OCT 1 2 1999 MAY 1 2 2003 (SD ENZYME CYTOLOGY ENZYME CYTOLOGY Edited by D. B. ROODYN Department of Biochemistry, University College London, England 1967 ACADEMICPRESS ' LONDON AND NEW YORK A» ACADEMIC PRESS INC. (LONDON) LTD. Berkeley Square House Berkeley Square London, W.l. U.S. Edition published by ACADEMIC PRESS INC. Ill Fifth Avenue New York, New York 10003 Copyright © 1967 by Academic Press Inc. (London) Ltd. All Rights Reserved No part of this book may be reproduced in any form by photostat, microfilm, or any other means, without written permission from the publishers Library of Congress Catalog Card Number: 66-30440 ERINDALE COLLEGE LIBRARY printed in great BRITAIN BY PAGE BROS. (NORWICH) LTD. LIST OF CONTRIBUTORS Norman G. Anderson, Molecular Anatomy Section, Biology Division, Oak Ridge National Laboratory and Technical Division, Oak Ridge Gaseous Diffusion Plant, Oak Ridge, Tennessee, U.S.A. C. DE. DuvE, The Rockefeller University, New York, U.S.A. and Universite de Louvain, Laboratoire de Chimie Physiologique, Louvain, Belgium. [ David Elson, The Weizmann Institute of Science, Rehovoth, Israel. G. P. Georgiev, Institute of Molecular Biology, Academy of Sciences of the U.S.S.R, Moscow, U.S.S.R. John G. Green, Molecular Anatomy Section, Biology Division, Oak Ridge National Laboratory and Technical Division, Oak Ridge Gaseous Diffusion Plant, Oak Ridge, Tennessee, U.S.A. D, O. Hall, Department of Botany, University of London, King's College, England. ^ E. Reid, Department of Biochemistry, University of Surrey, London, England. D. B. RooDYN, Department of Biochemistry, University College London, England. W. Straus, Division of Metabolic Research, Institute for Medical Research, The Chicago Medical School, Chicago, Illinois, U.S.A. F. R. Whatley, Department of Botany, University of London, King's College, England. EDITOR'S FOREWORD The bringing together of enzymology and cytology is relatively recent and has occurred in a somewhat disjointed, and at times confusing manner. Although the techniques for staining cells for enzymes have been considerably improved in the last fifteen years, the majority of enzyme-catalysed reactions still cannot be satisfactorily followed by histochemical means. The greater part of our knowledge of the enzy- mology and metabolism of cell components is therefore derived from studies on fractions isolated from cell homogenates, almost invariably by centrifugal procedures. The technique of "differential centrifugation", as it is usually called, is readily carried out by relatively unskilled workers, who certainly need no special training in cytological technique. The flexibility of the method, and the ease with which the isolated sub-cellular fractions can be subjected to standard chemical and enzymological procedures, has resulted in a great outburst of new information about the properties of cell components. Improved methods of cell disruption and centrifugal fractionation have resulted in greater precision and reproducibihty and the method has now been apphed to a very wide range of biological materials. Concurrently with these developments, the application of electron microscopy has resulted in a massive increase in our knowledge of cell structure. With improved instrument design, embedding, staining and sectioning techniques, the electron microscopist has led us into an intriguing world of fine detail that reaches down to the molecules themselves. Magnificent pictures are now obtained showing exquisite detail of the nuclear membrane, chloroplast lamellae, mitochondria) membranes, ribosomes, cytomembranes and so forth. The convergence of these two powerful techniques, electron microscopy and the centri- fugal fractionation of homogenates, has brought about a revolution in our understanding of the cell. However, the progress has not always been even. Biochemists and electron microscopists are often uneasy collaborators. The tempo of biochemical research is different from that of cytological investigation. The biochemist tends to convert the cell into the abstractions of instrument readings, tabulated data and graphs, whereas the cytologist is essentially concerned with visual phenomena, and may easily feel that Michaelis constants and metabolic maps are rather second hand, remote ways of describing the cell. Also, the electron microscopes themselves vii Viii ENZYME CYTOLOGY are frequently housed in anatomy, botany, zoology or cytology departments, and it is still rather unusual for biochemists to have very easy access to electron microscopy. Some research centres have deliberately worked for an integration of biochemical and cytological studies, with excellent results. Usually, however, the relationship is not so close, and detailed electron microscope characterization of isolated sub-cellular fractions is still far too infrequent. There are other difficulties arising from the fact that the technique of centrifugal fractionation of homogenates is still relatively new. Rigorous attitudes, or even terminology, have not yet been worked out. There are few "standard" procedures, and in some fields (for example the isolation of nuclei) until very recently, almost every research worker seemed to use his own method. There are still serious problems of cross- contamination of fractions, of enzyme adsorption and elution during fractionation, of structural damage by homogenizers and isolation media as well as great technical difficulty in obtaining vaUd enzyme assays in the complex mixtures of enzymes present is isolated fractions. Many of the results obtained with radioactive tracers give essentially qualitative answers which are not amenable to accurate expression in "balance sheets". For reasons of convenience, there has been undue emphasis on certain tissues (such as rat-liver) and also on certain enzymes which are easy to assay but are not necessarily of great importance. In spite of these difficulties, there is now Httle doubt that enzyme cytology is a new scientific discipline. As Professor de Duve says below; "It is a true hybrid, which after the inevitable growing pains and search for its own identity, has now come of age as a vigorous individual in its own right . . . . " One has only to survey the major findings reported in each chapter of this book to reahze the magnitude of the advance that has been made in such a short period. The role of the nucleus in nucleic acid synthesis and protein synthesis has been revealed, and our know- ledge of the molecular basis of gene action greatly enriched. In addition, the general enzymology of the nucleus has been explored in detail. There have been great advances in our understanding of the structure and function of the mitochondrion. Its role in ATP synthesis, hpid, amino acid and carbohydrate metabohsm, ion transport and other processes has been elucidated in great detail in a flood of pubhcations. The discovery of mitochondrial (and chloroplast) DNA has resulted in an awakening of interest in the problem of the mechanism of biogenesis of cell structures. Also, research on the structure and function of the chloro- plast has been most fruitful, and in addition to the most detailed elucidation of the fine structure of this organelle, its role in COg fixation, photophosphorylation and a wide range of other activities has been EDITOR'S FOREWORD ix examined most fully. A whole class of lysosomal particles has been discovered and these particles have been show^n to be involved in many different physiological and pathological processes. It is hard to believe that the discovery of the lysosome v^as as recent as 1955. The properties of cell membranes, and in particular the endoplasmic reticulum, have been studied in detail, and a great variety of membrane-associated enzymes has been revealed. The wealth of information that we now have of the structure and function of the ribosome is quite striking. When one considers the contemporary literature on ribosomes, polysomes, messenger RNA and the genetic code, again it is almost unbehevable that the term "ribosome" was only introduced into the literature in 1958! Finally, the soluble phase of the cell has been shown to have an important role in many metabolic processes, including glycolysis, fatty acid synthesis, nucleotide metabolism, and the pentose shunt pathway. Thus, in spite of the many imperfections of technique and the obvious gaps in our knowledge, it cannot be denied that a series of findings of the utmost significance to biology have been made in this field. Most of these results have come in the last decade, and a good proportion in the last 2 or 3 years. This book is an attempt to bring these findings together, covering the whole field of enzyme cytology in one volume. After an introductory chapter on the principles of the subject, we have, so to speak, worked from the bottom of the centrifuge tube upwards, dealing with nuclei, mitochondria, peroxisomes, chloroplasts, lysosomes, membranes, ribosomes and cell sap. The general design of the chapters is more or less the same, in that an attempt has been made in each case to survey the whole field in a reasonably detailed fashion, but without excessive emphasis on the authors' own interests, or on last month's research reports. The text is brought together by a rather lengthy subject index, and a list of the enzymes discussed is added at the end of the book. Some suggestions for terminology in enzyme cytology have also been included as an appendix. Although the editor consulted the various contributors for their views on this, and indeed has taken some terminology directly from the text in some cases, these suggestions are best regarded as the private prejudices of the editor, put forward more to stimulate discussion on the important semantic problems in the field than to lay down firm rulings. It is hoped that the reader will not be too impatient with the length of the book, and will appreciate the great difficulty in compressing what are now very active fields of research into chapters of a manageable size. The burden of new literature is heavy, and even as we write, our ideas must be modified to adapt to the ceaseless flow of new information. It is inevitable that much of what is written below will soon become X ENZYME CYTOLOGY outdated. However, if we are not to be overwhelmed by the outpourings of our electron microscopes, ultracentrifuges and automatic spectro- photometers, some attempt must be made, at one point of time, to stand back from the work and to assess the progress so far made. Perhaps such an effort is futile, and one should allow the subject to fragment into a series of specialities of an increasingly esoteric nature, presented in a language that is more and more incomprehensible to the general reader. However, it would be unfonunate if this process occurred simply because of the inabihty of the individual worker in the field to acquaint himself with the major findings in all aspects of the subject. However complex the cell appears to be, and however detailed and abstruse are the descriptions of its component parts, one must always hope that a unifying pattern exists, which will ultimately be comprehen- sible to a single individual. If this book is of some use in the quest for this pattern, the labours of its contributors will not have been in vain. November, 1966 D. B. Roodyn EDITOR'S ACKNOWLEDGEMENTS The Editor would like to express his sincere thanks to the following for permission to reproduce material, the loan of plates and diagrams, and for helpful discussion and correspondence: Dr. A. D. Bangham, Professor B. Chance, Dr. J. B. Chappell, Dr. R. S. Criddle, Professor C. de Duve, Professor H. Fernandez-Moran, Professor S. Fleischer, Professor D. E. Green, Dr. R. Gustafsson, Dr. H. W. Heldt, Professor M. Klingenberg, Professor H. A. Krebs, Professor A. L. Lehninger, Dr. D. J. L. Luck, Professor F. Lynen, Dr. P. Mitchell, Dr. E. Mugnaini, Professor M. J. Nadukavukaren, Dr. M. M. K. Nass, Dr. L. Packer, Dr. L. D. Peachey, Dr. C. S. Rossi, Professor D. R. Sanadi, Dr. P. Siekevitz, Professor E. C. Slater, Dr. R. T. Ward, Dr. E. P. Whitehead and Dr. V. P. Whittaker. He is also grateful to Dr. R. Bellairs for electron micrographs of mitochondria. The Editor is grateful to Dr. H. W. Heldt for the following recent comments. Since publication of the scheme given in Fig. 21 of Chapter 3, on "The Mitochondrion" [Fig 21. is adapted from Fig. 7 of Heldt (1966) and Fig. 15 of Khngenberg and Pfaflf (1966)], it now appears that the inner compartment of rat-liver mitochondria contains a GTP - AMP - phosphate transferase, and Dr. Heldt and his colleagues no longer maintain that adenylate kinase is also present in the inner compartment [see Heldt, H. W. and Schwalbach, K. (1966). Fed. Europ. Bioch. Soc. Abstr. Warsaw, p. 134]. The following have kindly given permission to reproduce material in the various figures plates and diagrams: The American Chemical Society, The American Physiological Society, Annual Review of Bio- chemistry, W. A. Benjamin, Inc., The Biochemical Society, Journal of General Microbiology, The Elsevier Publishing Company, Journal of Biological Chemistry, The National Academy of Sciences and the Rockefeller University Press. In cases where the photographs were reproduced from printed halftone copy, the quality of the results should not be taken as representative of the originals, to which the reader is referred. Other acknowledgements are given in the text in the various chapters, and the source of all plates and figures is marked in the appropriate legends. The Editor trusts that due acknowledgements have been made in all cases and apologizes for any omissions that may have been made inadvertently. xi Xii ENZYME CYTOLOGY The Editor is most grateful to his wife for arduous work in the preparation of the subject index and to the contributors to "Enzyme Cytology" who have kept their patience in what has been a rather complex and time-consuming affair. He is also most grateful to the staff of Academic Press who have coped with the production of quite complicated textual material. D. B. R. CONTENTS List of Contributors ........ v Editor's Foreword . . vii Editor's Acknowledgements xi Abbreviations Used in the Text xix Chapter 1 GENERAL PRINCIPLES, C. de Duve I. The Historical Significance of Enzyme Cytology. II. The Objectives and Methods of Enzyme Cytology '^11. The Conduct of Fractionation Experiments A. From Tissues and Cells to Homogenates . B. From Homogenates to Subcellular Fractions C. From Subcellular Fractions to Analytical Results IV. The Interpretation of Fractionation Data . A. From Analytical Results to Subcellular Fractions B. From Subcellular Fractions to Homogenates C. From Homogenates to Cells and Tissues . V. Conclusion ........ VI. Appendix: Graphical Presentation of Results References ......... 1 1 3 5 6 8 15 17 17 18 20 21 22 24 Chapter 2 THE NUCLEUS, G. P. Georgiev ....... 27 I. Introduction .......... 27 II. Methods of Isolation and Fractionation of Nuclei ... 28 A. Procedures Involving the Use of Organic Solvents . . 29 B. Aqueous Methods of Nuclear Isolation . . . . 31 III. Structure and Methods of Sub-fractionation of the Nucleus. . 35 A. Structure and Ultrastructure of Nuclei .... 35 B. Chemical Fractionation ....... 44 C. Mechanical Methods of Fractionation .... 46 IV. Auto-Reproduction (Duplication) (DNA Biosynthesis) . . 49 A. Nuclear DNA Polymerase ...... 50 B. Mechanism of DNA Synthesis in Chromosomes. . . 52 V. Transcription (RNA Biosynthesis) ...... 56 A. Intracellular Localization of RNA Synthesis ... 56 B. RNA Polymerase and its Intracellular Localization . . 60 C. Chromosomes and mRNA Synthesis ..... 66 D. Synthesis of Ribosomal RNA and sRNA and the Function of the Nucleolus ........ 72 E. Other Enzymes Involved in Synthesis and Breakdown of Polynucleotides ........ 74 VI. Translation (Protein Biosynthesis) ...... 76 A. Protein Synthesis in Nuclear Sap Ribosomes ... 77 B. Protein Synthesis in the Nucleolo-Chromosomal Complex . 79 xiu XIV CONTENTS VII. Enzymes of Energy Metabolism 81 A. Glycolysis 81 B. Citric Acid Cycle 83 C. Oxidative Phosphorylation 84 VIII. Other Enzymes (Enzymes of Intermediary Metabolism) . . 86 A. Nucleotide Metabolism 87 B. "Special" Enzymes and Widely Distributed Enzymes . 89 C. Occurrence of Enzymes of Different Cytoplasmic Structures in the Nucleus ........ 91 D. The Nuclear Membrane and Permeases .... 93 IX. Conclusion: The Function of the Cell Nucleus and Nuclear Structures 94 References 95 THE Chapter 3 MITOCHONDRION, D. B. Roodyn I. Introduction .... II. Survey of the Morphology and Metabolic Activity of Mitochondria A. Essentials of Mitochondrial Morphology . B. Problems in Surveying Metabolic Activity of Mitochondria C. Tricarboxylic Acid Cycle D. Oxidation of Fatty Acids. E. Respiratory Chain . F. Oxidative Phosphorylation G. Ion Transport H. Anabolic Reactions . I. Other Reactions J. Summary and Conclusions III. The Structural Basis of Mitochondrial Function , A. Mitochondrial Sap ..... B. Mitochondrial Inclusions: DNA C. Mitochondrial Inclusions: Dense Granules D. The Mitochondrial Membrane System E. Enzymes or Enzyme Systems Associated with the Membrane F. Chemical Composition of the Membrane . G. Stalked Bodies and Elementary Particles . H. The Mitochondrial Membrane as an Integrated Structure IV. Addendum : The Structure and Function of Peroxisomes References .......... 103 103 104 105 107 111 114 117 119 128 129 132 138 138 139 145 150 153 154 160 165 169 172 175 THE CHLOROPLAST, D. O. Chapter 4 Hall and F. III. Introduction ..... Chloroplasts and Other Plastids A. Chloroplasts .... B. Chromatophores C. Non-photosynthetic Chromoplasts D. Leucoplasts .... Methods for Isolation of Chloroplasts A. Isolation in Aqueous Medium . B. Isolation in Non-aqueous Medium R. Whatley 181 181 182 182 186 187 187 188 189 193 CONTENTS XV C. Comments on the Localization of Enzyme Activity IV. Enzymes of Chloroplasts A, Enzymes for Assimilatory Power B. Enzymes for CO 2 Fixation Cycle C, Additional Enzyme Reactions . D. Induced Enzyme Formation V. Comparison of Oxidative and Photosynthetic Phosphorylation References ......... Chapter 5 /LYSOSOMES PHAGOSOMES AND RELATED PARTICLES W. Straus I. Introduction II. Properties of Lysosomes ....... ~^. Enzymes ........ B. Non-Enzymic Components of Lysosomes . C. Lysosomal Membrane and Activation of Enzymes III. Occurrence of Lysosomes ...... A. Lysosomes in Epithelial Cells ..... B. Lysosomes in Reticulo-Endothelial Cells and Blood Cells C. Lysosomes in Different Species. .... D. Lysosomes in Tissue Culture Cells .... E. Lysosome-like Granules in Plant Cells IV. Origin and Formation of Lysosomes and Lysosomal Enzymes V. Lysosomes and Ingestion Processes (Phagosomes and Phago Lysosomes) ..... A. Microscopic and Sub-Microscopic Aspects of Ingestion Processes . "B. Uptake of Proteins C. Uptake of Lipids . . . D. Uptake of Nucleic Acids and Viruses E. Uptake of Other Macromolecular and Colloidal Material F. Uptake of Vital Dyes G. Factors Influencing Permeability, Uptake, and Transport H. Metabolism of Uptake Processes .... I. Secretion as Reversed Pinocytosis .... J. Degradation of Ingested Macromolecular Material, Especially of Proteins .... K. Accumulation of Poorly Digestible Material in Phago Lysosomes (Residual Bodies) ..... VI. Autolysis and Autophagy A. Physiological Autolysis B. Autophagy ........ VII. Relationship of Lysosomes and Phagosomes to Immune Processes A. Antibody Formation B. Uptake of Antigen-Antibody Complexes . C. Antigen and Antibody Reactions at the Cell Surface . D. Recognition of "Self" ...... VIII. Pathological Changes Related to Lysosomes and Phagosomes A. Pathological Enlargement of Lysosomes and Phagosomes B. Release of Lysosomal Enzymes In Vivo C. Extrusion of Lysosomes and Lysosomal Enzymes 195 197 197 208 217 230 230 232 239 240 243 243 250 251 253 253 256 257 257 258 258 261 262 267 271 273 274 275 277 281 282 283 285 286 286 288 289 289 291 293 293 294 294 296 296 XVI CONTENTS IX X D. Possible Role of Lysosomes in Inflammation, Necrosis, Shock, and Virus Infection Appendix: Methods for the Study of Lysosomes and Phagosomes ' A. Isolation of Lysosomes ..... B. Cytochemical Reactions for Lysosomal Enzymes C. Markers for Pinocytic and Phagocytic Vacuoles . Concluding Comments Acknowledgements ....... References ........ MEMBRANE SYSTEMS, E. Chapter 6 Reid I. Introduction ...... A, Nature of Membrane Systems . B. Cytomembranes II. Cytochemical Observations A. Methodology ..... B. Enzymes Associated with Membranes 4' ^ III. Biochemical Observations A. Isolation and Characteristics of Hepatic Microsomal Fractions B. Isolation and Characteristics of Hepatic Cytomembrane Fractions with Few Ribosomes C. Membrane-containing Fractions (Vertebrate) other than Hepatic Cytomembrane Fractions D. Membrane-containing Fractions (Invertebrate) IV. Correlations and Functional Aspects A. Normal Tissues B. Pathological Tissues V. Summary Acknowledgements References Chapter RIBOSOMAL ENZYMES, D. Elson I. Introduction .... II. The Structure of the Ribosome A. The Isolation of Ribosomes B. Properties of Ribosomes . C. Ribosomal RNA . D. Ribosomal Proteins E. The Structure of the Ribosome F. Non-bacterial Ribosomes III. The Function of the Ribosome IV. Ribosomal Enzymes — General Considerations V. Ribosomal Enzymes — Enzymes of Protein Synthesis VI. Ribosomal Enzymes — Nascent Enzymes . A. Pancreatic Enzymes and Zymogens . B. Other Studies of Nascent Enzymes . C. Summary ..... VII. Ribosomal Enzymes — Ribonuclease (2. 7. 7. -) CONTENTS XVll A. Pancreatic Ribosomal RNase ..... B. E. coli Ribosomal RNase ..... C. Biological Function of RNase ..... VIII. Ribosomal Enzymes — Other Enzymes which Act on Polynucleotides A. Deoxyribonuclease (3. 1. 4. -) . B. DNA Polymerase {2.1.1.1) . . . C. Polyribonucleotide Polymerases (2. 7. 7. -) D. Aminoacyl-sRNA Synthetases (6. 1 . 1 . -) . E. RNA Methylases F. Polynucleotide Phosphorylase (2. 7. 7. 8) . C. Phosphodiesterases (3. 1. 4. 1) . H. The Binding to Ribosomes of Enzymes which Act on Polynucleotides ....... IX. Ribosomal Enzymes — Other Examples .... A. Phosphatases (3. 1. 3. -) . B. Peptidases ........ C. NAD Pyrophosphorylase (2. 7. 7. 1) D. Adenosine Triphosphatase (3. 6. 1. 3) E. Other Enzymes Found in Ribosome Preparations X. Summary ......... References 439 440 447 450 450 453 454 454 454 455 455 455 456 456 456 457 460 460 461 468 Chapter 8 THE SOLUBLE PHASE OF THE CELL, N. G. Anderson and J G. Green I. Introduction ..... II. Definition of the Soluble Phase A. Classification of Components . B. Interpretation of Results . III. The Composition of the Soluble Phase A. Micromolecules B. Mesomolecules C. Macromolecules IV. Enzymes of the Soluble Phase . A. Enzyme Groups of the Soluble Phase B. Problems in Interpretation of Enzyme Data C. Control of Enzymic Activity in the Cell V. Physiology of the Soluble Phase A. The Efl'ect of Heat . B. The Efl'ect of pH . C. The Role in Membrane and Particle Formation VI. Conclusions ..... References . . . Appendix Author Index Subject Index .... List of Enzyme Numbers 475 475 476 477 481 483 483 484 491 496 496 497 499 500 500 501 503 503 504 511 519 547 583 ABBREVIATIONS USED IN THE TEXT '^ Energy-rich intermediate A Angstrom unit ACTH adrenocorticotrophic hormone ADP adenosine diphosphate S-ALA 8- amino laevuhnic acid AMP adenosine monophosphate ATP adenosine triphosphate ATPase adenosine triphosphatase CDP cytidine diphosphate CE chloroplast extract CMP cytidine monophosphate CMU 3-(4-chlorophenyl)- 1 , 1 -dimethyl urea CoA coenzyme A CoA.SH free coenzyme A CTP cytidine triphosphate DEAE diethylaminoethyl DNA deoxyribonucleic acid DNase deoxyribonuclease DNP deoxyribonucJeoprotein D-RNA RNA capable of hybridizing with DNA Eq standard oxidation reduction potential EAA extractable amino acids EDTA ethylene diamine tetra-acetate ETP electron transfer particle FAA free amino acids FAD flavin adenine dinucleotide FDP fructose diphosphate FMN flavin mononucleotide GDP guanosine diphosphate GTP guanosine triphosphate I intermediate in oxidative phosphorylation IDP inosine diphosphate Km MichaeUs constant mRNA messenger RNA NAD nicotinamide adenine dinucleotide NAD+ oxidized NAD NADH reduced NAD xix XX ABBREVIATIONS USED IN THE TEXT NADP nicotinamide adenine dinucleotide phosphate NADP+ oxidized NADP NADPH reduced NADP Pi inorganic phosphate P-Pi pyrophosphate RNA ribonucleic acid RNA-P phosphorus in RNA RNase ribonuclease RNP ribonucleoprotein R-RNA RNA with a base composition of ribosomal RNA S Svedberg unit SP soluble phase SPAA soluble phase amino acids sRNA soluble RNA Tm melting temperature TMPD tetramethylparaphenylenediamine tRNA transfer RNA tris 2 amino-2 hydroxymethylpropane,l-3 diol UDP uridine diphosphate UDPase uridine diphosphatase UDPG uridine diphosphoglucose UMP uridine monophosphate UTP uridine triphosphate UTPase uridine triphosphatase W-^ intermediate in oxidative phosphorylation X,X'-^I,X'^P intermediates in oxidative phosphorylation. I Chapter 1 GENERAL PRINCIPLES C. DE DUVE The Rockefeller University, New York, U.S.A., and Universite de Louvain, Laboratoire de Chimie Physiologique, Louvain, Belgium The Historical Significance of Enzyme Cytology , The Objectives and Methods of Enzyme Cytology The Conduct of Fractionation Experiments . A. From Tissues and Cells to Homogenates . B. From Homogenates to Subcellular Fractions C. From Subcellular Fractions to Analytical Results The Interpretation of Fractionation Data A. From Analytical Results to Subcellular Fractions B. From Subcellular Fractions to Homogenates C. From Homogenates to Cells and Tissues . Conclusion ........ Appendix : Graphical Presentation of Results References 1 3 5 6 8 15 17 17 18 20 21 22 24 p. The Historical Significance of Enzyme Cytology Enzyme cytology was born some twenty-five years ago from the cross ferti- lization of two flourishing disciplines, dynamic biochemistry and descriptive cytology, which traditional differences in outlook and methodology had long kept entirely separated. It owes its inception largely to the vision and efforts of a few isolated investigators who laboured for many years to break down a barrier which was taken for granted by most of their contemporaries. The names of Martin Behrens, Robert Bensley, Albert Claude and George Gomori remain attached to this historical breakthrough. At the time when these pioneering studies were being made, biochemistry was almost exclusively occupied with metabolic reactions occurring in soluble extracts, still riding the sweeping wave on which it had been launched at the turn of the century by the Biichner discovery that yeast juice carries out the complete fermentation of sugar. This preoccupation is betrayed in many publications from that period by the sentence: "The extract was clarified by centrifuging and the insoluble residue discarded." Few biochemists probably gave a second thought to this residue: cells and tissues obviously had to be kept together by something and the residue was "it", no more than the inert shell within which living processes take place under the influence of soluble enzymes. 1 2 C. DE DUVE It is true that Warburg had already demonstrated in 1913 that cellular respiration is associated with sedimentable particles, but this finding had attracted little attention and it took almost thirty years before the interest of biochemists became focused on the "residue". By 1948, the analysis of this system had already brought to light the existence of an integrated multi- enzyme complex, the "cyclophorase" of Green, Loomis and Auerbach, and it is conceivable that further studies along these lines might eventually have led to the correct biochemical concept of a mitochondrion. But it is difficult to guess how many false trails biochemists unaided by enzyme cytology would have explored, or might still be exploring, before appreciating the importance of isolating the particles in such a way as to preserve their elusive and apparently paradoxical metabolic inertness. Surely, the development at that time of methods specifically directed towards the isolation of intact intracellular components played a decisive role in opening what has now become one of the most important areas of research in biochemistry. The demonstration by Schneider and Potter (1949) and by Kennedy and Lehn- inger (1949) that the mitochondria are the main sites of cellular oxidations and of the associated phosphorylations, and the subsequent discovery by Kielly and Kielley (1951) and by Lardy and Wellman (1952) that mitochondrial respiration is controlled by the rate of regeneration of ADP from ATP are essential landmarks in our understanding of oxidative phosphorylation and of respiratory control. They can be traced back directly to the efforts of Bensley and Hoerr (1934), Claude (1946a, b) and Hogeboom, Schneider and Palade (1948). Another question which may well be asked is how far our knowledge of protein synthesis would have progressed without cell fractionation. It is sometimes forgotten that the triumphant road leading to the genetic code starts with the early experiments of Borsook, Deasy, Haagen-Smit, Keighley and Lowy (1950a, b) and of Hultin (1950) which brought to light the important role of the microsomal fraction in the new formation of proteins. The later identification of the ribosomes as the main centres of protein synthesis was again the result of combined cytological and biochemical studies (Littlefield, Keller, Gross and Zamecnik, 1955; Palade and Siekevitz, 1956; Siekevitz and Palade, 1958). Many other important developments of biochemistry owe a similar debt to cell fractionation techniques. In fact, the introduction of the four-fraction scheme has provided such spectacular returns that few biochemists have felt impelled to penetrate further into the intricacies of cell structure. Bio- chemistry has moved from the "extract-residue" era to that of "nuclei, mitochondria, microsomes and supernatant" ; it has not, except in the speci- alized field of enzyme cytology, attempted to move much further. Far from reflecting unfavourably on the adventurousness of biochemists, this fact emphasizes the richness of the field which has been opened to biochemical I 1. GENERAL PRINCIPLES research through the first contributions of enzyme cytology, as well as the Vrnificance of the latter discipline. The impact of enzyme cytology on descriptive morphology is hardly less important. We need only think of how sterile our vision of even the most beautiful present-day electron micrographs would be, were it not illuminated, sometimes even with direct cytochemical illustration, by our knowledge of the enzymic attributes of intracellular structures. How revealing this knowledge has been to our understanding of mitochondria and of ribosomes has already been mentioned. Another example of this revelation is provided by the wealth of data which have been derived from studies on acid phosphatase (3.1.3.2), one of the first enzymes to be successfully detected in tissue sections. Thanks to a combination of all the resources of enzyme cytology, this enzyme has helped to clarify the nature of numerous subcellular entities and to trace down both in time and in space the intricate digestive processes in which they are involved (Novikoff", 1963a; de Duve and Wattiaux, 1966). The role of the endoplasmic reticulum and of the Golgi apparatus in protein secre- tion (Palade, Siekevitz and Caro, 1962), the origin of mitochondria (Luck, 1963a, b; 1965a, b), the function of microbodies (de Duve and Baudhuin, 1966) are other problems which morphological research alone would have been quite unable to solve. An important conclusion suggested by this brief historical survey is that the function of enzyme cytology is not simply to serve as a connecting link between its parent disciplines. It is a true hybrid which, after the inevitable growing pains and search for its own identity, has now come of age as a vigorous individual in its own right, with specific properties that are much more than a simple combination of parental traits. It is a separate discipline with its own objectives, rules and methods, and it is capable of making unique contributions to our understanding of cell structure and function which neither biochemistry nor morphology could possibly achieve within their respective boundaries. II. The Objectives and Methods of Enzyme Cytology Enzyme cytology proposes to establish the exact intracellular localization of enzymes, justifying this objective by the rational argument that the work- ing of a machine can be understood only from a knowledge of both the functional properties and the spatial organization of its component parts. In order to achieve this purpose, it has to draw heavily on the resources and acquisitions of dynamic biochemistry and of descriptive cytology; but it must refrain from any dogmatic adherence to all their pronouncements. The reconstructions of the biochemist are not all established facts that stand no correction; neither are electron micrographs faithful representa- tions of all that is to be known about the fine structure of cells. If any revision is needed in either area, enzyme cytology is best placed to do so, and its v 4 C. DE DUVE searches should be conducted with a sufficient lack of bias so as to allow this important role to be played. Nothing could be more stultifying, especially when exploring a field where so much remains to be discovered, than an approach that leaves nothing to the unexpected. These aims and warnings can be translated into two simple rules. In the first place, one should aim as much as possible at localizing the site of single step enzymic reactions rather than of multi-enzyme systems. Truly, this may not always be feasible, nor even commendable, and one can think of many examples where work on complex systems has greatly helped our under- standing. However, the danger in doing this is that the association of enzymes found by the biochemists to be operative in disrupted systems is implicitly taken to be biologically meaningful and even to have a morphological counterpart. Such is not always the case and the verification can come only from a careful biochemical dissection of the system into its individual enzymic units, followed by localization of the latter by means of the tools of enzyme cytology. This necessity was clearly realized already in the early days of centrifugal fractionation, when a number of investigators found the mitochondrial fraction to be the main site of oxidative processes. As pointed out by Schneider and Hogeboom (1951), what the experiments actually proved was the unique localization of the terminal oxidase in this fraction \J and not, for instance, that of all the dehydrogenases many of which are present outside the mitochondria (See Chapter 3). A similar mistake was made in visual cytochemistry when the tetrazolium method was introduced for the localization of dehydrogenases : the mitochondria were found to stain selectively with all substrates, not because they contain all the dehydrogenases, but simply because they happen to contain the diaphorase necessary for the reduction of tetrazolium (Novikoff, 1963b). These are not isolated instances and it is now known that many polyenzymic processes require the co-opera- tion of two or more intracellular sites (de Duve, Wattiaux and Baudhuin, 1962). The second rule is to consider the cell as being still largely uncharted territory. Our present picture of cellular structure is delineated by such molecular associations, most of them of lipoprotein nature, which when fixed and stained in certain ways provide suitable contrast in the electron microscope. Impressive as the image developed in this manner may seem, there is little doubt that it would look very gross against a detailed enzyme map of the cell in which, for instance, each individual enzyme were painted a different colour. No doubt, the basic delineation by cytomembranes would remain clearly recognizable, but it would appear in a variety of colours and shades. In addition, the uniform areas between the membranes might well reveal an unsuspected mosaic of multicoloured patterns. To establish such a map is the main purpose of enzyme cytology. The most obvious and direct way of accomplishing this purpose is to let 1^ 1. GENERAL PRINCIPLES 5 each individual enzyme signal its location by means of reliable staining tech- niques applicable to intact cells or tissue sections. Like the individual plates of multichrome prints, the images obtained in this manner should bring out one by one the elements of the pattern we wish to reconstruct. This approach has generally been followed by the investigators who have entered enzyme cytology from the side of morphology. Working against great odds, they have succeeded in developing staining procedures for a fairly wide variety of enzymes. Unfortunately, very few of these procedures answer the require- ments of applicability at the level of the electron microscope, which is the only instrument providing the degree of resolution necessary for accurate enzyme cytology. The only detailed enzyme maps available today are those of some hydrolytic enzymes, especially phosphatases, and the resulting pic- ture we have of the cell is still woefully sketchy and monochromatic. It is even fair to state that the images would be much less enlightening than they actually are, were it not for the additional information obtained by other methods and allowing a single enzyme to serve as a marker for a whole group of others. When such information is available, the staining proce- dures become extremely valuable tools, as illustrated by the usefulness of the acid phosphatase method in the study of lysosomes. They also have the advantage of being simple and easily applicable to a variety of biological materials and are thereby well suited for histological controls and for com- parative work. But they have not yet been developed to a point where they can by themselves play any significant role in mapping out the enzymes of the cell. Up to the present time, most of this work has been done with the much more indirect procedure of ceU fractionation wjhich^tilljsnjains the mgjorjool of enzymejytolQgy, The rest of this chapter will be devoted to a discussion of this technique. It constitutes a somewhat expanded version of a previous article on the sub- ject (de Duve, 1964) and draws heavily on the frustrations and satisfactions experienced in our laboratories over the last fifteen years. The manner in which these experiences have progressively shaped our approach has been recounted in a recent review (de Duve, 1965). Some of the views expressed may seem controversial, especially if they are misunderstood. I have taken some pains to avoid this, even at the risk of being repetitious, and wish to emphasize again that what I am discussing specifically is cell fractionation as a method of enzyme cytology and not all the applications of the technique, for instance in biochemistry where, as pointed out by Roodyn (1965), it has many important uses that do not require the same rigorousness as when it serves to localize enzymes in cells. III. The Conduct of Fractionation Experiments All fractionation experiments proceed in three successive steps ijiomo^ genization, fractionation and analysis. The sequence is obligatory in that B» 6 C. DE DUVE order: we fractionate, not cells or tissues, but preparations obtained by disrupting them in an artificial medium; we analyse, not intracellular organelles, but fractions separated from homogenates. A. FROM TISSUES AND CELLS TO HOMOGENATES Homogenization is the necessary prerequisite of all mass fractionation experiments. It results in a considerable loss of morphological information, but such is the price we pay for being able to apply henceforth the innumera- ble techniques of biochemistry. Our problem is to conduct the operation in such a manner as to lose no more information than is needed, while making what is left retrievable with as little distortion as possible. Unfortunately, homogenization is still the most empirical of all technical procedures used in tissue fractionation. Numerous mechanical devices have been constructed (see: Anderson, 1956; Allfrey, 1959), but the principles on which they operate are not clearly known and the manner in which they are handled varies greatly from one investigator to the other. For lack of a satisfactory theoretical basis and of adequate standardization methods, homogenization is still largely an art. This is unfortunate since it is the critical step which determines the quality of our starting material. Here is an area which undoubtedly would repay a more comprehensive investigation of what is, after all, largely an engineering problem. The choice of an adequate medium creates an even more difficult problem since there is practically no other way of solving it except by empirical methods. Sucrose, first advocated by Hogeboom et al. (1948), is now widely used as the main ingredient, but there are innumerable individual recipes differing by such details as sucrose concentration, removal or neutralization of CO2, addition of buffer, of EDTA, of traces of specific cations, of macro- molecular substances, etc. In addition, it is now clear that sucrose solutions are not always superior to ionic media, as was originally believed (de Duve and Berthet, 1954). Salts tend to promote agglutination in liver homogenates, but the reverse is true with spleen preparations which actually show more agglutination in 0-25 m sucrose than in 0-2 m KCl (Bowers, 1964). Another controversial subject relates to the use of non-aqueous media, as introduced by Behrens (1932). The advantages and disadvantages of this method have been discussed in detail by Allfrey (1959). In general it may be stated that each biological material poses a specific homogenization problem, to be solved largely by trial and error. But this requires that we define first the qualities which we should strive for in a homo- genate as well as the control methods whereby they are best assessed. In discussions of this question, stress is often put on morphological criteria and the phase contrast microscope is mentioned as the main tool to be used for controlling homogenates. While recognizing the importance of microscopic examinations to ascertain the degree of disruption achieved as well as gross 1. GENERAL PRINCIPLES 7 alterations of organelles or agglutination artifacts, I submit that the most useful instrument for estimating the quality of a homogenate is the centrifuge. The best homogenate is the one which lends itself most successfully to frac- tionation. Or to put it colloquially, the proof of the homogenate is in the fractionating. This point is of more than trivial importance; it reflects a rather basic attitude which is that adopted by our group. In our opinion, cell fractionation is not a routine standardized technique, to be set up once and for all and then applied as such whenever desirable; it is a never-ending exploration of the organization of cells, every phase of which invites reappraisal as more information becomes available. When applied in a routine fashion, all it can do is to reveal the manner in which an enzyme is distributed amongst the isolated fractions; it rarely brings to light the true intracellular localization of the enzyme, the objective of enzyme cytology. To attain this objective, it is generally necessary to use a multiplicity of approaches, following the self-correcting technique of the chemist, whereby the information gained is continuously fed back into the experimental design until satisfactory resolu- tion is achieved. This, at least, is how hepatic enzymes such as glucose-6- phosphatase (3.1.3.9) (Hers, Berthet, Berthet and de Duve, 1951), cyto- chrome oxidase (1.9.3.1) (Hogeboom, Schneider and Striebich, 1952) or acid phosphatase (Appelmans, Wattiaux and de Duve, 1955) were first identified as specific components of certain intracellular structures although their dis- tribution between the fractions isolated by the routine procedure was quite heterogeneous. The resolution of catecholamine-containing granules from mitochondria in preparations from the adrenal medulla (Blaschko, Hagen and Hagen, 1957), that of nerve endings from other cytoplasmic components in brain homogenates (de Robertis, 1963; Whittaker, 1963), that of peroxi- somes (microbodies) from mitochondria and lysosomes (Beaufay, Jacques, Baudhuin, Sellinger, Berthet and de Duve, 1964; Baudhuin, Beaufay and de Duve, 1965) and many other separations were brought to a successful conclusion by adhering to the same principle. The control of homogenization procedures has become increasingly easier as our knowledge of cellular organization has grown. For instance, we now know enough about the general properties of mitochondria to feel ^entitled, even with an unknown material, to use cytochrome oxidase or succinate dehydrogenase (1.3.99.1) as marker enzymes for these particles, or respiratory control as a test of mitochondrial integrity. Again, the well- established distribution of lysosomes in a wide variety of animal cells allows the latency of acid hydrolases to serve as an indicator of the fate suffered by these fragile particles. The usefulness of this easy and convenient test as a means of controlling homogerrization damage is illustrated by the work of Greenbaum, Slater and Wang (1960) on mammary gland and by those of Conchie, Hay and Levvy (1961) and of Bowers (1964) on spleen. The binding 8 C. DE DUVE and latency of catalase (1.11.1.6) can serve a similar purpose in peroxisome- containing tissues such as liver and kidney (Baudhuin et al., 1965). These tests, together with suitable morphological examinations, may already go a long way in helping to devise a homogenization procedure appropriate to the material under study. However, it still remains to be seen whether the degree of disruption achieved and the physical environment offered are such as to allow satisfactory resolution. As already mentioned, this verification requires fractionation, preferably by several different tech- niques, and characterization of the fractions. Here again, much information can be gained by using marker enzymes for known organelles to appreciate the efficiency of fractionation, and thereby the quality of the homogenate. Most fractionation experiments are performed on solid tissues and this introduces two additional complications which have not yet been considered. One is the cellular heterogeneity of the material, which multiplies the diffi- culties mentioned by the number of different cell types present. The other is due to the connective framework of the tissue, the disruption of which may necessitate mechanical stresses highly traumatic to subcellular constituents. A similar difficulty is encountered with single cells when they are encapsu- lated. In this respect, tissue fractionation may gain a great deal from the numerous attempts that are being made with increasing success to prepare suspensions of single cells from solid tissues by gentle mechanical or enzymic techniques. If the cells can be obtained in viable and non-leaky form, a result which seems very difficult to achieve, one can even consider the possibility of separating the cell populations before homogenization, thus reducing the problems created by cellular heterogeneity (see: Matey ko and Kopac, 1963). Even imperfect techniques of this type may sometimes help to settle an ambiguity created by the results obtained on the whole tissue. It must be noted that the randomizing effect of disrupting a heterogeneous tissue does not necessarily lead to an irretrievable loss of the relevant informa- tion. For instance, homologous components of two different cell types may differ sufficiently in their physical properties to be separable by appropriate techniques; if so, it may be further possible to identify their respective host- cells by suitable morphological or biochemical criteria. For instance, recent work on spleen homogenates has disclosed the presence of several lysosome populations which appear to originate from different cell types (Bowers and de Duve, 1967). B. FROM HOMOGENATES TO SUBCELLULAR FRACTIONS ,7. Technical Procedures (^ The centrifuge is by far the most widely used instrument for the fractiona- tion of homogenates. Thanks largely to its important applications in the f" analysis of macromolecules, centrifuging has received exhaustive theoretical treatment from both the engineering and the physico-chemical point of view. 1. GENERAL PRINCIPLES 9 Excellent instruments are available and efforts are constantly being made to construct better machines. The behaviour of particles in a centrifugal field and the various factors which affect their rate of sedimentation have been studied in great detail and excellent monographs and reviews have been published on the subject (Svedberg and Pedersen, 1940; Ogston, 1956; Schachman, 1959). While it may not be necessary to master all the intricacies of analytical centrifuging to be able to perform good fractionation experiments, a thorough understanding of the physical processes involved and also of the construction characteristics of the instruments used must be considered essential. Several papers have dealt with these theoretical and practical aspects of the technique, as related more specifically to tissue fractionation (Pickels, 1943; de Duve and Berthet, 1953, 1954; Anderson, 1956; Allfrey, 1959; de Duve, Berthet and Beaufay, 1959; Beaufay and Berthet, 1963). The latter two papers and that of Anderson (1956) should be consulted particularly with respect to the use of density gradients. Workers searching for a suitable solute for the preparation of density gradients may be interested in the long fist compiled by Mateyko and Kopac (1963). A detailed discussion of techniques and procedures falls beyond the scope of this chapter, but one point of general interest deserves to be stressed. The centrifugal separation of subcellular particles takes advantage either of differences in density^^ or of differences in sedimentation coefficient, itself _a function of the density, size and shape of the particles. All these parameters are dependent on the properties of the medium. This is of course true of the sedimentation coefficient which is an explicit function of the density and viscosity of the medium; but it also applies to the physical characteristics of the particles owing mainly to the influence exerted by the composition of the suspension medium on the exchanges of water and solutes between particles and medium. These exchanges are themselves regulated by such intrinsic properties of the particles as hydration, membrane permeability and internal osmotic pressure. Particles belonging to different populations may differ significantly in some of these properties and may therefore respond in a different manner to changes in the composition of the medium. Although they may not be separable in a given medium, they may become so in another and it is therefore extremely useful to try a variety of approaches. We have discussed this principle in several publications (de Duve et al., 1959; Beaufay and Berthet, 1963; de Duve, 1964, 1965) and demonstrated its practical validity and usefulness (Beaufay et al., 1964). An extension of this approach is to subject the animals or the isolated preparations to treatments which modify the physical properties of some particles. For instance, it is now known that the size and density of the lysosomes can be altered to a con- siderable extent by injecting the animals with substances which accumulate in these particles. Injection of egg-white (Straus, 1954), Triton WR-1339 10 C. DE DUVE (Wattiaux, Wibo and Baudhuin, 1963), sucrose (Wattiaux, Wattiaux-De Coninck, Rutgeerts and Tulkens, 1964) and dextran (Baudhuin et al, 1965) have been found to produce such changes. The density and chemical com- position of mitochondria can also be altered experimentally under certain conditions (Greenawalt, Rossi and Lehninger, 1964; Luck, 1965a, b). Other techniques besides centrifuging have been applied to the fractionation of homogenates. Chromatography has been used successfully for the separa- tion of viruses and also of cell particles (Riley, Hesselbach, Fiala, Woods and Burk, 1949). Albertsson (1960) has developed a number of two phase systems which have served mostly for the separation of macromolecules and viruses, but have also been applied to cell particles and even whole cells. The application of electrophoresis to fractionation of homogenates has been explored recently by Davenport (1964) and by Hannig (1964). On the whole, a wide choice of procedures and approaches is now available for the fraction- ation of homogenates. It is the task of the investigator to determine which is best suited to the solution of his particular problem. No rule can be set, except that more than a simple four-fraction separation is likely to be required. 