented by Hille (1971, 1972), again on the basis of permeability studies in myelinated nerve. The permeability sequence of some cations was N a = hydroxylamine > hydrazine > NH4 = formamide = guanidine = hydroxyguanidine > aminoguanidine > > methylamine. Other cations [N-methylhydroxylamine, methylhydrazine, methylamine, ｭ･ｴｨｾｬｧｵ｡ｮｩ､Ｌ＠ acetamidine, dimethylamine, tetramethylammonium, TEA, ethanolamine, choline, tris(hydroxymethyl)aminomethane, imida-

Table VII Relative Cation Permeability Data Derived for the Potassium Channel in Frog Nerve u Pore diameter'

Permeability ratio

Cation

(A)

(PJPK)C

Lithium Sodium Potassium Thallium Rubidium Ammonium Hydrazine Hydroxylamine Cesium Methylamine Formamide Guanidine

1.20 1.90 2.66 2.80 2.96 3.0 3.3 3.3 3.38 3.6 3.6 4.8

< 0.018 < 0.01

<