2. The Preparative Approach Fractionation methods were first developed with the aim of isolating specific intracellular constituents in pure form and then studying their chemical and functional properties. They are still most widely used for this purpose today, especially in the field of biochemistry where it is often advantageous to start with the preparation of "nuclei", "mitochondria" or "microsomes" as a preliminary to metabohc or enzymological studies. Even in the specific field of enzyme cytology, some workers, like Allfrey (1959), tend to consider the "analysis of single fractions isolated under conditions which permit an estimate of their nature and purity" as an ap- proach allowing a "more definite answer to the question of the relationship between the function of an intracellular structure and its enzymatic com- position" than the analytical approach as exemplified by quantitative enzyme distribution studies. Statements to the opposite effect have frequently been expressed by our group and the causes of this disagreement deserve some attention. To examine this question, let us consider the electron micrograph of Fig. 1. It shows part of a rat liver cell and has been selected so as to illustrate most of the known constituents of this cell, including some of the rarer cytoplasmic particles. Assuming that we have no prior knowledge of the functional properties of any of the objects that we see, we may be tempted to seek such knowledge by the straightforward method of separating a chosen constituent from all the others and then determining its enzymic equipment. However, to attain this goal in practice, we must be able to monitor our purification attempts. Since we can only rely on morphological examination 1. GENERAL PRINCIPLES 11 * -. f ■^w^..:,f^msic,^ .'M5^>'-' Nu •if" cjl ^^^^|B^^^^-r J^RandGo ^'^ * "Rf^ r ■Lyf; ' .. ** • ••v^' •■' '■*■ * • i^^^^^^G^'. ' ) 1 • * ■% *• M«. .'^ •■ Fig. L Electron micrograph of ultrathin section through parenchymal cell of rat liver. Image shows part of the nucleus (Nu) in the upper left corner and of the cell membrane (CM) in the lower right corner, numerous mitochondria (Mt), several pericanalicular dense bodies or lysosomes (Ly), two microbodies or peroxisomes (Mb), areas of rough-surfaced endoplasmic reticulum (RER) and of smooth-surfaced endoplasmic reticulum (SER) and elements of the Golgi apparatus (Go). Small rosette figures are probably polysomes. Very rare rosettes made up of thicker particles are glycogen particles. Two particles with single membrane and finely granular matrix, amongst mitochondria in upper third and near cell membrane, are probably microbodies whose dense core lies outside the section. Courtesy of Dr. J. D. Jamieson, Rockefeller University, New York. 12 C. DE DUVE for this purpose, our search is virtually restricted to recognizable objects that are sufficiently large or numerous to be easily identified in complex mixtures. We may validly set out to purify nuclei or mitochondria, but should we wish to isolate the pericanalicular dense bodies or the microbodies, we would find it almost impossible to do so owing to the difficulty of assessing the frequency of our chosen material in the early steps of our purification attempt and consequently of devising a suitable preparative procedure. When it comes to subunits that cannot even be seen in electron micrographs — multi-enzyme systems like the fatty acid synthetase of Lynen (1961) could be of this type — there is of course no way of even defining the problem. Even if we decide to aim our attempt at a relatively accessible target, such d§ for iil'stahcfe the purification of mitochondria, we will nevertheless encounter the limitations of morphological techniques when we become faced with the problem of controlling the purity of our preparations. Obviously, objects which for one reason or another do not lend themselves to purification on the sole basis of morphological examinations, will for the same reason escape accurate detection when ihey occur as contaniinants. Biochemical methods do not suffer from these limitations. Especially in the form of specific cnzymic analyses, they can serve to detect and even estimate quantitatively any cell constituent, whether visible or invisible, rare or abundant, provided the enzyme measured is associated exclusively with this constituent and is known to be so. In our opinion, the latter information can come only from detailed studies of the distribution of enzymes amongst all the fractions that can be isolated from homogenates, in other words from analytical fractionations. For this reason, we consider purification an ultimate rather than an immediate aim in the field of enzyme cytology, and have in fact arrived at it in this manner in our own work. It is true, as will be pointed out below, that morphological methods are open to considerable improvement and could be developed so as to provide relatively accurate quantitative information, at least for components that can be detected and identified under the electron microscope. However, even with such information available, it would still be very difficult to appreciate its significance with respect to the biochemical properties of the preparation without analytical data to draw on. If we find, for instance, that a preparation of mitochondria contains one microbody for every twenty mitochondria and also possesses some catalase activity, we will still be unable to correlate these two findings unless we can relate ihem to other observations bearing on the frequency of microbodies and on the catalase activity in the homogenate and in other subcellular fractions. This again subordinates the preparative to the analytical approach The point at issue, therefore, is not whether the preparative approach can or cannot provide a more definitive answer to a problem than the analytical 1. GENERAL PRINCIPLES 13 approach, but rather whether a preparative attempt can be validly under- taken "under conditions which permit an estimate of the nature and purity" of the resulting preparation without a considerable amount of prior analytical work to guide it. It has been our contention that it cannot. There will probably be little quarrel with this opinion as applied to the rarer cytoplasmic particles with which most of our investigations have been concerned. But it could easily be argued that we are strongly biased by this very experience and that if workers engaged in the study of the major cell constituents had shared our concern, biology would be much less advanced than it is today. Had they waited, for instance, until they had available biochemically pure mitochondria or nuclei, we would still be largely ignorant of oxidative phosphorylation or of nuclear protein synthesis. This is an obvious truth. But so is the fact that if nuclear or mitochondrial fractions had been taken "at their face value", we would still credit the nucleus with a remarkable array of metabolic activities, while our functional representation of mitochondria would include a strange association of oxidative phosphorylation, acid digestion and hydrogen peroxide metabolism. If the tree is to be judged by its fruits, we can only conclude that both approaches have their merits as well as their limitations and that the richest returns may be expected if both are pursued simultaneously, drawing con- stantly on each other and on all the resources of morphology and cyto- chemistry for further improvement. Inadequately controlled preparative fractionation has been a highly successful shortcut to a great deal of im- portant information; but it involves something of a calculated risk and is related in this respect to the usual methods of dynamic biochemistry. Analytical fractionation is slower and less immediately rewarding, but it surrounds itself with greater precautions before merging its aims with those of the preparative approach. It is more specifically a tool of enzyme cytology. 3. The Analytical Approach Any quantitative fractionation, whether or not it is conducted on the basis of a priori morphological considerations, is an analytical experiment. Or at least it can be so if the investigator chooses not to commit himself as to the cytological composition of the fractions. It is perhaps easier to do so when the fractionation scheme is an arbitrary one, as in most forms of density gradient centrifuging, but the cuts do not have to be arbitrary to make the experiment an analytical one. Quantitative work and a certain mental attitude are the essential conditions. In an analytical experiment, we divide a homogenate in a certain number of fractions and assay each fraction as well as the original homogenate for one or more biochemical constituents, mostly enzymes. For clearer visualization, the results of the measurements are represented graphically as distribution patterns, or preferably, if the experimental design permits it, as frequency I 14 C. DE DUVE distribution histograms or curves of the enzymes as a function of the para- meter, sedimentation coefficient or density, which has served as a basis for the separation. Quantitative recovery is considered very important and the distributions are taken to be significant only if the sum of the activities of the fractions corresponds reasonably well to the activity of the original homogenate. To proceed further, it is necessary to assume some kind of relationship between the enzymes measured and the subunits with which they are asso- ciated in the homogenate. The word subunit is used here on purpose in order not to confuse the issue by any premature introduction of cytological terms. The simplest hypothesis is to assume that all subunits of a given population have the same enzyme content, as related for instance to their mass or total protein. We have called this assumption the postulate of biochemical homo- geneity, emphasizing its hypothetical nature, but pointing also to the theoreti- cal and experimental arguments which tend to support its validity. Taking this postulate as a working hypothesis, we may now tentatively consider the distributions of the enzymes as representing those of the sub- units to which they are attached. If two enzymes show sufficiently different distributions, we conclude that they are likely to belong to different subunits. If they show the same distribution, we take this as an indication but by no means as proof that they may be associated together with the same subunit. In order to distinguish between coincidental superimposition and true physical association, the fractionation is repeated under a number of different con- ditions. If the association is found to persist under each new set of conditions, the conclusion that it is real and not spurious becomes progressively strengthened. It will be clear from this schematic description of the analytical approach that it can in itself lead only to conditional interpretations whose accept- ability depends on the validity of the underlying postulate. Their final verifi- cation comes when enough information has been gathered to warrant a preparative attempt and a correlation of the biochemical results with morpho- logical observations. This aim has been achieved in a number of cases, thus providing an experimental justification for the approach and for the hypo- thesis on which it rests. Obviously, the analytical approach can be successful only if the procedures employed do not cause the disruption of the subunits themselves. Otherwise, one would eventually be led to identify as many subunits as there are enzymes in the cell. However, additional information of a very valuable kind may often be obtained by the deliberate use of disruptive procedures which may bring to light other similarities or dissimilarities between enzymes, based this time on the relative fragility of the subunits to which they belong. For instance, we may by comparative latency studies obtain new sets of curves which express the manner in which individual bound enzymes are 1. GENERAL PRINCIPLES 15 released by the treatment applied. Such curves complement the distribution curves and may be similarly interpreted, although with greater caution since there is no compelling reason why two enzymes associated with the same subunit should necessarily be released together when the subunit is subjected to graded disruption. However, the information obtained in this manner may be of great help in the interpretation of distribution data and may also serve to characterize further the subunits under study in terms of their physico-chemical properties. As pointed out by many authors, analytical fractionation is by no means a fool-proof method and is exposed to many artifacts. However, most of the latter are due to the rearrangement of contituents as a consequence o homogenization and exposure to an artificial medium. We have avoided discussing them here by defining the objective of the fractionation as a characterization of the subunits present in the homogenate. How these subunits are related to cytological entities becomes a matter of interpretation; it will be examined later. C. FROM SUBCELLULAR FRACTIONS TO ANALYTICAL RESULTS /. Biochemical Measurements Once the fractions are isolated, they fall almost entirely within the area of competence of the biochemist. Some cytological aspects have to be kept in mind, especially with regard to the influence structural integrity may have on enzyme activities. Many particle-bound enzymes are shielded by barriers which restrict their accessibility to the substrates used for their assay. This phenomenon must be investigated and methods ensuring the complete release of the enzymes for assay purposes must be worked out. The dissociation of structurally linked multi-enzyme systems may pose other problems, especially if we adhere to the rule that enzyme cytology should deal with single enzymes whenever it is at all possible. If those difiiculties can be dealt with satis- factorily, we are left with straightforward enzymological problems. This does not mean that they are easily solved or free from hidden pitfalls. The assay of enzymes in complex mixtures is rarely easy and thorough kinetic experiments are necessary in order to define conditions of temperature, pH, substrate concentration and medium composition under which the reaction velocity or its integral over a period of time provides a valid and reproducible measure of the number of enzyme molecules present. These kinetic experiments have to be repeated on every new biological material and also on the various isolated subfractions, for homologous enzymes / often have different kinetic properties in different tissues and they may be / accompanied in them or in separated subfractions by varying amounts of \ inhibitors or activators. J It is also of great importance to investigate the assay method from the K~^ 16 C. DE DUVE point of view of the number of enzymes involved. It often happens, for instance, that a given substrate is attacked simultaneously by more than one enzyme. If the enzymes involved are associated with different subunits, complex distribution curves will be obtained, possibly leading to false interpretations unless the source of the complexity is correctly traced down to the enzymes themselves. As already mentioned, the reaction measured may involve two or more enzymes acting in succession, leading to other possible artifacts. These problems have been discussed in more detail by de Duve and Berthet (1954) and by AUfrey (1959). 2. Morphological Examinations A number of investigators have successfully combined morphological examinations with enzymic measurements in analytical fractionations and have been able in this manner to correlate enzyme distributions with the observed frequency of certain structural components. As they have been applied until now, the morphological methods have been at best grossly semi-quantitative, but there is no reason why they could not, with a little effort, be improved very significantly. Accurate morphological assessment of the coniposition of a fraction depends on three conditions : (1) Perfect random sampling, a very important requirement in view of the extreme minuteness of the sample actually examined; (2) Reliable criteria of identification of the various components present; (3) A measuring device allowing the investigator to put the findings in quantitative terms. The condition of random sampling is not satisfied by current techniques, most of which rely on centrifuging for collecting the material and furnish stratified pellets. These can be examined at different levels, but this method is either inaccurate or very time-consuming. Baudhuin, Berthet and Evrard (1967) have recently developed a collection technique by MilUpore filtration which seems to obviate this difficulty. The condition of identification is likely to be the most difficult one to fulfill, especially since fractions isolated by centrifuging frequently contain damaged particles and variable amounts of unrecognizable debris. As to the means whereby the results can be quantitated, simple counting of profiles is not sufficient since the number of profiles furnished in thin sections by any given component depends on the volume and shape of the component. It is neces- sary, therefore, to make the measurements in such a manner as to be able to estimate the contribution of the component to the total volume or mass of the preparation. Loud, Barany and Pack (1965) have recently described a tech- nique for measuring volume ratios on tissue sections which could also be applied to isolated fractions ; these authors also discuss a number of technical problems associated with the use of the electron microscope for quantitative work. Dry mass distributions can be estimated by means of a method 1. GENERAL PRINCIPLES 17 developed by Bahr and Zeitler (1962). This method does not allow identifi- cation of the individual particles, but it provides sufficient statistical infor- mation to reveal in some cases the presence of more than one particle population. IV. The Interpretation of Fractionation Data The three successive steps followed in the conduct of fractionation ex- periments have to be retraced backwards in the process whereby the analytical results are converted into information on the intracellular localization of enzymes. Each of the three steps requires the investigator to assume, so to speak, a different personality and it is very important that they should be taken in succession and in the right order. A. FROM ANALYTICAL RESULTS TO SUBCELLULAR FRACTIONS /. Validity Control: the Balance-Sheet Our first concern is to examine the results of the assays strictly from the point of view of the enzymologist who has been given a certain number of unknowns to analyse. The question raised here is whether the raw data are faithful expressions of enzyme activities. Attention has already been called to the necessity of performing adequate kinetic experiments and if this precaution has been taken the results should inspire confidence. But provision must be left for the unexpected or the accidental. One of the most valuable controls is to add up the activities found on the various fractions and to compare this sum with the activity observed on the starting material. This presupposes that the fractionation has been conducted in a quantitative fashion and that no fraction has been discarded. Not all workers agree on the need of such a balance-sheet. Our own opinion coincides with that expressed by Schneider and Hogeboom (1951); we consider the balance-sheet essential. In the first place, it provides an almost indispensable test of the validity of the results. When the recovery is distinctly deficient or excessive, something must have gone wrong somewhere and it is better to know it. The cause may be accidental and traceable, and then a mistake will have been avoided; or it may be untraceable, but it will at least be known that the results are not reliable. If the defect in the balance-sheet is systematic, it becomes very significant and may either help to correct an unsuspected error or reveal a complicating phenomenon which may turn out to be of great biological interest. The latency of lysosomal enzymes was originally made manifest by a 200% recovery (Berthet and de Duve, 1951). The balance-sheet is also important at further stages of the interpretation, for instance in judging the validity of enzyme assays for purity assessment in preparative experiments. A purified fraction cannot be declared pure simply 18 C. DE DUVE because it lacks the characteristic enzymes of contaminants; the enzymes must be shown to have been actually removed rather than simply inactivated. The form in which they have been removed is also important. For enzymes that are easily released in soluble form from their host-particles, evidence that ihey are still particle-bound in the discarded fractions is necessary before their absence in purified fractions can be taken as proof that a true separation of particles has actually been achieved. Finally, the balance-sheet provides an essential element of information when it comes to deciding whether a given enzyme activity should be assigned to the subcellular component with which it is found associated or to a contaminant. The point at issue here is not whether one does or does not consider significant the association of let us say 10% of the total activity of an enzyme with 100% of the nuclei (AUfrey, 1959). This is a matter of opinion and the author is of course entitled to his own. But so is the reader, and he can only arrive at one if he is given the relevant facts. Surely it cannot be considered irrelevant to this question to know whether the activity displayed by isolated nuclei corresponds to 100, 10 or 0-1 % of the activity found in the corresponding amount of whole tissue. 2. Presentation of Results Graphical representation of the results often helps to bring out their significance more clearly than simple rows of figures. Especially in enzyme distribution studies, there is considerable advantage in following methods which may eventually allow the results to become represented in the form of histograms or frequency distribution curves which may serve to extract new information (see: IV. b. 2) and may even be amenable to statistical analysis. Examples of such methods are described in the appendix. B. FROM SUBCELLULAR FRACTIONS TO HOMOGENATES 7. Biochemical Characterization of Subunits Once a clear and reliable picture has been obtained of the manner in which enzymes and other biochemical components are distributed between the fractions, it now becomes necessary to interpret the results in terms of their association with one or more distinct populations of particulate entities. As already explained (III. b. 3), our main guiding principle here is the postulate of biochemical homogeneity. On the basis of this postulate, we identify as many subunits as there are clearly distinct distribution patterns, and keep in mind further the possibility that the procedure followed may not resolve all the subunits present and therefore that two enzymes showing the same distribution may nevertheless belong to different subunits. As evidence accumulates that two or more enzymes are truly inseparable, I ..^^ . I^Kme sort of biochemical picture of the subunit as an enzyme complex begins ^To emerge. Although we have not yet reached the stage where the subunit can be regarded as a true cytological component, this picture may be very ^Klpful in suggesting further experiments. In our own investigations, both ^me lysosome and the peroxisome concepts were first suggested by such enzyme associations and they then directed our further search towards acid hydro- ies in the former case and towards oxidases in the latter. Comparison of the distribution of enzymes with that of proteins and the computation of specific activities are also very helpful, indicating whether le hypothetical subunits represent a major or a minor component. Finally, studies of enzyme latency may serve to characterize the subunits further by indicating the mode of binding of the enzymes. They may also, as has been mentioned, help to distinguish different populations of subunits. 2. Physical Characterization of Subunits Having tentatively identified one or more subunits, we may try to derive additional information concerning their physical properties from the manner in which they become distributed under the influence of the fractionation procedure. In order to do this, it is necessary to know accurately the con- ditions under which the operation has been conducted, and also to ascertain that no artifacts such as convection or drop sedimentation have vitiated the experiment. Provided these conditions are met, the Svedberg equation can be used to estimate one or more physical properties of the subunits. If these calculations are based on enzyme distributions, it is obvious that their validity depends on the applicability of the postulate of biochemical homogeneity. Since fractionations are based either on differences in sedimentation coefficient or on differences in density, the most immediate information which can be extracted from the observed distributions relates to the distri- bution of these two particle parameters. The manner in which such graphs can be constructed is explained in the appendix. As shown elsewhere (Beaufay and Berthet, 1963; Beaufay et al, 1964; de Duve, 1965), when sufficient data are available concerning the density and sedimentation coefficient of the subunits in different media, one may attempt to fit them to the theoretical equation worked out by de Duve et al. (1959) for a model which seems applicable in first approximation to several types of cytoplasmic particles. These calculations which are generally performed on the median values of the distribution diagrams make it possible to estimate numerous additional physical parameters of the subunits, including their size, their dry mass, their content in osmotically active solutes and the dimensions of their water compartments. Complementary data on the osmotic behaviour of the subunits are sometimes provided by the results of enzyme latency studies. I 20 C. DE DUVE C. FROM HOMOGENATES TO CELLS AND TISSUES This is the final and most critical step in the interpretation. Having, through the process outlined above, arrived at some kind of robot picture of certain particulate subunits present as resolvable components in the homogenate, we are now required to answer the question whether these subunits exist as such within the cells or whether they are to a greater or lesser extent the product of homogenization artifacts. Granting this question can be answered satisfactorily, we must finally identify the intracellular entities from which the subunits originate and locate them at the cytological and at the histological level. The most direct way of achieving this result is through morphological examination of the fractions, especially if the analysis can be made quantitative (see: III. c. 2) or if a reasonable degree of purity has been attained. Some- times, careful examination of electron micrographs of the intact tissue may already be quite revealing. One of the arguments supporting the identification of the subunits characterized as peroxisomes in hepatic homogenates with the microbodies seen in parenchymal cells was that no other intracellular organelle fitted the robot picture derived from the fractionation experiments (Baudhuin et al, 1965). Cytochemical staining reactions may also be very helpful sometimes in correlating biochemistry with morphology, as illustrated by the results of Essner and Novikoff'(1961) and Holt and Hicks (1961) which confirmed in a definitive manner the lysosomal nature of the hepatic peri- canalicular dense bodies. However, the confirmation was to some extent mutual in this case, since the biochemical data also helped to estabUsh the validity of the staining method. Finally, there are already a number of instances where morphological examination can be largely dispensed with and where enough evidence is available to validate the use of specific enzymes as markers for their host-particles. All these procedures depend in their final accuracy on how successful we can be in eliminating or recognizing artifacts. There is no doubt that enzymes get reshuffled to a considerable extent and are also sometimes altered in their catalytic properties from the time cells and tissues are first removed from their natural environment up to the moment when subcellular fractions are separated and subjected to analysis. When complete translocations occur, they may escape recognition entirely until some new observation turns up to alert our suspicions. When the translocation is a partial one, the enzyme appears as a component of more than one population of subunits or is present partly in the supernatant fraction. The resulting experimental situation is identical with that which would obtain, without artifact, if the enzyme had a true multiple location. To distinguish between the two possibili- ties, the only logical attitude is always to suspect an artifact when a complex distribution is encountered, since it is the only attitude which results in 1. GENERAL PRINCIPLES 21 positive action. Guided by this working hypothesis which we have called the postulate of single location, we can then carry out a variety of experiments designed to reveal an artifact if there is one. This may lead us to experiment with different grinding devices and suspension media, to try new fractionation procedures, to investigate further the mode of binding of the enzymes, to search for biochemical differences between homologous activities associated with different subunits, and so on. If all these approaches fail, the ambiguity will remain, but at least we will have eliminated some possibilities and thereby strengthened the hypothesis that the observed distribution actually reflects multiple location. The rule is to accept this interpretation only to the extent that the alternative explanation invoking an artifact can be excluded on the basis of the experimental facts. -^ V. Conclusion The pathway described in this chapter is long, arduous and circuitous. It is not likely to attract the pragmatist who sees many shortcuts leading to what appears to be the same destination. Neither is it our contention that it is the only road to success. Our attempts at reconstructing the cell all proceed by a series of approximations and the approach presented is no exception. Our final goal is the understanding of cellular function, and enzyme cytology itself is only one of the multiple ways leading to it. To be fruitful, its findings must be integrated (with all the information that can be obtained through the other biological disciplines. They become truly validated only to the extent that the behaviour of living cells is found to be both compatible with and illuminated by our enzyme maps. However, there is no doubt that enzyme cytology has a particularly im- portant role to fulfill in this joint venture and it is essential therefore that those who practise this discipline remain critically aware of the nature of the risks which they accept when adopting a given approach. We believe that the analytical approach, as it has been outlined here and followed in our own investigations, involves fewer risks than more direct approaches and that its laborious character is justified on this account. Whether one chooses to follow it or not, the principles set out may help to define the limitations which are accepted and to facilitate communication between investigators engaged in connected areas of research. As has been pointed out elsewhere (de Duve, 1964), the confusion of operational with cytological terms has created some- thing of a nomenclature problem in enzyme cytology and has led in some cases to errors of interpretation. This is easily avoided provided cytological terms such as nuclei or mitochondria are never used in reference to subcellular fractions unless adequate evidence is available that the entities referred to are truly the organelles so designated in descriptive cytology. I 22 C. DE DUVE VI. Appendix: Graphical Presentation of Results GENERAL PRINCIPLE Let Q be the absolute amount of an enzyme or other biochemical component found in a subcellular fraction (eventually corrected for known sampling losses, see: Beaufay et al., 1964). Provided the sum EQ of the amounts found on all the fractions is not too different from the amount found on the starting material (satisfactory recovery) ; Then QI^Q may be taken as representing the fractional amount of the com- ponent present in the fraction (percentages (GIOO/Z'Q) may be used as an alternative). Graphs are constructed as shown schematically in Fig. 2, by aligning in a suitable order dictated by the experimental conditions along an abscissa x rectangles of width Jx and of height (Q/^Q-^x) [or (QlOOj^QAx)]. Fig. 2. Figure illustrates principle of graphical representation of distributions. For sign- ficance of symbols, see text. The surface area of each rectangle is then equal to the fractional (or percentage) amount of the component. The surface area of the whole diagram is always equal to unity (or to 100) if all the fractions are represented. With sufficient fractions, the dia- gram can be smoothened out to a continuous curve with the same total unitary integral and with fractional integrals equalling as closely as possible the surface areas of the individual rectangles. It is possible to program a computer to do this and also to differentiate the curve when it has a cumulative significance (differential density gradient centrifuging: boundary method). If jc is a particle parameter, the diagrams correspond to frequency distribution histograms or curves. If of normal shape, the latter may be expressed in statistical terms. APPLICATIONS WITHOUT PHYSICAL CHARACTERIZATION 7. Any fractionation Ax is arbitrary and equal for all fractions. This type of representation is not very informative, but may be necessary, for instance when type 2 cannot be used because 1. GENERAL PRINCIPLES 23 of too high relative specific activity values or for lack of information on protein or nitrogen distribution. 2. Fractionation by differential sedimentation Ax is the fractional (or percentage) amount of protein or nitrogen present in the fraction: Pl^p [or iP.\QG)lSP]. Then the ordinate (Q.2P)/i2Q.P) represents the relative specific activity of the enzyme and measures the degree of purification achieved over the homogenate. The fractions are aligned along the abscissa in the order, from left to right, in which they are isolated, i.e. in the order of decreasing sedimentation coefficient (For examples, see: de Duve, Pressman, Gianetto, -Wattiaux, and Appelmans, 1955). J. Density gradient centrifuging Ax is the fractional (or percentage) volume occupied by the fraction in the gradient, expressed in terms of the total volume of the gradient: VjSV [or (K100)/2'K|. With cylindrical tubes, fractional heights may be used similarly, with the total height taken as the distance between the meniscus and the bottom of the tube, minus one-third of the radius to correct for the sphericity of the bottom (Beaufay, Bendall, Baudhuin, Wattiaux and de Duve, 1959; Beaufay et ah, 1964). The ordinate (Q.2'fO/-^Q.f^) represents the relative concentration CjCi of the com- ponent, Q being the initial concentration when the material is homogenously distributed in the gradient at the beginning of the experiment, or the concentration it would have should it become so distributed, when the material is initially layered on top of the gradient. The fractions are aligned along the abscissa in the order in which they occur in the tube (For examples, see: Beaufay et al., 1964). APPLICATIONS WITH PHYSICAL CHARACTERIZATION 1. Density distribution If the final shape of the gradient is known in experiments of isopycnic centri- fuging, the intervals of density Ap and their limits are easily computed graphically. The diagram is plotted on a density scale with Ax = Ap. It represents a density distribution histogram or the corresponding frequency distribution curve, provided the conditions of the experiment are such that density equilibration has been achieved (For examples, see: Beaufay et al., 1964). 2. Distribution of sedimentation coefficient Ax equals As , the interval of sedimentation coefficients (in 0-25 m sucrose at 0") spanned by the fraction. To compute As^ and its absolute limits, it is necessary to know accurately the conditions of centrifuging, especially the geometry of the rotor and tubes and the time integral of the squared angular velocity IV = JJ, co^ d/ (de Duve et al., 1959). In ordinary differential sedimentation experiments, one can calculate from the conditions of isolation of each fraction the quantity .'^min (de Duve and Berthet, 1953; Appelmans et al., 1955), the sedimentation coefficient of the lightest particle to be sedimented completely. If the fractions have been washed twice or more, As^ may be taken in first approximation as being limited by the Smin of the fraction and by that of the fraction preceding it. In practice, such diagrams are not very useful owing to the small number of fractions and to the width of the intervals As^. Density gradient experiments provide much more accurate informa- tion, but the computation of As^ is more difficult. It has been treated in detail by de Duve et al. (1959) (For examples, see: Beaufay et al., 1959). I 24 C. DE DUVE 3. Size distribution Assuming a uniform density and a spherical shape for the particles, one can convert a sedimentation coefficient distribution into a size distribution by means of the Svedberg equation. Alternatively, one can also convert a density distribution into a size distribution by assuming a uniform sedimentation coefficient. Since both parameters are statistically distributed, we have not constructed such diagrams and have performed further calculations using the median values (or exceptionally the modes) of the distribution histograms (Beaufay and Berthet, 1963; de Duve, 1965). An example of a size distribution computed from sedimentation values can be found in the paper by Kuff, Hogeboom and Dalton (1956). References Albertsson, P.-A. (1960). "Partition of Cell Particles and Macromolecules" John Wiley, New York. AUfrey, V. (1959). In "The Cell" (J. Brachet and A. E. Mirsky, eds.) Vol. I, p. 193, Academic Press, New York and London. Anderson, N. G. (1956). In "Physical Techniques in Biological Research" (G. Oster and A. W. Pollister, eds.) Vol. Ill, p. 300, Academic Press, New York and London. Appelmans, F., Wattiaux, R. and de Duve, C. (1955). Biochem. J. 59, 438. Bahr, G. F. and Zeitler, E. (1962). /. Cell Biol. 15, 489. Baudhuin, P., Beaufay, H. and de Duve, C. (1965). /. Cell Biol. 26, 219. Baudhuin, P., Berthet, J. and Evrard, P. (1967). /. Cell Biol. (In press). Beaufay, H., Bendall, D. S., Baudhuin, P., Wattiaux, R. and de Duve, C. (1959). Biochem. J. 13, 628. Beaufay, H. and Berthet, J. (1963). Biochem. Soc. Symp. 23, 66. Beaufay, H., Jacques, P., Baudhuin, P., Sellinger, O. Z., Berthet, J. and de Duve, C. (1964). Biochem. J. 92, 184. Behrens, M. (1932). Z. Physiol Chem. 209, 59, Bensley, R. R. and Hoerr, N. L. (1934). Anat. Rec. 60, 449. Berthet, J. and de Duve, C. (1951). Biochem. J. 50, 174. Blaschko, H., Hagen, J. M. and Hagen, P. (1957). /. Physiol. {London) 139, 316. Borsook, H., Deasy, C. L., Haagen-Smit, A. J., Keighley, G. and Lowy, P. H. (1950a). J. biol. Chem. 184, 529. Borsook, H., Deasy, C. L., Haagen-Smit, A. J., Keighley, G. and Lowy, P. H. (1950b). /. biol. Chem. 187, 839. Bowers, W. E. (1964). /. Cell Biol. 23, 13A. Bowers, W. E. and de Duve, C. (1967). /. Cell Biol. (In press). Claude, A. (1946a). /. exp. Med. 84, 51. Claude, A. (1946b). /. exp. Med. 84, 61. Conchie, J., Hay, A. J. and Lewy, G. A. (1961). Biochem. J. 79, 324. Davenport, J. B. (1964). Biochim. biophys. Acta 88, 177. de Duve, C. (1964). /. theoret. Biol. 6, 33. de Duve, C. (1965). In "The Harvey Lectures" Series 59, p. 49, Academic Press, New York and London, de Duve, C. and Baudhuin, P. (1966). Physiol. Rev. 46, 323. de Duve, C. and Berthet, J. (1953). Nature, Lond. 172, 1142. de Duve, C. and Berthet, J. (1954). Int. Rev. Cytol. 3, 225. de Duve, C, Berthet, J. and Beaufay, H. (1959). Prog. Biophys. biophys. Chem. 9, 325. de Duve, C, Pressman, B. C, Gianetto, R., Wattiaux, R. and Appelmans, F. (1955). Biochem. J. 60, 604. 1. GENERAL PRINCIPLES 25 de Duve, C. and Wattiaux, R. (1966). Ann. Rev. Physiol 28, 435. de Duve, C, Wattiaux, R. and Baudhuin, P. (1962). In "Advances in Enzymology" (F. F. Nord, ed.) Vol. 24, p. 291, Interscience Publishers, New York, de Robertis, E. (1963). In "Methods for the Study of Pharmacological Effects at Cellular and Subcellular Levels" (O. H. Lowry, ed.) p. 49, Pergamon Press, Oxford. Essner, E. and Novikoff, A. B. (1961). /. biophys. biochem. Cytol. 9, 773. Green, D. E., Loomis, W. F. and Auerbach, V. H. (1948). J. biol. Chem. Ill, 389. Greenawalt, J. W., Rossi, C. S. and Lehninger, A. L. (1964). /. Cell Biol. 23, 21. Greenbaum, A. L., Slater, T. F. and Wang, D. Y. (1960). Nature, Lond. 188, 318. Hannig, K. (1964). Z. physiol. Chem. 338, 21 1. Hers, H. G., Berthet, J., Berthet, L. and de Duve, C. (1951). Bull. Soc. Chim. biol. 33, 21. Hogeboom, G. H., Schneider, W. C. and Palade, G. E. (1948). /. biol. Chem. Ill, 619. Hogeboom, G. H., Schneider, W. C. and Striebich, M. J. (1952). J. biol. Chem. 196, 111. Holt, S. J. and Hicks, R. M. (1961). /. biophys. biochem. Cytol. 11, 47. Hultin, T. (1950). Expl Cell Res. 1, 376. Kennedy, E. P. and Lehninger, A. L. (1949). /. biol. Chem. 179, 957. Kielley, W. W. and Kielley, R. K. (1951). /. biol. Chem. 191, 485. KuflF, E. L., Hogeboom, G. H. and Dalton, A. J. (1956). /. biophys. biochem. Cytol. 2,33. Lardy, H. A. and Wellman, H. (1952). /. biol. Chem. 195, 215. Littlefield, J. W., Keller, E. B., Gross, J. and Zamecnik, P. C. (1955). /. biol. Chem. Ill, 111. Loud, A. v., Barany, W. C. and Pack, B. A. (1965). Lab. Invest. 14, 996. Luck, D. J. L. (1963a). /. Cell Biol. 16, 483. Luck, D. J. L. (1963b). Proc. natn. Acad. Sci. U.S.A. 49, 233. Luck, D. J. L. (1965a). /. Cell Biol. 24, 445. Luck, D. J. L. (1965b). /. Cell Biol. 24, 261. Lynen, F. (1961). Fed. Proc. 20, 941. Mateyko, G. M. and Kopac, M. J. (1963). Ann. N.Y. Acad. Sci. 105, 183. Novikoff, A. B. (1963a). In "Lysosomes" (A. V. S. de Reuck and M. P. Cameron, eds.) p. 36, J. & A. Churchill, Ltd., London. Novikoff, A. B. (1963b). In "Abstracts of the First International Congress of Histochemistry and Cytochemistry" p. 465, Pergamon Press, New York. Ogston, A. G. (1956). In "Physical Techniques in Biological Research" (G. Oster and A. W. Pollister, eds.) Vol. II, p. Ill, Academic Press, New York and London. Palade, G. E. and Siekevitz, P. (1956). /. biophys. biochem. Cytol. 1, 671. Palade, G. E., Siekevitz, P. and Caro, L. G. (1962). In "The Exocrine Pancreas" (A. V. S. de Reuck and M. P. Cameron, eds.) p. 23, J. &. A. Churchill, Ltd., London. Pickels, E. G. (1943). /. gen. Physiol. 26, 341. Riley, V. T., Hesselbach, M. L., Fiala, S., Woods, M. W. and Burk, D. (1949). Science, N.Y. 109, 361. Roodyn, D. B. (1965). Int. Rev. Cytol. 18, 99. Schachman, H. K. (1959). "Ultracentrifugation in Biochemistry", Academic Press, New York and London. 26 C. DE DUVE Schneider, W. C. and Hogeboom, G. H. (1951). Cancer Res. 11, 1. Schneider, W. C. and Potter, V. R. (1949). /. biol. Chem. Ill, 893. Siekevitz, P. and Palade, G. E. (1958). /. biophys. biochem. Cytol. 4, 557. Straus, W. (1954). J. biol. Chem. 207, 745. Svedberg, T. and Pedersen, K. O. (1940). "The Ultracentrifuge". Clarendon Press, Oxford. Warburg, O. (1913). Pfluger's Arch. ges. Physiol. 154, 599. Wattiaux, R., Wattiaux-De Coninck, S., Rutgeerts, M.-J. and Tulkens, P. (1964) Nature, Lond. 203, 757. Wattiaux, R., Wibo, M. and Baudhuin, P. (1963). In "Lysosomes" (A. V. S. de Reuck and M. P. Cameron, eds.) p. 176, J. & A. Churchill, Ltd., London. Whittaker, V. P. (1963). In "Methods for the Study of Pharmacological Effects at Cellular and Subcellular Levels" (O. H. Lowry, ed.) p. 61, Pergamon Press, Oxford. Chapter 2 THE NUCLEUS G. P. GEORGIEV Institute of Molecular Biology, Academy of Sciences of the USSR, Moscow, USSR Introduction ......... Methods of Isolation and Fractionation of Nuclei A. Procedures Involving the Use of Organic Solvents B. Aqueous Methods of Nuclear Isolation [I. Structure and Methods of Sub-fractionation of the Nucleus A. Structure and Ultrastructure of Nuclei B. Chemical Fractionation ...... C. Mechanical Methods of Fractionation Auto-Reproduction (Duplication) (DNA Biosynthesis) . A. Nuclear DNA Polymerase ..... B. Mechanism of DNA Synthesis in Chromosomes . fV. Transcription (RNA Biosynthesis) ..... A. Intracellular Localization of RNA Synthesis B. RNA Polymerase and its Intracellular Localization C. Chromosomes and mRNA Synthesis .... D. Synthesis of Ribosomal RNA and sRNA and the Function of the Nucleolus ....... E. Other Enzymes Involved in Synthesis and Breakdown of Polynucleotides ....... ^I. Translation (Protein Biosynthesis) ..... A. Protein Synthesis in Nuclear Sap Ribosomes B. Protein Synthesis in the Nucleolo-Chromosomal Complex IL Enzymes of Energy Metabolism ..... A. Glycolysis ........ B. Citric Acid Cycle ....... C. Oxidative Phosphorylation ..... VIII. Other Enzymes (Enzymes of Intermediary Metabolism) U^B A. Nucleotide Metabolism ...... I^K B. "Special" Enzymes and Widely Distributed Enzymes . I^H C. Occurrence of Enzymes of Different Cytoplasmic Structures I^P in the Nucleus ....... D. The Nuclear Membrane and Permeases IX. Conclusion : The Function of the Cell Nucleus and Nuclear Structures References ........... 27 28 29 31 35 35 44 46 49 50 52 56 56 60 66 72 74 76 77 79 81 81 83 84 86 87 89 91 93 94 95 W T Introduction The metabolic processes which are known to occur in the cell involve four main types of reaction: firstly, reactions concerned with energy transfer; 27 28 G. P. GEORGIEV secondly, reactions of intermediary metabolism which result in the con- struction of building blocks for the synthesis of macromolecules and co- enzymes including the synthesis of biopolymers that do not require a tem- plate; thirdly, catabolic reactions; and finally reactions of polymer synthesis that require templates. The most important of these is the synthesis of nucleic acids and proteins. Reactions of the fourth type may in turn be divided according to Spiegelman and Hayashi (1963) into three groups, namely those related with the auto- reproduction or duplication of genetic information (synthesis of DNA), with the transcription of genetic information (RNA synthesis) and with translation of this information (synthesis of proteins). This conditional classification is useful in the discussion of the functions of subcellular structures. The reactions mentioned above are localized in different cellular structures. Thus mitochondria and chloroplasts are mainly concerned with the reactions of energy metabolism. Many enzymes of intermediary metabolism are present and function in the cell sap. The reactions of protein synthesis occur in ribosomes and associated structures and finally the duplication and trans- cription of genetic information take place in chromosomal structures of the nucleus. Therefore the synthesis of nucleic acids is the basic and specific function of the nucleus. This division of functions among cellular structures is, however, somewhat imprecise and not absolute. The synthesis of nucleic acids is not the only function of the nucleus, since reactions of protein synthesis, phosphorylation, synthesis of coenzymes and other reactions of intermediary metabolism also occur in the nucleus. But it is the synthesis of DNA and RNA that distinguishes the activities of the nucleus from those of other cell structures. The complexity of the function of nucleus is reflected in its complex organization. The nucleus from all types of cell is known to have chromo- somes, nucleoli, nuclear sap and a nuclear membrane. Different nuclear structures in turn have different and specific functions as will be evident from the ensuing discussion. II. Methods of Isolation and Fractionation OF Nuclei It is well known that no fractionation procedure can completely exclude the possibility of artifacts, e.g. the acquisition or loss of components or the inactivation of enzymes. The nucleus is not an exception in this regard. The principal sources of artifacts are the following: (1) contamination of nuclear preparations with whole cells; (2) contamination with cytoplasmic enzymes: (3) loss of some of the nuclear proteins and nucleic acids; (4) degradation and inactivation of nuclear components. In practice, there is no method available 2. THE NUCLEUS 29 at present which is free from all these drawbacks and different procedures have to be used for isolation of nuclei to avoid different artifacts. Several excellent reviews on the methods of isolation of nuclei have been published in the past few years (Dounce, 1955; Allfrey, 1959; Roodyn, 1959, 1963; and Siebert and Humphrey, 1965). Therefore, we will only consider the principles which one should adopt in looking for a method to use for a specific purpose and only briefly characterize the procedures most often employed. There are two principal groups of procedures used for isolation of nuclei: those which involve the use of aqueous solutions, mainly containing sucrose, and those making use of organic solvents. We will consider the use of organic solvents first. A. PROCEDURES INVOLVING THE USE OF ORGANIC SOLVENTS A non-aqueous method for the isolation of cell nuclei was first put forward by Behrens (1932, 1938). Further modification of the technique was made by Dounce, Tishkoff, Barnett and Freer (1950), Allfrey, Stern, Mirsky and Saetren (1952) and recently by Siebert (see Siebert, 1961 and Siebert and Humphrey, 1965). There are no important differences between the various modifications of non-aqueous methods of nuclear isolation. All of them include (1) lyophilization of tissue; (2) homogenization of lyophilized material by treatment in ball mill (as suspension medium, petroleum ether or some other light organic solvent is usually used) and (3) differential centrifuging. Mixtures of two organic solvents with high and low density are used to perform the centrifuging (for instance, CCU-benzene or brom- benzene-cyclohexane). By varying the ratio of solvents one can obtain mix- tures of different density. Subsequent use of these mixtures causes the nuclei to sediment or conversely to float, thus gradually separating them from cytoplasmic contaminants and whole cells which differ from nuclei in density. In Fig. 1(a) a preparation of clean nuclei obtained by non-aqueous methods is presented. In practice only non-aqueous methods are completely guaranteed from the loss of nuclear proteins and nucleoprotein components. In any aqueous method some of the nuclear protein and RNA may be washed out of nuclei. Therefore the results obtained with non-aqueous methods should be taken as the most reliable guide to the final localization of a particular enzyme in cellular sub-structures. The most obvious example supporting the use of non-aqueous methods in the investigation of enzyme localization is the study of the distribution of DNA polymerase (2.7.7.7.) in the cell. While after isolation of nuclei by aqueous methods, this enzyme was found exclusively in the cyto- plasm, the application of non-aqueous procedure gave quite opposite results: most of the DNA-polymerase activity was found in the nuclei (Keir, Smellie 30 G. P. GEORGIEV ■*7»IV ~ Fig. 1. Smears of isolated nuclei in light microscope, (a) Nuclei of avian erythrocytes obtained by non-aqueous method and stained with haematoxylin-eosin. < 1750 (Allfrey, 1959). (b) Rat liver nuclei isolated in concentrated sucrose — glycerophosphate medium and stained with methylene blue. Numerous nucleoli are clearly visible, x 1500 (Georgiev et al. 1960). and Siebert, 1962). Furthermore the use of non-aqueous methods prevents chemical binding of soluble proteins by nuclear nucleoproteins. Finally these methods are quite useful in the study of intracellular distribution of such small molecular weight substances as intermediary glycolytic metabolites, free nucleotides and amino acids. 2. THE NUCLEUS 31 The principal drawbacks of non-aqueous procedure are as follows: (1) it is not always possible to isolate nuclei without contamination; (2) the ultrastructure of nucleus may often be damaged thus interfering with subse- quent fractionation of sub-nuclear components; (3) the denaturation of enzymes in organic solvents is not completely ruled out. (Although the activity of an enzyme studied may still be high after isolation, one should always determine the effect of the isolation method on the enzymic activity), (4) non-aqueous methods cause the solubilization of nuclear lipids, (5) finally, in comparison to aqueous methods, the non-aqueous technique is always less reproducible and more troublesome. Nevertheless for the most part only the use of non-aqueous methods allows one to draw final conclusions as to the presence or absence of an enzyme in nuclei. IH B. AQUEOUS METHODS OF NUCLEAR ISOLATION ■™^ Aqueous methods of nuclear isolation are more commonly used than non-aqueous, and more have been described. They do not suffer from the above mentioned drawbacks. Since they are not so tedious and time-con- suming they are more often used in everyday practice. An example of nuclei isolated in an aqueous medium is given in Fig. lb. Two main stages are distinguished in any procedure which involves the use of aqueous media: homogenization and differential centrifuging. As a rule, the disruption of tissues and cells (homogenization) is carried out in the same medium that is used later in the procedure. To disrupt cells, earlier workers used either a high-speed blender (Waring Blendor) or a bacterial mill (see Dounce, 1955). Since considerable breakage of nuclei and mitochondria was always observed in these procedures, these machines are now only rarely used. However, in some exceptional cases, nuclei prepared in the Waring Blendor are of good quality (Allfrey, Mirsky and Osawa, 1957). The glass Potter-Elvehjem homogenizer (Potter and Elvehjem, 1936), often fitted with a teflon pestle, and the somewhat less popular Dounce homogenizer (Dounce, Witter, Monty, Plate and Cottone, 1956) are widely used now. Together with these simple tools, more complicated apparatus is also in use; for example, the Bonner apparatus for the disruption of plant tissues between two rotating rolls (Rho and Chipchase, 1962), or devices where tissues are forced through narrow apertures (Emanuel and Chaikoff, 1957). These mechanical methods gave quite satisfactory results when applied to different tissues but in almost no case was it possible to achieve complete disruption. They were found to give unsatisfactory results in the disruption of free cells (ascites tumours, cell cultures). The search for some other method has shown that hypotonic shock may be more successful: when placed in 32 G. P. GEORGIEV hypotonic medium or water, these cells swell and burst (Samarina, 1961, Fisher and Harris, 1962; and Martin, Malec, Coote and Work, 1961). Depending on the cell resistance, these procedures may involve only hypo- tonic shock in water, additional more or less intensive homogenization of cells or, finally, the use of detergents such as Tween-80 or saponin. The homogenization is usually followed by differential centrifuging at relatively low speed (500-1000 g) to separate nuclei from cytoplasmic material. Many media originally used for the isolation of nuclei contained citric acid (Dounce, 1943). This however led to aggregation and condensation of nuclear material as well as to a considerable extraction of nuclear proteins (Dounce, 1955). As a more mild procedure for general tissue fractionation, sucrose media of low ionic strength were introduced by Hogeboom, Schneider and Palade (1948) and Schneider (1948) and later used for nuclear isolation with some modifications by most investigators. The addition of Ca^^ ions to the medium was shown to have a favourable effect during the isolation procedure (Schneider and Petermann, 1950). Other workers also suggested layering the suspension of nuclei over more concentrated sucrose solutions before centrifuging (Wilbur and Anderson, 1961 ; Hogeboom, Schneider and Striebich, 1952; Roodyn, 1956). The use i)f low 4enie strength solutions of sucrose that have relatively high osmotic pressure significantly reduces the loss of nuclear proteins. There is no difference between the content of protein and RNA in thymus nuclei isolated in sucrose media or by non- aqueous methods (Stern and Mirsky, 1953). On the other hand, intact ultra- structural organization of nuclei, as observed by electron microscopy, is also preserved in sucrose isolation media (Georgiev and Chentsov, 1960, 1963; Maggio, Siekevitz and Palade, 1963a). (See Figs. 2-4.) Further progress in the development of sucrose methods was made by Chauveau, Moule and Rouiller (1956), who used concentrated sucrose solutions (2-2 m) for nuclear isolation. The density of these solutions is lower than that of nuclei but exceeds the density of whole cells and cytoplasmic structures. Hence during centrifuging of homogenates in these solutions nuclei move down the centrifuge tube, whereas all the cytoplasmic con- taminants go up. Using the original Chauveau technique or its modifications (Georgiev, Yermolaeva and Zbarsky, 1960; Gvozdev, 1960) it became possible to isolate intact nuclei without contamination (Fig. 2b). A further development of the Chauveau procedure was the use of a layering technique in which a homogenate prepared in more dilute sucrose (0-88 m, for example) is then layered on 2-2 m sucrose. In this method, nuclei reach the bottom of the tube during centrifuging, and whole cells and cytoplasmic contaminations are left in the upper layer or interphase, (Maggio et al., 1963a) (Fig. 3). The use of concentrated sucrose solutions for the isolation of nuclei is so far the only method by which to obtain a nuclear preparation with no whole cells present. 2. THE NUCLEUS 33 I Fig. 2. Electron micrograph of intact nucleus of Walker carcinoma. One can see a nucleolus (n), and double membrane (m). The nucleolus consists of nucleolonemata (nl) that are made of numerous small granules. Nucleoplasma contains elements of chromosomes and nuclear sap, not separated one from another. Microfibrils and granules 100-200A in diameter are visible. Chromosomal elements are present in the nucleolar zone. Nuclei fixed according to Palade. x 16,500 (Muramatsu et al. 1963). In addition to sucrose media glycerol media have been described for isola- tion of nuclei (Schneider, 1955). These somewhat reduce the loss of nuclear proteins. The high viscosity of these solutions, however, makes it difficult to isolate pure nuclei. The diversity of aqueous methods for isolation of nuclei, and the ease and simplicity which make it possible to obtain relatively pure and intact nuclei suitable for further fractionation into sub-nuclear components strongly favours the use of aqueous procedures for studies of nuclear metabolism. However, to exclude completely the danger of the loss of water-soluble components in these procedures, one should perform control experiments with nuclei isolated with organic solvents. 34 G. P. GEORGIEV 2. THE NUCLEUS 35 III. Structure and Methods of Sub-fractionation koF THE Nucleus In addition to experiments with intact nuclei the problem of further iractionation of nuclei and the isolation of subnuclear structures often irises. This problem was first studied many years ago. In 1943 Claude and *otter isolated nuclear material which was shown by microscope studies to oe chromatin threads. Later these threads were identified as interphase ■chromosomes (Mirsky and Ris, 1951), but this conclusion is not completely correct and the above mentioned threads are probably broken pieces of chromosome. In 1952 Vincent described a technique for isolation of nucleoli Krom oocyte nuclei of the starfish. In 1960 methods for the isolation of nuclear ibosomes were developed in a number of laboratories (Frenster, AUfrey and kiirsky, 1960; Samarina and Georgiev, 1960). Recently quite a few papers lave been published on systematic studies of nuclear fractionation with the purpose of isolating of various nuclear structures. The composition of the "factions is usually controlled chemically and by electron microscopy. Two main groups of fractionation procedures are being developed at resent: chemical fractionation and mechanical fractionation. A. structure and ultrastructure of nuclei o evaluate these methods let us first briefly recall what is known about ;he structure and ultrastructure of nuclei. By light microscopy it is possible ;o distinguish in the interphase nucleus nucleoli, chromatin (chromosomes) and nuclear sap. However, if unfixed preparations are used, it is not possible i^_to differentiate chromatin and nuclear sap in interphase nuclei. Uncoiled ^fchromosomes fill the whole nucleus and one can only see the nucleolus and the nucleoplasm (Ris and Mirsky, 1949) (see Fig. 1). The same situation is i ^-observed in the study of nuclear structure by electron microscopy. After Hfexation of the tissue with OSO4 the nucleolus zone may be clearly seen in electron micrographs. The rest of the nucleus is filled with fibrillar or granular material whose organization is not clear (Fig. 2). Aldehyde fixation (form- aldehyde, acrolein, glutaraldehyde) or fixation in acid media lead to the separation of chromosome material and nuclear sap (Swift, 1963; Maggio et a/., 1963a) (Fig. 4). This picture however does not apparently reflect the distribution of nuclear material in the intact cell. The nuclear envelope is easily seen in electron micrographs of nuclei. It 'iG. 3. Electron micrograph of liver nuclei obtained by centrifuging homogenate in 0-88 m 'sucrose/ 1-5 niM CaCl2 in discontinuous density gradient (the homogenate was layered over 2-2 M sucrose-0-5 mM CaCh). Intact (Ni) and partly damaged (N2) nuclei and rare cyto- plasmic impurities are recorded, (n) — -Nucleoli. Pellet fixed in 3-3 m for- maldehyde in 01 M phosphate buffer, pH 7-6 and stained in Pb(OH)2 :■: 4900 (Maggio etal., 1963a). 36 G. P. GEORGIEV Fig. 4. The same as in Fig. 3 at higher magnification. One can see (1) nucleoli (n) with well defined nucleolonemata; (2) chromatin masses (ch) localized around the nucleolus and intranucleolar, at the periphery of nucleus and scattered throughout the rest of nucleus; (3) interchromatin regions (nuclear sap) occupied by aggregated small granular material (gn); and (4) nuclear membranes (shown in arrows) x 17,000 (Maggio ei a/., 1963a). 2. THE NUCLEUS 37 consists of two membranes. The external membrane is essentially an extension of the endoplasmic reticulum (Hartmann, 1953; Watson, 1955). So-called pores are seen in zones where the two membranes of nuclear envelope are joined together (Callan and Tomlin, 1950; Watson, 1959; (Fig. 5)). Although electron microscopy of nuclei did not help much in the elucida- tion of the three-dimensional structure of chromosomes and nucleoli it led to the characterization of the main structural elements which form these structures. The basic structural elements of the nucleus are more or less coiled strands about 100 A in diameter, called elementary fibrils (Ris, 1957, 1961; Yasuzumi, 1955; Kaufmannand McDonald, 1956; Bopp-Hassenkamp, 1959; Georgiev and Chentzov, 1960, 1963; Swift, 1963). These strands have been observed by practically all the authors who have studied nuclear structure in situ as well as in isolated nuclei (Figs. 5-7). The fibrils are found Fig. 5. Electron micrograph of normal section through nuclear envelope. Om — outer, im — inner nuclear membrane. PP, op — pores of nuclear membrane ("plugged" or "open"). Inside the nucleus DNP fibrils, x 47,000 (Maggio et al. 1963a). distributed in the nucleoplasm after osmium fixation or concentrated in chromosomal zones after aldehyde fixation. They may also penetrate the nucleolar zone (Figs. 4 and 6). The diameter, degree of coiling and aggregation of elementary fibrils depend on their source and on the method of prepara- tion. The ionic strength of solutions used during the fixation of nuclei plays a most important role. The fibrils are localized in the zones rich in DNA, they are sensitive to DNase, dissolve in strong salt solutions and aggregate in the presence of 0-14 m NaCl and bivalent cations. These facts demonstrate their deoxyribonucleoprotein nature (Ris, 1957, 1961; Georgiev and Chentsov, 1960, 1963). Therefore they will be referred to below as "DNA-protein fibrils" or "DNP fibrils." The molecular structure of "DNP fibrils" is still quite obscure. Zubay and Doty (1959) have shown that the diameter of a DNA protein (DNP) molecule 38 G. P. GEORGIEV J. *4 ■'■■. ;^' nn_-ch Fig. 6. Electron micrograph of rat liver nucleus, from which nuclear sap is removed by salt extraction. One can see a nucleolar zone, that consists predominantly of nucleonemata (nn). Bundles of nucleonemata composed of nucleolonemata (ni). DNP fibrills (D) penetrate into the nucleolar zone. They fill the whole nucleus. In some part of it nucleonemata of chromosomes (nn-ch) are shown. Nuclei isolated in 2-2 m sucrose- 1 % glycerophosphate, extracted by 014 m NaCl, then placed in sucrose-glycerophosphate medium again and fixed by Os04. Slices are stained in uranyl acetate, x 26,500 (Georgiev and Chentszov, 1960, 1963). Fig. 7. Electron micrographs of deoxynucleoprotein fibres, (a) Nuclei of red blood cells of Trituras pyrohogaster disintegrated by ultrasonic vibrations, smeared on planchets and shadowed, f— lOOA fibrils, N, F — fibrils of the higher order of complexity. 10,000 < (Yasu- zumi, 1955). (b), (c) — Calf thymus nucleohistone fibrils from chromosomes prepared in 007 M NaCl-0001 m EDTA pH 6-5 and dispersed in water. lOOA-fibrils are visible (b) X 85,000). (d) The same as in (b), (c), but DNP isolated was performed at pH 80. One can see the separation of lOOA fibril into two 40A fibrils, x 130,000 (Ris, 1960). (e) DNP fibril of rat thymocyte, stained with uranylacetate. The separation of DNP fibril is visible (Struchkov, 1964). 2. THE NUCLEUS 39 40 G. P. GEORGIEV containing DNA and histone in the ratio 1 :1 is significantly less (30 A) than the diameter of DNP fibrils (Fig. 8). However nuclear DNP contains more protein than purified nucleo-histone and includes some proteins of non- histone nature (Mirsky and Pollister, 1946; Zbarsky and Debov, 1951). Fig. 8. Electron micrograph of nucleohistone molecules (obtained by the method of Zubay and Doty (1959) and of free DNA. Diameter of fibrils are DNP 30 A, (top) and DNA 20 A, (bottom). Zubay and Doty (1959). According to Ris (1961) 100 A strands separate into 30-40 A strands under the conditions favouring the removal of some protein from nucleo-protein (e.g. at high pH, or after removal of histone by extraction with HCl). In electron micrographs 30-40 A strands resemble nucleo-histone molecules (Fig. 7). It is possible that each DNP strand consists of two nucleo-histone molecules held together by additional histone or non-histone protein. 2. THE NUCLEUS 41 In addition to DNP fibrils some other structures may be distinguished in the nucleus. First of all, there are ribosome-like granules 150-200 A in I diameter. Some of these are not associated with fibrillar structures and lie freely in the nucleoplasm (^Georgiev and Chentzov, 1963). They are easily observed in interchromosomal regions (Swift, 1960) (Fig. 4). These free granules might correspond to the ribosomes which have been isolated from nuclei (see below). Similar granules are also found in nucleoli and chromosomes. In electron micrographs, using OSO4 stain, the nucleolus looks like a tangle of thick densely stained threads 500-1000 A in diameter, called "nucleolonemata" (Zivago, 1948; Estable and Sotelo, 1951) (Fig. 2, 4). Nucleolonemata in I turn consist of more thin fibrils (approximately 100 A in diameter) and ribosome-like granules. Fixation of isolated nuclei with OSO4 at low ionic strength causes the nucleolonemata to loosen, thus disclosing their inner structure (Fig. 6). The basic structural elements of the nucleolonemata are 100 A strands with attached ribosome-like granules (Georgiev and Chentzov 1960, 1963). The existence of granules as well as fibrils (which could resemble granules in thin section) is supported by the fact that after removal of osmium, IB the strands are no longer visible and the nucleolus looks like an accumulation of granules (Swift, 1960). The term "nucleonemata" was suggested for this structural complex of 100 A strands associated with ribosome-like granules, and we will use this term in our review. Although nucleonemata are mainly located in the nucleolus, chromosomes also contain them. After specific extraction procedures chromosomal nucleonemata are aggregated and become visible in the light microscope (Georgiev and Chentzov, 1960, 1963) (Fig. 9). Thus one can distinguish the following structural elements by electron microscopy: (1) DNP fibrils; (2) nucleonemata; (3) free ribosome-like granules; (4) double-layer membranes with pores. In addition (5) large granules (300-350 A in diameter) have been described in chromosomes (Swift, 1963). Finally, as mentioned earlier, (6) non-structural or small grained material — (pars amorpha) — is found in the nucleolus associated with nucleolonemata. At present, the nature of the last two components is unknown. Data on the distribution of these components among basic nuclear struc- tures are summarized in Table I. It is evident that the same elements, of apparently similar chemical composition, are found in various nuclear structures. It therefore follows that there are two principal approaches to the problem of the fractionation of the nucleus: (1) The chemical approach for the separation of nuclear ultrastructures which differ in chemical composition. (2) The mechanical approach — for the separation of fundamental nuclear macrostructures which in themselves are highly complex in organization. IP I n I 42 G. P. GEORGIEV Table I. Chemical fractionation of nuclear iiltrastructiiral elements Name of fraction Extraction medium Basic ultra- structural components released from nucleus Chemical constituents Nuclear localization (major localization and any addi- tional site) Globulin fraction 005-0-2 M neutral salts pH 60-70 Free particles 150-200 A in diameter Deoxynucleo- prolein fraction Residual fraction pH 7-8-80 2-2-5 M NaCl DNP fibrils Ribosomes (single or aggregates) sRNA soluble proteins ribonucleo- proteins containing mRNA DNP consists of DNA, histone and non-histone protein (2:3:1) Nucleonemata: fibrils (100 A diam.) and ribosome-like granules attached to them Nuclear membrane In nuclear sap (sRNA partly, from nucleolus) Chromosomes In chromo- somes (including material penetrating the nucleo- lar zone) In nucleolus (Partly in chromo- somes "resi- dual chro- mosomes") 'Acid" protein Ribosomal RNA. Probably in ribosomes or ribosomal precursors "Residual" protein (may be purified by extraction of nucleonemata by dilute alkali) Fig. 9. Electron micrograph of liver nucleus from which nuclear sap and deoxynucleo- protein have been removed. One can see nucleoli (n) consisting of nucleonemata (nn) and chromosomal nucleonemata (nn-ch). Aggregated chromosomal nucleonemata are visible in the light nicroscope as "residual chromosomes". The double membrane (m) with pores (p) is visible. Nuclei were treated successively by DNase, 0-14 m NaCI and 2 m NaCI and fixed by Os04 in 2 m NaCI. (Georgiev and Chentsov, 1963). 2. THE NUCLEUS 43 44 G. P. GEORGIEV B. CHEMICAL FRACTIONATION The chemical method of fractionation includes extraction of isolated nuclei with dilute salt solutions (0-05 m tris or phosphate buffer; 0-15 m NaCl and other more complex mixtures) and strong salt solutions (1 •0-2-5 m NaCl). Various modifications of this method were used for a long time for the separation of proteins and nucleic acids of the nucleus (Mirsky and Pollister, 1946; Zbarsky and Debov, 1951 ; Allfrey et al., 1957; Georgiev et ai, 1960). The initial extraction removes free particles from the nucleus, and thread-like elements (DNP fibrils and nucleonemata) are left (Georgiev and Chentsov, 1960, 1963). A certain amount of RNA and soluble nuclear proteins are present in the extracts obtained. Ribonuc!eoprotein particles were isolated from nuclear extracts by ultracentrifuging (Frenster, Allfrey and Mirsky, 1960; Samarina and Georgiev, 1960). These were later identified as ribosomes by their structural and metabolic properties (Pogo et al., 1962; Wang, 1963) (Fig. 10). sRNA was also found in this fraction. The fraction also contains a X' m.'^ • '■*»»■ #'* V. • • * * * • • * • 4 - ^ • • ♦ Fig. 10. Purified ribosomes from nuclear sap of calf thymus nuclei. ^ 76,800 (Wang, 1961). considerable amount ofprotein which corresponds in composition to soluble cytoplasmic protein (see Section VI). Hence the extract of nuclei obtained by dilute salt solution originally called the globulin fraction (Dounce, 1955) resembles cytoplasmic sap in its composition. In other words this globulin 2. THE NUCLEUS 45 fraction corresponds to "the nuclear sap fraction" (see Appendix: "Some Suggestions on Terminology"). This statement, of course, is somewhat arbitrary since it is not yet known from which part of the nucleus the com- ponents of the nuclear sap fraction originate. For example, ribosomes present in the nuclear sap fraction are not only derived from free ribosomes of the nuclear sap but may come from the nucleolus which also contains some ribosomes not firmly associated with nucleonema strands. This possi- bility also holds true for other components which may be associated in situ with some structures and are thus unequally distributed throughout the non-structural material. For example, some components although not struc- turally bound, may still only be found in the region of the nucleolus. Finally some components of the fibrillar elements may be cleaved off" and brought into solution during the extraction procedure. Thus one may obtain in this fraction ribonucleo-proteins containing messenger RNA (mRNA) originating from chromosomes by suitable modification of the extraction procedure (Samarina et al., 1965). All these factors complicate the comparison of results where extracting mixtures of different composition were used. Nevertheless the fact that the composition of the nuclear "globulin" fraction and that of cytoplasmic sap are similar proves that the globulin fraction is not an artifact but that it is derived mainly from the soluble phase of nuclei (i.e. nuclear sap). After extraction of nuclear sap, DNP fibrils and nucleonemata are left in the nucleus. By extraction of this residual material with 1 •0-2-5 m NaCl, DNP fibrils from chromosomes and the nucleolus region may be removed. In the extract DNA, histones and non-histone protein are present in the ratio 2:3:1, Some RNA is also present but the greater part of chromosomal RNA is recovered in the dilute salt extract. Unfortunately, strong salt solutions cause the DNP fibrils to dissociate and the DNP fraction thus obtained is not actually a suspension of DNP fibrils but rather a suspension of the chemical components of which these fibrils consist. Simple procedures have been developed for further fractionation of DNP fraction into histones, non- histone protein and DNA (Mirsky and Pollister, 1946; Zbarsky and Debov, 1951 ; Allfrey et al., 1957; Georgiev et al, I960). The material left after removal of chromosomal material with strong salt solutions represent structures consisting of nucleonemata (Georgiev and Chentsov, 1960): nucleoli and "residual chromosomes" (Mirsky and Ris 1951) and was termed the "nucleolus fraction" by, Allfrey et al. (1957), although this definition is not quite correct. Nucleonemal granules contain ribosomal RNA and seem to be ribosomes or the'r precursors (Lerman et al., 1964). By extraction with 0-01 n KOH nucleonemal material can be separated from membranes but this is accompanied by partial degradation. Thus by salt fractionation it is possible to separate and study the com- position of the basic structural components of nuclei: nuclear sap, DNP fibrils and nucleonemata. However, this extraction procedure is not free 46 G. p. GEORGIEV from artifacts since some extraction of material from other nuclear com- ponents may occur and there is a certain disruption of the structural or- ganization of the nuclei. C. MECHANICAL METHODS OF FRACTIONATION This group of methods are carried out as follows: nuclei are generally suspended in a medium of low ionic strength and relatively high pH (around 7-5). Most of the authors usually add Ca2+ ions since otherwise some destruc- tion of nucleoli may occur after disruption of the nuclei. Various techniques of mechanical disruption are then used: sonication, treatment in a Waring Blendor or forcing through a small hole. The suspension of broken nuclei so formed is fractionated by differential centrifuging in dilute or concentrated sucrose solutions. Several fractions may be obtained after centrifuging: (1) intact nuclei; (2) the nucleolus fraction; (3) one or two fractions con- taining broken chromosomes (the chromatin fraction); (4) free ribosomes; (5) and the final supernatant fraction (Vincent, 1952; Johnston, Setterfield and Stern, 1959; Birnstiel, Chipchase and Bonner, 1961; Birnstiel, Rho and Chipchase, 1962; Muramatsu, Smetana and Busch, 1963; Maggio, Siekevitz and Palade, 1963b). These methods are of special importance for the isolation of nucleoli since their ultrastructure and particularly their nucleolonemal structure are well preserved under these conditions (Fig. 1 1). One should remember, however, that material so obtained contains not only nucleolonemata but all components of the "nucleolus zone" and the chromatin adjacent to nucleolus (Fig. 11). In other words nucleolar ribo- nucleoproteins are isolated together with adjacent chromatin or DNP fibrils. This is apparently a result of the close juxtaposition RNPand DNP- containing nuclear ultrastructures. Hence isolated nucleoli usually contain approximately equal amounts of DNA and RNA, as well as histone that is completely derived from the DNP fibrils. It is not yet known whether chromatin associated with nucleoli is different from the rest of the cellular chromatin. RNA isolated from mechanically prepared nucleoli contains typical ribosomal RNA components (Chipchase and Birnstiel, 1963), in good agree- ment with the data on the nature of the RNA of nucleonemata. Furthermore, after treatment of nucleoli with deoxycholate, Birnstiel, Chipchase and Hyde (1963) were able to isolate ribonucleoprotein particles with properties similar to ribosomes. The sedimentation coefficient of these particles was found to be approximately 80 S and they could be reversibly dissociated into 40 S and V,60 S subunits by changing the Mg2+ ion concentration in the medium. There were more 60 S than 40 S subunits. Rates of protein synthesis as measured by amino acid incorporation in a cell-free system with nucleolar ribosomes are lower than those observed with cytoplasmic ribosomes. 2. THE NUCLEUS 47 Nucleonemal ribonucleoprotein particles of the nucleolus are thus actually ribosomes and/or ribosomal precursors (Birnstiel et a/., 1963a; Lerman, Mantieva and Georgiev, 1964). In addition to ribosomal RNA nucleoli contain a considerable amount of soluble RNA (sRNA) (10 to 30% of the total nucleolar RNA) as shown by its physical-chemical properties, its metabolic behaviour and its ability to accept amino acids (Vincent and Baltus, 1960; Birnstiel, Fleissner and Borek, 1963b). Since sRNA has not been detected in nucleonemata it is probably localized in the amorphous part of the nucleolus. The information on the chromatin fractions which are obtained after removal of the nucleoli is quite scanty. During homogenization in media of low ionic strength some breakage of the original structure of DNP-fibrils &: Fig. 11(a) [see p. 49 for legend] 48 G. P. GEORGIEV 'IT ^^ r k • # i #1 ■ • 1 V ^ 4 1 ' 4 chi ^ ch, 1 ' *r^^ Bi 2. THE NUCLEUS 49 with subsequent aggregation probably occurs, since thick threads are present in the precipitate (Maggio et a/., 1963b). Almost all the DNA and a significant part of the RNA of the nucleus are present in the chromatin fraction. Even less is known about the composition of the other fractions obtained by mechanical fractionation of nuclei. It is evident that mechanical methods of fractionation of nuclei are not sufficiently developed. Nevertheless they have already made possible the study of the function of the nucleolus and particularly of nucleolar enzymes. The principal drawback of mechanical methods is that it is not possible to isolate the different subnuclear structures free from contamination one with another. Also it is not possible to rule out the dangers of extraction and adsorption of various substances on isolated structures during fractionation as occurs during isolation of nuclei in aqueous media. Further improvement of methods of fractionation of nuclear structures and control of their purity is therefore needed. Some combination of mechanical and chemical methods of fractionation of nuclei may probably be the most fruitful future develop- ment. IV. Auto-Reproduction (Duplication) (DNA Biosynthesis) The primary process which provides for the transmission and continuity of hereditary information in the cell is the duplication of the cellular genetic apparatus or, in biochemical terms, the synthesis of DNA. Apart from oocytes and certain plant cells the greater part of the DNA of the cell is located in the chromosomes of the nucleus. It is to be expected, therefore, that enzymes participating in DNA synthesis would also be localized in chromosomal structures. Numerous experiments in vivo with labelled DNA precursors, and particularly ^H-thymidine, have demonstrated that these precursors are specifically incorporated into the nuclear chromosomes* It is now known that incorporation of thymidine into DNA also takes place in mitochondria and chloroplasts. This incorporation, however, does not reflect the synthesis of a chromosomal DNA precursor, but rather the synthesis of the DNA of these structures. The base composition of cytoplasmic DNA differs from chromosomal DNA and it probably carries information for the synthesis of at least some of the proteins of the mitochondrion or chloroplast, as the case may be. In cells where the DNA content of the cytoplasm is extremely low incorporation of ^H-thymidine is almost ex- < Fig. n. Electron micrographs of liver nucleoli isolated (a) by Muramatsu el al. (1963) and (b) by Maggio et al. (1963b) ( ■ 60,000). The nucleolonemal structure of nucleoli is well defined. In preparation (b) stained by uranyl acetate and Pb(OH)2 nucieolus-associated chromatin located around nucleoli (chi) or in nucleolar lacunae (cha) is visible. 50 G. P. GEORGIEV clusively into the nucleus. (See Chapter 3 for a fuller account of mitochondrial DNA.) Since chromosomal incorporation of ^H-thymidine is an established fact, let us consider the nature of enzymes which participate in DNA synthesis. A. NUCLEAR DNA POLYMERASE In 1956 Kornberg demonstrated the presence of an enzyme in bacterial extracts which catalysed the synthesis of DNA from deoxyribonucleoside triphosphates. This enzyme was called DNA polymerase or DNA deoxy- nucleoside triphosphate nucleotidyltransferase (Kornberg, Lehmann, Bess- man and Simms, 1956). The enzyme catalyses the following reaction: n (deoxyribonucleoside triphosphate) \ DNA, Mg*+ (deoxyribonucleoside monophosphate)^ + n pyrophosphate It was found that for the synthesis of DNA to proceed all four deoxy- nucleoside triphosphates and DNA had to be present. DNA has a template function in the reaction, that is the sequence of bases in the synthesized DNA is determined by that of the added template DNA (Kornberg, 1960). In other words, complementary synthesis is observed. The synthesis of a com- plementary strand and "repair" of single-stranded DNA into double- stranded form take place. Double-stranded DNA is less effective as a primer in the reaction. Although net synthesis of DNA in the presence of double- stranded DNA does occur, anomalous DNA and in particular branched molecules are formed (Richardson, Schildkraut and Kornberg, 1963). Not long after the discovery by Kornberg and co-workers of bacterial DNA polymerase a similar enzyme was isolated from animal tissues (Mant- savinos and Canellakis 1959a,b; Bollum, 1959, 1960a,b; Smellie, Keir and Davidson, 1959; Smellie et a/., 1960). It was demonstrated that tissue extracts of different origin stimulate the incorporation of ^^c or a-^^P labelled deoxy- nucleoside triphosphates into an acid insoluble product with the properties identical to native DNA. As with the bacterial extracts, the reaction required all four nucleoside triphosphates, Mg2+ ions and DNA (as a primer). The composition of the product was also determined by the nucelotide com- position of the primer DNA. In experiments with partially purified animal enzymes it was found that there was an obligatory requirement for single-stranded DNA as primer (for example heat denatured DNA, or DNA from the (f)X 174 virus). Native double-stranded DNA does not possess primer activity in the reaction (Bollum, 1959, 1960b). Synthesis is again complementary and results in the "repair" of single-stranded DNA into a double-stranded molecule. I 2. THE NUCLEUS 51 When this is achieved the synthesis ceases. Thus in contrast to E. coli DNA polymerase, purified polymerase from higher organisms possesses absolute specificity with respect to single-stranded primer. Apparently some factor is lost during purification of DNA polymerase which is responsible for the uncoiling of DNA strands. At first it appeared paradoxical that althought all DNA is to be synthesized in the nucleus all the DNA-polymerase activity was present in the supernatant of cytoplasmic extracts after centrifuging at 105,000^ (Mantsavinos and Cannelakis, 1959a,b; Bollum, 1959, 1960a, b; Smellie et a/., 1959). Nuclei did noi reveal any polymerase activity. Furthermore Smellie and Eason (1961) studied calf thymus nuclei isolated by non-aqueous method and found no polymerase activity in these nuclei either. Using the same material, Krakow, Coutsogeorgopoulos and Canellakis (1962) demonstrated the presence of an enzyme which catalyses the attachment of deoxynucleotides to the end of DNA strands ("end DNA nucleotidyl transferase"), but again DNA polymerase was absent from the nuclei. However, it was later shown that in different extracts of nuclei in addition to DNA polymerase there are deoxyribonucleases whose activity depends on the method of extraction. These deoxyribonucleases mask the presence of the DNA polymerase (Keir and Aird, 1962). Taking these facts into account, new experiments were performed with nuclei isolated in non-aqueous media from regenerating rat liver (Keir, Smellie and Sicbert, 1962) hepatoma, (Behki and Schneider, 1963) and calf thymus (Smith and Keir, 1963). It was found that in addition to the enzyme catalysing end attachment, nuclei do contain a true DNA polymerase and nuclear extracts which had no DNase activity or from which DNase had been removed, were found to have a higher activity than cytoplasmic extracts. The activity of DNA polymerase in crude extracts may vary depending upon the method of extraction but after removal of DNase all nuclear extracts had approximately the same activity. DNA polymerase is readily soluble in water and practically any solution (with pH ^ 4-5) removes this enzyme completely from the nuclei. In this respect the original failure to localize the DNA polymerase in the nucleus is a good example of the necessity of control experiments with nuclei isolated in non-aqueous media. At the same time Mazia and Hinegardner (1963) have demonstrated that nuclei of sea urchin oocytes isolated in the presence of mM MgCl2 contained practically all the cellular DNA polymerase. Bivalent cations were also shown to prevent the washing of this enzyme out of calf thymus nuclei (Main and Cole, 1964). It is of great interest that a significant part of the DNA polymerase in the bacterial cell is closely associated with DNA primer (Billen, 1962, 1963). Thus nuclei contain the enzyme DNA polymerase which catalyses the complementary repair of single-stranded DNA into a double-stranded molecule. Although no rigorous proof is yet available, it is very hkely that 52 G. P. GEORGIEV DNA polymerase participates in DNA replication in vivo. It is worth men- tioning that the activity of this enzyme correlates well with the rate of DNA synthesis in different tissues. In order to discuss this question more thor- oughly, let us consider some macromolecular aspects of the reproduction of the cellular genome. B. MECHANISM OF DNA SYNTHESIS IN CHROMOSOMES In 1953 Watson and Crick put forward the hypothesis of a semi-conserva- tive mechanism of DNA replication. According to this idea the DNA molecule formed contains one old and one newly synthesized strand. This concept was proved in the well-known experiments of Meselson (1960). He showed the presence of one preformed and one newly formed strand in E. coli DNA by combining the heavy isotope technique with density gradient centrifuging in caesium chloride of isolated native and denatured DNA's. The experiment of Meselson was repeated with DNA from other organisms with the same result (Sueoka, 1960; Simon, 1961). Recently this mechanism of DNA replication has acquired new support from the experiments carried out by Cairns (1963). By using the methods of molecular autoradiography and electron microscopy it was possible to demonstrate that the bacterial genome is a continuous double-stranded structure 400 A long. Synthesis of DNA is accompanied by the formation of a "fork", i.e. the division of one chain into 2 strands with both daughter strands containing newly formed material (see Fig. 12). The proposed semiconservative mechanism for DNA replication in vivo is in good agreement with the data existing on the mechanism of the DNA- polymerase reaction. The latter is known to involve the formation of double- stranded from single-stranded molecules, i.e. each new double-stranded molecule contains one old and one newly formed strand. If DNA polymerase does participate in DNA reproduction the question arises as to what factors are responsible for the unwinding of double-stranded DNA that must occur before the nucleotidyl-transferase reaction. First of all, does unwinding of a considerable length of the DNA molecule occur before the replication starts? Some indirect data, for example the action spectra of u.v.-light at different stages of phage formation in bacteria (Setlow and Setlow, 1960) may be interpreted as some proof for the existence of single- stranded DNA. On the other hand, all direct attempts to isolate single- stranded DNA from different organisms have failed (Georgiev and Mantieva, 1962; Rolfe, 1963). Experiments by Cairns (1963) also show that a region of intensive incorporation or DNA synthesis exists following the point of unwinding of DNA molecule into two strands. There is no lag between separation of the two strands and the subsequent formation of a double- stranded structure. Probably these two processes are coupled, and not 2. THE NUCLEUS 53 ENZYME First replicotion rst generation cycle) Second replication (second generotion cycle) (b) Fig. 12a. Autoradiograph of E. coli DNA following incorporation of (^H) thymidine for a period of two generations. The arrow shows the point of replication. One of newly formed strand is twice as active as the other preformed strand. Scheme explaining the sequence of labelling is given in Fig. 12(b). x 2200 (Cairns, 1963). separated in time and space. However the concomitance of the unwinding and doubling processes does not resolve the nature of factors controlling the unwinding. It has been suggested that nuclear deoxyribonucleases may be these factors. The nature of DNases present in the nucleus is at present quite uncertain. Experiments with nuclei isolated in non-aqueous media demonstrate that there is no DNase I (3.1.4.5) in nuclei (Stern and Mirsky, I 54 G. P. GEORGIEV 1952). However, according to Keir and co-workers (Keir and Aird, 1962; Keir and Smith, 1963) nuclei isolated in non-aqueous media exhibit other DNase activities, and the DNases of nuclei from different tissues are different. Some DNases are known to enhance the activity of primer DNA (Bollum, 1963). As a rule, however, their action is not specific. This activation largely results from the appearance of single-stranded DNA and oligonucleotides in the course of DNA degradation by DNase. Single-stranded DNA, as well as oligonucleotides, are both good primers. It is therefore probable that nuclear DNases are not functionally connected with DNA unwinding, although such a possibility cannot be completely ruled out at the moment. It is more likely that the factors controlling unwinding in vivo are closely related to DNA polymerase per se. It is possible that the interaction of DNA polymerase with DNA or the proteins in the DNP-complex results in local unwinding of the double strand and rapid subsequent synthesis of comple- mentary strands. An attractive hypothesis for the possible participation in this process of iron atoms with variable valency was recently suggested by Ivanov (1965). According to this Fe2+ ions are bound to adenine of DNA and may cause the transition of adenine from the amino- into the imino-form and as a result a weakening of the double helix occurs. What are the cytological ultrastructural features of chromosome re- production in the cell? DNA synthesis is known to occur at the so-called S phase of the cellular cycle. This is a phase of interphase which is preceded and followed by inactive states: the presynthetic Gi phase and postsynthetic G2 phase. During S phase different chromosomes and even different chromo- somal regions duplicate in certain specific order so as to double their DNA content. Since incorporation of ^H-thymidine is observed in mitotic chromo- somes and DNA synthesis occurs during interphase, experiments on the order of DNA synthesis in chromosomes do not allow us to correlate DNA synthesis with definite changes in chromosome structure. In the S-phase of the interphase nucleus ^H-thymidine is incorporated in all regions of the nucleus with some concentration of label in the region adjacent to the nucleolus. A more convenient experimental situation is observed in the macronucleus of Euploteus where the nucleus is elongated and a narrow zone of DNA synthesis moves from one end of the nucleus to the other. It is important to note that in the region where DNA synthesis occurs, RNA synthesis is completely suppressed. In other words, there is competition between DNA polymerase and RNA polymerase (2.7.7.6) for the template (Prescott and Kimball, 1961). In contrast to the rest of the nucleus, DNP fibrils in the zone of synthesis appear in a de-spiralized and extended state (Fig. 13) (Fure-Fremiet, Rouiller and Ganchery, 1957). Hence it is necessary for DNA autoreproduction that there is transition of DNP from the con- densed to the extended state. Similar results were obtained by Littau, Allfrey, Frenster and Mirsky (1964), who used ^^C-thymidine to label thymus nuclei; J 2. THE NUCLEUS 55 10 f^ Fig. 13. Micrograph of Euplotes eurystomus macronucleus stained by the Feulgen method and an electron micrograph of the same nucleus. The following zones are seen: (a) zone of condensed non-duplicated chromatin; (b) zone of chromatin reorganization; (c) non- stained zone and (d) second zone of condensed chromatin (replicated). Arrows indicate the corresponding zones in the electron micrograph. Mi — aggregates of condensed deoxy- nucleoprotein; Ch — transformation of DNP material into the extended state; DNA duplication takes place in zone (c) (as shown by O'H), thymidine incorporation (Prescott and Kimball, 1961); M2 — aggregates of condensed DNP in post-duplication zone (Fure- Fremiet et al. 1957). after labelling, the nuclei were disrupted and separated into material which contained condensed and extended chromatin DNP fibrils. Almost all the label was found in the extended fraction. It is of interest to note that DNA polymerase is inhibited by excess histone (Bazil and Philpot, 1963 and Billen and Hnilica, 1964). These are only the first experiments in the elucidation of factors responsible for the proper interaction of DNA polymerase with the DNA template. In conclusion it may be seen that the auto-reproduction of genetic material (or DNA synthesis) is one of the basic metabolic functions of the nucleus. 56 G. P. GEORGIEV DNA synthesis in the nucleus is carried out by the DNA polymerase present in high concentrations in the nucleus. Synthesis is likely to be semiconserva- tive. However a number of questions are still not answered. First of all it is not known whether only one enzyme participates in the synthesis of DNA or whether the process of repair into the double-stranded form by DNA polymerase is preceded by the action of another enzyme that controls the unwinding of the double helix. What is the function of deoxyribonucleases present in the cell nucleus? What factors determine the entry of DNP into the duplicative phase? What is the role of the interaction of the soluble enzyme with the DNA primer in this process? This list of unsolved problems, although far from complete, demonstrates that the principal stages of the process of auto-reproduction are not understood at present, except for the last stage carried out by DNA polymerase. V. Transcription (RNA Biosynthesis) In addition to the system which provides for the duplication of genetic material there exists another system in the cell nucleus responsible for supplying genetic information in a form suitable for controlling the synthesis of proteins. This role of information transfer from the genetic apparatus to the protein-synthesizing centres is played by messenger RNA (mRNA). The enzymic system participating in mRNA synthesis is the system that provides the supply of information from the genome. As suggested by Spiegelman and Hayashi (1963) this process is called "transcription". However, mRNA alone is not enough for the expression of genetic in- formation. A system is required which provides for the transition from the nucleotide sequence of mRNA to the amino acid sequence of the polypeptide chain that is synthesized. This is the stage of "translation". Two other types of RNA take part in the process of translation: ribosomal RNA (organized into the RNP particle or ribosomes) and soluble RNA (sRNA) which functions in this process as an "adaptor" or "translator". The synthesis of these RNA molecules, or in other words the creation of the translation system for protein synthesis, is also the function of the cell nucleus. All this demon- strates that RNA synthesis is one of the basic and specific functions of the cell nucleus which determines its role as regulator of cellular function. We will therefore discuss this question in detail. (Note that the mechanism of protein synthesis is also discussed in Chapter 7.) A. INTRACELLULAR LOCALIZATION OF RNA SYNTHESIS The nuclear origin of RNA was first suggested by Caspersson (see Caspers- son, 1950). This hypothesis became widely accepted when evidence accu- mulated that the hereditary information for protein structure is encoded in I 2. THE NUCLEUS 57 DNA molecules while protein synthesis per se does not require DNA but is controlled by RNA. It was natural to suggest that DNA determines the structure of proteins not directly but rather through some intermediary RNA. In other words RNA molecules are synthesized on DNA in the nucleus and then RNA moves to protein-synthesizing structures primarily in the cytoplasm. The question became somewhat more complex when the hetero- geneity of cellular RNA was established. In particular ribosomal, soluble and informational RNA's were shown to exist in the cell. However, it was further demonstrated that all three types of RNA are synthesized in the nucleus, in the same reaction catalysed by RNA polymerase. Let us recall briefly the principal facts which demonstrate the nuclear origin of RNA, before we start the discussion on RNA-polymerase itself. First of all there are kinetic data on the incorporation of labelled RNA precursors into the nucleus and cytoplasm. Bergstrand et al. (1948) and Marshak and Calvet (1949) were the first to show that after short exposure of cells to 32Pj the specific activity of nuclear RNA was more than ten or even hundred times higher than that of cytoplasmic RNA. This important fact was later confirmed by many investigators using various cells and different precursors (Jenner and Szafarz, 1950; Smellie, Mclndoe and Davidson, 1953; Smellie, Mclndoe, Logan, Davidson and Dawson, 1955; Fresco and Marshak, 1953). Similar results were obtained in biochemical investigations of RNA fractions, isolated from nuclear and cytoplasmic preparations, as well as in autoradiographic experiments with tritiated precursors (Fitzgerald and Vinijchaikul, 1959; Goldstein and Micou, 1959; Felling, 1959; Woods, 1959; Zaiokar, 1959, I960). In the majority of animal and plant cells after short incubation (1 hr or less) with labelled RNA precursors more than 90% of label incorporated into RNA is found in the nucleus and in some cases no label at all is found in cytoplasmic RNA. It was further shown that isolated nuclei retain the ability to incorporate purines, pyrimidine nucleosides and even ^^Pj into RNA (Allfrey and Mirsky, 1957; Rho and Bonner, 1961) under conditions where phosphorylation of nuclear nucleotides takes place. These data unequivocally demonstrate the existence of RNA synthesis in the nucleus although they do not solve the problem of the origin of cytoplasmic RNA. More recent studies on the incorporation of labelled precursors into nuclear RNA have dealt with the question of what type of RNA is synthesized in the nucleus. It has been found that in nuclei there is rapid incorporation of label into all three types of RNA (informational, ribosomal and soluble) so that synthesis of all three types of RNA does occur in the nucleus (Geor- giev and Samarina, 1961 ; Georgiev and Mantieva, 1962). What kind of experiments demonstrate the origin of cytoplasmic RNA? Numerous biochemical and autoradiographic studies on the kinetics of 58 G. P. GEORGIEV precursor incorporation into RNA fractions have been carried out using conditions of cold chase, i.e. after pulse labelling (for 10 min to 2 hr) the cells were transferred to non-radioactive medium and incubated for a further period of time. The principal drawback of this method is that the labelled precursor stays in the nucleus and continues to be incorporated into RNA a long time after transfer of the nuclei into the non-radioactive medium. This necessarily complicates the analysis of the transport of label. Kinetic data on specific and total radioactivity obtained in this kind of experiment are consistent with the assumption that cytoplasmic RNA is synthesized in the nucleus. This holds true not only for total RNA, but also for the main types of RNA : mRNA, ribosomal RNA, and transfer RNA (tRNA) (Amano and Leblond, 1960; Perry, 1960; Georgiev, 1961). However, the interpretation of kinetic data is based on a number of assumptions (for example that there is a common pool of precursors for nucleus and cytoplasm) and hence such results cannot be considered as conclusive. Thus on the basis of kinetic experiments some authors came to the conclusion that the synthesis of cytoplasmic RNA and the degradation of nuclear RNA were independent (Harris, 1961). Another approach to the study of the origin of cytoplasmic RNA is to investigate cytoplasmic synthesis after inactivation or removal of the nucleus. In experiments with amoeba and tissue culture of human amnion cells it was demonstrated (Prescott, 1959; Goldstein, Micou and Crocker, 1960) that the removal of the nucleus leads to immediate cessation of RNA synthesis in the anuclear cellular fragments. In fragments containing nuclei, normal syn- thesis of RNA was found to occur. The cessation of RNA synthesis in anuclear fragments of cells is a specific reaction, since the incorporation of amino acids into the proteins of anuclear fragments continues normally for a long time. In experiments by Perry and Errera (Perry, 1960; Perry, Hell and Errera, 1961) it was found that the inactivation of the nucleolus by a micro- beam of u.v. light results in 70% decrease in the incorporation of labelled precursors into the RNA of the cytoplasm. On the other hand enucleation of Acetabularia does not result in cessation of RNA synthesis in the anucleated fragments (Brachet, 1957). It was later demonstrated that this synthesis is completely dependent on the presence of chloroplasts (Naora, Naora and Brachet, 1960), structures of plant cells which are quite autonomous organelles and which contain their own DNA. A third possible approach to the problem is to prepare artificial systems of labelled nuclei and normal cytoplasm. Goldstein and Plant (1955) transferred nuclei containing labelled RNA into non-labelled amoeba and followed the flow of label from the nucleus to cytoplasm. Similar phenomena were ob- served by Scholtissek and Potter (1960) and Schneider (1959) who mixed isolated rat liver-nuclei and non-labelled cytoplasm. They were able to show the flow of label into free RNA and into ribonucleoprotein particles 2. THE NUCLEUS 59 in the cytoplasm. These experiments, however, are not very conclusive since the procedure of homogenization and transfer may disturb the normal functions of the nucleus. The fact that for all types of RNA molecule there are complementary DNA molecules in the cellular genome strongly favours the nuclear origin of RNA. This v^^as demonstrated in experiments where specific double- stranded hybrids between mRNA, ribosomal RNA, sRNA and denatured homologous DNA were obtained (Goodman and Rich, 1962; Yankofsky and Spiegelman, 1962; Hoyer, McCarthy and Bolton, 1963). Since almost all cellular DNA is localized in the nucleus these experiments speak in favour of the nuclear origin of cytoplasmic RNA. Finally a method has been suggested recently that uses actinomycins which specifically inhibit DNA-primed RNA synthesis (Goldberg, Rabinovitz and Reich, 1962; Harbers and Muller, 1962; Reich, Franklin, Shatkin and Tatum, 1962). It was shown that these inhibitors completely stop the incorporation of label into all three types of RNA in the nucleus as well as in the cytoplasm. Hence the synthesis of cytoplasmic RNA depends on DNA and occurs on a DNA template. In later experiments cells, after pulse labelling, were treated with actinomycin D in doses which completely blocked RNA synthesis ("actinomycin chase-experiments"). The purpose of these experiments was to follow the fate of newly synthesized RNA under conditions where there was no further synthesis of cellular RNA. It was found however that the inhibition of RNA synthesis lead to disturbances in its transport and removal from the site of formation. Nevertheless, in some cases, it was possible to follow the transfer of labelled RNA from nucleus to cytoplasm under conditions of actinomycin D block, although the transfer was not complete (Georgiev, Samarina, Lerman and Smirnov, 1963; Girard, Penman and Darnell, 1964). While each group of experiments is not conclusive per se taken together they unequivocally prove the nuclear origin of at least a considerable part of all three types of cytoplasmic RNA. Two points, however, require special mention. Firstly, not all the RNA synthesized in the nucleus is transferred to the cytoplasm. As "cold-chase" and "actinomycin-chase" experiments demonstrate, a certain amount of the newly synthesized RNA is degraded in the nucleus (Harris, 1963a, b; Lieber- man, Abrams and Ove, 1963). The amount broken down may vary consider- ably in different cells. This phenomenon will be discussed below. Secondly, some RNA synthesis occurs in those cytoplasmic structures which contain DNA. Thus RNA is synthesized in chloroplasts which have been shown to have DNA. A similar situation probably exists in mitochondria (see Chapter 3). One may think that RNA synthesized in chloroplasts is necessary for the synthesis of chloroplast proteins. However, the main part of cellular RNA which participates in the synthesis of cellular proteins is of nuclear origin. (It is only after viral infection that most RNA synthesis is localized in the 60 G. P. GEORGIEV cytoplasm, but under these conditions viral and not cellular RNA is syn- thesized.) In bacteria, which do not usually possess well defined nuclei, more active incorporation of labelled RNA precursors is also observed into the nuclear zone. After short exposures to radioactive precursors the greater part of the label was found in the nuclear region or in bacterial "nucleoids" in auto- radiographic experiments (Caro and Forro, 1961), as well as after fractionation of bacterial cells into nuclear and cytoplasmic material (Godson and Butler, 1962). Thus in different organisms the nucleus has been shown to be the organelle whose specific function is the synthesis of cellular RNA. In other words, the nucleus determines the amount of protein synthesis (via the ribosome-soluble RNA system) and its specificity (via the messenger RNA). B. RNA POLYMERASE AND ITS INTRACELLULAR LOCALIZATION Thus different RNAs are synthesized in the nucleus and then are trans- ported to the cytoplasm. What enzymes participate in these processes? In 1959 Weiss and Gladstone obtained an enzyme preparation from rat liver nuclei containing practically all the nuclear DNP. The enzyme catalyses the transfer of labelled uridine 5' monophosphate from uridine 5' triphosphate into internucleotide positions in RNA strands. The incorporation occurred only if all four nucleoside-triphosphates were present. Incorporation was inhibited by DNase (Weiss and Gladstone, 1959; Weiss, 1960). Soon after these experiments enzyme preparations with similar properties were isolated from different sources: thymus nuclei (Biswas and Abrams, 1962), HeLa cells (Goldberg, 1961), pea seedlings (Huang, Maheshwari and Bonner, 1960) and various bacteria (Furth, Hurwitz and Anders, 1962; Chamberlin and Berg, 1962). All these preparations had similar properties regardless of the source of isolation. The enzyme from E. coli was studied more fully than the others. The rational systematic name for the enzyme is DNA-dependent ribo- nucleic acid-nucleoside-5'-triphosphate-nucleotidyl-transferase (2.7.7.6) since it catalyses the following reaction n (nucleoside-5'-triphosphate) t (nucleoside-5'-monophosphate)M + n Pyrophosphate As a trivial name the term RNA nucleotidyltransferase is recommended. However use of this term may lead to confusion of two different enzymes, namely the DNA-dependent cellular enzyme and the RNA-dependent enzymes induced by RNA viruses. Therefore we will use the well-known term "RNA polymerase" for the cellular enzyme in distinction from the 2. THE NUCLEUS 61 "RNA synthetase" induced by viruses. Recently the term "transcriptase" has been suggested for RNA polymerase, which more precisely reflects its functional significance. There is an absolute requirement for the following components for the action of RNA polymerase: (1) all four nucleoside triphosphates (if DNA and not a homodeoxypolynucleotide is used as template), (2) DNA and (3) Mg2+ and/or Mn2+ ions. The pH optimum of the enzyme is in the slightly alkahne region (pH 7-5-8-0). The presence of DNA is absolutely essential for RNA polymerase. If the purified enzyme preparation does not contain DNA the reaction occurs only when DNA is added to the reaction mixture (Furth et al, 1962; Furth and Loh, 1963, 1964; Ballard and Williams- Ashman, 1964). The product of the reaction is a polyribonucleotide with 3',5'-phospho- diester bonds having quite a high sedimentation constant (10-20 S) (Hurwitz and August, 1963; Bremer and Konrad, 1964). The nucleotide sequence of the product is wholly determined by template DNA. In fact, RNA synthesized in the reaction has a base composition analogous to that of the DNA added. It can form double-stranded hybrids with this DNA and it has the same nearest-neighbour frequencies as the DNA primer. Thus the RNA synthesized is complementary to the DNA present in the reaction mixture (Geiduschek, Nakamoto and Weiss, 1961; Hurwitz and August, 1963). RNA polymerase is localized predominantly in the nucleus. In the study of RNA polymerase from animal and plant sources the enzyme was purified from nuclear preparations isolated by various aqueous methods. Treatment of isolated nuclei with dilute salts (0- 1-0-4 m) does not lead to the extraction of the enzyme from nuclei (Weiss, 1960; Goldberg, 1961 ; Biswas and Abrams, 1962). After disruption and subsequent fractionation of nuclei most of the enzyme is present in the fraction containing chromosomal fragments (Huang and Bonner, 1962). Thus the enzyme is probably a part of chromosomal structures. Solubilization of DNP in 1 m NaCl makes RNA polymerase come into solution; if water is added to the solution the enzyme is precipitated with DNP (Goldberg, 1961). In the experiments by Huang and Bonner (1962) chromosomal DNP of pea seedlings was centrifuged in 4 m caesium chloride. In concentrated CsCl, DNP dissociated and most of the proteins including all the histones came to the surface. Only a small part of the undissociated protein sedimented with DNA (the protein : DNA ratio of the sediment was 1:24). This non-histone type of protein attached to DNA was found to contain most of the RNA polymerase activity. Hence nuclear RNA poly- merase is a part of DNP fibrils and appears to be firmly bound to DNA. In the experiments of Huang and Bonner (1962) the possibility of contamina- tion of nuclei with cytoplasmic enzyme was minimized since the CsCl used in the centrifuging effectively removes absorbed proteins from nucleic acids 62 G. P. GEORGIEV or nucleoprotein structures. Thus the nuclear locahzation of RNA poly- merase seems to be well established at present. Recently Reid, El-Aaser,, Turner and Siebert (1964) have demonstrated the presence of RNA polymerase in nuclei isolated in non-aqueous media. In addition to RNA polymerase bound to DNP (the so-called "aggre- gated enzyme") some activity is found in cellular extracts (Furth and Loh, 1963, 1964). Recently RNA polymerase was partially purified from extracts of seminal vesicles; these enzyme preparations do not contain DNA and addition of DNA is necessary to activate the enzyme (Ballard and Williams- Ashman, 1964). "Soluble RNA polymerase" is probably bound to the nucleus (or is in the nuclear sap) but no conclusive evidence for this is yet available. It is likely that DNA-bound enzyme is "functional" RNA poly- merase and the soluble enzyme represents "reserve" RNA polymerase of the nucleus. That most of the RNA-polymerase activity is associated with DNA has also been demonstrated for bacteria. Treatment by streptomycin or prota- mine was needed to dissociate the RNA polymerase from DNA (Hurwitz and August, 1962). Therefore in vivo a part of the RNA polymerase is always bound to the template which takes part in transcription. Let us now consider some aspects of the mechanism of the RNA-poly- merase reaction. It is very important for the elucidation of chromosome function to establish the mechanism of the participation of DNA in the RNA-polymerase reaction. Does it follow the conservative or semiconserva- tive mechanism and what happens to the DNA molecule during RNA synthesis ? It is now established that DNA strands do not separate along any con- siderable length of the molecule in the course of RNA synthesis. DNA participates in RNA synthesis as a double-stranded molecule. This statement is based on the following facts. In systems studied in vitro, RNA synthesis by bacterial RNA polymerase is primed by native as well as by denatured DNA (Chamberlin and Berg, 1962). When native DNA is used in this system it is not altered in the course of reaction ; no intermediary complexes consisting of one DNA strand and one RNA strand are formed (Hurwitz and August, 1963). The reaction follows the conservative scheme of replication. The DNA template remains un- changed after completion of the cycle. When denatured DNA is used a DNA-RNA hybrid is first formed which then serves as template or primer in RNA synthesis. Continuous replacement of RNA in the hybrid molecules occurs, i.e. the semiconservative mechanism is operating, and at the end of the cycle, the double-stranded template contains one old and one new strand (Chamberlin and Berg, 1963). However, in vivo it was not possible to find double-stranded hybrids containing one RNA and one DNA strand and in which the RNA was characterized by a high rate of turnover (Mantieva, 2. THE NUCLEUS 63 1963). The only report of natural hybrids of this kind was from a bacterio- phage-bacterial system (Spiegelman, Hall and Storck, 1961), but this was not later confirmed by Konrad and Stent, (1964). ^ In experiments with soluble RNA polymerase from animal sources it ' was shown that only native double-stranded DNA is used as primer (Furth and Loh, 1964). Single-stranded DNA is not able to serve as primer. Finally actinomycin D was found to inhibit RNA synthesis only when double-stranded DNA was used as primer in experiments in vitro. Synthesis of RNA on single-stranded DNA is considerably less sensitive to this anti- biotic (Strelzoff, 1963). At the same time RNA synthesis in vivo is very sensitive to actinomycin and the effect is comparable to the sensitivity of RNA synthesis on double-stranded DNA observed in vitro. Thus double-stranded DNA performs the function of primer in the RNA- polymerase reaction in vivo and this process is not accompanied by un- winding of DNA along any significant part of the DNA molecule. Since, however, strand separation is obligatory for complementary synthesis, one may postulate that some restricted separation (e.g. of several nucleotide pairs) does take place and a separation wave moves along the DNA molecule participating in the reaction. Hypothetical mechanisms of this kind for RNA synthesis have been suggested by a number of authors, for instance by Butler (1963), Fig. 14. X I NZYME Fro. 14. A schematic mechanism for the RNA polymerase reaction (Butler, 1963). According to this scheme, during the process of RNA synthesis in chromo- somes in vivo a complex is formed containing double-stranded DNA and single-stranded RNA, linked by some protein (probably by RNA poly- merase). The existence of such complexes in vivo and in vitro has been shown by several investigators. Schulman and Bonner (1962) have isolated from Neurospora material containing native DNA and RNA which could not be separated by various methods. In experiments by Bonner, Huang and Maheshwari (1961) the incorporation of label into nuclei from pea seedlings was studied in vitro. They found that newly synthesized RNA is bound to the chromosomes and may be detached from them by treatment with detergent, DNase or heating to 60°. Since phenol does not extract newly formed mRNA at temperatures below 55-60° (Georgiev and Mantieva, 1962), it is likely that the thermal phenol fractionation of cellular RNA is 64 G. P. GEORGIEV also based on the existence of this type of complex. In the course of studying RNA synthesis with a cell-free system, Bremer and Konrad (1964) observed the formation of a complex containing DNA, RNA polymerase and newly formed RNA. Each DNA molecule was associated with several molecules of enzyme, each of which in turn formed one RNA molecule. One may speculate that just as the ribosome moves along the mRNA molecule "reading off" the information for the synthesis of protein, the RNA-polymerase molecule moves along the length of the DNA, sequentially copying it. This process results in the gradual increase of RNA chain length. If several molecules of RNA polymerase participate in the transcription of the DNA molecule, a structure should result containing a DNA strand (or DNP) with molecules of RNA polymerase and synthesized RNA attached to it. This structure should resemble polysomes in its morphology. The molecular weight of RNA polymerase is approximately 6 x 10^. This means that RNA-polymerase molecules can be readily visualized by electron microscopy. Although such structures have not been found in the nucleus, so far nobody has searched for them. In electron micrographs of DNP fibrils dense zones along the length of the molecules are often observed (see Fig. 7). The above arguments show the importance of detailed study of the organization of chromosomal DNP fibrils in general in order to elucidate the structural localization of RNA polymerase. Recently, Fuchs, Zillig, Hofschneider and Preuss (1964) have examined such a complex by electron- microscopy. They used a cell-free system with T2 phage DNA as a primer, and E. coli RNA polymerase. According to these authors, RNA polymerase consists of a cylindrical array of 6 sub-units. DNA probably passes through the axial hole of this cylinder. The problem of whether one or two strands of double-stranded DNA serve to generate genetic messages is of great importance. A number of observations demonstrate that in vivo only one DNA strand is transcribed into RNA. First of all, all mRNA isolated from phage-infected bacteria, or newly formed RNA isolated from nuclei of animal cells is single-stranded. Attempts to obtain RNA-RNA hybrids from naturally occurring RNA have not been successful either (Bautz, 1963; Samarina, Lerman, Tumanjan, Ananieva and Georgiev, 1965). This demonstrates that no complementary strands are present in RNA as would be expected if both DNA strands had participated in RNA synthesis. Furthermore, mRNA synthesized in bacteria after phage infection is complementary only to one strand of phage DNA, and no hybrids can be formed between mRNA and the second strand (Spiegelman and Hayashi, 1963; Tocchini-Valentini et al. 1963; Marmur and Greenspan, 1963). In hybridization experiments with agar-trapped DNA it was found that only half of the bacterial DNA could form hybrids with homologous mRNA. (McCarthy and Bolton, 1964). Thus RNA synthesis in vivo has an assymmetrical character, i.e. only one DNA strand is transcribed. 2. THE NUCLEUS 65 In experiments in vitro one can observe both types of synthesis: asym- metrical and symmetrical. Geiduschek, Moohr and Weiss (1962) observed the transcription of both strands of DNA and the accumulation of double- stranded RNA. Hayashi, Hayashi and Spiegelman (1964) were able to demon- strate an asymmetrical RNA synthesis by using the "replicative form" of DNA of phage 0 X 174 to prime the reaction. It is interesting that ^ X 174 replicative DNA is a circular, double-stranded molecule. However, with sonicated DNA (containing opened strands) both DNA strands were copied. Hence in this case the cyclic state of the DNA molecule is necessary for the asymmetrical character of RNA synthesis. This fact is probably not of uni- versal significance, however. Geiduschek, Tocchini-Valentini and Sarnat (1964) were able to observe synthesis of RNA on one strand of DNA using opened strands of T2 phage DNA and a crude enzyme preparation from Bacillus mega- terium. On the other hand. Green (1964) and Luria (1965) obtained asym- metrical synthesis with a purified enzyme from E. coli. Only DNA denatura- tion altered the character if the RNA synthesis in these experiments. The studies in vitro of factors that determine asymmetric RNA synthesis hinge on the problem of the initial sites of transcription. Some experiments on complex formation between DNA and RNA polymerase may indicate a role of free 3'-OH ends in this process. RNA polymerase competes with DNA poly- merase for the template. It also inhibits the action of DNA of 5'-exonu- cleases but does not inhibit the action of endonucleases (Berg, Kornberg, Fancher and Dieckmann, 1965). It is probable that only one strand of DNA has available initial sites of transcription and this would explain why only one strand has template activity. Modifications of DNA or of the enzymes that takes place in the above experiments may render the corresponding sites of another strand available to the enzyme. The studies on the problem of the initial sites and precise character of transcription seem to offer great possibilities for understanding the morphological organization of the func- tional unit of the genome. We have devoted so much attention to the macromolecular aspects of the RNA-polymerase reaction and to the role of DNA in this reaction because this enzyme plays a key role in RNA synthesis and consequently in the most important and specific function of the nucleus, namely the generation of genetic messages. This important role of RNA polymerase will become especially evident when we consider the types of RNA which are made in the RNA-polymerase reaction. It is relatively certain that RNA polymerase catalyses mRNA synthesis since the RNA synthesized in this system exhibits many characteristic properties of mRNA. Firstly it has a DNA-like nucleotide composition and shows complementarity towards DNA (see above). Also, this RNA stimulates amino acid incorporation into proteins in ribosomes (Chamberhn and Berg, 1962) and synthesis of specific protein in ribosomes (Bonner, Huang and 66 G. P. GEORGIEV Gilden, 1963). Participation of RNA polymerase in the synthesis of ribo- somal RNA and sRNA is less certain. However some results indicate that these RNA types are also synthesized on DNA by the RNA-polymerase reaction. These are for example, the experiments that show the inhibition of all RNA synthesis by actinomycin (which inhibits the functioning of the DNA template in the RNA-polymerase reaction) and experiments demonstrating the complementarity of all RNA types to DNA (Reich et al., 1962; Yankof- skyand Spiegelman, 1962, and Goodman and Rich, 1962). Thus normally all types of cellular RNA are probably synthesized by the RNA-polymerase reaction although it is not yet known whether there is only one enzyme for the synthesis of all RNA or whether several different iso- enzymes of RNA polymerase exist in the cell. We will now consider the role of different nuclear structures in the synthesis of the various types of RNA. C. CHROMOSOMES AND mRNA SYNTHESIS Let us first consider experiments which demonstrate the chromosomal synthesis of mRNA. It was already noted that the base composition of chromosomal RNA differs from that of ribosomal RNA and is similar to that of DNA (Edstrom, 1960; Edstrom Grampp and Schorr, 1961 ; Edstrom and Gall, 1963). DNA-like RNA isolated from nuclei by phenol fractionation is also of chromosomal origin (Georgiev et al., 1963). Further data on the intracellular localization of mRNA synthesis are obtained from the study of the nature of newly synthesized RNA. It has been shown that all newly formed RNA (i.e. RNA labelled after short incubation of cells with suitable radioactive precursors of RNA) is localized in the nucleolo-chromosomal complex (Sibatani, Yamana, Kimura and Okagaki, 1959; Georgiev and Mantieva, 1960). Newly formed or pulse- labelled RNA is a mixture of molecules with DNA-like base composition (D-RNA) and RNA with base composition of ribosomal RNA (R-RNA) (Georgiev and Mantieva, 1962; Lerman, Mantieva and Georgiev, 1963). By its properties (base composition, ability to form hybrids with homo- logous DNA, and its stimulatory effect on the amino acid incorporation activity of isolated ribosomes) D-RNA has been identified as newly formed messenger RNA (Samarina et al., 1965b). Consequently the localization of newly formed D-RNA would correspond to the site of mRNA synthesis. In autoradiography experiments it was demonstrated that the synthesis of nucleolar RNA is much more sensitive to actinomycin D than RNA synthesis in the chromosomes (Perry, 1962; Sirlin, Jacob and Kato 1962). On the other hand, low concentrations of actinomycin D first cause inhibition of R-RNA synthesis (Lerman et al, 1963). It is possible to find a concentration of actinomycin such that all the label would be found only in the chromosomes; under these conditions all newly formed RNA would be D-RNA (Georgiev et al., 1963). These facts clearly demonstrate that mRNA is synthesized in 2. THE NUCLEUS 67 w hromosomes. However, the possibility cannot be excluded that other types f RNA are also synthesized in the chromosomes (see below). It is well known that in higher organisms only a certain part of the genome active in mRNA synthesis. Most of the genome is suppressed and these strons do not participate in RNA synthesis. This allows differentiation of lis in an organism so that there may be somatic differences between various cellular types with the same genome. This is well illustrated cytologically by the following facts. In the giant chromosomes of the salivary glands of rosophila so-called "puffs" appear at different stages of morphogenesis in iome chromosomal disks. Although puffs differ in their chemical character- istics and metabolic properties, in many cases the essential feature of puffs is that they are the site of intensive RNA synthesis (Fig. 15) (Rudkin and ^FiG. 15. The incorporation of T'H) cytidine into puff regions of salivary gland chromo- somes of Drosophila melanogaster. The position of two puffs in sections 58 and 60 of chromosome 2R are shown by arrows. ■ 1250 (Rudkin and Woods, 1959). One figure is stained and the other shows the autoradiogram. I 68 G. P. GEORGIEV Woods, 1959). It is of special significance that at certain stages of cellular differentiation puff formation always taices place in the same chromosomal region. On the other hand differences in puff localization are observed in cells at different stages of development (Breuer and Pavan, 1955). Conse- quently at different stages of morphogenesis RNA is synthesized in different parts of the genome and on different molecules of DNA, Another illustration of this idea is given by experiments on the hybridiza- tion of DNA from animal organisms with mRNA isolated from different organs of the same species and on the competition between these mRNAs for DNA cystrons. By this approach it is possible to find out whether same or different genes are active in different tissues. It was shown that a significant proportion of active genes are indeed different in different tissues (McCarthy and IJpyer, 1964). The question therefore arises as to what factors are responsible for the regulation of DNA activity during RNA synthesis. Let us again consider the cytological aspect of the problem. It seems to be well established that the difference between mitotic and interphase chromosomes lies in the fact that the former represent completely spiralized and condensed structures whereas the latter have at least partially lost this configuration. RNA is not synthesized during mitosis (Prescott and Bender, 1962). It is possible therefore that the spiralized and condensed state of the deoxynucleoprotein fibrils in the mitotic chromosome prevents the participation of DNA in RNA synthesis. A very attractive system to study chromosome function are the so-called "lampbrush chromosomes" which are meiotic chromosomes of amphibian oocytes. During growth and development of the oocytes, side loops are branched off the axis of the chromosomes which consists of chromomeres. In these loops the DNP fibrils are in a stretched state and have despiralized whereas in the chromomeres the fibrils are condensed. The loops are formed in a sequential order and different chromomeres form loops of varying length. Thus in different chromomeres different DNP fibrils undergo the transition from the condensed into the extended configuration. As soon as growth and development of oocytes is over these loops disappear. Gall and Callan (1962) have found that RNA synthesis takes place in these loops. (Fig. 16). Thus the study of lampbrush chromosomes demonstrates that some regions of chromosome may become stretched while the main part of chromosome stays in relatively condensed state. It is stretched and non- spiralized zones in loops that participate in RNA synthesis, i.e. are "active cistrons". In experiments by Frenster, Allfrey and Mirsky (1963) thymus nuclei were incubated with labelled RNA precursors, then disrupted by sonication and eventually preparations of "condensed" and "extended" chromatin were isolated by differential centrifuging. It was found that the specific and total radioactivity of the "stretched" chromatin was considerably higher than that 2. THE NUCLEUS 69 I Fig. 16. The incorporation of uridine-H^ into loops ol iampbiu->li Xll chromosome of Triturus cristatiis. The giant granular loops are shown by arrows. The incorporation is concentrated in loops and is negligible in axis. < 2000 (Gall and Callan, 1962). of the condensed chromatin. A similar distribution of label between con- densed and extended chromatin was observed in autoradiography studies (Littau et al., 1964). Thus the transition from the state of repression to the state of active func- tion and RNA synthesis is accompanied by cytological changes i.e. by transition of DNP fibrils from the condensed state with a high degree of spiralization into the extended, despiralized form. What is the molecular basis of this phenomenon? What chemical changes lead on to the one hand to despiralization and on the other to "switching on" of the cistron? In 1947, Stedman and Stedman suggested that inhibition of gene activity or gene "switching off" may be explained by the action of histones, principal constituents of chromosomal structure. The first experi- mental support to this idea came from experiments by Huang and Bonner (1962) who demonstrated that histones inhibited RNA-polymerase activity in chromosomes from pea-seedlings. The removal of histones from the chromosomes led to 25-fold increase in their RNA-polymerase activity. The addition of histones had the opposite effect. I 70 G. P. GEORGIEV Similar results, i.e. 200 % increase in the RNA synthesis was observed by Allfrey, Littau and Mirsky (1963) with isolated calf thymus nuclei after digestion of histones with trypsin. RNA synthesis in isolated nuclei was inhibited by histones. The site of histone action is the DNA template. No immediate effect of histones on enzyme activity itself was observed (Bonner and Huang, 1963). In order to demonstrate that the histone mechanism of repression is used in the selection of active genes in vivo, the following experiments were carried out (Bonner et a/., 1963). Chromatin from pea-seedlings was added to E. co/i RNA polymerase and E. coli ribosomes. The synthesis of mRNA occurred which in turn led to protein synthesis. Chromatin from parts of the plant which are known to synthesize globulin in vivo was shown to induce the synthesis of globulin in vitro whereas chromatin isolated from non-synthesiz- ing parts did not stimulate globulin formation. If DNA isolated from both parts of the plant was added to the system, little synthesis of globulin was observed in either case. Thus proteins of the nucleoprotein complex may be considered as specific inhibitors of the template in the RNA-polymerase system or in biological terms as the repressor of the genetic activity of DNA. However, the problem is actually much more complex than it seems at first sight. Histones are known to be a very heterogeneous group of proteins, and there are a large number of individual histones, usually divided into three main groups: arginine-rich, moderately lysine-rich and lysine-rich. (See Mirsky and Osawa, 1961). Even the question of which of these types of histone inhibit RNA synthesis is not yet answered. According to Bonner and Huang (1963) it is the lysine-rich histones that inhibit, but Allfrey et al. (1963) ascribe this function to the arginine-rich histones. Recently, Huang and Bonner (1965) have observed that histone molecules are bound to a new type of RNA, characterized by a very low molecular weight (equivalent to only 40 nucleotides) and a high content of dihydrouracil. The authors sug- gested that the complex between this RNA and histone is a true repressor. The presence of RNA allows for the recognition of base sequences in DNA. If histones do regulate gene activity it is necessary to assume the existence of two types of DNP in chromosomes: one more saturated and one less saturated with histone. Nucleohistone was found to melt at a temperature 10-12° higher than DNA. Chromatin isolated from different sources gives a melting profile consisting of two phases: the first small increase in optical den- sity at the melting temperature {Tm) of DNA and the larger second increase at the Tm of nucleohistone. The probable explanation of these results is that DNP contains a small proportion of free DNA, or DNA with less than the normal amount of histone (Bonner and Huang, 1963; Bonner et al. (1963). Soluble DNP isolated from nuclei according to Zubay and Doty (1959) behaves on melting like pure nucleohistone, i.e. "free" DNA seems to 2. THE NUCLEUS 71 remain in the nuclear residue. The latter indeed contains less histone (Bonner and Huang, 1963). It is necessary to point out however that the fractionation of DNP and histones needs further study. In particular, individual histones, and not the total histone fraction, should be studied. In addition it is important to remem- ber that not only histones but also certain basic compounds of low molecular weight considerably affect the RNA-polymerase system. Thus some poly- amines stimulate RNA synthesis on double-stranded DNA (Krakow, 1963). Finally the most complex problem is the mechanism of the "switching on" and "off" of genes during cellular differentiation and even during the cell cycle. Experimental data concerning this problem are very scanty. A possible approach to the question has been made in experiments by Allfrey, Faulkner and Mirsky (1964), who showed that the arginine-rich histone fraction retains the ability to combine with DNA and to change its melting tempera- ture after chemical acetylation or methylation, but is no longer able to inhibit the RNA-polymerase reaction. Acetylation and methylation reactions were also discovered in the nucleus. Whether these reactions are related to the process of "switching on" of the gene is not known at the moment. Frenster (1965) suggested that non-histone proteins of the chromosomes or some part of the chromosomal DNA may act as de-repressors by making DNA cistrons available to the action of RNA polymerase. Finally one must consider the results that indicate that the nucleus of a cell that is not actively synthesizing nucleic acids begins to be so after it has been transplanted into the cytoplasm of active cells. For example, erythrocyte nuclei begin to synthe- size not only RNA but also DNA after association with tumour cells (Harris 1965). These results indicated the role of cytoplasmic factors in determination of gene activity. In conclusion, let us consider the problem of the removal of mRNA from the template-enzyme complex and its transfer into the cytoplasm. Samarina et al. (1965a., 1966) isolated from cell nuclei ribonucleoprotein particles containing mRNA. The particles sedimented as a Very homogeneous peak with sedimentation constant of 30 S. It was also possible to observe the formation of an mRNA-protein complex in vitro by addition of free mRNA to isolated nuclear 30 S particles. Some indirect experiments indicate that these particles take part in the transfer of mRNA from the chromosomal template to the sites of protein synthesis. Similar particles with sedimentation constant of 40 S have been observed in the cytoplasm of animal cells (Joklik and Becher, 1965). It was suggested that these were small ribosomal sub-units involved in the transport of mRNA (Samarina et al., 1966; Henshaw et al., 1965; Joklik and Becker, 1965). However, the possibility cannot now be excluded that the 30-40 S par- ticles involved in the transport of mRNA are special protein entities and not ribosomal sub-units. Recent results show this to be more probable. i 72 G. P. GEORGIEV D. SYNTHESIS OF RIBOSOMAL RNA AND sRNA AND THE FUNCTION OF THE NUCLEOLUS In addition to mRNA the synthesis of R-RNA, i.e. RNA with ribosomal RNA base composition also occurs in the nucleolo-chromosomal apparatus. The molecular weight of newly formed R-RNA is higher than that of ribo- somal RNA and therefore it sediments faster (35-45 S) than ribosomal RNA (18 S and 28 S) (Hiatt, 1962, Scherrer and Darnell, 1962; Georgiev et al., 1963). A number of investigators have shown that the heavy R-RNA (35-45 S RNA) is a metabolic precursor of ribosomal RNA. Thus if a short pulse of radioactive precursor is given, the major part of the newly formed RNA is heavy R-RNA. If this is followed by addition of actinomycin to inhibit further RNA synthesis, the heavy non-ribosomal R-RNA completely dis- appears after several hours and all the label is then found in typical ribosomal RNA (Perry, 1962; Sherrer et al. 1963). By thermal phenol fractionation it can be demonstrated that there are two components in heavy R-RNA with sedimentation coefficient 45 S and 35 S. The 35 S component is probably the precursor of 28 S RNA and the other (45 S) is the precursor of 18 S ribosomal RNA (Georgiev and Lerman, 1964; Lerman, Vladimirzeva, Terskich and Georgiev, 1965). Thus there are two stages in the synthesis of ribosomal RNA: synthesis of heavy R-RNA precursors, and their subsequent cleavage into ribosomal RNA. Both processes take place in the nucleolo-chromosomal apparatus. In what part of this structure do these synthetic processes occur? A number of experiments demonstrate that synthesis of R-RNA and its transformation into ribosomal RNA takes place in the nucleolus. As already mentioned (Section III) the nucleolus contains ribosome-like particles and the greater part of nucleolar RNA is ribosomal RNA. Inactivation of the nucleolus by a microbeam of u.v. light strongly inhibits incorporation of label into cyto- plasmic RNA (Perry, 1960; Perry et al, 1961). These data suggest that the nucleolus is concerned in the synthesis of ribosomal RNA. Recently, the following more direct evidence has been obtained : (1) Low concentrations of actinomycin only inhibit the synthesis of R-RNA and this is accompanied by selective inhibition of incorporation into nucleolar and not chromosomal RNA (Perry, 1962; Lerman et al, 1963). (2) Newly formed RNA of nucleoli (or more precisely of the nucleonemal material) was prepared by subsequent salt extraction of isolated nuclei and its base composition was studied. It was found that newly formed RNA of nucleonemata is actually R-RNA. Since after short pulses it has the highest specific activity of the various nuclear RNA fractions, nucleonemata are the original site of localization of newly formed R-RNA (Lerman et al., 1964; Steele, Okamura and Busch, 1965). 2. THE NUCLEUS 73 ^^ad been treated with phenol-0-14 m NaCl at different temperatures indicate that solubiHzation of heavy R-RNAs (35 S and 45 S) was followed by loss of label from nucleoli. Thus the localization of the bulk of newly formed precursors of ribosomal RNA is the nucleolus. (4) Mutants of Xenopus laevis which do not have nucleoli cannot synthe- size ribosomal RNA, although mRNA synthesis occurs normally (Brown and I^urdon, 1964). ^P (5) The distribution has been studied of DNA complementary to ribo- somal RNA in chromatin fractions obtained after mechanical disruption and fractionation of cell nuclei. In HeLa cells the content of complementary DNA in the nucleolar fraction was 5-6 times higher than in the rest of the chromatin (McConkey and Hopkins, 1964). Most of the DNA with base composition similar to that of ribosoma. RNA was also found in the nucleolar fraction of the aquinus fungus Blastogladuella emersonii (Comb and Katz, 1964). Ritossa and Spiegelman (1965) observed in mutants of Drosophila that lack of one, two or three nucleoli results in a corresponding decrease in the amount of DNA complementary to ribosomal RNA. Hence in these cells, R-RNA is apparently synthesized in nucleoli (on DNP fibrils of the nucleolar region) or in chromatin adjacent to the nucleolar surface. On the other hand, in pea-seedlings, cistrons complementary to R-RNA are equally distributed amongst the various nuclear fractions (Chipchase and Birnstiel, 1963). One should emphasize, however, that nucleonemata are concentrated not only in nucleoli but are found in considerable amount in chromosomes. Probably synthesis of ribosomal RNA takes place not only in the nucleolus but in nucleonemata in general (see above) and this may explain these contradictions. (6) Liau, Hnilica and Hurlbert (1965) have observed the synthesis of R-RNA in nucleoli isolated from hepatoma cells. (7) It was already pointed out that nucleonemata contain newly formed R-RNA. Actinomycin chase experiments show that the transformation of the R-RNA precursor into true ribosomal RNA occurs in those structures in which synthesis of R-RNA took place; newly formed ribosomal RNAs are present in the same fractions in which heavy precursors of ribosomal RNA were found prior to addition of actinomycin (Lerman et al., 1965). The following conclusions may be drawn from the above. The accumu- lation of newly formed R-RNA occurs in nucleoli and probably in chromatin nucleonemata. The conversion of precursor R-RNA into true ribosomal RNA also takes place in the nucleolus. The synthesis of R-RNA occurs on the DNA template of chromosomal DNP fibrils of the nucleolar region or of the adjacent zone and possibly on DNP fibrils bound to extra-nucleolar nucloenemata, although the latter point is not yet rigorously proved. Thus the nucleolus is the organelle participating in the synthesis of ribosomal RNA. The next question is the localization of sRNA synthesis in the nucleus. A I 74 G. P. GEORGIEV number of facts suggests that the nucleolus has a certain role in this process. Nuclear sRNA exhibits high specific activity after pulse-labelling (Georgiev and Samarina, 1961). A considerable part of this nuclear sRNA is found in the nucleolus. (Birnstiel et al., 1963 a, b). pHl Pseudo-uridine, a specific precursor of sRNA, is incorporated initially into the nucleolus (Sirlin, Kato and Jones 1961). A specific form of low molecular weight RNA which differs from sRNA by the absence of methylated bases and is considered to be the precursor of sRNA, is found in the nucleolus. (Comb and Katz, 1964). These data suggest that, in addition to the synthesis of ribosomal RNA, sRNA is also synthesized in the nucleolus. In addition, the nucleolus has been shown to participate in the methylation of sRNA and probably ribosomal RNA. It is well known that in addition to the four principal bases some rare or minor bases (mostly methylated deriva- tives of purines and pyrimidines) are encountered in nucleic acids. sRNA is especially rich in such minor components and their content in ribosomal RNA is considerably less. The process of base methylation occurs at the level of the macromolecules and is catalysed by the enzyme adenosylmethionine- RNA-methyltransferase (Fleissner and Borek, 1962). This enzyme was found in preparations of nucleoli from pea-seedlings by Birnstiel et al. (1963) and the nucleolar enzymic activity was much higher than that of the chromo- somes. The incorporation of methyl groups occurred mainly into low mole- cular weight RNA, evidently sRNA. The formation of the methylated bases 1-methylguanine and 6-methyl-adenine was demonstrated. Sirlin, Jacob and Tandler (1963) have studied incorporation in vivo of "C-methionine into the salivary gland cells of Chironomus in which protein synthesis has been inhibited by puromycin. The label incorporated dis- appeared after treatment with RNase. This fact demonstrated that this incorporation was due to methylation of RNA. It was found that after short incubation times the label is found mainly in the nucleolus. Thus from experiments in vivo and in vitro one can conclude that at least the final stages of ribosomal RNA formation and methylation of sRNA take place in the nucleolus. E. OTHER ENZYMES INVOLVED IN SYNTHESIS AND BREAKDOWN OF POLYNUCLEOTIDES In addition to RNA polymerase which plays a key role in the synthesis of different RNAs a number of enzyme catalysing the incorporation of nucleo- side phosphates into polynucleotides have also been found in the nucleus. These enzymes are found in nuclear extracts and stay in the supernatant after ultracentrifuging. They catalyse the incorporation of the nucleotidyl residue of nucleoside triphosphates into homopolynucleotides (i.e. poly- 2. THE NUCLEUS 75 nucleotides that contain only one type of base). The most thoroughly studied is the enzyme polyadenylate-adenosinotriphosphate-nucleotidyltransferase (polyadenylate-synthetase : 2.7.7.19.) found by Edmunds and Abrams (1960, 1962) in the supernatant fraction of nuclear extracts. The presence of primer (RNA or polyadenylate) is obligatory for the enzyme activity. Similar en- zymes which can catalyse the synthesis of homopolymers and also the addition of nucleotidyl residues on to the end position of polynucleotide chains have been described by other authors (Burdon, 1963; Smellie, 1963). The functional role of these enzymes is not yet understood. 1^^ It is of interest that RNA degradative enzymes have been discovered in the ^■cleus, since RNA breakage apparently takes place in vivo. Harris and co-workers (Harris, 1961; Harris, 1963a, b, Harris, Fisher, Rodgers, Spencer and Watts, 1963) and other authors (Lieberman et al., 1963) have demon- strated that a fraction of RNA synthesized in the nucleus also undergoes breakage there. In different cells, and in similar cells under different con- ditions, variable amounts of the newly formed RNA were degraded. An attempt to elucidate the nature of the enzyme responsible for the degradation of this RNA in HeLa cells was made by Harris et al. (1963). The enzyme was found in chromosomes and it appears to hydrolyse only newly formed RNA. 5'-nucleotides were found as the only reaction products. Orthophosphate seems to activate the reaction whereas ADP exerts the opposite effect. If RNA labelled with ^^C adenine is used in the reaction, labelled ADP is formed. RNA degradation is accompanied by incorporation of 32p into ADP. These results suggest that degradation of newly formed RNA in cells is the result of polynucleotide-phosphorylase activity (poly- nucleotide-nucleosidediphosphate-nucleotidyltransferase) (2.7.7.8). Data on the presence of polynucleotide-phosphorylase in rat liver cell nuclei were obtained earlier by Hilmoe and Heppel (1957). Recently ribonuclease activity (2.7.7.16) has been observed in nuclei isolated in non-aqueous media (Reid et al., 1964). The functional significance of RNA degradation in the nucleus is not yet clear. The most probable explanation is that the degraded RNAs do not differ fundamentally from those RNAs which are not degraded, but are transformed into various stable RNAs (Harris, 1964; Lerman et al., 1965). It has been suggested that the bottleneck in the formation of stable cellular polynucleotides is not the synthesis of RNA, but their subsequent incorpora- tion into ribonucleoproteins and transport to their functional sites. Therefore these latter processes regulate the transfer of RNA into the cytoplasm and any excess of RNA synthesized in the nucleus is degraded. It cannot be ^eluded that this "excess" nuclear RNA plays some specific role. H At present the problem of degradation of nuclear RNA and the character- ization of the enzymes involved is not yet solved, but it is undoubtedly of great interest. The possible role of nuclear polynucleotide-phosphorylase in II 76 G. P. GEORGIEV the process of degradation might throw some light on the role of this enzyme in other systems. VI. Translation (Protein Biosynthesis) The information sent by the genome is encoded in the nucleotide sequence of mRNA and for its expression it must be converted into an amino acid sequence by the process of protein synthesis in the ribosomes. To denote this process the term "translation" was therefore suggested. The process of protein synthesis takes place in the ribonucleoprotein particles, the ribosomes, and is described in Chapter 7. Here we will consider the experiments which show that protein synthesis occurs not only in the cytoplasm but also in the nucleus and will discuss some details of this nuclear protein synthesis. The first indications of protein synthesis in nuclei were obtained by Caspersson and by Kedrowsky (see Caspersson, 1950; Kedrowsky, 1959). Later, experiments with labelled amino acids showed that the rate of incor- poration in vivo into nuclear proteins is similar to that into cytoplasmic proteins (Daly, AUfrey and Mirsky , 1952; Smellie etal., 1953; Zbarsky and Pere- voshchikova, 1956). In non-proliferating cells little incorporation of label into histones was observed while the rate of incorporation into histones of pro- liferating cells was much higher. Independent synthesis of proteins in nuclei was finally demonstrated by the study of protein synthesis in isolated nuclei. Incorporation of labelled amino acids into protein was found after incubation of nuclei isolated in sucrose from thymus, lymphosarcoma and some other tissues. In control experiments this incorporation was demonstrated to represent net protein synthesis. In particular, the distribution of activity between various nuclear fractions was the same in experiments in vivo and with isolated nuclei. Peptides containing labelled amino acids were isolated from hydroly sates of nuclear proteins (Allfrey et al, 1957). Further it was demonstrated that the protein synthesis observed does occur in nuclei and is not due to contamination of nuclear preparations with whole cells or cytoplasmic constituents. In contrast with protein synthesis in cell suspensions, amino acid incorporation into isolated nuclei is sensitive to_ DNase and requires Na+ ions. Na+ is necessary for amino acid transport into! nuclei. Degradation of DNA by DNase addition interferes with ATP synthesis in nuclei necessary for protein synthesis. This may explain the somewhat unusual requirements for amino acid incorporation in isolated nuclei. On the other hand these requirements demonstrate that the synthesis does occur in nuclei themselves (Allfrey, et al., 1957). That the label was really incorporated into nuclei free of contamination was also shown by autoradiography (Logan, Ficq and Errera, 1959). Labelled amino acids are incorporated into protein in the nuclear sap as well as in the nucleolo-chromosomal complex. i 2. THE NUCLEUS 77 A. PROTEIN SYNTHESIS IN NUCLEAR SAP RIBOSOMES As already mentioned we mean by the term "nuclear sap" nuclear proteins and nucleoproteins not bound firmly to thread-like structures of nucleolo- chromosomal-complex and therefore extracted from nuclei by dilute salt solutions. Protein synthesis is known to involve two principal stages. The first is ;tivation of an amino acid and its attachment to sRNA. ATP + Amino acid ^ E-AMP --^^ Amino acid + PPi (1) E — AMP '-«-' Amino acid + sRNA ^ sRNA - Amino acid + E + AMP (2) Enzymes (E) catalysing this reaction are called aminoacyl sRNA-synthe- tases (6.1.1.1-11) (Hoagland, 1960). Hopkins (1959) has found that calf thymus nuclei and pig liver nuclei could catalyse amino acid-dependent ATP-pyrophosphate exchange, reac- tion (1). It is to be noted that amino acid activation was also observed with nuclei isolated from pig kidney by a non-aqueous method ; this seems to prove the nuclear origin of the enzyme. In the same report it was demonstrated that transfer of amino acid to sRNA (2) also takes place in nuclei since the incorporation of labelled amino acids into RNA was shown to occur. Some- what later amino acid activation was demonstrated for plant cell nuclei (Webster, 1960) and rat liver nuclei (Gvozdev, 1960). All the aminoacyl-sRNA synthetase activity of nuclei may be extracted with dilute salt solutions. After uUracentrifuging of these extracts the activity stays in supernatant and may be precipitated on lowering the pH to pH5. In other words, the enzyme is similar in its behaviour to the corresponding enzyme from the cytoplasm (Hopkins, 1959). We have already noted that nuclear sap contains low molecular weight sRNA (see Section III). It is this RNA to which amino acids are transferred by the aminoacyl-s;RNA-synthetase of cell nuclei. No complex is formed between amino acids and the nucleolo-chromosomal RNA. Cytoplasmic and nuclear aminoacyl-sRNA-synthetases, as well as sRNAs, do not differ from each other. In particular cell-free systems consisting of nuclear (or cytoplasmic) enzymes and sRNA, nuclear enzymes and cyto- plasmic sRNA and cytoplasmic enzymes and nuclear sRNA all have the same activity, so that no specificity has been found (Khessin, Gvozdev and Astaurova, 1961). To establish the exact localization of the enzyme in nuclei autoradiography experiments were performed on the distribution of the enzyme amongst nuclear structures of oocytes. The incorporated amino acid removed by hydroxylamine or ribonuclease treatment was used as a measure of the 78 G. P. GEORGIEV amount of sRNA-amino acid complex. Homogeneous distribution of label in the nucleus was observed without any relation to structural elements (Platova, 1962), This fact is consistent with data on the localization of the enzyme in the soluble nuclear phase or nuclear sap. The next stage of protein synthesis is the transfer of the aminoacyl-sRNA to the ribosome and the subsequent attachment of amino acid to the — COO-terminal end of the growing polypeptide chain. The incorporation into proteins seems to require two different enzymes, GTP, ATP and Mg2+ ions. It was mentioned above that ribonucleoprotein particles found in nuclear sap (Frenster et al., 1960; Samarina and Georgiev, 1960) were later identified as ribosomes (Frenster et al., 1961; Pogo et al., 1962; Wang, 1961). The particles isolated from nuclear sap by ultracentrifuging originally contain 15-20% of RNA but by treatment with deoxycholate particles identical to cytoplasmic ribosomes were obtained (Wang, 1961 ; Allfrey, 1963). Ribosomes of the nuclear sap actively incorporate label in vivo (Samarina and Georgiev, 1 960) and also during incubation in vitro of isolated nuclei (Frenster et al., 1961). The nuclear origin of ribosomes found in nuclear sap is well established. Although extracted from nuclei, the ribosomes retain the ability to incorporate amino acids and the requirements of the system are similar to those for cytoplasmic ribosomes. In addition to sRNA, amino acids and aminoacyl-sRNA-synthetase (or i.e. in addition to aminoacyl sRNA) this system requires a cytoplasmic or nuclear supernatant enzyme, ATP, GTP and a ATP-generating system. (Frenster et al., 1961). Thus nuclear sap contains a complete system for protein synthesis, including ribosomes, sRNA and aminoacyl-sRNA synthetase, which is active in vivo and in vitro. Probably in vivo this system participates in the synthesis of soluble proteins of the nuclear sap. Thus, for example, if slices from rat hver labelled in vivo with radioactive amino acid are incubated in a medium con taining non-radioactive amino acid, one observes a fall in radioactivity o: nuclear sap ribosomes and an equivalent increase in the radioactivity of th soluble proteins of the nuclear sap (Zbarsky and Samarina, 1962). On the other hand some non-nuclear proteins and particularly some cell specific functional proteins are probably synthesized in the nuclear sap. Thus in erythropoetic cells, haemoglobin synthesis takes place in nuclei, the haemoglobin being transferred later to the cytoplasm (Hamel and Bessman, 1964). In a number of other cells with a high nuclear/cytoplasmic ratio nuclei also appear to participate in the synthesis of cytoplasmic proteins (Brodsky, 1961). The amount of protein synthesis in nuclear sap may vary quite significantly (Hopkins, 1959; Brodsky, 1961). This synthesis is apparently not a basic and specific function of the nucleus and is largely analogous to protein synthesis in cytoplasmic ribosomes. ;r 2. THE NUCLEUS 79 B. PROTEIN SYNTHESIS IN THE NUCLEOLO-CHROMOSOMAL COMPLEX The existence of protein-synthesizing enzymes in chromosomes and the nucleolus is not yet established. Both in vivo (Daly et al, 1952; Smellie et al., 1953; Zbarsky and Perevoshchikova, 1956; Samadna, 1961) and in experi- ments with isolated nuclei incubated with labelled amino acids (Allfrey, et al., 1957) the label is incorporated into proteins of the nucleolo-chromo- somal-complex. The highest rate of incorporation is observed in the non- histone protein of DNP and in "acidic protein" ("residual protein"). In non-dividing differentiated cells incorporation into histones is lower than in dividing cells, though some labelling of histones does occur. After chemical fractionation of cell nuclei, the ability of the nucleolo- chromosomal-complex to synthesize proteins is lost. However some syn- thetic ability is still left after mechanical fractionation of nuclei (Birnstiel et al., 1961; Birnstiel and Hyde, 1963). The first stage of protein synthesis, i.e. the formation of aminoacyl-sRNA needed for protein synthesis in the nucleolo-chromosomal-complex probably takes place in the nuclear sap. Since the soluble phase is distributed through- out the nucleus, aminoacyl-sRNA molecules are available for all nuclear structures. The second stage of protein synthesis, the formation of peptide bonds, is probably localized in fibrillar structures of the nucleolochromo- somal-complex. Since this activity of the nucleolo-chromosomal-complex has not yet been studied in a cell-free system, it is not possible to discuss the enzymic aspects of the problem, and we shall only briefly comment on some important aspects of the question. 1. Role of Ribosomes Since the nucleolo-chromosomal complex has a complicated struc- tural and chemical organization and contains proteins associated with DNA and at least some of the proteins are synthesized synchronously with it (Bloch and Godman, 1955; Chalkley and Maurer, 1965) the question arises: what is the mechanism of protein synthesis in this complex and particularly in the DNP fraction? Is it related to the mechanism involving ribosomes or does it follow some non-ribosomal scheme ? It is well known that the antibiotic, puromycin, inhibits protein synthesis in ribosomes and it may be used to rule out artificial incorporation. It was found that puromycin is equally effective towards all proteins of cell nuclei including histones (Schweiger, Master and Alivisatos, 1964; Gvozdev, 1963). Although lysine-rich histone synthesis is somewhat more resistant to puro- mycin its synthesis is also inhibited. These observations strongly suggest a ribosomal mechanism of synthesis for all nuclear proteins. In other experi- ments the transfer of labelled amino acid from aminoacyl-sRNA to nuclear protein in the presence of ATP has been studied. With isolated liver nuclei, the incorporation occurred in all proteins except the histones. However, in II 80 G. P. GEORGIEV Erlich ascites tumour cells damaged by hypotonic shock there was transfer of amino acid from aminocyl-sRNA to histones (V. A. Gvozdev, personal communication). One can conclude therefore that proteins of nucleolo-chromosomal-com- plex are probably synthesized on ribosomes and this process involves enzymes similar to cytoplasmic enzymes. 2. Role ofmRNA Protein synthesis in ribosomes is known to require the participation of mRNA. A possible approach to this question is to study protein synthesis after inhibition of RNA synthesis. This is difficult to carry out in animal cells because of the stability of mRNA. However, two hours after actino- mycin inhibition of RNA synthesis, a 50% decrease in incorporation of amino acids into nuclear DNP (including histones and non-histone protein) occurred (Honig and Rabinovitz, 1964; Zbarsky and Gauze, 1964). It is possible that the life-time of mRNA for proteins of the DNP-complex is comparatively short. The fact that cell nuclei contain large amounts of informational (DNA-like) RNA with high metabolic heterogeneity is of interest (Samarina et al., 1965b). One may suppose therefore that nuclear mRNA contains mRNA for chromo- somal proteins in addition to newly formed mRNA due to be transferred into the cytoplasm. Since the synthesis of chromosomal proteins and particularly histones is related to the regulation of gene expression (see Section IV) further investigation of this problem is of considerable importance. 5. Nucleolar Protein Synthesis Nucleolar proteins are actively labelled by radioactive amino acids both in vivo and in vitro during the incubation of isolated nuclei with label. This demonstrates the existence of intensive protein synthetic activity in the nucleo- lus (Allfrey, et al., 1957; Samarina, 1961 ; Birnstiel et al., 1961 ; Birnstiel and Hyde, 1963). Which proteins are formed in the nucleolus? It was already mentioned that it has been found that ribosomal RNA is synthesized in the nucleolus. Also, ribosomes attached to nucleonemal fibrils are one of the major nucleolar components. It is natural to suggest that not only synthesis of ribosomal RNA, but also the synthesis of ribosomal protein and the formation of ribosomes occur in the nucleolus. Some indirect experiments support this view. Thus ribosomes isolated from the nucleolus are heterogene- ous and incorporate amino acids less actively than cytoplasmic ribosomes. This may be a consequence of their "incompleteness" (Birnstiel et al., 1963a). A significant part of the nucleolar ribosomal RNA was found in particles smaller than ribosomes which could not be collected by centrifuging at 105,000g (Zbarsky and Samarina, 1962). It was also demonstrated by Tamaoki and Mueller (1965) that in chase experiments after addition of 2. THE NUCLEUS 81 actinomycin some label in RNA and in proteins of the nucleolochromosomal complex was transferred to RNA and proteins in free ribosomes. These authors also demonstrated the presence in nuclei of ribonucleoproteins con- taining heavy R-RNA. This indicates that R-RNA combines with protein before it is cleaved into the shorter chains of ribosomal RNA. It is evident of course that more direct experiments are needed to elucidate the function of the nucleolus in the synthesis of ribosomal proteins and the formation of ribosomes. In conclusion it should be emphasized that the study of protein synthesis in the nucleolo-chromosomal-complex is extremely important since it is here that the synthesis of proteins that probably regulate gene activity and the synthesis of ribosomal proteins occur, i.e. the basic and specific function of the nucleus is related to the synthesis of proteins in the nucleolochromosomal- complex. Unfortunately no conclusive information on the mechanism and localiza- tion of these processes is at present available. VII. Enzymes of Energy Metabolism It has been shown in the preceding sections that many synthetic reactions in the cell nucleus, such as DNA, RNA and protein formation require a constant supply of energy in the form of high energy bond nucleoside tri- phosphates. Energy is consumed by many other processes as well, for example the complex nuclear reorganizations that occur during the cell cycle. There- fore the question arises as to where these high energy bond compounds (primarily ATP) are formed and by which mechanism. Do they travel to the nucleus from the cytoplasm, for example from mitochondria, or does their synthesis take place in the nucleus itself? The first question cannot be answered definitely at the present, although one should take into account that the membrane of the isolated nucleus is impermeable to ATP. The second question may be answered in the affirmative, since isolated nuclei when incubated at 37° without any additions of nucleotides to the medium can phosphorj'late nucleoside monophosphates to nucleoside triphosphates (Osawa et al., 1957). A number of investigations have been undertaken in order to identify the system supplying nuclei with energy. A GLYCOLYSIS It has been shown in a number of papers that cell nuclei from various tissues contain a number of glycolytic enzymes (Dounce and Beyer, 1948; Bounce, 1953; Stern and Mirsky, 1952, 1953; Stern, Allfrey, Mirsky and Saetren, 1952; Roodyn, 1956a, b). It was shown that nuclei could catalyse the entire sequence of glycolytic reactions, that is the formation of lactic acid 82 G. P. GEORGIEV from glucose, (Le Page and Schneider, 1948). Lang and Siebert (1951) how- ever, observed lactic acid formation only with fructose- 1,6-diphosphate as a substrate but not with metabolites preceding it in the glycolysis sequence. In these experiments nuclei were isolated in an aqueous medium and therefore loss of a number of enzymes could not be excluded. Decisive experiments with nuclei isolated in non-aqueous media were performed by Siebert and his collaborators (Siebert, 1960, 1961, a, b; Siebert, Bassler, Hannover, AdlofF and Beyer, 1961) and later by McEwen, AUfrey and Mirsky (1963b). It was found that nuclei possess a complete system of glycolysis and that they can catalyse the formation of lactic acid from glucose. The presence of a number of glycolytic enzymes in nuclei was demonstrated: these were hexokinase (2.7.1.1), phosphofructokinase (2.7.1.11), aldolase (4.1.2.7), triosephosphate isomerase (5.3.1.1), triosephosphate dehydrogenase (1.2.1.13), glycerophos- phate dehydrogenase (1.1.1.8), phosphoglycerate kinase (2.7.2.3), lactate dehydrogenase (1.1.1.27), and pyruvate kinase (2.7.1.40). A number of important features in the distribution of these enzymes have been observed. The specific activities of glycolytic enzymes in nuclei and in cytoplasm are approximately equal. Consequently the relative activities of different enzymes in nuclei and in cytoplasm are very close. The high specific activity of enzymes in nuclei excludes the possibility of contamination with cj^oplasmic enzymes, especially if one takes into account that the nuclei were isolated in non-aqueous media. These enzymes are found in the soluble fraction both in the nuclei and in the C3^oplasm. In the cytoplasm glycolytic enzymes are found in the final supernatant after ultracentrifuging (see chapter 8). A portion of the nuclear glycolytic enzymes can be extracted by solutions of very low ionic strength, and another portion can be extracted with saline. After ultracentrifuging of nuclear extracts these enzymes are found in the supernatant (Roodyn, 1956b, Siebert, 1960). Thus all nuclear glycolytic enzymes are locaHzed in the nuclear sap. The specific activities of nuclear and cytoplasmic extracts based on their nitrogen content are equal, as mentioned above. The presence of glycolytic enzymes in nuclei is characteristic of all organisms studied, both animal and plant (Siebert, 1961a; McEwen et al, 1963b; Stern and Mirsky, 1952). Nuclear glycolytic enzymes are inhibited if isolated nuclei are treated with deoxy- ribonuclease. There is no inhibition if whole cells or cytoplasmic extracts are treated with DNase. This is an additional proof of the true intranuclear localization of these glycolytic enzymes. The phenonenon is jprobably due to the liberation of histones by DNase treatment, since subsequent addition of DNA or other poly-anions releases the inhibition (McEwen et al., 1963b). The presence of virtually all the glycolytic enzymes does not prove, how- ever, that glycolysis really proceeds in nuclei. In order to prove this Siebert, et al. (1961) and McEwen et al. (1963b) studied the content of low molecular metabolites of glycolysis in nuclei isolated in non-aqueous media. It was 2. THE NUCLEUS 83 found that all the metabolites studied could be detected in nuclei in approxi- mately the same concentrations as in cytoplasm. This was demonstrated for such metabolites as glucose-6-phosphate, fructose- 1 ,6-diphosphate, dihydroxy- acetone phosphate, 3-phosphoglyceric acid, glycerol- 1 -phosphate, phospho- enolpyruvic acid and lactic acid, as well as for AMP, ADP and ATP. Thus one may conclude that glycolysis really proceeds in cell nuclei in vivo. Further proof for the existence of glycolysis in nuclei, as well as of its importance in nuclear energy metabolism, comes from experiments with inhibi- tors (McEwen, AUfrey and Mirsky, 1963c). lodoacetic acid, an inhibitor of triosephosphate dehydrogenase, and NaF, an inhibitor of phosphopyruvate hydratase (or enolase: 4.2.1.11), decrease nuclear respiration, formation of i'*C02 from [6-^'*C]-glucose, as well as the phosphorylation of nucleotides. In anaerobic conditions when oxidative reactions in nuclei are suppressed, addition of glucose to isolated nuclei stimulates glycolysis and leads to the partial restoration of phosphorylation. An indirect indication of the fun- tional importance of glycolysis in vivo is the increase in the content of glycolytic enzymes during the activation of nuclear macromolecular syntheses, preceding cell division (Siebert, 1961a). Another enzyme system for carbohydrate breakdown, the hexose mono- phosphate shunt, is also present in cell nuclei. Such enzymes as glucose-6- phosphate dehydrogenase (1.1.1.49) and 6-phosphogluconate dehydrogenase (1.1.1.43) are found in nuclei isolated in non-aqueous media, (Siebert, 1961; McEwen et al., 1963b). These enzymes play a part in the formation of a pool of pentose compounds necessary for nucleic acid synthesis and for this reason this metabolic pathway may be important. B. CITRIC ACID CYCLE The presence of a number of enzymes of the citric acid cycle has been described in nuclei isolated both in aqueous and non-aqueous media. These enzymes are isocitrate dehydrogenase (1.1.1.41), malate dehydrogenase (1.1.1.37) and succinate dehydrogenase (1.3.99.1) (Siebert, 1961b; McEwen et al, 1963b). The specific activities of these enzymes and ratios of their activities are identical in nuclei and in cytoplasm. Similarly, corresponding cytoplasmic enzymes found in the supernatant, i.e. such enzymes as iso- citrate dehydrogenase or malate dehydrogenase, are found in the nuclear sap. Since they have been demonstrated in nuclei isolated in organic solvents their origin is certainly nuclear. Less certain is the situation with succinate dehydrogenase, a typical mitochondrial enzyme. In organic solvents this dehydrogenase is inactivated, while in work with nuclei isolated in aqueous media the danger of enzyme adsorption from the cytoplasm always exists. McEwen et al. (1963) presented some evidence for the existence of a truly nuclear succinate dehydrogenase. For example, unlike the enzyme in whole cells or mitochondria, the nuclear enzyme is inhibited by deoxyribonuclease. •E 84 G. P. GEORGIEV The results presented, however, are not decisive and new experiments are necessary. The concept of the existence of a complete and functional tri- carboxylic acid cycle in nuclei is supported by the following facts: the presence of corresponding metabolites such as a-oxoglutarate, succinate and malate in nuclei isolated in non-aqueous media (McEwen et al., 1963b); the sensitivity of nuclear phosphorylation to fluoroacetic acid, an inhibitor of aconitate hydratase (4.2.1.3), and to iodoacetic acid, an inhibitor of succinate dehydrogenase ; the ability of isolated nuclei to catalyse the conversion of 2-i4C-acetate and 3-i4C-pyruvate to i'*C02. The latter is possible only if all the stages of the citric acid cycle are present. This process is suppressed by the inhibitors mentioned above. Finally one should mention the high content of NAD in nuclei, which is a co-factor of many reactions in the above mentioned metabolic sequences (Stern and Mirsky, 1952). In addition to NAD, a peptide-bound flavin, a coenzyme of succinate dehydrogenase, was found in nuclei isolated in non-aqueous media (McEwen et al., 1963b). One therefore can draw the general conclusion that cell nuclei contain enzymes of glycolysis, the citric acid cycle and the hexose monophosphate shunt ; these enzymes enable them to accomplish the first stages of carbohydrate oxidation, up to the formation of succinate, NADH2 and NADPH2 and the formation of high energy bond compounds, such as ATP. Most of the enzymes mentioned, except succinate dehydrogenase, are found in the nuclear sap, that is in the soluble phase of the cell nucleus which can be extracted from nuclei with dilute salt solutions or even with solutions of low ionic strength. In the cytoplasm these enzymes are found in the final supernatant fraction, that is in soluble phase also (see Chapter 8). Some of them are also found in the matrix of the mitochondrion, but not attached to lipoprotein membranes — the main structural and functional component of mitochondria (see Chapter 3). Thus the nuclear sap is the functional analogue of other soluble cellular components, primarily of the hyaloplasm. It has been suggested that nuclear sap is a continuation into the nuclei of the soluble phase of the cell (Siebert, 1961a). This question however is rather complex and will be discussed in the following section. C. OXIDATIVE PHOSPHORYLATION In this section the question will be considered as to whether enzymes of terminal oxidation exist in nuclei. These enzymes effect phosphorylation during transfer of electrons from NADH2 and succinate to oxygen via a number of stages. The bulk of older results, obtained with purified nuclei, indicated that enzymes of this group are absent from the nuclei. The presence of small amounts of succinate oxidase, NAD-cytochrome c oxidoreductase (1.6.99.3.), 2. THE NUCLEUS 85 cytochrome c and cytochrome oxidase (1.9.3.1) was explained by mito- chondrial contamination. Additional purification of nuclei from mito- chondria and whole cells sharply decreased the activities of these enzymes (Stern and Timonen, 1954; Schneider and Hogeboom, 1950; Hogeboom et al, 1952; Schneider, 1946; Siebert and Smellie, 1957; Roodyn, 1956a, 1959). Residual activity can be explained by the adsorption of protein onto the nuclei, since Beinert (1951) has shown that nuclei in sucrose medium strongly adsorb added cytochrome c. Nuclei isolated in non-aqueous media are unsuitable for studies of oxidative enzymes since some of them are inactivated under such conditions. Nevertheless the absence of a number of them from nuclei isolated in non-aqueous media has been demonstrated (for example of NAD-cytochrome c oxidoreductase, Stern and Timonen, 1954). There is no doubt, therefore, that the chain of electron transfer enzymes described for mitochondria is absent from nuclei. An exception are the nuclei of erythrocytes, which contain a number of oxidative enzymes, for example the cytochrome oxidase system (Rubinstein and Denstedt, 1953). It was suggested in the above papers that anaerobic metabolism is a charac- teristic property of nuclei in general. However, this conception was revised after the work of Allfrey et al. (1957), who showed that the phosphorylation of nucleotides in nuclei depends on respiration. Inhibitors of electron transport and uncoupling agents (cyanide, antimycin, dinitrophenol and anaerobic conditions of incubation) inhibited phosphorylation of nucleotides in isolated calf thymus nuclei. Somewhat later it was shown that isolated calf thymus nuclei consume oxygen; however, for purified nuclear preparations a qo2 of 0-5 was obtained (McEwen et al., 1963a; Gaizhoki, 1963). Further experiments are necessary however, since much of the observed qo2 was due to cytoplasmic contamination. In Mirsky's laboratory some experiments have shown that oxidative phosphorylation in nuclear preparations is really nuclear and is not due to contamination. Although the nuclear and the mitochondrial processes are similar in their sensitivity to certain inhibitors, considerable differences were observed in other properties. For example, carbon monoxide, Ca2+ ions, methylene blue, Janus green and dicumarol inhibit mitochondrial but do not affect nuclear oxidative phosphorylation. Also, nuclear oxidative phos- phorylation, like many other intranuclear processes, is sensitive to DNase. Destruction of DNA by DNase strongly inhibits nuclear phosphorylation, and subsequent addition of DNA or poly-anions restores it. The inhibition is probably due to histones liberating during DNA digestion. DNase does not affect phosphorylation either in whole cells or in isolated mitochondria. Unlike the mitochondrial process, the addition of substrates, such as suc- cinate, does not stimulate nuclear oxidation. This is due to the impermeability of the nuclear membrane to dicarboxylic acids (Allfrey and Mirsky, 1957; 86 G. P. GEORGIEV McEwen et al, 1963, a, b, c; Rees and Rowland, 1961; Rees, Ross and Rowland, 1962). Thus oxidative phosphorylation observed in nuclear preparations is very probably a truly nuclear process, different from mito- chondrial oxidative phosphorylation. However there is no information at the present time about the enzymes taking part in this process. The nuclear structures participating in this process are also obscure. It is possible that the nuclear membrane is some- how involved. The question of the universality of the oxidative phosphoryla- tion system described for calf thymus nuclei is also uncertain. Rees and Rowland (1961) and Rees et al. (1962) described a similar enzymic system in liver nuclei and identified a number of individual enzymes, e.g. NAD- cytochrome c oxidoreductase and cytochrome oxidase. In these experiments however there were no controls for purity of the nuclei. On the other hand Creasy and Stocken (1959) have confirmed the presence of oxidative phos- phorylation in thymus nuclei and also in nuclei of spleen, lymph glands, bone marrow and intestine mucosa, but failed to detect it in nuclei of liver, kidney and pancreas. This however may be due to greater damage to these nuclei during their isolation. It is of interest that nuclear oxidative phos- phorylation is extremely sensitive to X-ray irradiation. Thus, the cell nucleus possesses all the machinery necessary for the break- down of carbohydrates via glycolysis and the citric acid cycle to CO2, NADH and succinate. An enzymic system of electron transport from NADH and succinate to molecular oxygen probably also exists in nuclei. However there is no information on its components. Recently, Conover and Siebert (1965) have observed cytochrome b^ and cytochrome c in nuclei isolated in aqueous and non-aqueous media, but the authors suggested that the presence of these cytochromes may be an artifact, due to cytoplasmic contamination. VIII. Other Enzymes (Enzymes of Intermediary Metabolism) Apart from enzymes necessary for the reproduction and expression of genetic information and enzymes of energy supply, a large group of enzymes of intermediary metabolism are also found in nuclei. Among them are enzymes participating in the formation of building blocks such as activated amino acids and nucleotides for polymer synthesis; in the biosynthesis of coenzymes; in the synthesis of cell polymers by non-template mechanisms, and some other enzymes. Studies of the distribution of these enzymes between subcellular structures have only rarely clearly established their nuclear localization. Many of them are found in nuclei isolated from one kind of tissue but not from another so that their nuclear localization is not a general 2. THE NUCLEUS 87 rule. Probably these enzymes are not concerned with specific nuclear func- tions. Therefore results with this group of enzymes will be discussed briefly and only enzymes of nucleotide metabolism will be described in more detail. r A. NUCLEOTIDE METABOLISM An important group of enzymes of intermediary metabolism found in nuclei is that involved in nucleotide metabolism. In the preceding section devoted to oxidative phosphorylation we have already mentioned the pre- sence in nuclei of enzymes catalysing the phosphorylation of nucleoside monophosphates to di- and triphosphates. This group of enzymes has not been studied in detail, but it is clear that the formation of the triphosphates of different nucleosides requires not only a system of energy generation but also the presence of suitable kinases. The presence of adenylate kinase (2.7.4,3.) in nuclei isolated in non-aqueous media was demonstrated by Miller and Goldfeder (1961). Reid et al. (1964) have reported active phosphorylation of UMP to UDP and UTP by rat-liver nuclei isolated in non-aqueous media. Cell nuclei from various sources contain high concentrations of another enzyme of nucleotide metabolism — nucleoside phosphorylase (2.4.2.1.) cata- lysing the reaction: X-ribose + Pi % X + ribosephosphate, where X is a purine or pyrimidine base. The concentration of nucleoside phosphorylase in nuclei is much higher than in the cytoplasm. Results obtained with nuclei isolated in aqueous and non-aqueous media were similar (Stern et al., 1952; Stern and Mirsky, 1953). Experiments on the localization of NAD synthesizing enzymes are of considerable interest. It has been shown that the synthesis of NAD from nicotinamide proceeds via the following stages (Preiss and Handler, 1957; Kornberg, 1950): Nicotinamide + 5'-Phosphoribosylpyrophosphate t Nicotinamide mononucleotide + Pyrophosphate. Nicotinamide mononucleotide + ATP % NAD -f Pyrophosphate. The first of these enzymes (nicotinamide phosphoribosyl-transferase (2.4.2.12) is found in mitochondria but not in nuclei (Morton, 1961). The second enzyme, NAD pyrophosphorylase (2.7.7.1) or NAD synthetase is found exclusively in cell nuclei. This was first shown by Hogeboom and Schneider (1952) with rat liver nuclei isolated in a sucrose medium. Later NAD pyrophosphorylase was found in nuclei from various sources, isolated by different methods, including the non-aqueous technique (Branster and Morton, 1956, 1957; Morton, 1958, 1961). This enzyme was not found in the cytoplasm. The above mentioned experiments proved unequivocally the exclusive nuclear localization of NAD pyrophosphorylase. Morton purified 88 G. P. GEORGIEV this enzyme from the hog liver nuclei and obtained a crystalline preparation with very high specific activity (Morton, 1961). It should be noted that nuclei contain high concentrations of NAD (Stern and Mirsky, 1952). The precise intranuclear localization of this enzyme is at present still unknown. Treatment of nuclei in sucrose media with sonic vibrations or extraction with dilute salt solutions (Morton, 1961) solubilizes NAD pyrophosphorylase ; this probably means that it is not bound to nucleolo-chromosomal structures and is localized in the nuclear sap, and that the nuclear membrane is imper- meable to this enzyme. On the other hand Baltus (1956) detected NAD pyrophosphorylase in nucleoli from the oocytes of starfish. The problem deserves further investigation but in any case it is certain that NAD pyro- phosphorylase, the terminal enzyme of NAD synthesis, is concentrated exclusively in the cell nucleus. It is known that NAD is a coenzyme of a large number of dehydrogenases, catalysing the early stages of oxidation of many substrates. Therefore the cell nucleus may regulate the level of many cellular oxidative processes via NAD synthesis. Among other enzymes of coenzyme synthesis one should note the finding in nuclei of glucose- 1 -phosphate uridylyl transferase (2.7.7.9.). This enzyme catalyses the synthesis of UDP-glucose : Glucose- 1 -phosphate -f UTP % UDPG + Pyrophosphate (Smith, Munch-Petersen and Mills, 1953; Mills, Ondarza and Smith, 1954). It is known that UDPG is a substrate necessary for many synthetic reactions, including glycogen synthesis. The nucleus also contains up to 80% of the total cellular adenylate cyclase, an enzyme catalysing the conversion of ATP to adenosine-2'-3' cyclic phos- phate, which is a regulator of phosphorylase activity (Sutherland, Rail and Menon, 1962). A large number of investigations in enzyme cytochemistry are devoted to phosphatases. Older experiments demonstrated a high level of phospha- tases in the nucleus, but this was subsequently found to be due either to enzyme adsorption during isolation of nuclei in aqueous media or to re- distribution of the reaction product during the histochemical reaction. Experiments with nuclei isolated in non-aqueous media (Stern et al., 1952) have shown that acid phosphatase (3.1.3.2), alkahne phosphatase (3.1.3.1.) and ATPase are absent from the nuclei. Somewhat later, however, Fisher, Siebert and Adloff (1959) and Siebert (1961a) demonstrated the presence of ATPases (3.6.1.-) in nuclei. In these experiments nuclei were also isolated in non-aqueous media. These workers found two ATPases A and B, differing somewhat in certain properties such as solubility, pH optimum and affinity to the substrate. ATPases, especially ATPase B, are tightly bound to the DNP 2. THE NUCLEUS 89 chromosomal complex; during salt reprecipitation (dissolving in 1 m NaCl with subsequent dilution) the enzyme remains in complex with DNA. Apart from its chromosomal localization, nuclear ATPase differs from mito- chondrial by its low substrate-specificity. The reaction is equally rapid with the triphosphates of adenosine, guanosine, cytidine and uridine as well as with deoxynucleoside triphosphates. Moreover the affinity of enzyme for the deoxynucleoside triphosphates is higher than that for the ribonucleoside triphosphates. Therefore nuclear ATPases are non-specific nucleoside tri- phosphatases. However the existence of several enzymes with different specificity cannoc be excluded. The role of these enzymes in the nucleus is obscure. Their wide action spectrum and chromosomal localization suggest that they may regulate nucleic acid synthesis in chromosomes (Siebert, 1961). It is possible, however, that the phosphatase activity is a side effect of some other unknown reaction. It is clear from the previous comments that cell nuclei are rich in enzymes catalysing the release of pyrophosphate from a range of substrates (e.g. various nucleotidyl transferases, nucleoside triphosphatases and amino-acyl sRNA synthetase). It has been found that the levels of inorganic pyro- phosphatase (3.6.1.1) and other polyphosphatases (3.6.1.-) in the nucleus are higher than in the cytoplasm (Grossman and Lang, 1962). The removal of pyrophosphate by the pyrophosphatases makes the reactions mentioned above practically irreversible. One should note that the majority of the enzymes listed were studied with the nuclei isolated from a limited number of sources so that the universal nuclear occurrence of these enzymes is not well proven. Studies with nuclei isolated from various sources have only been made with NAD-ATP nucleo- tidyl transferase (NAD pyrophosphorylase) and in all cases this enzyme was found to be strictly nuclear. In summary, one should stress that nuclei contain many enzymes of nucleotide metabolism, and among them enzymes catalysing the synthesis of a number of key compounds of cellular metabolism. These findings en- hance our concept of the regulatory function of the nucleus in cellular metabolism. B. SPECIAL ENZYMES AND WIDELY DISTRIBUTED ENZYMES The major experiments devoted to enzymes described in previous sections were usually done with nuclei isolated from a variety of sources. Data on the distribution of these enzymes are in general in accordance with our concept of the metabolic function of the cell nucleus. The nuclear localization of these enzymes has been proved by different methods corroborating each other. However there are no such decisive results for many other enzymes. The experimental material is often scant, controls with non-aqueous nuclei I 90 G. P. GEORGIEV have been made in only a few cases and the data on the distribution of these enzymes are difficult to correlate with the functions of nuclei common to different types of cells. For this reason we shall not discuss these experiments in detail. Only two general aspects will be considered. The first is whether the nuclear enzymic machinery depends on the tissue from which the nuclei were isolated. In other words, are nuclei differentiated? The second question is: which enzymes characteristic of cytoplasmic structures are present in nuclei and which are not. For other works on the enzymology of the nucleus one may consult the summary table in the review by Roodyn (1959). The first of the questions raised has been studied in detail by Stern et al. (1952) and by Stern and Mirsky (1953), who worked exclusively with nuclei isolated in non-queous media so that adsorption or loss of enzymes was excluded. Nuclei were isolated from different sources and both "special enzymes", that is, enzymes present only in certain types of organs and absent from others, and "widely distributed enzymes" present in most of the organs studied were investigated. The distribution of "special" enzymes indicated (1) that the cell nucleus, like the cjrtoplasm, is differentiated; (2) the differentiation of the nucleus and the cytoplasm are not necessarily similar and hence (3) the enzymic pattern of the cell nucleus may not reflect the enzymic pattern of cytoplasm. Arginase (3.5.3.1), for example, is present in the nuclei of calf, horse and chicken liver as well as in calf kidney, but it is absent from chicken kidney nuclei, while it is found in the chicken kidney cytoplasm. Catalase (1.11.1.6) is present in the nuclei of horse liver but not of horse kidney. Haemoglobin is found in the nuclei of the avian erythrocyte and mammalian erythroblasts (Carvalho and Wilkins, 1954), while another haemprotein, myoglobin is absent from heart nuclei. The nuclei of pancreas do not contain lipase (3.1.1.3), amylase (3.2.1.1) and deoxyribonuclease I. In other words "special enzymes" may be present in nuclei or may not. It is difficult to draw any general conclusion, since even in nuclei of a particular organ from different species a given enzyme may be present or not (see for example arginase). Another group of enzymes, studied in the above mentioned work from Mirsky's laboratory, are those enzymes that are widely distributed in dif- ferent tissues. Again there are no general rules as to the distribution of this group of enzymes. Some enzymes were absent in the nuclei of all the tissues studied; these were alkaline phosphatase, adenylate 3'- and 5'-phosphatases, jS-glucuronidase (3.2.1.31). Other enzymes were absent from the majority of types of nuclei studied but were found in a few. For example esterase was found in calf heart and liver nuclei but it was absent from kidney and thymus nuclei. DNase II (3.1.4.6) was found in liver nuclei, but not in nuclei of a number of other organs. Finally several enzymes, such as adenosine 2. THE NUCLEUS 91 deaminase (3.5.4.4) and nucleoside phosphorylase are found in high con- centrations in the nuclei from many organs (liver, heart, pancreas) but they are absent in nuclei from a few other prgans (e.g. nuclei of intestinal mucosa). Thus cell nuclei contain a number of enzymes of intermediary metabolism, as well as catabolic enzymes. Nuclei from different tissues and from different species have different enzyme patterns. Sometimes, but not always, these differences reflect the differences between tissues themselves. Generally the picture is rather confused, however, and no fruitful conclusions can be made at present. These enzymes are probably unrelated to the specific nuclear functions common to all different tissues. C. OCCURRENCE OF ENZYMES OF DIFFERENT CYTOPLASMIC STRUCTURES IN THE NUCLEUS It is clear from the other chapters in this book that different cytoplasmic structures differ sharply in their enzymic pattern. Let us now consider how frequently enzymes characteristic of different cytoplasmic structures are found in the cell nucleus. The most evident fact is that enzymes typical of the structural elements (or membranes) of mitochondria are absent from nuclei (see Chapter 3). Thus, nuclei are devoid of the enzymes of the mitochondrial chain of electron transfer. The only exception to this is succinate dehydro- genase found in nuclei from a variety of tissues (McEwen et al, 1963). The enzymes of lysosomes are also absent from nuclei ; these are such catabolic enzymes as alkaline nucleases, hyaluronidase, phosphatases and some others. Cell nuclei are also devoid of enzymes of secretory granules: amylase, lipase, trypsinogen, chymotrypsin and others (Stern et al, 1952). Nuclei do not contain many enzymes, typical of the endoplasmic reticulum, for example alkaline phosphatase, which is abundant in Golgi bodies. However a typical membrane enzymes, glucose-6-phosphatase (3.1.3.9) is found in the cell nucleus but in a lower concentration than in the cytoplasm (Moule and Chauveau, 1959; Roodyn, 1959). This may be explained by the fact that the nuclear membrane, at least in its outer layer, is a continuation of the endo- plasmic reticulum and hence contains reticulum enzymes. It is also possible that the synthesis of membranes of the reticulum begins in the nucleus. It may be interesting in this connection that the ribosomal pellet isolated from the nuclear sap contains a large amount of protein. The metabolic activity of this protein, as shown from amino acid incorporation experiments is different from that of the supernatant protein of the nuclear sap (Allfrey, 1963). Like proteins of the microsomal membranes this protein can be hsolubilized and separated from the ribosomes by deoxycholate treatment (Allfrey, 1963; Wang, 1963). The questions of the site of the synthesis of reticulum membranes and the nature of the nuclear membrane are very important and therefore the study of the presence and localization in the 92 G. P. GEORGIEV nucleus of various proteins and enzymes typical of cytoplasmic structures is of considerable interest. Finally enzymes present in the soluble phase of the cytoplasm are widely distributed in nuclei. It has been already mentioned that enzymes of glycolysis, the hexosemonophosphate shunt and the citric acid cycle are present in the nucleus. These enzymes are usually found in the soluble phase of the cyto- plasm (many enzymes of the citric acid cycle are found in mitochondria, but in the matrix and not in the membranes). Most of the enzymes discussed in the preceding section (b.) are also in the soluble phase. Thus, the cell nucleus contains many enzymes usually found in the soluble phase of the cytoplasm. In the nucleus they are also found in the soluble phase and either come into solution during extraction of nuclei with solvents of low ionic strength, or may be extracted with dilute salt solutions having ionic strengths of 0- 1-0-2. This is why the soluble phase of the cell nucleus may be regarded as analogous to that of the cytoplasm. Siebert (1961a) regards the nuclear sap as a continuation of the cytoplasmic soluble phase into the nucleus, so that there is free exchange between nuclear sap and soluble phase of the cytoplasm. This suggestion was. based on the following facts. Firstly the enzymic pattern of the nuclear sap and the hyaloplasm, as shown by the specific activities of the enzymes and the ratios of activities of different enzymes, and the content of metabolites are roughly similar. Secondly it was suggested that the nuclear membrane is permeable to proteins. The first point has already been discussed and only some exceptions will be mentioned. For example, Sibert et al. (1961) have shown that at certain stages of liver regeneration the activity of nuclear glycolytic enzymes increases while that of the cytoplasmic enzymes does not. This indicates that immediate exchange between nuclear sap and cytoplasm may be difficult. Also it has been shown that nuclear and cytoplasmic lactate dehydrogenases are not identical enzymes but are in fact isoenzymes (Vesell and Beam, 1962). This raises some doubt about the identity of nuclear sap and hyaloplasm. The second point about the permeability of nuclear membranes to proteins is rather difficult to prove. The membranes of isolated nuclei are certainly permeable to proteins, since some soluble proteins are lost even in solutions of low ionic strength. The association of many soluble proteins with nuclei in media of low ionic strength depends on electrostatic interaction between these proteins and deoxynucleoprotein but not on the impermeability of nuclear membrane. Roodyn (1957) has shown that if nuclei are disrupted by ultrasonic vibrations in a solution of low ionic strength aldolase remains in the nuclear sediment, but if undamaged nuclei are extracted with 0-14 m NaCl they lose this enzyme. If nuclei extracted with 0-14 m NaCl are placed in a sucrose extract of cytoplasm they reversibly bind cytoplasmic protein. The amount of bound protein is roughly equal to the protein content of the nuclear globulin fraction (Burton, 1960). The permeability of the nuclear 2. THE NUCLEUS 93 membrane to protein in isolated nuclei can hardly be an artifact, due to its damage, since this membrane is at the same time impermeable to many low molecular substances (see below). On the other hand, if oocytes are placed in the solution of fluorescent y-globulin or albumin these proteins readily accumulate in the cytoplasm, but not in the nucleus. This means that the nuclear membrane in vivo is not permeable to all proteins (Harding and Feldherr, 1959; Feldherr and Feldherr, 1960). The reasons for the difference between the permeability in vitro and in vivo have not been fully investigated. To summarize, nuclear sap may be regarded as analogous to the cyto- plasmic supernatant. Free exchange between these two soluble phases of the cell may occur under certain conditions but not under others. The inter- relation between protein components or enzymes of the nuclear sap and the hyaloplasm probably depends on many factors. Among these are whether a particular enzyme is synthesized in nuclear or cytoplasmic ribosomes, whether it can migrate rapidly through the nuclear membrane, its electro- static properties, and the size of the soluble phase in the particular type of nucleus. These factors may differ considerably in nuclei of different tissues and this is why the enzymic pattern of nuclear sap is extremely variable. However in spite of all these limitations, it may be concluded that nuclear sap is probably functionally and structurally similar to the hyaloplasm. D. THE NUCLEAR MEMBRANE AND PERMEASES In the preceding section the passage of proteins through the nuclear mem- brane was discussed. At the present time, the mechanism of protein and nucleoprotein transport from the nucleus to the cytoplasm is unknown; it is not even certain whether it is an energy dependent process. We have already mentioned that the passage of proteins through the membrane of isolated nuclei is a non-enzymic process. On the other hand, the penetration of some low molecular substances through the nuclear membrane probably involves some enzymic reactions. For example, as mentioned above, the membrane of isolated nuclei is impermeable to such substances as dicar- boxylic acids and nucleotides. Amino acids do penetrate into isolated nuclei, but this process is an endergonic one and depends on the presence of sodium ions in the medium. Only L-amino acids can penetrate, and competition between different amino acids for incorporation into the intranuclear pool has been described. Similar phenomena have been described for a number of other low molecular weight compounds, such as nucleosides and purine bases (Allfrey et al, 1957; AUfrey et al., 1961). Thus one can consider the possibility that the transport of low molecular weight precursors of proteins and nucleic acids into the nucleus depends on permeases, attached to the nuclear mem- brane. Future investigation into the existence of different kind of permeases 94 G. P. GEORGIEV and study of their properties and specificity will help us to understand the precise role of the nuclear membrane in cell metabolism. IX. Conclusion: The Function of the Cell Nucleus and Nuclear Structures Consideration of the nuclear enzymes, the nuclear structures and their related metabolic processes makes it possible to draw certain conclusions about the role of the nucleus in cellular metabolism. The most important and specific structural feature of the nucleus is the nucleolo-chromosomal complex, the functional genetic apparatus of the cell. Accordingly the major and specific aspect of nuclear metabolism is the conservation, reproduction and expression of genetic information. The key nuclear enzymes are therefore DNA polymerase, for DNA duplication and RNA polymerase, for DNA transcription. Both processes take place in the nucleolo-chromosomal-complex. The synthesis of certain cellular proteins, for example protein components of the ribosomes and deoxynucleoprotein fibrils, and other proteins bound to nucleic acids, also takes place there. It has been mentioned above that chromosomes and nucleoli probably have different functions. The formation of the non-specific machinery of protein synthesis, i.e. the synthesis of ribosomal RNA, assembling of ribo- somes, and probably the synthesis of sRNA takes place in nucleoli and probably also in nucleonemata of the chromosomes. The synthesis of infor- mational RNA programming this machinery proceeds in the chromosomes. Thus the nucleolo-chromosomal complex is a complete system, producing the protein synthesizing machinery of the cell. The cell nucleus may regulate both the rate of protein synthesis and its specificity. Another fundamental nuclear component, nuclear sap, has non-specific functions and this is why its enzymic pattern may differ in different cells. Nuclear sap is a medium in which reactions take place such as the synthesis of nuclear and some cellular proteins, high energy bond formation and many steps of intermediary metabohsm. 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Biol. 1, 1. Chapter 3 THE MITOCHONDRION D. B. ROODYN Department of Biochemistry, University College, London, England I. Introduction .......... 103 II. Survey of the Morphology and Metabolic Activity of Mitochondria . 104 A. Essentials of Mitochondrial Morphology .... 105 B. Problems in Surveying Metabolic Activity of Mitochondria . 107 C. Tricarboxylic Acid Cycle . . . . . . .111 D. Oxidation of Fatty Acids . . . . . . .114 tE. Respiratory Chain . . . . . . . . 117<^ F. Oxidative Phosphorylation 119 G. Ion Transport 128 H. Anabolic Reactions . . . . . . . .129 I, Other Reactions . . . . . . . .132 J. Summary and Conclusions . . . . . .138 III. Structural Basis of Mitochondrial Function 138 A. Mitochondrial Sap . . . . . . . .139 B. Mitochondrial Inclusions: DNA . . . . .145 C. Mitochondrial Inclusions: Dense Granules . . . .150 D. The Mitochondrial Membrane System . . . .153 E. Enzymes or Enzyme Systems Associated with the Membrane . 154^ F. Chemical Composition of the Membrane . . . .160 G. Stalked Bodies and Elementary Particles . . . .165 H. The Mitochondrial Membrane as an Integrated Structure . 169 IV. Addendum: The Structure and Function of Peroxisomes . . .172 References ........... 175 I. Introduction The intention of this article is first to conduct a broad survey of the major metabolic activities of the mitochondrion, and then to consider the relation- ship between mitochondrial structure and function. It is on this question that the great sciences of biochemistry and cytology have come closest together, and the problem of the elucidation of the organization of mito- chondrial enzyme systems has become identical to that of the elucidation of the structure of mitochondrial membranes. 103 104 D. B. ROODYN An aspect that is not considered in detail here is the behaviour of the mito- chondrion in the living cell. Unfortunately, the mitochondrion has been considered as a static structure for too long. In spite of many clear indications that mitochondria are dynamic, changing structures, capable in certain cases of growth and differentiation, the major interest of the great schools carrying out research into mitochondria has until very recently been almost exclusively into the elucidation of the detailed mechanisms of mitochondrial oxidations, energy transfer reactions, ion transport and related phenomena. For example it is most surprising that even now we have only fragmentary evidence as to the half-Ufe of mitochondria in the cell and no idea of the cellular site of synthesis of such important enzymes as cytochrome oxidase (1.9.3.1.) or cytochrome c. The recent discovery of DNA in mitochondria, however, has awakened interest in the challenging problems of mitochondrial growth and replication and there is Uttle doubt that there will be greater emphasis on these aspects of the problem in the near future. The question of the mechanism of biogenesis of the mitochondrion is considered in detail elsewhere (Roodyn and Wilkie (1966a). A short addendum on peroxisomes (microbodies) has been included at the end of the chapter. The results with these particles are still very new and no doubt our views of their function may well change in the near future ; how- ever, the fascinating implications of their role in extra-mitochondrial oxida- tions fully merit inclusion of a section on them in an article on mitochondria. Because of the sheer number of pubUcations on the subject, it is now no longer possible to give a comprehensive and detailed account of the enzym- ology of the mitochondrion in the space of one chapter of a book. As a result, in this article many important aspects have been dealt with in a rather cursory manner and have often been illustrated merely by reference to one or two typical experiments. The detailed aspects of such sections are best followed in the many excellent books, monographs and review articles cited in the text. It is hoped that the reader will therefore regard this article as a rather subjective essay, or excursion, into a vast rapidly expanding field of contem- porary biology, and will excuse the many factual omissions that are in- evitable in such an approach. II. Survey of the Morphology and Metabolic Activity of Mitochondria This section is intended to give a general picture of the major aspects of mitochondrial metabolism. To provide a basis for the account, however, a very brief summary of the essentials of mitochondrial structure is given at the beginning. The mitochondrion is described in a most erudite and compre- 3. THE MITOCHONDRION 105 hensive fashion in the excellent book by Lehninger (1965). Recent develop- ments in research into the mitochondrion are in the account on the Sym- posium on "The Regulation of MetaboHc Processes in Mitochondria" held recently in Ban, Italy (see Tager, Papa, QuagHariello, and Slater, 1966) and recent accounts of oxidation and phosphorylation are given by Ernster and Lee (1964) and Slater (1966). A more general account of the mitochondrion, with particular emphasis on cytological aspects, is given Novikoff (1961) in a rather extensive article. In order to obtain a comprehensive list of mito- chondrial enzymes, with specification of the tissues studied, the reader is recommended to consult the general survey of the distribution of enzymes in subcellular fractions of animal tissues by de Duve, Wattiaux and Baudhuin (1962). A quantitative survey of the sub-cellular distribution of enzymes in mammalian liver is given in Roodyn (1965a) and may be of use on obtaining precise information for the distribution of mitochondrial enzymes in this tissue. (Enzymes are listed according to their classification numbers of the Commission on Enzyme Nomenclature in both these articles, which usefuly complement each other.) There are many other reviews and monographs on mitochondria, respiratory enzymes and oxidative phosphorylation (see for example Ernster and Lindberg, 1958; Green and Fleischer, 1960; Chance, 1963; Sanadi, 1965) There is certainly no shortage of reading matter in this subject. A. ESSENTIALS OF MITOCHONDRIAL MORPHOLOGY The essential structural features of the mitochondrion are the double mem- brane, and the characteristic cristae. The reader is referred to Palade (1953) for what may now be regarded as a "classical" analysis of the main features of mitochondrial structure. As first stressed by Palade (1953) the number of cristae and their conformation varies considerably from one type of mito- chondrion to another. However, the elements that appear to be common to all mitochondria are an outer membrane, an inner membrane, cristae, mitochondrial "sap" or "matrix", dense granules and DNA. The mitochon- drial "sap" or "matrix" is generally regarded as being the space between the cristae and the inner membrane, and is sometimes called the "inner com- partment". The space between the inner and outer membranes is best called the "outer compartment". The cristae are double-membrane structures, and the space between these membranes is in connection with the outer compart- ment. During mitochondrial swelling, this space and the outer compartment are greatly expanded. The nomenclature just used is based on that of Whit- taker (1966) and a diagram showing these relationships is given in Fig. 1. The variations in the morphology of cristae is discussed in detail in Lehnin- ger (1965). They may have the appearance of simple stacked membranes, as 106 D. B. ROODYN Outer compartment Matrix Unit membrane / Protein Protein External membrane Fig. 1. Diagram illustrating the main structural features of the mitochondrion (from Whittaker, 1966). in liver and kidney mitochondria, or may be tube-like, branched or have a variety of other shapes. A particularly clear electron micrograph showing stacked cristae in isolated heart muscle mitochondria is given in Fig. 2. The inner layer of the mitochondrial membrane is generally assumed to be in continuity with the membranes of the cristae, which may be regarded as invaginations of this inner membrane. However, these invaginations may only occur at certain regions of the inner membrane. The connection between the inner and the cristal membrane is particularly well shown in the section of a chicken oocyte mitochondrion given in Fig. 3. The sectioning tech- nique, of course, will only give a true picture of the shape of the mitochondrion if it is lying in one plane, and is sectioned through its long axis. Whittaker (1966) has made elegant use of the technique of negative staining by applying it to fixed, intact mitochondria. He was able to demonstrate very clearly the elongated form of the liver mitochondrion (Fig. 4) It is interesting that this form is retained in mitochondria isolated in 0-44 m sucrose, but not in 0-25 M sucrose. DNA may now be considered to be a universal constituent of mitochondria as a result of the extensive studies of Nass, Nass and Afzelius (1965) who showed the occurrence of DNA fibrils in mitochondria from a very diverse range of biological materials (The matter is discussed more fully in Section III. B. below). The dense granules are most probably insoluble aggregates 3. THE MITOCHONDRION 107 Fig. 2. Stacks of cristae in rabbit-heart muscle mitochondria isolated in 0-88 m sucrose- 001 M EDTA. X 25,000 (from Deshpande, Hickman and Von Korff, 1961). consisting mainly of calcium and magnesium phosphates, and this is dis- cussed fully in Section III. c, below. B. PROBLEMS IN SURVEYING METABOLIC ACTIVITY OF MITOCHONDRIA There is great difficulty in giving an accurate, but reasonably concise account of the metabolic activity of the mitochondrion for several reasons. A rigorous account would have to consider the many differences in biochemi- cal constitution of mitochondria from different sources, since there is little doubt that the range of morphological variation mentioned above is reflected by a similar range of metabolic variation. Although a great deal of work has been done with rat-liver mitochondria, it is certainly unsafe to assume that all these findings are of general significance. Indeed, even liver mitochondria isolated from different mammals are by no means identical. For example there are diff"erences between rat-liver and rabbit-liver mitochondria. Cer- tainly there are major differences between the enzymic constitution of, for F* 108 D. B. ROODYN -^^rT"^;- w^ n *SZl_St£ •" . i '. ^ ^s;., 1 ^'^^. ■ -^i- ^■'^^ \ V / r ^ : ■^ti Fig. 3. Mitochondria taicen from an oocyte in the ovary of a domestic fowl (Gallus bankiva). Fixed in Os04, stained in lead citrate. Note invaginations of inner membrane to form cristae, and also frequent dense granules, x 72,000. (This electron micrograph was kindly supplied by Dr. R. Bellairs.) example, yeast, insect flight muscle and rat-liver mitochondria. The fact that the general features of the respiratory and phosphorylative systems appear to be similar in mitochondria of diverse origin has tended to obscure the many differences that exist. A second difficulty is essentially due to technical reasons. Because of the ease of disruption of liver, and the abundance of mitochondria in the liver "^ ^ cell, it is relatively easy to obtain liver mitochondria in reasonable yield, without excessive damage or non-mitochondrial contamination. (The problem of contamination of liver mitochondria with other sub-cellular components should not be under-estimated, however. Neither should the possible presence Fig. 4. Elongated rat-liver mitochondrion isolated in 0-44 m sucrose and negatively stained in sodium phosphotungstate after fixation. The electron micrograph is a composite one of a number of high magnification micrographs of different portions of the mitochondrion (from Whittaker, 1966). 110 D. B. ROODYN of bacteria be ignored: Roodyn, Reis and Work 1961). With other tissues, such as brain or ascites tumours, it is far more difficult to obtain reasonable preparations of mitochondria. Some early preparations of so-called "brain mitochondria" were in fact grossly contaminated with other structures, and special procedures are needed to obtain even a moderately pure preparation. The techniques used for the disruption of tumour cells, such as osmotic shock, may very easily result in osmotic shock to the mitochondria as well. Also, liver is a relatively homogeneous tissue as regards cell-type, since it consists mainly, but by no means entirely, of columnar cells. Preparations of mito- chondria, for example, from chick embryos, or plant tissues are clearly derived from mixed populations of cells and interpretation of metabolic studies in such cases is difficult. The third difficulty is more fundamental, and has been discussed extensively elsewhere (Roodyn, 1965a). It is that there is considerable variation in the aims and attitudes of the various investigators of mitochondrial metabolism. The early pioneer work of Claude, Schneider, Hogeboom and their associates was aimed at establishing the mitochondrial localization of certain enzymes. However, much of the subsequent work on mitochondria has lost contact with the aim of establishing intra-cellular localizations of theenzymes studied, but has concentrated on isolated mitochondria as if they were a tissue per se. This work is generally carried out without any concomitant examination of other sub-cellular fractions, or even of the total homogenate from which the mitochondria were derived. As a result, we have a truly massive literature describing the properties of isolated mitochondria, but a far smaller literature concerned with establishing whether the enzymes studied are in fact truly localized in the mitochondrion. An excellent example of the dangers of such an approach is given by studies on catalase (1.1 1.1.6.). There are a large num- ber of papers published between 1950 and 1960 concerned with the catalase activity of mitochondria. However, it is now clear that this enzyme is in fact present in peroxisomes (microbodies) and not in mitochondria (see Section IV.) In order to avoid such difficulties in the future, the author has suggested the adoption of five criteria that should be met before it can be said with certainty that an enzyme is truly associated with a sub-cellular component isolated from a tissue homogenate (Roodyn, 1965a). These are as follows: 1. The assay should be such that a single enzyme is measured. If a multi- enzyme system is studied, the components of the system should be known. 2. The enzymic activity should be compared in some way with the activity of the homogenate. 3. During the fractionation of the homogenate, no serious activation or inactivation of the enzyme should have occurred. Thus only those fractiona- tions in which a reasonable recovery of enzyme is obtained are valid. 3. THE MITOCHONDRION 1 1 1 ponent is not due to contamination with some other component. 5. A clear association between the sub-cellular component in question and the enzyme should be demonstrated in a variety of preparations of varying purity. It may be noted that these criteria are insufficient to establish that the ob- served activity of the isolated cell component corresponds to the activity of that component in the living cell, but only refer to the structure present in the homogenate. (See Chapter 1 for a general discussion of these issues.) It is a disturbing fact that the greater part of the enzyme studies on isolated mitochondria do not satisfy one or other of these criteria. In some cases, none of the criteria are met at all. As a result, whilst we can say in many cases that such and such an enzyme is probably in the mitochondria, it is far more diffi- cult to establish this without any reasonable doubt. Some may argue that there is no certainty in biology. Nevertheless there is sufficient information on the sub-cellular distribution of cytochrome oxidase to satisfy all the above criteria and so to establish without doubt that this is a truly mitochondrial enzyme. If similar information were available for other enzymes, our knowledge of the enzymology of the mitochondrion would be on a firmer footing than at present. As a result of these considerations, it is certain that a rigourous account of the properties of the mitochondrion would require a careful examination, for each tissue used, of the evidence establishing which enzymes are truly mito- chondrial. Space does not allow such an account, which would probably run to many hundreds of pages. The alternative is therefore to present a general picture of the major metaboUc activities that appear to be common to mito- chondria. C. TRICARBOXYLIC ACID CYCLE The details of the tricarboxylic acid cycle are given in Fig. 5 and the reader is referred to Krebs and Lowenstein (1960) for an account of the essential features of the cycle. Demonstration of the presence of a functional citric acid cycle in isolated mitochondria was one of the early achievements in the field. The basis of these findings may be found in Schneider and Hogeboom (1956). If was found that isolated mitochondria could completely oxidize pyruvate and various citric acid cycle intermediates to carbon dioxide and water when incubated in relatively simple media (for example in the presence of buffer, Mg2+ ions, sucrose and inorganic phosphate). Thus the entire complex of enzymes and co-factors necessary for the cycle is present in the mitochondrion. The striking nature of these findings has tended to distract attention from the important quantitative question as to whether the enzymes of the citric 112 D. B. ROODYN CHj-CO-S-CoA+HaO HS-CoA COOH 1 CO 1 2H CHo 1 ^ X COOH COOH y^ OxQlacetate HCOH 1 CH 1 COOH /i-Malate COOH 1 CH 1 COOH Fumarate , ?.H, COOH I CH2 CH2 I COOH Sjccinate GTP + HS-CoA GDP+Pi COOH I CHo I CHg I CO-S-CoA Succinyl coenzyme A COOH COOH CHz CH2 CO I COOH HC'COOH I CO I COOH Oxalosuccinote CO 2 a-Ketoglutarote CO2+2H HS-CoA Fig. 5. The tricarboxylic acid cycle:* Citrate can yield isocitrate without c«-aconitate as a free intermediate;! isocitrate can yield a-ketoglutarate without oxalosuccinate as a free intermediate;! Succinyl-CoA can also yield succinate by the action of succinyl-CoA deacylase (3.1.2.3), and succinyl CoA transferases (from Krebs and Lowenstein, 1960: see the original text for full references to the above comments). acid cycle are exclusively localized in the mitcKhondrion. It is interest- ing that the "dogma" of the unique mitochondrial localization of these enzymes was questioned as early as 1956 by Schneider and Hogeboom in a review at that time (Schneider and Hogeboom, 1956). Although there is little evidence at the moment for the existence of a functional 3. THE MITOCHONDRION 113 tricarboxylic acid cycle in a cell component other than the mitochon- drion, many of the enzymes of the cycle show a dual localization, appearing both in the soluble fraction and the mitochondrial fraction. The relative partition of enzyme observed between these two fractions may depend to a certain extent on the degree of damage inflicted on the mito- chondria during isolation, if the enzyme is present in the mitochondrial sap. However, it is certain that not all the activity in the soluble fraction can be ascribed to leakage from the mitochondrion. The surest example of this is that of malate dehydrogenase (1.1.1.37.) where the "soluble" and "mito- chondrial" enzymes have been shown to differ in many respects and are undoubtedly different proteins. For example, soluble and mitochondrial malate dehydrogenases from beef-heart have very different amino acid compositions (Siegel and Englard, 1962). The dual location of hver malate dehydrogenase is discussed in Roodyn, Suttie and Work (1962). Examples of other enzymes of the cycle that show such a dual location are aconitase (4.2.1.3.), iso-citrate dehydrogenase (1.1.1.41), fumarase (4.2.1.2.) and pos- sibly citrate synthase (4.1.3.7.) although it has not been established in every case that the mitochondrial and soluble enzymes are different proteins. The existence of "mitochondrial" and "soluble" enzymes that catalyse the same reaction but are different proteins is by no means confined to the citric acid cycle. (See Section II. i. and Chapter 8). The widespread nature of this duality has recently been stressed by Greville (1966) in a discussion of the factors affecting the utilization of substrates in the cell sap by mito- chondria. Dare one speculate that the reason for this duality lies in the evo- lutionary origin of mitochondria, i.e. that they were originally symbiotic parasites within a host cell, and that the soluble enzymes are those of the original host ? Arguments in favour of the "bacterial" theory of mitochondrial origin have become more credible recently and the matter is discussed criti- cally in Roodyn and Wilkie (1966a). The reason for our confidence in assigning the functional tricarboxylic acid cycle to the mitochondrion is that certain key enzymes of the cycle, namely the a-oxoglutarate dehydrogenase system, and succinate dehydrO' nase (1.3.99.1.) appear to be uniquely associated with mitochondria. The enzymes of a-oxoglutarate oxidation (and the closely related enzymes of pyru- vate oxidation) are arranged in organized enzyme assemblies of considerable particle weight. The complex processes involved in these oxidations require coenzyme A, lipoic acid, thiamine pyrophosphate and NAD amongst the co-factors. The detailed enzymology and organization of these systems is described fully by Sanadi (1963) and Massey (1963), and is also discussed in Sections II. f. and III. e. below. (See Figs. 13 and 34, below). Succinate dehydrogenase is the marker enzyme par excellence for mitochondria, and has been demonstrated in mitochondria from a very wide range of tissues. Since the venerated Keihn-Hartree muscle preparation is capable of oxidizing 114 D. B. ROODYN succinate, it is probably safe to say that succinate dehydrogenase was the first enzyme of the citric acid cycle to be assigned to a respiratory particle. An interesting exception to the unique mitochondrial location of succinate dehydrogenase is the finding of Rossi, Hauber and Singer (1964) that there are at least three enzymes in yeast that catalyse the conversion of succinate _lQ_fumarat&j One is the "classical" mitochondrial succinate dehydrogenase and is absent from yeast grown under anaerobic conditions and from respir- tory deficient ("petite") mutants of yeast (see also Roodyn and Wilkie, 1966b). The other two are non-mitochondrial enzymes and are still present in anaero- bic and respiratory-deficient yeast. They probably function more as fumarate- reductases than succinate dehydrogenases, and are possibly concerned in supplying succinate for synthetic reactions in the absence of a functional citric acid cycle. These findings illustrate very clearly the dangers pointed ^ut above of arguing exclusively from the results with mammalian liver / In conclusion, it may be said that mitochondria contain all the enzymes — i of the tricarboxylic acid cycle, but that some of these are not tightly bound to the mitochondrial structure and also exhibit a dual distribution between \^e mitochondrial and soluble fractions. D. OXIDATION OF FATTY ACIDS Another "classical" property of the mitochondria is the ability to oxidize fatty acids, again when incubated in relatively simple reaction media. The details of the ^-oxidation "spiral" of fatty acid oxidation are given in Fig. 6, and the reader is referred to Lynen (1955) for an account of the enzymes involved in this process. Since the terminal product of the spiral, acetyl-Co A, feeds directly into the tricarboxyUc acid cycle by means of the enzyme Phosphatides, Fatty acids-* fats CoAShX ATP -CHa-CHe-CHg-CO-SCoA FAD-s^FADH) Ethylenereductose (Acyl— CoA— Dehydrogenos«) -CHg-CO-SCoA CHa— CO-SCoA p -Ketothlolase HSCoA i -CHg— CH=CH— CO-SCoA -CH^-CH-CHj-CO-SCoA^— CH=CH-CH2-CO-SCoA I >/ +H2O \V OH — CH2-CO-CH2-CO-SC0A {NADi^NADH + H"^) ^-Keforeduclas* {/J -Hydroxyacyl-CoA-Dehydrogeno»«) Fig. 6. The j3-oxidation "spiral" for fatty acid oxidation (from Lynen, 1955). 3. THE MITOCHONDRION 115 citrate synthase, mitochondria can completely degrade the added fatty acid to carbon dioxide and water. They also provide a magnificent experimental tool for examination of the mechanism of interaction of carbohydrate and fat metabolism. The work of Lehninger and his colleagues, in particular, revealed in the 1950's the complex metabohc interactions that can still occur in isolated mitochondria. Recently, there has been considerable interest in the mechanism of transfer of fatty acids and two-carbon fragments across the mitochondrial membrane. Interest has focused on the growth factor carnitine which can form esters with fatty acids. For example, Bremer (1963) suggested that palmityl-CoA formed from the result of fatty acid activation by the reaction: Palmitate + CoA + ATP > Palmityl-CoA + AMP + P-Pi can then react with carnitine to form palmityl-carnitine : Palmityl-CoA + carnitine -> palmityl-carnitine + CoA A palmityl-CoA : carnitine palmityl transferase (2.3.1.-.) was isolated from calf-liver mitochondria by Norum (1964) Acyl carnitine esters and palmityl carnitine transferase are apparently concerned in the transfer of fatty acyl derivatives across mitochondrial membranes. A scheme showing the possible intra-mitochondrial location of some of these reactions was suggested by Fritz and Marquis (1965) and is given in Fig. 7. This is the first example in this article in which we have had to consider the problem of the mechanism Site A (Cristoe space?) Palmitofe ATP + CoA . „g2+ AMP+PPj ■* ,, Palmityl CoA Carnitine CoA T Palmityl cornitine ' Barrier (Inner membrane) Palmityl carnitine Site B (matrix?) CoA Carnitine Palmityl CoA J3 Oxidation I 8 Acetyl CoA Fig. 7. Possible intra-mitochondrial location of reactions of carnitine metabolism. The "barrier" is per^e&bie to pahnitylcamitine, but not to palmityl-CoA (adapted from Fritz and Marquis, i965). ^ 116 D. B. ROODYN of transport of metabolites across the mitochondrial membrane. In connec- tion with such problems, Chappell and Crofts (1965b) have examined the effect of atractylate, which interferes with the transport of adenine nucleotides into the mitochondrion (see Section II. i. below). The scheme suggested by these authors for the role of carnitine in transport across the membranes is similar to that of Fritz and Marquis, but in addition gives the site of inhibition of atractylate (Fig. 8). Atractylate barrier Cn-^H 1 "^ Cn R-CO-CoA-«-H----— 1 R-C02H ■ Atractylate: ATP- R-CO-Cn- Cornitine borrier 1 R-CO-CoA- CoA*- Compartmentl R-CO-Cn y3-Oxidation -R-COCoA yv CoA Compartment 2 Fig. 8. Site of action of atractylate in fatty acid oxidation. Cn: carnitine, => site of inhibition by atractylate (from Chappell and Crofts, 1965b). Many other reactions concerned with fatty acid metabolism occur in mitochondria. One interesting example is the cycUc process concerned in the conversion of acetoacetyl-CoA to free acetoacetate. The acetoacetyl-CoA is not apparently hydrolysed directly to the free acid under normal conditions but reacts with a molecule of acetyl-CoA to form the compound 3-hydroxy- 3-methylglutaryI-CoA. This is then broken down to form acetoacetate, with the regeneration of acetyl-CoA. The two enzymes involved are 3-hydroxy- 3-methyl-glutaryl-CoA synthase (4.1.3.5.), followed by 3-hydroxy-3-methyl- glutaryl-CoA lyase (4.1.3.4.) (Bucher, Overath and Lynen 1960). The cycle operates as shown in Fig. 9. The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (1.1.1.34) which is on the pathway to cholesterol synthesis, is pre- dominantly found in the microsomal fraction (Bucher et al., 1960). Related to these reactions are those concerned with the metaboHsm of 3-hydroxy- butyrate and acetoacetate. For example, mitochondria contain dehydrogen- ases to oxidize 3-hydroxybutyrate to acetoacetate (1.1.1.30.). Mitochondria from mammalian kidney and heart can form acetoacetyl-CoA from this acetoacetate by transacylation with succinyl-CoA by means of the enzyme 3-ketoacid CoA-transferase (2.8.3.5.). The acetoacetyl-CoA can then react with free CoA in the terminal "thioclastic" reaction of the j8rOxidation spiral and thus be oxidized to carbon dioxide and water. Liver mitochondria, 3. THE MITOCHONDRION 117 however, cannot form acetoacetyl-CoA from free acetoacetate, and hence oxidize 3-hydroxybutyrate to acetoacetate and no further. This fact was of considerable use in determining the yield of ATP from the oxidation of NADH in liver mitochondria, since 3-hydroxybutyrate could act as an CHsCOCHgCO-S-CoA (Acefoacetyl CoA) CHsCO-S-CoA (Acetyl CoA) CH3COCH2COOH (Acetoocetote) CH2COOH HO-C-CH, + I CHz-CG-S-CoA ( 3 Hydroxy 3 methyl Glutary! CoA ) CoA-SH Fig. 9. The hydroxy-methylglutaryl cycle for the conversion of acetoacetyl-CoA to aceto- acetate in mitochondria (adapted from Bucher et ai, 1960). excellent source of NADH, uncomplicated by further oxidations. (The differ- ence between liver and heart mitochondria is an important example of differences between mitochondria of different origin). E. RESPIRATORY CHAIN The respiratory activity of the mitochondrion is generally regarded as its central property. However, again we must be wary against assuming that all cellular oxidations occur in the mitochondrion. There are several more or less defined extra-mitochondrial oxidative systems. For example, it appears from recent evidence (given in Section IV.) that the peroxisome is an important example of such a system. The reader is referred to the recent review of de Duve and Baudhuin (1966) for an account of the role of extra-mitochondrial oxidation pathways. Fortunately, however, the terminal enzyme of the respiratory chain in mitochondria, namely cytochrome oxidase, is established without doubt as being exclusively localized in the mitochondria. The pioneer experiments of Schneider and Hogeboom rigorously established that the enzyme was associa- ated with mammalian liver mitochondria. (See for example Schneider, 1946, and Schneider and Hogeboom, 1950). In addition it was shown by quantitative fractionation experiments that the cytochrome oxidase activity in the nuclear fraction was entirely due to mitochondrial contamination (Hogeboom, Schneider and Striebich, 1952). Studies of this enzyme in a wide range of 118 D. B. ROODYN tissues have consistently shown that it is exclusively concentrated in the mitochondrial fraction. In addition, the application of more sophisticated fractionation methods, in particular by the Louvain School, has firmly established cytochrome oxidase as a primary enzyme marker for mitochon- dria. (Beaufay, Bendall, Baudhuin, Wattaiux and de Duve, 1959). Because of this fact, it must follow that any oxidative process that is measured by oxygen uptake and proceeds through the terminal enzyme of the / respiratory chain will have the same apparent localization in the cell as cyto- r/ chrome oxidase. Thus many so-called "oxidases", which were in fact systems of enzymes linked to the terminal oxidase, were originally found to have an exclusively mitochondrial location. For example, isocitrate oxidase activity was detected by Hogeboom and Schneider (1950) only in the mitochondrial fraction. When they measured the isocitrate dehydrogenase directly, however, they found activity both in the mitochondrial and soluble fractions. The fact that the sub-cellular distribution of the component enzymes of a multi-en- zyme system need not be identical to that of the terminal enzyme of the system is often not fully appreciated. In a recent survey of enzyme distributions in sub-cellular fractions (Roodyn 1965a) it was found that only a small propor- tion of the studies on multi-enzyme systems satisfied the first criterion men- tioned in Section II. b. above. This is that it is only possible to obtain quanti- tative distribution patterns if single enzymes, or multi-enzyme systems of known constitution, are studied. It is therefore necessary to exercise a certain amount of caution when interpreting the massive literature on mitochondrial oxidations. If, for exam- ple, glutamate oxidation is only observed in mitochondria, it cannot be inferred that the primary dehydrogenase, glutamate dehydrogenase (1.4.1.3,4) is necessarily a mitochondrial enzyme. A small amount of the total cellular enzyme may well have been sufficient to catalyse the first stage of the oxida- tion to carbon dioxide and water, which would be the actual reaction measured by the "oxidase" assay. Unfortunately, as stated above, a critical assessment of the literature on the sub-cellular distribution of oxidases would be far too long for this article. It must therefore be sufficient to stress that the oxidation of a large number of substances has been demonstrated with isolated mitochondria. These include many fatty acids of different chain length, ketoacids, dicarboxylic and tricarboxylic acids, several amino acids, glycerol- 1 -phosphate, various steroids, choline and various other N-methyl derivatives, amines and other compounds. These oxidations usually proceed by interaction of a primary dehydrogenase with the respiratory chain. How- ever, some oxidases may react directly with oxygen. (For example, this may apply to mono-amine oxidase (1.4.3.4.) which has been demonstrated in mitochondria by Baudhuin et al., 1964). The respiratory chain is formulated by Lehninger (1965) as shown in Fig. 10. The various substrates oxidized by it either react primarily with 3. THE MITOCHONDRION 119 NAD or NADP linked dehydrogenases or with various flavoproteins (FP) that then interact with the chain of cytochromes and ultimately with oxygen. It is difficult at the present time to present a formulation of the respiratory chain that is completely free of controversy, but that given in Fig. 10 is similar Malate L-y3-hydroxyacyl-CoA glufomote D-ji3- N Hydroxybutyrate Succinate Pyruvote NAD \ ® pp^ ® ® n-H.FP s-s FP — CoQ— ►cyt6-**-cyt c,— *-cyt c— vcyt o->-cyT03- NAD / r^5 v^ Arsenite / . Amytal pp / Antimycin A / Cyanide ' Bound NAD / «-Ketoglutorate / /'''* NAD / , / FP3 f NADP / of-Glycerophosphate / Fatty Gcyl-CoA Isocitrate glutamate Fig. 10. Respiratory chain, sites of ATP synthesis and sites of action of important inhibitors as formulated in Lehninger (1965) (FP: flavoprotein). to the recent formulation given by Sanadi (1965). Perhaps the most serious areas of contention are concerned with the roles of non-haem iron and of ubiquinone. The purification and characterization of many of the components of the respiratory chain has proved to be extremely difficult, because of their very tight binding to the mitochondrial membrane. As a result, there is still some uncertainty as to the relationship between the various enzyme prepara- tions obtained by different workers. For example, Machinist and Singer (1965a, b) have suggested that the enzymes NADH-cytochrome c reductase (De Bernard, 1957) and NADH-ubiquinone reductase (Pharo and Sanadi, 1964) are really degraded fragments of the enzyme NADH-dehydrogenase (1.6.99.3.). In spite of these difficulties, the scheme presented in Fig. 10 probably gives a sufficiently accurate summary of the constitution of the respiratory chain as it is at present understood. F. OXIDATIVE PHOSPHORYLATION It has been estabhshed for many years that the oxidation of various sub- strates by isolated mitochondria is accompanied by the disappearance of inorganic phosphate from the medium, with the formation of ATP. The detailed mechanism of this phenomenon has engaged the interest of many active groups for the past 10-15 years. The reaction can be formulated in a deceptively simple fashion by the formula: AH2 + i02 + ADP + Pi -> A + H2O + ATP •o 120 D. B. ROODYN (where AH2 is the substrate oxidized by the respiratory chain). The precise intermediates involved in this reaction have not been isolated, or even con- clusively characterized. Perhaps the search has been misguided and one should look to theories similar to the chemi-osmotic theory of Mitchell (1961) for a solution. In this theory, the coupling between electron transfer and phosphorylation is not by means of the synthesis of "energy-rich" chemical intermediates, but is due to the asymmetric or "anisotropic" localization of oxido-reduction and phosphokinase systems on a charge-impermeable "coup- ing membrane". Thus in the reaction: ATP + H2O -> ADP + Pi if the H+ and OH" ions of the water could be separated by a suitable membrane, the reaction could be reversed, viz : ADP + Pi -> ATP + H+ + [OH- where the symbol [ represents separation by a membrane. A model for such an anisotropic ATPase (3.6.1.3.) is given in Fig. 11. which shows how a OH" ion participating in the reverse process (i.e. ATP synthesis) may only be ATPase in section ADP- Zj© f ADP-C j Fig. 11. a proposed model for an "anisotropic" ATPase. The channel to the active centre on the left allows passage of ATP (ADP-0'-P+), phosphate (POH) and H+ but not OH'. The right hand channel allows the passage of OH' only. (The phosphorylium group P+ is thought to form an intermediate with a negative group S' in the enzyme) (from Mitchell, 1962). 3. THE MITOCHONDRION 121 generated on one side of the "enzyme". If such a system is present in a mem- brane and is coupled to an oxido-reduction system that is similarly aniso- tropic, except that it generates OH~ ions on the opposite side of the mem- brane, the oxidations may be used to drive the ATP synthesis, as shown in Fig. 12 (adapted from Mitchell, 1966a). In addition to the charge-impermeable Charge impermeable Coupling membrane Left-hond phase SHj 1) Anisotropic oxido-reduction system 2) Anisotropic phosphokinase system 3) Overall oxidative phos- phorylation system Right-hand phase 0 2 ^^ ^02 + H2' S + ZH""'*^^ 2 0H- ^ 2 H3PO4 2 0H"-*^^ 2HPO3 + 2H* SH2 \ ^ IO2-1-2H3PO4 S + H,0'«^^ 2 HPO,+ H,0 Fig. 12. Essentials of chemi-osmotic theory of oxidative phosphorylation. The over all P/O ratio in the reaction is 20 (adapted from Mitchell, 1966a). coupling membrane, and the two anisotropic enzyme systems, Mitchell pro- poses the existence of an exchange-diffusion system. This is to maintain the internal ionic composition and pH of the mitochondrion against the pro- duction of H+ ions on one side of the membrane, and OH" ions on the other. The elegance of Mitchell's theory is that studies on oxidative phosphorylation, ion transport, water transport and membrane structure are all brought to- gether as investigations of different facets of the same system, namely a functional membrane. (A full account of this theory is given in Mitchell, 1961, 1966a, b). Although discussion of Mitchell's theory may well cause some heart- searching amongst those seeking to isolate defined chemical intermediates of oxidative phosphorylation, they may well be encouraged by the great dividends yielded by the "classical" approach to complex systems i.e. sub- fractionation, purification of enzymes and isolation of intermediates as ap- plied to closely related problems. The detailed elucidation of the mechanism of ATP synthesis in the glycolysis system is usually taken as the most out- standing example of such success. However, it should not be forgotten that the mechanism of complex oxidation and energy transfer reactions occuring in the pyruvate oxidase and a-oxoglutarate oxidase complexes has been 122 D. B. ROODYN revealed in considerable detail (see Sanadi, 1963). In fact, the so-called "sub- strate-level phosphorylation" associated with the conversion of a-oxoglu- tarate to succinate proceeds by a series of reactions that are now relatively well defined, with known co-factors and enzymes being involved. The for- mation of acyl-CoA derivatives in these reactions is shown in Fig. 13. These 0 II DPT-C-R hDPT ,. ^S-CO-R CoASH •R— CO— S— CoA NADH Fig. 13. Possible mechanism of oxidative decarboxylation of a-keto acids. DPT: diphos- phothiamine.l)DPT-dependent decarboxylation,2),3) reductase-transacylase step, 4)trans- acylase,5 a-d) lipoamide dehydrogenase (from Sanadi, 1963). derivatives are then transferred to ATP. Thus in the case of succinyl-CoA, energy transfer reactions occur in the sequence : Succinyl-CoA -> Phosphohistidine -> GTP -> ATP the latter reaction proceeding via nucleoside diphosphate kinase (2.7.4.6.). These reactions are also discussed in Section III. e, below (see Fig. 21). The role of phosphohistidine, and phospho-proteins, is the subject of active investigation at the moment. Heldt (1966) showed that phosphohisti- dine was labelled with ^^p before GTP, ATP and ADP in substrate-level phosphorylation occurring during the oxidation of a-oxoglutarate. Sperti, Pinna, Lorini, Moret and Siliprandi (1964) had previously found evidence to suggest a role of phosphohistidine and phosphoproteins in substrate-level phosphorylation, although they could not exclude the possibility that some of the phosphoprotein was formed by phosphorylation of protein with ATP derived from respiratory chain phosphorylation. The role of phospho- histidine in respiratory chain phosphorylation has been the subject of some 3. THE'MITOCHONDRION 123 controversy, because of the relatively slow rate of turnover of this compound. However, Boyer (1965) has argued that this does not necessarily preclude the possibility that at least some of the phosphohistidine is involved in the major path of ATP synthesis. This is because of difficulties of interpretation of kinetic studies when complex pools exist. These conclusions were disputed by Slater (1965) however, Whatever the final outcome of these difficulties, there is now little doubt that phosphohistidine in particular, and phospho- proteins in general, are of considerable metabolic importance in mitochondria. The example of phosphohistidine illustrates the difficulties that arise in the search for defined chemical intermediates of oxidative phosphorylation. However, if we accept the view that such intermediates do in fact exist, one may present a minimum hypothesis for the mechanism of oxidative phos- phorylation. This is based on a large number of observations, best given in the many excellent reviews on the subject (see for example Chance and Williams, 1956; Racker, 1961; Lehninger, 1965; Slater, 1966). This highly specialized topic is still in a very dynamic state. Nevertheless, it seems that the essential basis of most "classical" theories of oxidative phosphorylation is as follows. During electron transfer along the respiratory chain, a suitable energy carrier (or carriers) reacts with the chain. There have been many symbols suggested for this carrier, and in this article we will use the most frequently quoted symbol I (see Chance and Williams, 1956). It is very likely that the formation of the complex between I and the respiratory carrier results in inhibition of electron transport. Whether I reacts with the carrier in its reduced or oxidized state is not so certain, although Chance and Wil- liams favour the former possibility. There are probably three sites of inter- action of I with the chain, and these are between NAD and flavoprotein, between cytochrome b and ci and between cytochrome c and oxygen. Using the symbol '-^ for any energy-rich intermediate, and assuming that there are different species of I at each site, and that these react with the reduced carriers, the first stage of oxidative phosphorylation is the formation of NADH '^ Ii, ferrocytochrome b r^h and ferrocytochrome c r^ I3. It is believed that a second type of energy carrier reacts with these complexes. Again, the nomen- clature is very confusing, but the symbol X is widely used (see Chance and Williams, 1956). It is not known if there are one, or three, species of X. The second reaction is then the formation of X -^ Ii, X '^ I2 and X -^ I3. These X -^ I compounds (or non-phosphorylated energy rich intermediates) then react with inorganic phosphate to form X -^ P, a phosphorylated carrier. Finally, X '^ P reacts with ADP to form ATP and liberate X. The essentials of such a scheme is given in Fig. 14 (from Packer 1961). The system is formu- lated slightly differently by Lehninger (1965) in that inorganic phosphate is thought to react directly with the complex between the respiratory carrier and I. (see Fig. 20 below). It must be stressed that none of the various -^ compounds have yet been 124 D. B. ROODYN ^ Oxygen Fig. 14. a scheme for oxidative phosphorylation. PN: pyridine nucleotides (NAD, NADP); FP: flavoprotein; b, ci, c, a, 03: cytochromes; I, X: intermediates in oxidative phosphoryl- lation; '~ indicates high-energy state (from Packer, 1961). isolated, or even identified, and there is probably little purpose in this article to delve into the fine arguments for and against various theories. The point that is generally agreed is that non-phosphorylated energy-rich intermediates are generated during oxidative phosphorylation. Although these intermedi- ates may be used to form ATP, they may also be used for a wide variety of energy-requiring reactions. These reactions have been discovered largely by the use of the antibiotic oligomycin which prevents the formation of ATP, but not of the non-phosphorylated energy rich intermediates. A detailed study of the mode of action of oligomycin, and particularly the inhibition of ATPase induced by a wide variety of agents, has recently been made Lardy, Connelly and Johnson (1964). The possibility was suggested that oligomycin combines with X '^ P, and so prevents phosphate transfer to any acceptor. These authors also proposed the existence of a special category of high-energy intermediate (called W r^) which is specifically concerned in ion transport, swelling and contraction phenomena, and is distinct from intermediates of oxidative phosphorylation, such as X '^ P and X '--' I. The variety of energy transfer reactions that occur in mitochondria are amply described in the report of a "Symposium on Energy-linked Functions of the Mitochondrion" (Chance, 1963). Interest in energy transfer reactions within mitochondria was greatly stimulated by the important discovery of the reversal of oxidative phosphoryl- ation and electron transport (see for example Klingenberg and Scholl- meyer, 1960; Chance and Hollunger, 1961). It is possible, for example, to bring about the thermodynamically unlikely reaction : Succinate + NAD+ -> Fumarate -\- NADH + H+ 3. THE MITOCHONDRION 125 I either by supplying exogenous ATP, or by using ATP or even non-phosphory- lated energy-rich intermediates generated by the mitochondria themselves. If the energy is generated by the mitochondria, the energy-linked reduction of NAD+ is lost after treatment that damage ~ production. By using suitable inhibitors and oxidizable substrates, it is possible to demonstrate that energy derived from electron transport along one part of the chain may be used to reduce another part of the chain. The effect of inhibitors on the respiratory chain and energy-transfer process was examined in detail by Chance and Hol- lunger (1963) and a summary of their findings is shown in Fig. 15. Electron Ironsfer inhibitors Oxyborbiturote--. Guanidines Methylene glycol .■ Progesterone ■' Uncoupling agents '^ -'Oligomycin.l Energy 'Qtroctyloter^°"sfer inhibitors ADP+Pi - ATP Fig. 15. Site of action of inhibitors of electron transport and energy transfer reactions. (l)fd), C2)(b) and (3)(c) represent the three sites of energy production, i.e. sites I, II and III (Atractylate is now thought to inhibit the transport of adenine nucleotides into mito- chondria: see text) DPN = NAD (from Chance and Hollunger, 1963). An elegant example of experiments on energy-transfer reactions in mito- chondria is given by the work of Tager and Slater (1963) on the synthesis of glutamate by isolated mitochondria. By following the formation of gluta- mate from a-oxoglutarate and NADH plus NH3, they were able to examine the energy-linked formation of NADH, e.g. by reduction of NAD+ with succinate. The energy for NAD+ reduction could be provided by ATP, by aerobic oxidation of succinate, or by the aerobic oxidation of tetramethyl- paraphenylene diamine (TMPD) which is oxidized by the terminal part of the respiratory chain. Tager and Slater examined many related energy-linked reactions, and perhaps it would be useful to illustrate this type of work with a specific example given by Slater and Tager (1963) as follows: The respiratory chain is represented in an abbreviated form by the diagram : NAD+- B .O2 Succinate TMPD 126 D. B. ROODYN where '^ are energy-rich intermediates, A is a respiratory carrier in the flavo- protein-ubiquinone region, B is a carrier in the cytochrome c region and TMPD is the substrate tetramethy-paraphenylene diamine. In the pres- ence of antimycin, succinate oxidation is prevented, However, succinate dehydrogenase can still reduce the carrier A by the reaction: Succinate + A % Fumarate + AH2 The reduced form of A is then used to reduce NAD+. However, this is an energy requiring process, since the oxidation of NADH by A normally results in the formation of one molecule of ATP. The source of energy is the oxidation of TMPD. This is achieved by the following reaction: TMPD + B % oxidized TMPD + BH2 BH2 is then oxidized by the terminal oxidase of the respiratory chain with the formation of an energy-rich compound with the carrier I3, which acts at the third site of phosphorylation in the chain: BH2 + i02 + I3 -> B '^ I3 + H2O This compound may then be used to provide energy for the reduction of NAD+ by AH2 by means of the following reactions B -^ I3 + NAD+ + Ii ^ B + I3 + NAD ^ Ii (where Ii is the carrier acting at the first site of phosphorylation in the chain), followed by: NAD ~ Ii + AH2 ^ A + NADH + Ii + H+ The overall reaction is therefore : Succinate + TMPD + ^©2 + NAD+->Fumarate + oxidized TMPD + NADH + H+ so that the reduction of one component of the chain has been driven by energy produced by oxidation in another. These, and other reactions studied by Tager and Slater are excellent examples of the subtle interactions of energy-yielding and energy-requiring processes that may be demonstrated in the mitochondrion. This is clearly shown in a figure from Slater and Tager (1963) which shows competition for energy-rich compounds by 2,4-dinitrophenol, ATP synthesizing reactions and NAD+ reducing systems (Fig. 16). f 3. THE MITOCHONDRION 127 Perhaps a word of caution is needed in the interpretation of experiments with oligomycin, which is frequently used to study energy-transfer reactions. Although oligomycin eflfectively prevents the formation of ATP from added ADP and inorganic phosphate, it should not be assumed that if a reaction A+I (i) (ii) -*-ADP-« >v^ Glucose -6- P AH2+ B+I-*-^BH2 + A~I < ^"^ »-A+I+ATP — -^^ Glucose Succinate ^< ,> »-NAD*-« \ /-^Glutomofe ■^ ^ADP -* ^^ Glue — ^"^ »-A + I+ATP — -^^ Glue '*\^^ »-NAD*-« v^-^GIutomofe -*^ XA + I + NADH ^^^ Oxoglutorate;!-! (iii) Fumcrafe -*^ XA + I + NADH ^^^ Oxoglutorate ;i-NH3 Fig. 16. Competing reactions for energy-rich compound (r^) formed during oxidative phosphorylation, (i) decomposition by 2,4-dinitrophenol, (ii) synthesis of ATP (as fol- lowed by formation of glucose-6-phosphate in the presence of glucose and hexokinase), (iii) the energy driven reduction of NAD+ by succinate (as followed by the formation of glutamate from a-oxoglutarate plus NH3). A and B are adjacent carriers of the respiratory chain (from Slater and Tager, 1963). observed in the intact mitochondrion is not inhibited by this agent, it must derive its energy from non-phosphorylated intermediates. A good example of this is found in some recent experiments of Kroon (1966). He had observed previously that amino-acid incorporation into protein in isolated mitochon- dria was not seriously inhibited by oligomycin. This had also been observed by Bronk (1963). However, if arsenite was added to inhibit substrate-level phosphorylation, it was possible to demonstrate inhibition by oligomycin. The failure to demonstrate inhibition previously was because the incorpora- tion system was able to use ATP produced by substrate-level phosphoryla- tion which was not inhibited by oligomycin. Thus ATP, and not non-phos- phorylated intermediates, is apparently required for incorporation. A final point of interest is the problem of whether oxidative phosphorylation only occurs in mitochondria. In general it is believed that extra-mitochondrial pathways for NADH and NADPH oxidation are not associated with any synthesis of ATP. However, there have been rather detailed studies of ATP synthesis in nuclei, apparently accompanying oxidative processes (e.g. Osawa, Allfrey and Mirsky, 1957). The nuclear phosphorylation differs in many of its properties from mitochondrial oxidative phosphorylation. The most important difference is that added ADP is not phosphorylated, but the nuclear ATP appears to be synthesized from a pool of endogenous adenine nucleotides. Also, certain inhibitors of oxidative phosphorylation, such as dicumarol, have no effect on nuclear phosphorylation. The matter is discussed in Chapter 2, and the safest interpretation of the present results is that although there is no doubt that isolated nuclei are able to synthesize i 128 D. B. ROODYN ATP, the exact relation to aerobic processes is uncertain. This is particularly so because of the absence of cytochrome oxidase (Hogeboom et al, 1952) and succinoxidase (Roodyn, 1956) from nuclei. G. ION TRANSPORT Recent research has indicated that the transport of ions across the mito- chondrial membrane is a reaction of fundamental importance and is inti- mately dependent on the structural and metabolic integrity of the mito- chondrion. The reader is referred to a recent article by Harris and Judah (1966) for an up to date account of ion accumulation by mitochondria. Mitochondria from a wide range of sources have been shown to be able to take up a variety of ions, including Ca^^, Sr^^, Mg2+, K+ and inorganic phosphate from the medium. It is now becoming increasingly clear that a complex, balanced ion-translocation system exists in the mitochondrial membrane and that ingress of one ion is frequently accompanied by egress of another. For example, Carafoli, Rossi and Lehninger (1964) observed that Na+ is taken up during uptake of Cd?^ and Mg2+ ions, and at the same time there is a loss of mitochondrial K+ into the medium. Another aspect of great interest is that, particularly in the case of Ca2+ uptake it has been possible to show that in certain circumstances there is a stimulation of respiration during ion uptake, and this stimulation bears a stoichiometric relationship to the amount of ion taken up. An excellent demonstration of this is shown in Fig. 17, which is from Rossi and Lehninger (1964). It can be seen that the 10 mw Mg* ,-Mg* Mito^ 10 mM Mg** 120 /.M\\ ^0 150 mm\ Ca + + \atoms 0 Ca^^ n\'^8-8 m/x vatoms 0 sec"*" I 24-4m/A atoms 0 f Fig. 17. Repeated stimulation of oxygen uptake by tightly coupled rat-liver mitochondria on addition of low concentrations of Ca^^ ions. Oxygen uptake indicated by a fall in the trace (from Rossi and Lehninger, 1964). 3. THE MITOCHONDRION 129 addition of low concentrations of Ca2+ results in a burst of oxygen uptake. This process can be repeated several times. A similar result may be obtained with Sr2+ uptake (Carafoli, 1965). The relationship between ion uptake, phosphate uptake and the dense granules of mitochondria is discussed further in Section III. c. below. ^ H. ANABOLIC REACTIONS There is increasing realization that the mitochondrion has considerable synthetic ability. Considering lipid synthesis first, there have been reports of the incorporation of radioactive precursors into the phospholipids of isolated mitochondria. For example, energy dependent uptake of phosphate into phospholipids was observed by Garbus, DeLuca, Loomans and Strong 0963). Using guinea pig liver mitochondria, Hajra, Seiffert and Agranoff (1965) showed that ATP labelled in the y position with ^^p was incorporated into phosphatidyl inositol phosphate. The incorporation observed with free 32Pi is probably via ATP. Hulsmarm (1962) demonstrated the incorporation of acetate into saturated and unsaturated fatty acids by isolated heart sarco- somes. He also observed incorporation of malonate into fatty acids, and concluded that synthesis of fatty acids proceeds via the malonyl-CoA path- way. The synthesis of malonyl-CoA from oxalosuccinate plus acetyl-CoA was reported in a preparation of sonically disrupted rat-heart sarcosomes. (Hiilsmann, 1963). Also, Hiilsmann and Dow (1964) obtained results that indicated that mitochondria from lactating mammary gland could also synthesize fatty acids from malonyl CoA. The role of mitochondria in lipid synthesis has recently been discussed by Hiilsmann etal. (1966). Apart from the enzymes described by Hiilsmann and his co-workers, there have been reports of other systems for lipid synthesis. For example, Webster, Gerowin and Rakita (1965) purified an enzyme from bovine-heart mitochondria that catalysed the synthesis of butyryl-CoA. The enzyme also showed activity towards proprionate, valerate and caproate. Also, the incorporation of i'*C-palmitate into triglycerides, choline and ethanolamine phosphatides by isolated rat-liver mitochondria has been described by Johnson and Kerur (1963). This is a far from complete account of the role of mitochondria in the synthesis of lipids, and there is little doubt these reactions are of consider- able importance, particularly in relation to problems of mitochondrial replication. There have been an increasing number of reports in the last two years of RNA synthesis in isolated mitochondria. The incorporation of ^^C-UTP, GTP, and ATP into the RNA of isolated rat-liver mitochondria has been demons- trated by Neubert and Helge (1965). In intact mitochondria, the incorpora- tion did not require added nucleotides, and was not sensitive to actinomycin C, however, swollen mitochondria that had enhanced permeability showed 130 D. B, ROODYN dependence on added nucleotides and inhibition by actinomycin C. The behaviour of the intact mitochondria was used to exclude the possibility that the observed activity was due to contamination with nuclear RNA polymerase (2.7.7.6.). The inhibition by actinomycin strongly suggested the presence of a DNA-dependent RNA polymerase in mitochondria. Additional evidence has been obtained recently for the presence of this enzyme in mito- chondria isolated from a variety of biological materials [eg. yeast: Winters- berger (1964), a variety of warm-blooded animals: Neubert, Helge and Merker, (1965), lamb-heart: Kalf (1964) and Neurospora crassa: Luck and Reich (1964)]. Kalf (1964) also observed that amino acid incorporation into protein was inhibited by actinomycin D, and concluded that mitochondrial protein synthesis is dependent on a continually functioning RNA polymerase. A similar conclusion was reached by Kroon (1965). The subject is developing very rapidly at the present moment, and there is little doubt that the next few years will see an intensive investigation of the mechanism of RNA synthesis in mitochondria. Although there have been several reports of the labelling of mitochondrial DNA in vivo by ^H-thymidine (for example in Tetrahymena pyriformis: Par- sons, 1965) the author is not aware of any reports of the demonstration of DNA synthesis in isolated mitochondria. Perhaps the best established example of anabolic activity of the mito- chondrion is the ability of isolated mitochondria to incorporate radioactive amino acids into protein. Incorporation of a range of labelled amino acids has been demonstrated in mitochondrial fractions from very diverse sources (See Roodyn, 1965b for a list of these). There is little doubt that this activity is a general property of the mitochondrion. The properties of the incorpora- tion system are similar in all mitochondrial fractions examined, and differ from microsomal incorporation in being insensitive to rather massive amounts of added ribonuclease (2.7.7.16,17.), showing complete dependence on oxidative phosphorylation, having more prolonged progress curves (i.e. in- corporation can continue for 1-3 hr, rather than 10-30 min) and being in- hibited by chloramphenicol. Although some of the early reported values of cts/min/mg protein were rather low, recent improved methods have given activities that are at least comparable to those obtained with microsomal systems, particularly if disrupted mitochondria are used (Kroon, 1965). The essential properties of the incorporation system in rat-liver were summarized recently (Roodyn 1966) and are given in Fig. 18. In the case of rat-liver, at least, it is reasonably certain that the incorporation is not due to bacterial, nuclear, lysosomal or microsomal contamination. Bacterial and microsomal contamination are also very unlikely in the case of muscle mitochondria. The incorporation is dependent on the maintenance of the structural integrity of the mitochondrial membrane system and its ability to carry out oxidative phosphorylation. With well-washed rat-liver 3. THE MITOCHONDRION 131 ■piitochondria, there is a clear requirement for added Mg2+ ions, Pi, adenine nucleotides and one of a range of oxidizable substrates. Not all preparations, however, have been reported to show a requirement for added substrates, d presumably these use endogeneous substrates. The major site of in- rporation is into insoluble proteins associated with the mitochondrial Exogenous amino acids 'AMP Mifochondria! membrane Endogenous amino acids incl. ALA GLU ASP i,_»^ Activated mino acids Mg2*^ ATP y:^ Endogenous FU substrate aOG OA /JOHBl cis AC ;]l\^_^/x~p ( Insol pr. RNA pr. Cell sap fRMAlf^EDTA Contaminant I Microsomes ADP Oxidized - Coenzymes Mg2*j Reduced - Coenzymes I ^ a c -RNcse - Poly U ^~^ -Sucrose for tonicity -HgO Exogenous substrate CO5 Structural attack ' Freeze ttiawing Detergent Higti speed blodes "iG. 18. Summary of factors affecting amino acid incorporation into protein by isolated rat-liver mitochondria. Thick lines represent direction of incorporation of labelled amino acids, thin continuous lines ancillary reactions involved in this, and broken lines (and question marks) areas of uncertainty. ALA: alanine, ASP: aspartate, cis AC: cw-aconitate, CT: citrate, FA: fatty acid, FU: fumarate, GLU: glutamic acid, MA: malate, aOG: a-oxoglutarate, j9-(OH)Bu: /3-hydroxybutyrate, OA: oxaloacetate, PP: pyrophosphate, PolyU: polyuridylic acid, PY: pyruvate (from Roodyn, 1966). I Ve I membrane. In Uver mitochondria it has been shown that the labelled material shows some resemblance to the insoluble structural protein described by Criddle, Bock, Green and Tisdale (1962) (see Section III. f. below). The in- soluble proteins were closely associated with the mitochondrial membrane and were extremely difficult to purify by conventional methods of protein fractionation (Roodyn, 1962). It was further shown that the enzymes cyto- chrome c, malate dehydrogenase and catalase, present in the mitochondrial fraction were not labelled in vitro (Roodyn et al., 1962). The physiological state of the animal affects the incorporation. In particular, thyroid hormones given in vivo stimulate the incorporation observed in vitro (Roodyn, Freeman and Tata, 1965). There are reports of other synthetic reactions in isolated mitochondria, for xample the synthesis of para-aminohippuric acid (Kielley and Schneider 1 950). The formation of protoporphyrin and haem has been reported by Rimington 132 D, B. ROODYN and Tooth (1961) and related to this is the report of the synthesis of 8-amino- laevulinic acid by isolated mitochondria (Granick and Urata 1963). Also, Nishida and Labbe (1959) have demonstrated the conversion of protopor- phyrin to haem by using ^spe as a tracer. Space does not allow a complete description of such reactions. However, the point to be stressed is that it is now clearly not correct to regard the prime function of the mitochondrion as the "power-house" of the cell, i.e. to see its role merely as a provider of ATP for endergonic reactions. Whilst there is no doubt that the catabolic and ATP-producing aspects of mitochondrial metabolism are of the greatest importance, the anabolic aspects of the mitochondrion seemed to have received scant attention until the last five years or so. It is hoped by the author, at least, that this bias is now corrected, and that the many fascinating impli- cations of examining the mitochondrion as an integrated biosynthetic organelle will now be fully explored. I. OTHER REACTIONS One could include under this heading a truly massive literature, and it is impossible to provide a full account of these reactions here. Instead a few interesting examples will be taken, that are particularly relevant to other aspects of mitochondrial metabolism discussed in the article. 1. Amino Acid Metabolism There are many enzymes in mitochondria involved in the various aspects of amino acid metabolism. Several amino-transferases have been reported in mitochondria, but it is interesting that as with certain of the enzymes of the tricarboxylic acid cycle, the activity is not uniquely localized in the mito- chondrial fraction, but also appears in the soluble fraction. There are good indications that the soluble enzymes are different proteins, and not simply released from the mtiochondria during isolation. For example the soluble and mitochondrial aspartate aminotransferases (2.6.1.1.) have many different properties (for example different pH-activity curves : Sheid, Morris and Roth, 1965). The soluble and mitochondrial alanine aminotransferases (2.6.1.2,12) also appear to be different proteins. The mitochondrial enzyme is more unstable and has a lower Km than the soluble enzyme (Swick, Barnstein and Stange, 1965). Morino, Kagamiyama and Wada (1964) extended their previ- ous observation that crystalline mitochondrial and soluble beef-liver aspartate amino-transferases differed in physico-chemical and kinetic properties by showing that rabbit antisera prepared against the mitochondrial enzyme did not react with the soluble enzyme and vice versa. They also made the inter- esting observation that mitochondrial enzymes from a variety of different tissues were inhibited by the anti-mitochondrial enzyme serum, and the corresponding soluble enzymes were inhibited by the anti-soluble enzyme I 3. THE MITOCHONDRION 133 kL y^i serum. TTius there was greater immunological similarity between enzymes from the same organelle in different tissues than between enzymes from different structures in the same tissue. Another interesting aspect of the aminotransferases is that the enzymes attacking the various amino acids are by no means equally distributed be- een mitochondria and the soluble fraction. For example, the tyrosine amino-transferase activity (2.6.1.5.) of rat-liver mitochondria is far less than the aspartate or alanine aminotransferase activities, (Litwack, Sears and Diamondstone, 1963; Kenney, 1962). Glutamate dehydrogenase is a key enzyme of mitochondrial amino acid metabolism. It has been known for many years that there is an active gluta- mate dehydrogenase in liver mitochondria (Hogeboom and Schneider, 1953). More refined fractionation methods have established without doubt that this enzyme is clearly associated with mitochondria, and in fact may be regarded as a "marker" enzyme for mitochondria (see Beaufay et al, 1959). Because of the presence of glutamate-linked aminotransferases in mitochondria, it be- came generally accepted that they were the site of what may be called the "classical" transamination cycle. This is described fully in Braunstein (1957) and is shown in Fig. 19a. It is this cycle which is responsible for the production of ammonia during the oxidation of amino acids. Recent work, however, particularly of Tager, Papa, Quagliariello and their associates has emphasized the importance of another mechanism that does not give rise to ammonia. This is shown in Fig. 19b. Although transamination Scheme (a) Of Amino ocids cOxo-acids Scheme (b) «Amino acids X aOxo-glutarate Glutamate 2H + NH3 H2O Amino transferase Glutamate dehydrogenase CO. «Ammo acics n /" aOxo-acids >V 1O2 cfOxo-glutarate Glutamate Succinate - [Tricarboxylic acid cycle [s^ io. X Aspartate Oxaloacetafe Fumorate ■H?0 Amino transferase Glutamate dehydrogenase Malate- FiG. 19. (a) "Classical" transamination cycle for de-amination of amino acids to form 1 ammonia (see Braunstein, 1957). (b) Alternative cycle for transamination of amino acids to form aspartate (see Quagliariello and Papa, 1964). I 134 D. B. ROODYN Still occurs, the glutamate is not reconverted to a-oxoglutarate by the gluta- mate dehydrogenase reaction, but reacts by means of aspartate aminotrans- ferase with oxaloacetate. This has been formed by oxidation of another molecule of a-oxoglutarate in the tricarboxylic acid cycle. The result is the formation of aspartate, and the regeneration of glutamate. This cycle is really an extension of the reactions studied by Krebs and Bellamy (1960). The effect of the "transamination cycle" is therefore to transfer the a-amino groups of amino acids to aspartate, which may then be used in synthetic reactions, or for the synthesis of urea in the urea cycle. QuagHariello, Papa, Saccone, Palmieri and Francavilla (1965) have recently shown that both the "classical" and "transaminase" routes for glutamate metabolism occur in rat Uver mitochondria. However, ammonia production decreases with time, and the transamination route is more important in prolonged incubations. One well-established observation that is relevant to this question is that in intact mitochondria the glutamate dehydrogenase activity is very low. In order to activate the enzyme fully, the mitochondria have to be disrupted by drastic procedures, such as by forcing under high pressure through a small orifice (Hogeboom and Schneider, 1953) or by exposure to high-speed blades, osmotic shock or detergents (Bendall and Duve, 1960). It may be noted that the phenomenon of latency of mitochondrial enzymes is not confined to this example, but has been observed with several other enzymes. For example the atency of rhodanese (2.8.1.1.) was the subject of a careful study by Greville and Chappell (1959). Another interesting aspect of glutamate dehydrogenase activity that has emerged recently is that although the purified enzyme can react equally with NAD or NADP, and it had previously been thought that the enzyme was in fact NAD-Unked (Hogeboom and Schneider, 1953), it is now thought Ukely that most of the oxidation of glutamate in intact mitochondria results in the formation of NADPH in the first instance. This is then used to reduce NAD+ by means of an energy-linked transhydrogenase reaction. This is discussed more fully in QuagHariello et al. (1965). There are numerous other reactions of amino acid metabolism in mito- chondria. For example the oxidation of proUne to Ai-pyrroline-5-carboxylate was demonstrated in rat-Hver mitochondria by Johnson and Strecker (1962) and the reaction was related to the known inter-conversion of proline to glutamic acid. The oxidation of proline has been the subject of several other studies. For example, Brosemer and Veerabhadrappa (1965) observed that the enzyme for the conversion of proline to Ai-pyrroline-5-carboxylate is tightly bound to the mitochondria, whereas that for the conversion of this compound to glutamate is more loosely attached. The mitochondrion is also capable of oxidizing methylated glycines, choline, betaine aldehyde and related compounds (see for example Williams, 1952a, b; Hoskins and MacKenzie, 1961; Hoskins and Bjur, 1964; Bianchi and Azzone, 1964). It is therefore I 3. THE MITOCHONDRION 135 well established that the mitochondrion is the site of complex interactions between amino acids and their various derivatives. 2. Hydrolases This is another group of enzymes of considerable importance in mito- chondria. However, because of the serious and technically difficult problem of lysosomal contamination of mitochondrial fractions, it is difficult to be certain which hydrolases are truly mitochondrial. Perhaps the best established case, however, is that of ATPase. Considerable ATPase activity has been demonstrated in mitochondria from a very wide range of tissues, and there is little doubt that it is a fundamental mitochondrial enzyme. The ATPase activity is generally latent in intact mitochondria but is greatly increased if the mitochondria are damaged by a wide variety of treatments, or by addi- tions of agents that uncouple oxidative phosphorylation, such as 2,4-dinitro- phenol. In the last 10 years there has been extensive research into the precise relation between ATPase activity, the structural state of the mitochondrion and the reactions of oxidative phosphorylation. It is quite likely that at least part of the ATPase activity observed in dam- aged mitochondria is due to reversal of the terminal reactions of oxidative phosphorylation, for example by hydrolysis of X '-«-' P. Thus instead of the reaction: X ~ P + ADP -> X + ATP hydrolysis of X '^ P causes reversal of this reaction by removal of one of the reactants i.e. X -- P + H2O -> X + Pi ATP + X -> ADP + X '-' P Sum: ATP + H20->ADP + Pi Net hydrolysis of ATP results. The effect could also be due to "discharge" of one of the non-phosphorylated energy rich intermediates, with resultant failure to form X '--' P. It could also be caused by the physical disorganiza- tion of the '^ P acceptor system, so that water is used as an acceptor, instead of ADP. The agent 2,4-dinitrophenol probably acts by the discharge of energy-rich intermediates, while activation of ATPase by such treatments as repeated freezing and thawing probably acts by the latter mechanism. An important finding in this respect is that oligomycin not only inhibits oxidative phosphorylation, but it also inhibits the dinitrophenol-stimulated ATPase (Lardy, Johnson and McMurray, 1958). As a result, the combined use of these two agents has been of great help in examining the terminal stages of oxidative phosphorylation, and of the so-called "partial reactions" (e.g. ATP/Pi exchange, ATP/ADP exchange and H2O/ATP isQ exchange). 136 D. B. ROODYN The relation between these reactions, ATPase and the sites of action of oligomycin and dinitrophenol is given in Fig. 20 (from Lehninger, 1965). The interesting theory of the role of ATPase in oxidative phosphorylation presented by Mitchell has been discussed above (see Section II. f.). Ared + I Pi-HzO exchange ATP- Pi exchonge and ATPase Oligomycin] Fig. 20. "Partial reactions" of oxidative phosphorylation and sites of action of 2,4-dinitro- phenol and oligomycin (from Lehninger, 1965). I In their attempts to fractionate the components of the respiratory chain phosphorylation system Penefsky, Pullman, Datta and Racker (1960) purified a soluble ATPase that had the ability to restore phosphorylation activity to suitably treated respiratory particles. It is an example of one of the several "coupling factors" described by this and other groups. The enzyme has the interesting and unusual property of being more unstable at 0° that at room temperature (Pullman, Penefsky, Datta and Racker, 1960). There have been many other investigations into the relationship between ATPase and the reactions of oxidative phosphorylation. An interesting example is from work done several years ago by Myers and Slater (1957). They studied the effect of pH on mitochondrial ATPase acitvity and obtained a complex pH-activity curve. They suggested that this was due to the super-imposed pH-activity curves of four different ATPases. Three of these, with pH optima at pH 6- 3,7-4, and 8-5 were stimulated by dinitrophenol and it was suggested that they were specific ATPases acting at the three sites of ATP production in the respiratory chain. Another important aspect of mitochondrial ATPase activity is its relation- ship to contraction of the membrane, and the possibility that there is a mem- brane bound actomyosin-like ATPase in mitochondria. In many ways there are similarities between the properties of the mitochondrial and muscular contraction systems (see Lehninger, 1965 for a discussion of this) and indeed there have been reports of the isolation of actomyosin-like "contractile pro- teins" from mitochondria (Neifakh and Kazakova 1963, and Ohnishi and 3. THE MITOCHONDRION 137 Ohnishi, 1962). Ohnishi, Kawamura, Takeo and Watanabe (1964) have exten- ded their studies to the isolation of two proteins from rabbit liver mitochon- dria, one resembling actin, and the other resembling myosin. It may be concluded at this point that mitochondrial ATPase is clearly closely involved in the function and structure of the mitochondrial membrane system. There have been reports of many other hydrolases in mitochondria and, as stated above, it is often difficult to assess the contribution of lysosomal contamination to these activities. One enzyme, however, is certainly not due to lyososomes present in the mitochondrial fraction. This is the alkaline DNase (3.1.4.5.). Unlike the acid DNase, which is a typical lysosomal enzyme, this enzyme clearly follows mitochondrial marker enzymes, such as cytochrome oxidase, in analytical gradient centrifuging (Beaufay et al., 1959). The role of mitochondrial DNase is far from clear, but it may be associated in some way with the metabolism of the recently discovered mitochondrial DNA (see Section III. b.). There is some doubt as to whether there are proteolytic enzymes in mitochondria. The classical "four-fraction" scheme of analysis (i.e. the isolation of nuclear, mitochondrial, microsomal and soluble fractions) revealed a wide range of proteolytic enzymes and amidases in the mito- chondrial fraction. (For example, see Maver and Greco, 1951). More sophisti- cated schemes of fractionation, however, revealed that catheptic activity was concentrated in the lysosome-rich mitochondrial fraction (de Duve, Pressman, Gianetto, Wattiaux and Applemans, 1955) and "cathepsin" (a somewhat loose term) is now regarded as a typical lysosomal enzyme. However, some proteolytic activity has been ascribed to mitochondria. For example a CoA, ATP activated enzyme hydrolysing serum albumin was reported in rat-liver mitochondria by Penn (1960). Alberti and Bartley (1963) ascribed the increase in concentration of free amino acids that occurs on incubation of mitochondria in vitro to the action of soluble intramitochondrial proteolytic enzymes, but it is not certain that they could exclude the possibility of lysosomal contamination. A similar conclusion was reached by Baird (1964). The level of free amino acids in isolated mitochondria is surprisingly high, even in mitochondria that have been thoroughly washed (Roodyn et al., 1961). This may be partly due to the action of proteolytic enzymes in the mitochondria, acting during the course of isolation. The production of amino acids by isolated rat-liver mitochondria has recently been examined in more detail by Alberti and Bartley (1965) and the results correlated with the metabolic state of the mitochondrion. One interesting conclusion of these workers was that release of amino acids during incubation of mitochondria in the presence of radioactive amino acids can cause dilution of the label, and an apparent fall in incorporation into protein. Thus when Alberti and Bartley (1965) corrected the incorporation curve obtained by Roodyn et al. (1961) for this effect, a straight line was obtained 138 D. B. ROODYN over a 2 hr period. Thus proteolytic activity in mitochondrial preparations may have an important effect on the results of metaboUc studies, particularly if long incubation periods are used. A most interesting aspect of the amino acid release by mitochondria is that it is inhibited by chloramphenicol (Alberti and Hartley, 1965) which is also a powerful inhibitor of mitochondrial protein synthesis. 3. Other Enzymes It is impossible to deal here with the many other enzymes that have been detected in mitochondria from various sources. Simply giving two random examples, one may mention the enzyme rhodanese which is clearly associated with liver mitochondria and exhibits interesting activation and latency properties (Greville and Chappell, 1959). Secondly, the enzyme phosphoenol pyruvate carboxykinase (4.1.1.32.) has been demonstrated in mitochondria from various tissues. The amount varies somewhat with different mitochondria. For example, rabbit liver mitochondria are far more active than rat-liver mitochondria (Nordhe and Lardy, 1963). The enzyme is relatively soluble and there is considerable enzyme activity in the soluble fraction of the cell. It is interesting that there is evidence that the mitochondrial and soluble enzymes have certain differences in their properties, and may well be two different enzymes (Holten and NordUe, 1965). J. SUMMARY AND CONCLUSIONS The author hopes that the reader will accept the many ommissions and oversimplifications in the above account of the metaboHc activity of the mitochondrion. A most salutary method of proving the extraordinary wealth of reactions that have been described in isolated mitochondria is to follow the entries against "mitochondria" in the Chemical Abstracts. Any system of indexing or literature abstraction soon breaks down under the assault of the volume of material. Perhaps it is now no longer reasonable to attempt such a survey, any more than it is reasonable to survey the properties of cells. However, if the reader is left with the impression that the mitochondrion is an organelle possessing a wide range of interacting metabolic activities, this survey has not been completely without value. However, the reader is recom- mended to consult the appropriate reviews and monographs cited in the text for fine details of the above reactions. III. The Structural Basis of Mitochondrial Function Surveying the sub-cellular distribution of enzymes (see Roodyn, 1965a) is made relatively easy by the fortunate circumstance that the "four-fraction" 3. THE MITOCHONDRION 139 ^^late 1940's and early 1950's was generally adopted by large numbers of ^^ workers. It is thus possible to list activities in the nuclear, mitochondrial, ^■microsomal and soluble fractions with relative ease, and to compare the ^^^ results of different workers. Unfortunately, no one scheme for the sub- ^^ fractionation of mitochondria appears to have been generally adopted, and it ^^pis frequently difficult to compare the results obtained with different "sub- ^^^ mitochondrial" particles. Also, centrifugal analysis of disrupted mitochondria does not result in such clear cut fractions as is the case with the fractionation of disrupted cells. There is a broad division between "soluble" and "insoluble" mitochondrial components, but the "insoluble" components actually include a range of sub-mitochondrial fragments (Watson and Siekevitz, 1956). Although there have been many studies of "compartmentation" in mito- chondria, it is difficult to establish the structural basis of these compartments K with a great degree of certainty. For these reasons, sub-mitochondrial struc- ^■ture will only be considered under the three main headings of mitochondrial ^Bsap, mitochondrial inclusions and the mitochondrial membrane system. A. MITOCHONDRIAL SAP It is well established that isolated mitochondria, even after several washes, contain many soluble components. These include a variety of nucleotides and phosphate esters, a considerable amount of free amino acids, many different cations, and many soluble enzymes. Work on mitochondrial fluid spaces has indicated that there are two separate spaces, one more accessible to added solutes than the other. It is generally believed that the easily accessible space lies between the inner and outer membranes (and thus corresponds to the "outer compartment" or "cristae space"), whereas the less accessible space is the true "matrix" of the mitochondrion and is bounded only by the inner membrane (i.e., it may be called the "inner compartment".) Unfortunately, it is not possible in all cases to allocate in a quantitative fashion the various soluble components of the mitochondrion to the inner or outer compart- ments. However, as a result of combined enzymic, chemical and electron- microscope studies, Klingenberg and co-workers have recently been able to go some way towards demonstrating more precisely the location of some mitochondrial components. A model suggested by these workers (Heldt, 1966; Klingenberg and Pfaff, 1966) is given in Fig. 21 which shows the posi- Ition of several enzyme systems and nucleotides, together with the supposed site of permeability barriers to these substances. Other workers have frequently assigned activities to "inner membranes" or "outer membranes", but generally the correlation that is obtained with the actual mitochondrial membranes is not as well established as in the above work. An example of a rather complex chart of this kind is that given 140 D. B. ROODYN Inner compartment (matrix Outer compartment (cristae) ATP- .AMP. Creatine ^ l^ kinase r„'^~' Creatine' ■ADP Creatine Succinyl CoA Fatty acid activation phosphoenol pyruvate corboxy kinase nhibition by atractyloside Fig. 21. "Compartmentation" of adenine nucleotide linked reactions of mitochondria (adapted from Heldt, 1966 and Klingenberg and Pfaflf, 1966). by Chappell (1961) to describe the suggested location of certain co-factors and enzymes involved in integrated oxidations in isolated mitochondria. This is given in Fig. 22. Several other similar diagrams have been presented to illustrate different aspects of mitochondrial metabolism (cf. Figs. 7 and 8). Outside Membrane Succinate dehydrogenase Fumarase Malate D NA[ Oxoioacetate i Pyruvate Inhibition — Glutomate, /3-Hydroxybutyrate dehydrogenases, Diaphorose Inside Isocitrate dehydrogenase Oxoloacetate i-* — -^ DNP, arsenate Phosphoenol - pyruvate Fig. 22. Postulated spatial relationships of some mitochondrial enzymes (DPNi, DPN2 are bound and free forms of NAD; D dehydrogenase; TPN = NADP) (from Chappell, 1961). 3. THE MITOCHONDRION 141 In spite of the attraction of such diagrams, the precise location of sub- mitochondrial components is certainly not as well known as generally thought. In the particular case of soluble proteins, definition of the term "soluble" is in fact rather difficult. (See Chapter 8 for a general discussion of this question.) One cannot simply state that a protein is either "soluble" or "insoluble" without more precise specification of the method of extraction of the protein. For example, it is well established that many proteins can be removed from mitochondria simply by washing them in salt media. For example, Dallam (1958) was able to extract 70% of the dry weight of rat-liver mitochondria by repeated washing with 0-16 m NaCl. At the other extreme, some proteins are only removed after total disruption of the mitochondria, for example by high concentrations of detergents. In a careful study of the mechanism of disruption of mitochondria by deoxycholate, Watson and Siekevitz (1956) showed that this does not occur by simple lysis of an all-or- none nature, with a sudden release of the soluble contents. Disruption is not simply bursting of a bag. By layering deoxycholate over a mitochondrial pellet and examining sections at different levels of the pellet, they were able to show that a series of changes occur under the influence of the disrupting agent. The mitochondria first swell greatly, and then they contract to about twice their original diameter. There follows dilution of the contents of the mitochondrion, followed by severe emptying and finally the formation of membranous elements. Thus the agent produces a series of changes, with progressive loss of mitochondrial material, rather than sudden lysis. These changes are shown in Fig. 23. Support for these results comes from studies of the disruption of mito- chondria with the Triton series of alkylphenoxypolyethoxyethanol detergents (Roodyn, 1962). Firstly, it was found that the ability to disrupt mitochondria depended greatly on the length of the side-chain of the detergent molecule. /For example, concentrations of Triton-X-lOO (with 9-10 ethylene oxide groups in the side chain) and Triton-X-305 (30 ethylene oxide groups) which had approximately the same surface active effect (i.e. would be expected to produce the same fall in surface tension) had very different effects on the mitochondrion. 0-1 % Triton-X-100 produced complete lysis, whereas 0-2% Triton-X-305 had no effect. One could conclude that the action of the deter- gent depended on specific interaction with binding sites on the membrane that had to be of suitable size. This interaction results in loosening of mem- brane structure. Therefore increase in detergent concentration should result in more binding and a progressive loosening of structure. This was then confirmed by so-called "titration" experiments, in which mitochondria were exposed to increasing concentrations of detergent, and the release of various fejomponents followed. It was found that there were a series of "extraction ■curves" for the various components studied (malate dehydrogenase, total protein, succinoxidase, phospholipid, RNA and protein labelled after 3. THE MITOCHONDRION 143 L.,.„„..„.., . ^Bn Fig. 24, and it is clear that there is no sudden jump from "intact" to ^^'disrupted" mitochondria, but rather a series of sequential extractions of the various components. 0-1 0-2 0-3 Concn. of detergent (°/o,v/v) MG. 24. Sequential extraction of components from rat-liver mitochondria by treatment with increasing concentrations of the neutral detergent, Triton-X-100. D malate dehydro- genase, ■ total protein, A phospholipid, A succinate oxidase, # radioactive protein after incubation in vitro with "C-valine, C RNA (from Roodyn, 1962). It is therefore reasonable to imagine that the mitochondrion is not a simple bag containing a "solution" of enzymes of equal solubility, but rather contains a spectrum of components with a wide range of affinity for the most insoluble of the mitochondrial structures (presumably the structural proteins of the membrane — see Section III. f. below). There therefore may be little meaning in simply listing the "soluble" enzymes of the mitochondrion. If we define the term soluble as meaning that the enzyme is released into solution after dis- ruption of the mitochondria, such a list would be quite large, and would include the enzymes involved in the /3-oxidation of fats, several aminotrans- ferases, several dehydrogenases, including malate dehydrogenase, isocitrate dehydrogenase, and glutamate dehydrogenase, fumarase, rhodanese, amino acid activating enzymes, certain phosphatases and phosphotransferases. Fig. 23. Changes during disruption of mitochondria by 0-3% deoxycholate. The deoxy- cholate was layered over a pellet of intact mitochondria and sections taken at diflferent levels of the pellet. (A) bottom of pellet shows intact mitochondria (wi), and some begin- ning to swell (ms); there is some microsomal contamination {p.). (B) middle of pellet, showing greatly swollen mitochondria. (C) top of pellet: mitochondria have shrunken somewhat and the matrix has become greatly diluted. (D) surface of pellet, showing free membranes with complete loss of matrix. / 30,000 (from Watson and Siekevitz, 1956). 144 D. B. ROODYN the ATP/ADP exchange enzyme, cytochrome c, choline oxidase (1.1.99.1), and other enzymes. However, the procedures used to solubilize these various enzymes are different in many cases, and in order to be accurate one would have to specify the precise conditions of extraction. In order to emphasize that extractability is a very arbitrary definition of solubility, it may be pointed out that the most insoluble of all mitochondrial proteins, the structural protein, may in fact be extracted by a concentrated mixture of suitable detergents (Criddle et a/., 1962). In a recent study of this problem, Klingenberg and Pfaff (1966) divided mitochondrial enzymes into three groups: 1. "easily associated", 2. "tightly enclosed" and 3. "structurally bound". These corresponded most probably to enzymes in the outer compartment, matrix and membrane, respectively. Enzymes in group 1 are easily extracted with salt media and include phospho- transferases, adenylate kinase (2.7.4.3.), creatine kinase (2.7.3.2.), the ATP/ ADP exchange enzyme and cytochrome c. Enzymes in group 2 may be dissociated from the mitochondrial structure by mechanical means, such as o o o o o o o o o o o o o o o o o o o o o Model (a) Model (b) Model (c) Membrane Membrane Fig. 25. Three models of mitochondrial structure showing possible binding of soluble proteins, (a) Proteins are either free or membrane-bound, (b) Intermediate situation in which proteins fall into several groups, including no binding, loose binding, and tight binding, and (c) Proteins have a continuous spectrum of binding. (Strength of binding is indicated by number of lines between molecules). 3. THE MITOCHONDRION 145 sonic vibrations, and include many NAD and NADP linked dehydrogenases (such as malate dehydrogenase, isocitrate dehydrogenase, glutamate dehydro- genase and lipoyl dehydrogenase (1.6.4.3.)), and a proportion of the amino- transferases, phosphotransferase, cytochrome c and ATPase. Finally the "structurally-bound" enzymes in group 3 are only released by detergent and include cytochromes a, 03, ci and b, succinate dehydrogenase, NADH dehydrogenase, and some of the ATPase. Thus the mitochondrion contains a number of components of varying solubility that can be released by more or less violent treatments. It is preferable not to talk simply of "soluble" or "insoluble" but to specify the treatment needed to detach the component from the least soluble structural elements of the mitochondrion. Three possible models of mitochondrial structure that illustrate this question are given in Fig. 25. In the first (Fig. 25a) the mitochondrion is depicted as consisting of a bag of soluble enzymes. Insoluble enzymes are attached to the inner surface of the bag, and represent the membrane-bound enzymes. In the second model (Fig. 25b) there are some completely soluble elements within the bag, but the walls of the bag are in fact multilayered with a range of more or less tightly bound components. In the third model (Fig. 25c) there are no "soluble" or free components at all, but rather a continous spectrum of binding from extremely tight binding to extremely weak binding. (These models ignore the question of compart- ments within the mitochondrion, and clearly may be drawn in a more compli- cated fashion). Of the three models that represent the intermediate situa- tion, Fig. 25b is probably the most accurate on present evidence. B. MITOCHONDRIAL INCLUSIONS : DN A The term "inclusion" includes any large ordered structure lying in the mitochondrial sap. The density of the matrix is generally rather high in intact mitochondria, and it is difficult to make out any fine structure within it. For this reason there is considerable uncertainty as to whether there are intra- mitochondrial ribosomes or not. There has been a tendency to regard the matrix as a rather homogeneous material. However, this may be false, and more refined electron microscopy techniques may reveal structured elements hitherto not noticed. For example, sections of mitochondria have been examined for many years, and yet it is only recently that the presence of DNA fibrils has been well established. DNA is the most important of the "inclusions". The history of the discovery of mitochondrial DNA is an interesting example of how an oversimplified view can easily develope into a false dogma that can then retard the develop- ment of further research. The false dogma in this case is that of the exclusively nuclear localization of DNA. Although there has been considerable genetic evidence for the existence of systems of cytoplasmic inheritance for many 146 D. B. ROODYN years and although there were several well-authenticated examples of cyto- plasmic DNA (see Gahan and Chayen, 1965), until very recently, most biochemists, at least, held the view that in "normal" cells, such as rat-liver, all the DNA was in the nucleus. Since this would have made DNA a very useful chemical marker for nuclei, the author was rather surprised to find in surveying the evidence for this view that in fact there were very few experi- ments in which it had been rigorously established that 100% of the DNA was in the nuclear fraction (Roodyn, 1959). Thus, in most fractionations in which the bulk of the DNA (90-95% of the total) had been recovered in the nuclear fraction, no analyses had in fact been carried out on the cytoplasmic fractions. Although there are clearly great technical difficulties in establishing, for example, that 1 % of the total DNA of the cell found in the mitochondrial fraction is not entirely due to contamination with nuclear fragments, it is an extraordinary illustration of the power of the dogma of the exclusive nuclear localization of DNA, that of the thousands of studies on mitochondria in the fifties and early sixties, there were extremely few reports of DNA assays in the mitochondrial fraction. Yet the discovery of mitochondrial DNA may well lead to a revolution in our thinking about mitochondrial function. Chevremont, Chevremont-Comhaire and Baeckeland (1959) studied tissue-culture fibroblasts by autoradiography and cytochemical means, and reported the presence of DNA in mitochondria in these cells. Later they found that treatment of these cells with DNase resulted in greatly increased DNA contents in the mitochondria, as judged by the Feulgen stain. (Chevre- mont, Baeckland and Chevremont-Comhaire, 1960). However, it was not until the demonstration by Nass and Nass (1963) that fibrous inclusions frequently observed in the matrix of mitochondria (e.g. Nass and Nass, 1962) were in fact DNA fibrils that serious attention was focused on the possible existence of mitochondrial DNA. The DNA fibrils, which were present either in a clumped or dispersed state, were observed in mitochondria from a wide range of biological material (Nass et al, 1965) and there can be little doubt that they are present in all mitochondria. A typical example of these fibrils (in mitochondria in a gastropod) is shown in Fig. 26. The conclusion that DNA is present in all mitochondria appears to be confirmed by the isolation of DNA from mitochondria from a wide range of organisms. Thus preparations of DNA that are distinct from nuclear DNA have been isolated from mitochondria from rat, rabbit, pigeon, chick-embryo liver, chick- embryo and beef-heart, a variety of plant tissues, Neurospora crassa, Tetrahymena pyriformis, Paramecium aurelia, and Saccharomyces cerevisiae. A full survey of these findings is given elsewhere (Roodyn and Wilkie, 1966a) Fig. 26. DNA-fibres in mitochondria of larval ectoderm of the gastropod Littorina littorea. Fixed in 2% Os04 in sea water. The fibres have clumped to form straight stubby rods (arrows) connected to the cristae by finer fibres x 64,000 (from Nass et al., 1965). I 3. THE MITOCHONDRION 147 148 D. B. ROODYN and it is clear that interest in mitochondrial DNA is growing very rapidly. The mitochondrial DNA is double-stranded and generally, but not always, has a lower specific gravity than nuclear DNA from the same organism. It has a different melting temperature and base composition. Caesium chloride density gradients of mitochondrial and nuclear DNA from chick embryo liver are shown in Fig. 27. These results are from Rabinowitz, Sinclair, ■707 1-686 1-698 Density, GM/CC Fig. 27. Sedimentation profiles in caesium chloride gradients of DNA from nuclear and mitochondrial fractions of chick-embryo liver. The band at /> = 1-740 is a marker DNA (from Rabinowitz et al., 1965). DeSalle, Haselkorn and Swift (1965). Mitochondrial DNA of considerable size has been isolated from Neurospora crassa by Luck and Reich (1964). It appeared under the electron-microscope as a thread of length 6-6 /x, corresponding to a molecular weight of about 13 x 10^. This rather striking thread is shown in Fig. 28. Tewari, Jayaraman and Mahler (1965) have recently made a careful study of DNA from yeast mitochondria, and have shown that mitochondrial DNA is very different in its properties (e.g. in its thermal transition properties) from DNA associated with purified yeast lactate dehydrogenase (cytochrome ^2) (1.1.2.3.) that is also associated with mitochondria. This DNA is a single strand of only 33 deoxyribonucleotides (Mahler and Pereira, 1962). In the slime mould Didymium nigripes, the DNA in the mitochondria is located in a characteristic "core" that appears to consist of tightly coiled fibrils, and certainly has all the appearance of an organized structure (Schuster, 1965). It is clear from this work and that of Nass and Nass that the DNA fibrils do not lie in a randomly mixed "bundle" within the mitochondrion. 3. THE MITOCHONDRION 149 Mitochondria from certain cells, particularly in the nervous system, have been found to contain definite fibrous elements in the matrix. A particularly striking example of this was found by Mugnaini (1964) in certain glial cells (classified as astrocytes). In some mitochondria, filaments with a very clear helical Fig. 28. Single molecule of DNA from mitochondria of Neurospora crassa. Length of molecule is 6-6 /x. x 55,000 (from Luck and Reich, 1964). structure almost entirely filled the intracristal space. A model of such a spiral filament is shown in Fig. 29. It is not yet certain whether these structures are related to DNA-containing fibrils or not. The relationship of DNA to the mitochondrial membrane is a question of some interest. Nass et al. (1965) observed that although the main mass of DNA fibrils appeared to lie in the matrix, it was frequently connected to the mitochondrial membrane or to the cristae by several fine fibrils. However it was not certain whether this was the result of fixation. Kroon (1965) has recently found that sub-mitochondrial particles prepared by digitonin lysis of beef-heart mitochondria were considerably enriched in DNA, relative to the intact mitochondria. This would suggest a rather close relationship be- tween DNA and the mitochondrial membrane. As it appears that the mem- brane is the chief site of protein synthesis in the mitochondria (see Section 150 D. B. ROODYN III. E.) and that DNA has a role to play in this synthesis, elucidation of the precise intra-mitochondrial locahzation of DNA is clearly of the greatest importance. 30 A 120 A 140S Fig. 29. Model of a helical filament observed in mitochondria from astrocytes in corpus striatum of the rat. The filament is about 30 A thick, the total diameter of the helix is about 140 A and the pitch is 120 A (from Mugnaini, 1964). C. MITOCHONDRIAL INCLUSIONS: DENSE GRANULES These granules are very obvious in electron-micrographs of mitochondria (see Fig. 3 for example). However, their nature was unknown for many years. It has been shown recently that they are insoluble inorganic salts. Probably they normally consist mainly of the insoluble calcium phosphate salt hydroxyapatite, which has the composition (Ca3(P04)2)3.Ca(OH)2. However, the precise composition of the granules depends on the salts to which the mitochondria have been exposed. For example, Peachey (1964) incubated smooth muscle cells from toad bladder in calcium-free Ringer's solution containing 2 mM barium acetate. He found the appearance of dense intra-mitochondrial granules, with a 40 A fine structure, and a characteristic hollow appearance. These were probably granules of barium phosphate (Fig. 30). Isolation of granules from Ca2+ loaded mitochondria has recently 3. THE MITOCHONDRION 151 been achieved by Weinbach and Von Brand (1965). The major constituents of granules isolated from mitochondria disrupted by deoxycholate were found to be Ca2+ 30-0%, Pi 18-5%, Mg2+ 3-9% and COs^- 4-0%. (These values are the % values of residual weight after ashing). The granules appeared I iG. 30. Intra-mitochondrial granules, probably of barium phosphate, from smaoth muscle cells of toad urinary bladder incubated in calcium-free Ringer's solution containing ' mM barium acetate. Lead acetate stain, k 210,000 (from Peachey, 1964). o contain some organic material, and their composition varied somewhat with the method of disruption of the mitochondria. it is possible that the granules act as stores for Ca2+, Mg2+ or even phos- phate ions. It may be noted that H+ ions would be liberated during the formation of calcium, magnesium, or even barium and strontium phosphates. For example, Brierley, Bachmann and Green (1962) formulated the uptake of Mg2+ and phosphate ions by beef-heart mitochondria by the reaction: MgCl2 + 2 MgHP04 % (Mg)3(P04)2 + 2 HCl with one mole of acid being produced per mole of phosphate bound. These reactions have been expressed in a general way by Chappell and Crofts (1965a) as one of two possibilities: 3M2+ + 2HPO42- -> M3(P04)2 + 2H+ or 3M2+ + 2H2PO4 -> M3(P04)2 + 4H+ (M is a general symbol for a divalent cation). 152 D. B. ROODYN Thus the H+ production per divalent cation taken up will depend on the pH. At pH 7-2, near to the second acid dissociation constant of orthophosphate, the ratio will be approximately 1 H+ per M2+ taken up. Chappell and Crofts (1965a) have exploited this fact to perform some ingenious experiments demonstrating the connection between ion transport, mitochondrial swelling and respiration. An interesting finding is that if excess Ca2+ is added, there is extensive swelling followed by release of Ca2+ into the medium. If this is then removed from the medium by a suitable chelating agent, the swelling is reversed (Crofts and Chappell, 1965). This reversal of swelling is dependent on respiration, and could be supported by electron transport through a limited portion of the respiratory chain. The contraction process is thought to be mediated by an intermediate acting at a site between X '-^ 1 and the respiratory chain. The site of action of various inhibitors, and the supposed sites of interaction of the ion transport, contractile and ATP synthesizing systems are given in Fig. 31, from Crofts and Chappell (1965). RoTenone Succinate Antimycin: Ascorbate - letramethyl -p- tK Cyt. c ohenylenediamine KjFeCCNJe-^ HCN NADH, ♦ Cyt. 6 Cyt.o Alkylguonidine Ion accumulation Oligomycin Atractyloside A ^ x~i * y >x-^p^.— -^ ►ATP \ \ \ \ \ \ Phosphate arsenate ADP Contractile system t 02 Fig. 31. Sites of action of inhibitors and associations of respiratory chain, contractile system, and ion accumulation as given by Crofts and Chappell (1965). To conclude this brief account of mitochondrial inclusions reference should be made to protein inclusions, sometimes present as crystals of striking size, that are occassionally found in mitochondria. An elegant example of this is given by Ward (1962) in a study of mitochondria from oocytes of Rana pipiem, and a picture of this enormous inclusion is shown in Fig. 32. Oberling (1959) has described protein-storage deposits in rat-kidney mito- chondria after injections of ovalbumin. 3. THE MITOCHONDRION 153 '■^ '^*T:m 'Sit Fig. 32. Hexagonal crystal of yolk protein (yolk platelet PY) present in mitochondrial cortex (MC) and surrounded by a membrane (C) believed to be the limiting membrane of a greatly enlarged crista. 100,000 (from Ward, 1962). D. THE MITOCHONDRIAL MEMBRANE SYSTEM Unfortunately, fractionation methods are not yet sufficiently refined to be able to separate the outer membranes, inner membranes and cristae mem- branes. Much of the evidence for localization in different parts of the mem- brane system is therefore essentially indirect. For example the greater number of cristae in mitochondria that have high respiratory activity per unit weight suggests that the respiratory system is located on the membranes of the 154 D. B. ROODYN cristae. The assignment of enzymes to specific parts of the mitochondrial membrane system is often based on indirect conclusions from metabolic studies and the effects of inhibitors (cf. Figs. 7, 8 and 22). Although some workers have presented rather detailed models of the structural organization of mitochondrial enzyme systems (see Section III. e. below) it should be stressed that at least some of these are still essentially conjectures or, at best, reasonable estimates, and are certainly not established with any great cer- tainty. For these reasons it is best to consider the mitochondrial membranes as a whole, under the general heading of "membrane system". E. ENZYMES OR ENZYME SYSTEMS ASSOCIATED WITH THE MEMBRANE 7. Respiratory Enzymes Many of the activities described in Section II. above are associated with the mitochondrial membrane. The most well known example of this is the respiratory chain. There is little doubt that all the elements of the respiratory chain concerned with the oxidation of reduced NAD, NADP and FAD are bound to the mitochondrial membrane. It is also of considerable importance that the terminal enzyme of the chain, cytochrome oxidase, is clearly associ- ated with the membrane (Siekevitz and Watson, 1956). In fact, it is generally believed that the respiratory enzymes are arranged in highly organized groups or complexes, usually called "respiratory assemblies", arranged along the surface of the mitochondrial membrane. There is still uncertainty as to the number, size and precise composition of these assemblies. However, it is generally agreed that in addition to the component enzymes of the respiratory chain (NADH dehydrogenase, cytochromes b, ci, c, a and ^3) there are "auxiliary" respiratory enzymes associated with cellular oxidations, namely succinate dehydrogenase, NAD/NADP transhydrogenase (1.6.1,1.), fatty acyl CoA dehydrogenase (1.3.99.3.) and various other enzymes. Also there is little doubt that factors involved in oxidative phosphorylation are also associated with the respiratory assembly. (An up to date account of such "coupling factors" is given in Sanadi, 1965). An estimate of the composition and possible particle weight of the assembly is given in Lehninger (1965), and values for the chemical and enzymic constitution of the "unit of electron transfer" or "elementary particle" are given by Blair, Oda, Green and Fernandez-Moran (1963). From these values, these workers calculated that the weight of the particle was about 2-1 x 10^. This is considerably higher than the value of 1-4 x 10^ which is the combined particle weights of the complexes thought to be present in the particle (see below). It was suggested that this discrepancy was due to the presence of a protein "not required for integrated electron transfer activity", probably corresponding to the structural protein of Criddle et al. (1962). Some estimates have been made of the number of respiratory assemblies or I 3. THE MITOCHONDRION 155 "units" per mitochondrion. The number is certainly of the order of tens of thousands. For example Green and Oda (1961) give an estimated number of 17000-18000 per heart mitochondrion. These authors also calculate that there are about 300 units per crista. The number of cytochrome a molecules per "average" liver mitochondrion has been estimated at about 17000 in an interesting paper by Estabrook and Holowinsky (1961). However, this is not equal to the number of units, since there are probably several cytochrome a molecules per unit. Thus the number of respiratory assemblies per liver mitochondrion is probably considerably less than the number per heart mitochondrion. This is quite consistent with the fact the cristae in liver mitochondria are much less densely packed than in heart mitochondria. Estabrook and Holowinsky also made the interesting calculation that there were about 130,000 molecules of "enzymically-reducible pigments" and about 500,000 molecules of pyridine nucleotide (NAD + NADP) in each liver mitochondrion. This gives us some idea of the potential complexity of the mitochondrial respiratory system, and also shows the magnitude of the task of determining the molecular structure of the mitochondrion. A question of considerable practical and theoretical importance is whether it is certain that the various components of the assemblies are present in stoichiometric amounts i.e. whether there are integral numbers of enzymes in each assembly, or whether the assembly has a somewhat loose composition with considerable variation in the relative number of components. The "integral", "quantal" or "stoichiometric" philosophy is satisfying intellec- tually and has been the driving force in the prolific studies on the organization of the mitochondrion that have come from the Madison School. In a large number of publications. Green and his co-workers have examined the detailed enzymology of a number of sub-mitochondrial preparations in order to determine the precise composition of the respiratory assembly and its relation to mitochondrial structure. (The relationship of the so-called "stalked-bodies" and the respiratory assembly will be discussed below: Section III. g.). As a result of these studies, Green and his colleagues have resolved the respiratory system into four complexes, and have attempted to define the exact number of molecules of each type of respiratory enzyme in each complex. The four complexes are I, NADH-coenzyme Q reductase; II, succinate-coenzyme Q reductase; III, reduced coenzyme Q-cytochrome c reductase, and IV, reduced cytochrome c oxidase. The molecular composition of these complexes and their inter-relations are given in Fig. 33, which was derived from Green and Fleischer (1963). The particle weights of the complexes are given in Blair et al. (1963) as follows: Complex I 530,000, Complex II 230,000, Complex III 200,000 and Complex IV 430,000. If the complexes were combined in a simple 1:1:1:1 ratio the complete system would thus have a particle weight of 1 -39 x 10^, which is less than the weight of isolated elementary particles (see above). 156 D. B. ROODYN Although there is still some discussion about the fine details of the compo- sition of the respiratory assembly, there can be little doubt that the formula- tion in Fig. 33 is a reasonable picture of the major aspects of organization Succinate NADH -(Ck FiG. 33. Proposed stoichiometry of complexes of respiratory chain. The number of mole- cules or atoms of each component is given as a subscript. Fs: succinate dehydrogenase; FD: NADH (DPNH) dehydrogenase; b, c\, c, a the various cytochromes; (Fe): non- haem iron; (Cu): copper; Q: coenzyme-Q. See text for names and molecular weights of the complexes I-IV (derived from Green and Fleischer, 1963). of the assembly. Associated with these components are other organized enzyme systems concerned with feeding NADH to the respiratory chain. The most intensively studied of these "primary dehydrogenases", as they have been called, are the complexes involved in the oxidative decarboxylation of pyruvate and a-oxoglutarate. The detailed mechanism of these reactions has been described in Fig. 13 and space does not allow a full discussion of recent research into the structural basis of these reactions. (See Sanadi, 1963). An interesting example of these studies is a recent theoretical paper by Whitehead (1965) in which he proposes a model for the pyruvate oxidase complex given in Fig. 34. This complex had been studied in detail by Koike, Reed and Carroll (1963). It was isolated as a particle of molecular weight about 4 X 10®. Koike and co-workers reversibly dissociated this into three sub-units which could be recombined with restoration of activity. The three sub-units were as follows: 1. A pyruvate-carboxylase that catalyses the reaction: pyruvate + [thiamine-PP] -> [acetaldehyde-thiamine-PP] + CO2 (The symbols [ ] indicate that the component is bound to the protein). 2. A lipoate reductase-transacetylase that catalyses the reaction: [acetaldehyde-thiamine-PP] + lip-S2 -^ [acetyl. S.lip.SH] + [thiaminePP] (where lip-S2 is lipoic acid and acetyl-S-lip.SH is S-acetyl-dihydrolipoic acid) and the subsequent reaction : [acetyl-S-lip.SH] + CoA.SH -> acetyl.S.CoA + [lip.SH2] (where lip.SH2 is dihydrolipoic acid). 3. THE MITOCHONDRION 157 3. A dihydrolipoate dehydrogenase that catalyses the reaction: FAD [lip.SH2] + NAD+ — > [Hp.S2] + NADH + H+ The spatial arrangement of these subunits given in Fig. 34 was derived by Whitehead on the basis of a general theory of the structure of multimolecular complexes. This system has been described in some detail in order to give an example of the sort of molecular organization that is possibly present in many regions of the mitochondrial membrane system. Fig. 34. Proposed structure of pyruvate decarboxylation complex. #: dehydrogenase sub-units; ^: carboxylase sub-units; O: lipoic reductase-transacetylase sub-units (see text for activity of sub-units) (from Whitehead, 1965). An interesting, and important, confirmation of the stoichiometric theory of respiratory chain structure has come from the work of Biicher, Pette and co-workers on the precise measurement of groups of mitochondrial enzymes (see for example, Pette, KHngenberg and Biicher, 1962; Klingenberg and Pette, 1962). It is found that if mitochondria from different sources are com- pared, some of the mitochondrial enzymes occur in groups of strikingly constant relative proportions, (the so-called "constant-proportion groups") For example, malate dehydrogense, succinate dehydrogenase, pyruvate oxidase and cytochrome a were always found in a certain ratio to each other, and also to the amount of cytochrome c + ci in the mitochondria. In addition to the constant proportion groups whose compositions are different in different types of mitochondria there are so called "specific proportion groups" that are characteristic of the type of mitochondrion. For example, insect flight muscle mitochondria have a high ratio of glycerol- 1 -phosphate dehydrogenase (1.1.99.5.) to glutamate dehydrogenase, whereas mammalain liver mitochondria have the converse. Such results suggest that the essential 158 D. B. ROODYN respiratory system common to all mitochondria consists of a number of rigidly determined units, which can vary in relative and absolute numbers in different mitochondria, but not in composition. "Ancillary" enzyme systems, however, may be present in different relative amounts, depending on the speciahzed function of the mitochondrion in question. 2. Enzymes Involved in Protein Synthesis Another group of enzymes clearly associated with the mitochondrial membrane are those involved in amino acid incorporation into protein. If rat-liver mitochondria are incubated with radioactive amino acids, the most radioactive proteins formed are those associated with the mitochondrial membrane (Roodyn, 1962). The soluble mitochondrial enzymes malate dehydrogenase and cytochrome c do not appear to become labelled in vitro (Roodyn et al, 1962). The radioactive product has many similar properties to the structural protein described by Criddle et al. (1962). The labelling of insoluble proteins in the membrane, rather than soluble proteins in the sap, may have some bearing on theories of the mechanism of mitochondrial replication, and it is interesting that similar labelling of insoluble proteins has been observed recently in the phylogenetically widely separated organisms, yeast (Wintersberger, 1965) and insect flight muscle (Biicher, 1965). It is therefore possible that labelling of membrane proteins by isolated mito- chondria is a general phenomenon. Studies of amino acid incorporation with sub-mitochondrial particles has confirmed that membrane-linked enzymes are actively involved in mito- chondrial protein synthesis (Truman, 1963; Kroon, 1965). In fact, the amino acid incorporation activity observed by Kroon in sub-mitochondrial particles was comparable to that observed by other workers with isolated microsomal systems. As a result of inhibitor studies with chloramphenicol and actino- mycin D, Kroon (1965) has suggested that mitochondrial protein synthesis is dependent on the continuous synthesis of RNA by mitochondrial DNA- dependent RNA polymerase. If this is so, the protein synthesis machinery in the mitochondrion would include DNA, RNA polymerase, some form of messenger RNA and enzymes involved in synthesis of peptide bonds. If all these are bound to the membrane, they would be in close proximity to the energy generating system of the mitochondrion. There is no doubt that there is a very close relationship between the protein synthesis and oxidative phos- phorylation systems of mitochondria (Roodyn et al., 1965; Roodyn, 1965b), and it would be most exciting if it could be shown that the mitochondrial membrane has at least some capacity for self-replication. Some of the advantages of such a limited self-replicating system are discussed in Roodyn and Wilkie (1966a). 3. Transport Systems Another system of enzymes undoubtedly associated with the mitochondrial 3. THE MITOCHONDRION 159 membrane are those concerned with the transport of metabolites and ions across the membrane. Interest in this field is rapidly expanding, and has been stimulated by investigations into the mode of action of the inhibitor of oxidative phosphorylation, the glycoside atractyloside. The drug is also called potassium atractylate and is extracted from the rhizome of a Medi- terranean member of the Compositae called Atractylis gummifera (Bruni, Contessa and Luciani 1962). Inhibition of oxidative phosphorylation was reversed competitively by addition of adenine nucleotides. However, inhi- bition by oligomycin was not reversed by this. It appears, therefore, that unlike oligomycin which reacts directly with some intermediate in oxidative phosphorylation, these agents prevent the transfer of adenine nucleotides across the mitochondrial membrane. Chappell and Crofts (1965b) have made a detailed comparison of the actions of oligomycin and atractyloside. The effect of the drug on adenine nucleotide transport has been fully confirmed by direct measurement of these nucleotides in mitochondrial "spaces" (Klingenberg and Pfaff, 1966). The site of action of atractyloside is shown in Figs. 8 and 21. The drug may therefore be of great use in delineating the enzyme systems involved in the transport of adenine nucleotides. Carnitine- linked transfer systems in fat metabolism have also been described in the membrane, as shown above in Figs. 7 and 8. It is still too early to assign such "permeases" to the inner or outer membranes with complete certainty, but there is little doubt that we will have a much clearer picture of their location in the very near future. There is little evidence to suggest at the moment that the transport of ions across the mitochondrial membrane is controlled by enzymes or "permeases" specific for each type of ion. However, it is certain that active ion transport is closely associated with the respiratory and energy-transfer systems, and is dependent on the physical integrity of the mitochondrial membrane. Also, there is little doubt that non-phosphorylated energy rich intermediates can provide the energy for ion translocation and accumulation (see for example Brierley, Murer, Bachmann and Green, 1963). [It should be mentioned here that it is important to distinguish between ion transport of relatively low concentrations of ions, and the rather massive uptake of Ca2+, Mg2^ and phosphate ions that is associated with the formation of dense granules. Rossi and Lehninger (1964) discuss the differences in properties of these two systems.] In a recent study, Chance (1965) suggests that Ca2+ ions can interact with the three energy-conservation sites in the respiratory chain, and the reaction is probably with the X ^>-^ I type of compound, although he does not completely rule out the possibility of reaction with X^P to form Pi and Ca^^ '^ X. The reactions proposed by Chance for the overall mechanism of calcium binding and uptake by mitochondria are shown in Fig. 35, and it can be seen that the essential postulate is the formation of an intermediate of the type 160 D. B. ROODYN (1) (2) (3) (4) Colcium Formation of Tronsport of Energy dependent binding carrier carrier calcium binding (5; Precipitation intermediate intermediate Co-^+ Co^ Ca3(P04)2 Co(OH)2 + 2H + ! J ^ — ^^ -car(x~i) m I T H2(X~I) +2H+ A i — CaJ-^(X~l)— —^-p- ;— r- ri ^ + 2H* CQ^+ Ca*+ 2P04|2H20 -— 2X+2I +4H+ (6) +2H''' Co Ca Neutralization and swelling 1 Ca^*(X~I)- -^^ ^^^. -r- I H2(X~I) +2H* \^^ T 1 Ca-^+ Co** \ Ca-' 4HAC + 4H20 Ca-^+ Ca+^ / \ / \ Fig. 35. Hypothesis for the accumulation of cations in mitochondrial membranes (from Chance, 1965). Ca2+(X ~ I). The subject is in a state of rapid development at the moment, but it would not be unreasonable to imagine that the close connection between ion transport, oxidative phosphorylation, and the swelling-contraction cycles of the mitochondrial membrane is a result of the fact that all these activities are dependent on the maintenance of the identical ordered structure. They cannot be separated by classical fractionation methods, as would be used for enzyme purification, because they represent different aspects of the behaviour of a single entity. This view of the mitochondrial membrane as an integrated structure that is dependent on the maintenance of relative positions of its component parts for correct functioning is discussed more fully in Section III. H. below. Some of these questions are dealt with in an informative article by Lehninger (1962) on the relationship between water uptake and extrusion by mitochondria and the structure and function of the mitochondrial membrane. The above account of the metabolic activity of the mitochondrial membrane system is far from complete, and is intended merely to present a picture of a series of complex systems that can interact both functionally and spatially. F. CHEMICAL COMPOSITION OF THE MEMBRANE Examination of the chemical composition of the membrane is essential to a correct understanding of its molecular organization. The most striking feature of the membrane is its high lipid content. The amount of lipid in isolated membrane fragments depends very much on the method of isolation used. However, in some cases it may be as high as 50 % of the total weight. I 3. THE MITOCHONDRION 161 Green and Fleischer (1963) have stressed the fundamental importance of lipids, and particularly phospholipids, in the maintenance of the integrity of the electron-transfer and oxidative phosphorylation systems. Indeed there is overwhelming evidence to support this view. Many respiratory enzymes have been shown to form complexes with phospholipids (e.g. cytochrome c\ Das, Haak and Crane 1965). Also a variety of agents that either remove or destroy mitochondrial lipids have been shown to damage or to disrupt completely the respiratory and phosphorylation systems. A good example of this is the observation of Sekuzu, Jurtshuk and Green (1961) that the tightly bound D(-)3-hydroxybutyrate dehydrogenase of beef heart mitochondria shows an absolute requirement for lecithin at all stages of purification after the initial extraction from the mitochondria. Failure to add lecithin either to the crude or purified enzyme resulted in complete loss of activity. (This type of phenomenon may well explain the difficulty frequently experienced in the purification of enzymes that are tightly bound to the membrane.) A more striking case is the experiment of Fleischer, Brierley, Klouwen and Slautterback (1962) in which intact mito- chondria were treated with aqueous acetone. This resulted in the removal of more than 80% of the total phospholipid, and was accompanied by almost total loss of respiratory activity, as measured by succinate-cytochrome c reductase for example. However, respiratory activity could be restored com- pletely by addition of phospholipid. The reactivation of succinoxidase activity in rat-liver mitochondria that had been treated by lecithinase was demon- strated by Casu and Modena (1964), who used lipid extracts of rat-liver as re-activating agents. It is interesting in this connection that there is an exact re'ationship between the loss of succinoxidase activity and the extraction of phospholipid during "titration" of mitochondria with Triton-X-100 (Roodyn, 1962, and Fig. 24 above). The changes produced by lipid-damage may sometimes be of a subtle nature. An interesting example is given by Machinist and Singer (1965a,b). Sub-mitochondrial particles (the so-called "electron-transfer" particles or "ETP") are able to reduce added coenzyme Q homologues by NADH (Complex I, Fig. 33 above). As in intact mitochondria, the oxidation of NADH was inhibited by rotenone and Amytal. However, if these prepara- tions were treated for a very short time with purified phospholipase A, the enzyme activity was lost. However, it could be restored by the addition of suitable phospholipids. If the phospholipase treatment was at all prolonged, however, it was no longer possible to restore activity by adding phospholipids. In addition, it is possible to obtain a preparation of purified NADH- coenzyme Q reductase from ETP. However, this preparation has a much lower phospholipid content than ETP, and in addition has lost the sensitivity to rotenone and Amytal. Thus subtle changes in metabolic activity, or even in response to inhibitors, 162 D. B. ROODYN can result from alterations to the mitochondrial hpids. It is not surprising, therefore, that there are frequent differences between the results obtained by different workers with various sub-mitochondrial fragments or purified enzyme systems. Most, if not all, methods of mitochondrial fragmentation and sub-fractionation would depend on the rupture of lipid-protein bonds of one type or another, and translocation or even complete extraction of lipids would not be unexpected. The same considerations apply to the purification of bound enzymes. It is quite likely that the reason why such a large number of compounds can uncouple oxidative phosphorylation is that any agent which disturbs the precise inter-relationship of lipid and protein in the membrane will result in disturbance of the energy-transfer system. Thus any surface active or lipophilic agent is likely to act as an uncoupler. When one considers the complexity of enzyme systems present in mitochondria and the probability that lipid arrays play an important part in maintaining the correct spatial relationship of these systems, one realizes the magnitude of the task of obtaining a precise picture of the molecular organization of the membrane. This is particularly so because our methods of analysis un- doubtedly disrupt these arrays. Another important factor in the maintenance of membrane structure is undoubtedly the presence of insoluble proteins. The structural framework of the mitochondrion does not depend only on the presence of lipids, since Fleischer et al, (1962) have demonstrated the retention of reasonable fine structure in beef-heart mitochondria even after removal of most of the phospholipid by aqueous acetone (Fig. 36). It has been known for many years that most sub-cellular components contain insoluble "residual" pro- teins, which are not readily brought into solution, for example by repeated extraction with concentrated salt solutions. Levin and Thomas (1961) discuss the subject and describe the properties of insoluble lipoproteins in liver cell fractions. Interest in insoluble proteins responsible for maintaining the structure of cell components was stimulated recently by the isolation of the so-called "structural protein" of mitochondria (Criddle et al., 1962). This protein is extremely insoluble in water at pH 7 but dissolved in anionic detergents, or extremes of pH. In solution structural protein has a marked tendency to polymerize, thus forming insoluble aggregates. The monomer has the rather low molecular weight of 20,000 to 30,000. Its most interesting property is its ability to form complexes with specific respiratory enzymes, e.g. cytochromes b,ci and a. It does not interact with cytochrome c except in the presence of phospholipid. It also has the property of combining spon- taneously with phospholipid micelles at neutral pH (Richardson, Hultin and Fleischer, 1964). As a result of these properties, it was suggested that struc- tural protein has a central role in the binding together of mitochondrial enzymes. A diagram showing the supposed interaction of lipids, cytochromes and structural protein is presented in Fig. 37 (from Criddle et al., 1962). It is I 3. THE MITOCHONDRION 163 Fig. 36. Beef-heart mitochondria after removal of phospholipid by treatment with aqueous acetone. Note the retention of fine structure, x 86,000 (from Fleischer et al., 1962). thought that the chief interactions in these complexes are between non-polar regions of the molecules. It has also been suggested that similar proteins occur in all cell membranes and that the binding of phospholipids to structural protein is a phenomenon of general importance. For example, proteins resembling structural protein of mitochondria, and exhibiting binding prop- erties with phospholipid have been isolated from liver microsomal fractions, erythrocyte stroma and leaf chloroplast lamellae (Richardson, Hultin and Green 1963). The properties of structural protein would confer on it the possibility of being an "organizer" for mitochondrial assembly. One may speculate that the assembly of the mitochondrion may be controlled genetically by synthesis of 164 D. B. ROODYN S.P.-lipid-c-b-« S.P.-lipid-b -• — lipid .F'.-b S.P.-lipid-a-c-«- -S.P.-lipid-a -»-S.P-b-c. S.P.-lipid-c -* S. P. -lipid-* — I Structural protein | ^S-P.-c, S.P- ^S.P.-c,-a Fig. 37. Schematic summary of interaction of structural protein (S.P.) and cytochromes a, b, c, and a (from Criddle et ai, 1962). a specific mitochondrial "organizer protein" which would then form aggre- gates of the correct molecular organization by only complexing with those enzymes that had specific binding sites for the organizer protein. Thus if the protein has a region with affinity for a specific region of the cytochrome c molecule, and another region with affinity for part of cytochrome ci, it would be able to form an aggregate of cytochrome c and ci. The nature of this aggregate may be determined by complimentary sequences of amino acids in the peptide chains of the organizer and bound proteins. If the amino acid sequence of the organizer protein is itself determined by the nucleotide sequence in a specific type of "organizer" messenger RNA, which is itself encoded in mitochondrial DNA, one would have a genetically controlled system of reasonable simplicity for the ordered synthesis of the complex organization of the mitochondrial membrane. An intriguing aspect of such a hypothesis is the finding that most of the amino acid incorporation in isolated mitochondrial is into insoluble mem- brane-bound protein, similar in many ways to the structural protein of Criddle et al. (1962) (see Roodyn, 1962). The matter is also discussed in Sections II h. and III e. An interesting experiment has been reported very recently which possibly argues against a role of structural protein in the control of mitochondrial replication, however. Katoh and Sanukida (1965) have shown that structural proteins isolated from mitochondria of wild-type and respiratory deficient yeast have the same sedimentation coefficients, amino acid compositions and serological properties. They also appear to be present in identical amounts in the two kinds of mitochondria. Since there is a gross disturbance of mitochondrial function and organization in res- piratory deficient yeast, it seems that structural protein cannot have an important role in causing this. (The significance of this observation is dis- cussed in further detail in Roodyn and Wilkie, 1966a). However, it is difficult at the moment to assess work with structural proteins, because the techniques of isolating them and studying their properties are still rather primitive. Current improvements in this field (for example the observation of Mac- 3. THE MITOCHONDRION 165 Lennan, Tzagoloff and Rieske (1965) that succinylation of structural protein results in an increase in its solubility) may well lead to important discoveries, not only about mitochondrial structure but also about the molecular mech- anisms of mitochondrial formation. The observations that RNA appears to be associated with the mitochon- drial membrane appear to be relevant to this question (Roodyn, 1962; Kroon, 1966). Although there has been some doubt as to whether or not mitochondria contain RNA, it now seems reasonably certain that the small amounts of RNA found in the mitochondrial fraction are indeed due to mitochondria, and not to microsomal contamination (See Roodyn, 1966 for some recent views on mitochondrial RNA). Sub-fractionation of mito- chondria shows that there is very little RNA in the soluble fraction, and most of it remains associated with membranous material. It is very resistant to the action of ribonuclease, even after disruption of the mitochondria to allow access of the enzyme. Resistance to ribonuclease is a diagnostic property of mitochondrial amino acid incorporation into protein. It is interesting that RNA has been found in other "smooth-membranes" (see for example Chauveau, Moule, Rouiller and Schneebeli, 1962) although the amount per mg protein is far less than in ribosomes. Also, some "post-microsomal" membrane fractions have been shown to have amino acid incorporation activity that is not inhibited by ribonuclease (for an account of these see Hird, McLean and Munro, 1964). This may imply that there are non- ribosomal protein synthesis systems associated with certain membranes. It may be that a special type of "membrane RNA" exists in cell membranes and is concerned in the control of their replication. The question of membrane oriented protein synthesis is fully discussed in an interesting recent article by Hendler (1965). In conclusion, reference should be made to an interesting aspect of the chemistry of the mitochondrial membrane. Patterson and Touster (1962) reported the presence of sialic acid in mitochondria. However, the micro- somal fraction was rich in this material, and the possibility of cross-con- tamination should not be ruled out. Nevertheless the possible presence of such materials in the membrane certainly deserves further detailed study. G. STALKED BODIES AND ELEMENTARY PARTICLES The current state of knowledge of this subject has been discussed recently by Whittaker (1966), from which the following account has mainly been drawn. The application of the technique of negative staining to studies of mitochondrial sub-structure resulted in the discovery of rather striking stalked particles along the surface of the cristae. They were arranged in ordered rows along the membrane and appeared to be attached to it by a definite "neck" or "stalk". This is shown in Fig. 38, taken from Fernandez- Moran (1962). Particle studded membranes were observed by many other 166 D. B. ROODYN It* Z- .rf ^^v' .ur Fig. 38. Arrays of stalked particles in isolated beef-heart mitochondria, negatively stained in phosphotungstic acid, x 130,000. Insert shows fine structure of particles, with head piece, stalk and base piece, x 650,000 (from Ferndndez-Moran et al., 1964). 3. THE MITOCHONDRION 167 workers in negatively stained mitochondria from diverse sources. A par- ticularly clear example found in plant mitochondria by Nadakavukaren (1964) is shown in Fig. 39. Perhaps rather prematurely, these particles were identified by Blair et al. (1963) with the fundamental unit or "elementary particle" of the electron-transfer system. This was particularly premature because the isolated elementary particle had no attached stalk (Fernandez- Moran, Oda, Blair and Green (1964)). More careful analysis, however, suggested that the particles were too small to contain the entire respiratory assembly. Thus the minimum particle weight for the assembly is about 1-4 X 10^ (See Section III. e.). As the spherical head of the stalked body is l^konly about 90 A in diameter, it can be calculated that the particle weight ^^^ould only be about 230,000 if the particles had a density near to unity. Even if they had a density of 1-3, and the dimensions had been under- estimated by 12%, the value would still only be about 420,000 (Whittaker, 1966). Stoeckenius (1963) in fact suggested a maximum particle weight of 500,000. It has therefore been suggested by Fernandez-Moran et al. (1964) IH||that the knob, stalk and the base-piece embedded in the membrane all take ^^^part in formation of the respiratory assembly, as their combined weight is 1-3 X 106. IK However, there is further evidence that the stalked bodies are not res- piratory assemblies. For example, mitochondria that had been stripped of all inner membrane structure still retained their cytochromes (Chance, Parsons and Williams, 1964). Stasny and Crane (1964) observed that fractions of sonically disrupted beef-heart mitochondria that were richest in succinoxidase and cytochromes consisted of membranes that were free of attached stalked bodies. On the other hand, the sub-mitochondrial fraction that had the least respiratory activity was in fact richest in these sub-units. Chance and Parsons (1963) observed that mitochondria from Ascaris lumhricoides lack cyto- chromes c\, a and a^. Nevertheless, negative staining still revealed the presence of 80 A diameter particles along the surface of the membrane. Ogawa and Barnett (1965) used histochemical staining and electron microscopy to examine the localization of succinate dehydrogenase. Although clear deposits of formazans could be observed after staining, these did not correspond in position to the stalked particles. It has been suggested that the particles may be the Fi coupling factor of oxidative phosphorylation. Racker, Chance and Parsons (1964) observed that the isolated Fi factor consisted of 90 A diameter particles. Non-phosphory- lating sub-mitochondrial preparations were found to have about six particles per jLi of membrane. Addition of Fi restored the capacity for phosphorylation, and at the same time increased the number of attached particles to 30/[jl. (The number in beef-heart mitochondria is 60/jli). The reader is referred to Racker (1962) for a description of the coupling factors. Apart from these suggestions, there is the more worrying possibility that the particles are in 3. THE MITOCHONDRION 169 fact artifacts of the negative staining method. Whittaker (1966) discusses this problem and shows a very interesting electron-micrograph from Bangham and Home (1964) in which artificial lamellae of ovolecithin that had been treated with lysolecithin showed arrays of globular structures 70-80 A in diameter (Fig. 40). Although this does not establish conclusively that the stalked bodies are artifacts, it demonstrates the extreme caution that is needed in the interpretation of such structures. Fig. 40. Dispersion of purified ovolecithin after treatment with lysolecithin and negative staining in phosphotungstate. The particles are estimated to be 70-80 A in diameter. y 210,000 (from Bangham and Home, 1964). H. THE MITOCHONDRIAL MEMBRANE AS AN INTEGRATED STRUCTURE The above account demonstrates that the mitochondrial membrane system is the site of complex oxidation and energy transfer reactions, protein syn- thesis, ion translocation, water transport and probably the active transport of many metabolites into and out of the mitochondrion. There is a marked A Fig. 39. Stalked bodies on the cristae of mitochondria from Castor beans (Ricinus com- munis) negatively stained in phosphotungstic acid. 1. General view of preparation ; 2. larger magnification showing stalks (arrows) (from Nadakavukaren, 1964). 170 D. B. ROODYN inter-dependence of many of these reactions on one another. Sometimes the relationship is so close that it is difficult, if not impossible, to separate the two systems. Perhaps an example from the author's experience will show the difficulty that is often encountered in separating effects on interacting mitochondrial systems. It was found by Tata et al (1963) that treatment of thyroidectomized rats with thyroid hormone resulted in enhanced respiration and phosphoryla- tion activity per mg mitochondrial protein. As there was also a stimulation in amino acid incorporation in microsomal protein, it was suggested that the hormone had produced a stimulation of protein synthesis, and a net increase in the number of respiratory assemblies per mitochondrion. This thesis was confirmed in a most striking fashion by the electron microscope studies of Gustafsson, Tata, Lindberg and Ernster (1965) in which it was shown that there was a clear increase in the mitochondrial population and also in the ratio of cristae to matrix. A photomicrograph from this paper is presented in Fig. 41 in order to illustrate the striking mitochondrial hypertrophy that can Fig. 41. Hypertrophy of rat skeletal muscle mitochondria in rat rendered hypermetabolic by treatment with L-thyroxine for three weeks. The mitochondrion marked with arrows measures about three sarcomere lengths. The normal mitochondrion in rat skeletal muscle measures only \-\ sarcomere length (from Gustafsson et al., 1965). 3. THE MITOCHONDRION 171 l....,.^„.,..„.. .,., place in vivo in the morphology of mitochondria). Because of these observa- ■tions, it was therefore not surprising to find that similar treatment resulted in k stimulation of amino acid incorporation into protein observed in isolated mitochondria (Freeman, Roodyn and Tata, 1963). However, when the effect ■was studied in more detail it was not possible to establish in a conclusive fashion whether the increase in incorporation was due to specific stimulation of mitochondrial protein synthesis, or was simply because the enhanced ATP synthesis made more energy available for incorporation (Roodyn et a/., 1965). The problem could not be resolved because amino acid incorporation was seriously affected by any process that damaged the oxidative phosphorylation system and it was not possible to supply energy for incorporation from added ATP. Thus the two systems could not be separated, and it was not possible to localize the effect of thyroid hormones. There are many other examples of such unsuccessful attempts to separate closely associated processes in the mitochondrion. Attempts to sub-fractionate I or "purify" a complex system frequently fail when a critical amount of damage has been inflicted on the mitochondrial organization. Are we there- ifore advancing into a blind-end by the conventional analytical approach? In many ways the philosophy of biochemistry is totally alien to the concept that the whole can only be understood as a whole, and not deduced from the properties of its parts. Such a view would be condemned as "vitalism", a most sinful term. Admittedly, it is a most depressing prospect if it can be » shown that many of the integrated processes occurring in the mitochondrion biay only be detected in intact membranes, and are absent from membrane fragments, simply because they can only occur in a structure that has the correct spatial organization. Thus it may be just as hopeless to look for integrated processes in sub-mitochondrial fragments below a certain size as it would be to look for catalytic activity in small fragments of an enzyme L^_ (for example in dipeptides or free amino acids). The catalytic activity of the l^penzyme is entirely dependent on the spatial configuration of its component parts, and clearly could never be "restored" by mixing suitable proportions I of amino acids. These problems are discussed in a fascinating essay by Mitchell entitled "Metabolism, Transport and Morphogenesis: Which drives Which?" (Mitchell, 1962). His chemi-osmotic theory has been discussed above (Section II. E.) and there is still considerable discussion as to its significance. However, it seems to the author at least that the point of supreme issue is not whether this or that mechanism for oxidative phosphorylation is correct, but whether attempts to "sub-fractionate" many membrane systems are based on false premises and are inherently doomed to failure. One may argue from the rich harvest of results in the field of protein synthesis that the sub-fractionation approach is valid. Yet even here at least some have expressed doubts at the ^ 172 D. B. ROODYN validity of ignoring the role of the intact membrane (Hendler, 1965). What- ever the final outcome, there is little doubt that the elucidation of mito- chondrial function in molecular terms is a central challenge to modern biology and will require the most gentle dissection of the mitochondrial membrane by the most subtle research weapons. The days of the hammer are over. IV. Addendum: The Structure and Function OF Peroxisomes For many years the sub-cellular distribution of the enzymes catalase and urate oxidase (1.7.3.3.) were difficult to elucidate. The enzymes were at first thought to be in mitochondria but more refined fractionation methods sug- gested that this was incorrect. However, the enzymes did not show precisely the same sedimentation properties as lysomal enzymes. More detailed analysis strongly suggested that the enzymes catalase, urate oxidase and another enzyme, D-amino acid oxidase (1.4.3.3.), in fact belonged to a separate class of particle. In a detailed study, Beaufay et al. (1964) resolved rat-liver mito- chondrial fractions into three distinct groups of particles, mitochondria, lysosomes and the particles containing urate oxidase, catalase and D-amino acid oxidase. There was some difficulty in separating these particles from ysosomes, however. Fortunately, it was found by Wattiaux, Wibo and Baud- huin (1963) that injection of suitable doses of the neutral detergent Triton WR 1339 produced massive swelling of the lysosomes, but was without effect on the urate oxidase particles. By this means it was possible to obtain a reasonably pure preparation of these particles (Baudhuin, Beaufay and de Duve, 1965). Electron microscopy revealed that the liver particles had the characteristic morphology of the so-called "microbodies" previously des- cribed by Rouiller and Bernhard (1956). They were slightly smaller than mitochondria, had a single bounding membrane and contained a characteris- tic central core. There was a clear correlation between the number of micro- bodies in the preparation and the specific activities of urate oxidase, catalase and D-amino acid oxidase. (Because they were more numerous in regenerating liver, Rouiller and Bernhard (1956) had suggested that microbodies might be precursors of mitochondria. However, this now seems to be very unlikely (de Duve and Baudhuin, 1966)). A comparison of some properties of liver mitochondria and microbodies is given in Table I, taken from de Duve (1965). Most of these values were obtained from results of density gradient centrifuging experiments, using cytochrome oxidase as a marker for mitochondria, and urate oxidase as a marker for microbodies. Very recent developments in this field are discussed extensively in the 3. THE MITOCHONDRION Table I. 173 Mitochondria Microbodies Dry wt ([xg) 10-' 2-4 X 0-« Osmotically active solutes (milli osmoles/g dry wt) 0-157 0 Water compartments (cm^/g dry wt) Hydration 0-430 0-214 Sucrose space 0-905 2-51 Osmotic space 0-595 0 Total in 0-25 m sucrose 1-930 2-724 Sedimentation coefficient in 0-25 M sucrose (Svedberg Units) 10^ 4-4 X 10* Diameter in 0-25 m sucrose (y.) 0-8 0-54 Density in 0-25 m sucrose 1-099 1095 (Data computed from results of density gradient centrifuging using cytochrome oxidase as marker for mitochondria and urtea oxidase as marker for microbodies. From de Duve, 1965). ^aHjO R-CHOH-COO" R-CO-COO~ CHj-CHgOH CH3-CHO Fig. 42. Possible role of peroxisomes in extra-mitochondria! oxidation of NADH. The o-hydroxy acid oxidase produces H2O2 (e.g. by lactate oxidation) and this is used by catalase, in high concentration in the peroxisome, to oxidize a range of compounds, for example alcohol. Reduction of the product is achieved by NAD-linked dehydrogenases, and serves as a means of removing NADH produced by enzymes in the cell sap (e.g. triosephosphate dehydrogenase) (from de Duve and Baudhuin, 1966). 174 D. B. ROODYN review by de Duve and Baudhuin (1966). The enzyme L-a-hydroxy-acid oxidase has been added to the list of enzymes found in microbodies. In addition, particles with very similar enzymic properties to liver microbodies have been isolated from rat-kidney and Tetrahymena pyriformis. Vaes and Jacques (1965) found that catalase sedimented in a different way to mito- chondria from homogenates of rat bone, and from the activation properties of catalase Vaes (1965) concluded that the enzyme was present in particles similar to rat-liver microbodies. Since it is not certain that all these particles have identical morphology to rat-liver microbodies, the general name of "peroxisome" has been proposed (see Duve and Baudhuin, 1966). It is suggested that peroxisomes have an important role in extra-mitochondrial oxidaiions, by the mechanisms described in Fig. 42. The fascinating possi- bility that the particles are really "fossil organelles" of some primitive pre- mitochondrial oxidative system is discussed by de Duve and Baudhuin. The fine structure of the liver microbody is now being actively examined. The pronounced central core has a regular sub-structure, and there now seems little doubt the core is the site of location of urate oxidase (Baudhuin Fig. 43. Guinea-pig liver microbody siiowing hexagonal lattice in core. The spacing between the elements of the lattice is about 1 10 A. x 126,000 (from Baudhuin et al., 1965). I 3. THE MITOCHONDRION 175 et ah, 1965). The outer "sap" contains catalase and the other peroxisomal enzymes, and it may be noted that the catalase concentration appears to be very high. An electron-micrograph of a microbody from guinea-pig liver is shown in Fig. 43. 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Quagliariello and E. C. Salter, eds.). Vol. 7. Elsevier (BBA Library), Amsterdam, p. 1. Williams, J. N. (1952a). /. biol. Chem. 194, 139. Williams, J. N. (1952b). /. biol. Chem. 195, 37. Wintersberger, E. (1964). Z. Physiol. Chem. 336, 385. Wintersberger, E. (1965). Biochem. Z. 341, 409. I I. II. III. IV. Chapter 4 THE CHLOROPLAST D. O. HALL and F. R. WHATLEY King''s College University of London, London, England Introduction ...... Chloroplasts and Other Plastids . A. Chloroplasts ..... B. Chromatophores .... C. Non-photosynthetic Chromoplasts D. Leucoplasts ..... Methods for Isolation of Chloroplasts A. Isolation in Aqueous Medium . B. Isolation in Non-aqueous Medium . C. Comments on the Localization of Enzyme Activity Enzymes of Chloroplasts .... A. Enzymes for Assimilatory Power B. Enzymes for CO2 Fixation Cycle C. Additional Enzyme Reactions . D. Induced Enzyme Formation Comparison of Oxidative and Photosynthetic Phosphorylation References 181 182 182 186 187 187 188 189 193 195 197 197 208 217 230 230 232 I. Introduction The chloroplast occupies a particularly important place in the economy of the plant cell, being the site of the whole complex of reactions leading to the capture of light energy and its eventual storage in chemical form as carbo- hydrates. Isolated chloroplasts have proved to be convenient experimental objects for the study of many of the partial reactions which together lead to their overall metabolism. It was early found that certain of these processes were associated with the water-insoluble fraction of the chloroplasts and the enzymes catalysing them could be readily localized within the chloroplast. However, the detailed characterization of the water-insoluble enzymes has proved more difficult. On the other hand many of the enzymes thought to be originally present within the chloroplast are easily extracted by water or dilute salt solutions. These have frequently been characterized in some detail as soluble enzymes but their definitive localization within the chloroplast has proved to be 181 182 D. O. HALL and F. R. WHATLEY correspondingly more difficult. The technique of isolating the chloroplasts in non-aqueous solvents has provided a promising approach to proving the original distribution of many of these easily water-soluble enzymes. In this chapter we shall describe the structure of the chloroplasts (and other plastids) and discuss some of the evidence for the localization of a considerable number of enzymic components within chloroplasts. II. Chloroplasts and Other Plastids Plastids may be generally divided into two groups, (a) the chromoplasts or coloured plastids and (b) the leucoplasts or colourless plastids The chromoplasts may be conveniently divided into (i) chloroplasts and other photosynthetically active chromoplasts, which contain chlorophyll a plus varying accessory pigments (ii) chromatophores which contain the far-red absorbing bacteriochlorophyll (photosynthetically active) and (iii) non- photosynthetic chromoplasts which usually lack chlorophylls but contain carotenoids (Granick, 1961). This classification is convenient but by no means ideal since a particular plastid may occur as a colourless, green, red or yellow plastid at different periods of its existence. A. chloroplasts In this category we shall include the green plastids of green algae and higher plants where chlorophyll b is the accessory pigment; the brown or yellow plastids of the brown algae and diatoms in which fucoxanthin is the accessory pigment; the red plastids of the red algae where phycoerythrin is the accessory pigment ; and the green plastids of the blue-green algae which have phyco- cyanin and phycoerythrin as the accessory pigments (Haxo and OhEocha, 1960) This review will be primarily concerned with the chloroplasts of green algae and higher plants since most of the research in photosynthesis has been done with these organisms. Chloroplasts in higher plants are saucer shaped organelles 1 to Six in diameter. In algae the chloroplast may be of much more complicated shape, varying from the simple cup-shaped chloroplast of Chlorella to the stellate chloroplast of Zygnema or the spirally wound, ribbon-shaped chloroplast of Spirogyra. Chloroplasts possess a lamellar structure. Discrete chloroplasts are lacking in blue-green algae but a lamellar structure is diffused throughout the cell. In the light microscope chloroplasts can often be seen to contain grana, within which the pigment appears to be concentrated. Electron microscope studies have shown further details of the structure. As shown in Fig. la, representing an ultra-thin section of bean chloroplast, the organelle is bounded by a double membrane within which is contained the ground substance, the 4. THE CHLOROPLAST 183 >% i c .2 E _3 o _aj o a. X) 3 _c o O x: /— s X) H >< u S 2 o z (« a T3 2 — E Q'S.2 •^ ^ > t; o I"' ._ *> «J t c *-=■(/) c =3 -5 — •= '^ c u — 5 ^ — E_ 2-0 o o "^ il CS b X> ^ 184 D. O. HALL and F. R. WHATLEY Stroma, and various apparently lipid droplets visible only under certain fixation regimes. Embedded in the stroma are the lamellar structures, con- sisting of flattened discs, which in some cases are concentrated to form distinct "stacks" corresponding to the grana in the light microscope. Within the grana certain of the lamellae appear to end, but it will be observed (Fig. lb) that a considerable number extend beyond the limits of the grana, becoming stroma lamellae. The distinction between stroma and grana lamellae may or may not be significant. The highly magnified section shows the detailed lamellar structure to be quite complicated (see Weier, 1961; Weier and Thomson, 1962, for discussion). The discrete structure which can be observed along the double lamellae may be related to the elementary photosynthetic particles, the quantasomes. In Fig. 2a is shown a single lamella from a spinach Fig. 2(a). Chromium shadowed spinach chloroplast lamella (surface view) showing regular arrangement of quantasomes (Park and Biggins, 1964). 4. THE CHLOROPLAST 185 160 I Fig. 2 (b). Diagram of typical lamellar structure from blue green algae, Euglena and higher plants. (Park and Pon, 1963). grana fraction in surface view. The surface has become mechanically disrupted so ihat the regular orientation of particles underneath can be clearly seen. It is thought that these particles correspond to the quantasomes. Park and Pon (1963) have suggested a possible arrangement of the quantasomes along the lamellae (Fig. 2b) which may correspond to the particles observable in the very highly magnified section in Fig. lb. Figure 2b also suggests the evolution of the lamellar structure from that found in the blue-green algae (no distinct chloroplast, but well developed lamellae), through that found in green algae and many higher plants (in which the lamellae become stacked), to the condition in the chloroplasts of most higher plants (in which grana formation occurs). Chloroplasts contain ribonucleic acid, a component which is normally associated with ribosomes. Lyttleton (1962) has isolated a fraction corre- sponding to ribosomes by sonic disruption of isolated chloroplasts and subsequently centrifuging in a sucrose gradient, indicating the existence of these protein-synthesizing particles within the chloroplast. When starch is deposited within the chloroplast it is laid down between the grana lamellae, eventually pushing them apart and leading to a great disorganization of the structure — a phenomenon which may represent a mechanism for controlling the overall extent of starch synthesis. The chemical structure of chloroplasts of various higher plants has been determined (see Thomas, 1960; also Menke, 1962, 1963, for references). Chloroplasts contain 75% to 80% water and between 30% and 60% of the total protein in the leaf is associated with them. Zucker and Stinson (1962) exceptionally found that Oenothera chloroplasts contain 75% of the leaf protein. On a dry weight basis chloroplasts contain 25 % to 35 % lipid, 45 % I 186 D. O. HALL and F. R. WHATLEY to 60 % protein, 4 % to 7 % chlorophyll and a variable content of the caro- tenoids, jS-carotene and xanthophyll. About half the protein is readily ex- tracted by treatment with water, yielding aqueous extracts containing a considerable number of enzyme proteins, as is discussed in more detail in this chapter. The lipids in the chloroplasts contain only a small amount of neutral triglycerides, but are characterized by the presence of galactolipids (e.g. j8-galacto-pyranosyl diglycerides), the plant sulpholipid sulphodeoxyglycosyl diglyceride, and the phosphatidyl glycerols (Benson 1963). Small amounts of metabolically important substances are also concentrated within the chloro- plasts. These include catalysts of electron flow, e.g. ferredoxin, cytochrome/, cytochrome b%, flavoproteins, plastocyanin and pyridine nucleotides, various metals, e.g. iron, copper, manganese, magnesium and zinc, and a number of quinones, e.g. vitamin K, tocopherylquinone and the plastoquinones. A detailed chemical analysis of the composition of the purified lamellar fraction has been published by Park and Biggins (1964). The large amounts of several of the quinones and pigments suggests a structural role for these substances in the chloroplasts. The proposed model of Frey-Wyssling (1953) for the chloroplast lamellae has lipoprotein structures which is in accordance with this analysis. The chloroplast is the site of a number of important synthetic activities of the cell including photophosphorylation, CO2 fixation, carbohydrate interconversions as well as a number of important ancillary reactions. These activities are discussed in some detail in succeeding sections. B. CHROMATOPHORES Chromatophores are particles approximately 6OOA in diameter which can be isolated from photosynthetic bacteria by sonication or by mechanical disruption followed by differential centrifuging. This is an operational definition. It is now thought that the chromatophores isolated from the cells of purple bacteria broken by such methods represent fragments derived from an initially continuous cytoplasmic membrane and therefore, if we may quote, "chromatophores ... are structural artefacts produced by the disrup- tion of the bacterial cytoplasmic membrane" (Cohen-Bazire and Kunisawa, 1963). Thus a membrane bound vesicular system within the cytoplasm constitutes the intracellular site of the photosynthetic apparatus in these photosynthetic bacteria. The chromatophores when isolated contain all the photosynthetic pigments of the bacterial cells including the bacterio- chlorophyll which resembles chlorophyll a in plants although there is no accessory pigment similar to chlorophyll b or one of the phycobilins found in higher plants. Large amounts of carotenoids and phospholipoproteins are found, it having been observed by Newton and Newton (1957) that these constituents occur in the ratio bacteriochlorophyll icarotenoid cytochrome in the proportion 10 : 5 : 1. Protein constitutes approximately 40% on a 4. THE CHLOROPLAST 187 weight basis and 21 % is lipid, principally phospholipid. Chromatophores contain considerable quantities of non-haem iron. They also contain pyridine nucleotides, flavin, a considerable amount of a bacterial cytochrome which resembles cytochrome /of plants in many respects and a haem protein called Rhodospirillum haem protein. Isolated chromatophores are able to carry out photophosphorylation and the photoreduction of pyridine nucleotide and these reactions are presumably catalysed by the membrane system within the bacteria from which they are derived. The review by Gest and Kamen (1960) may be consulted for further details of the metabolism of photosynthetic bacteria. C. NON-PHOTOSYNTHETIC CHROMOPLASTS These non-photosynthetic plastids lack chlorophyll but often contain considerable amounts of carotenoids. The plastids contain pigments which are red or yellow and impart colours to some flowers, fruits, roots, fungi, etc. They are usually spindle- or needle-shaped bodies which occur either singly or grouped in bundles. They may also consist of droplets containing the carotenoids, each droplet representing a degenerated plastid. The colour may change from green due to the disappearance of chlorophyll and the unmasking of the carotenoid pigments; when this happens in existing chloroplasts the red or yellow coloured chromoplasts are obviously derived from original chloroplasts. Carotenoids commonly occurring in red and yellow coloured chromoplasts are a- and j8-carotene and the xanthophylls lutein, violaxanthin and nea- xanthin (Goodwin, 1960). The yellow chromoplasts of carrot roots develop from leucoplasts, and as the carotene is synthesized the starch contained by the leucoplast disappears. Older chromoplasts appear as large flat plates or as crystalline needles. Carotene may make up to 20% to 56% of the plastid on a dry weight basis. When the carotene content of the plastid is about 30% the other lipids may amount to 30 % to 40 % and the protein to some 1 5 % (Granick, 1961). D. LEUCOPLASTS This term is applied to all colourless plastids, the term proplastid desig- nating immature colourless plastids occurring in developing tissues (Granick, 1955; Steffen, 1955). Leucoplasts are predominantly storage plastids. Accord- ing to the product principally stored the leucoplasts are named amyloplast if starch predominates, elaioplast if oil predominates and aleuroplast when protein granules predominate. 188 D. O. HALL and F. R. WHATLEY 1. Amyloplasts These are mature leucoplasts filled with starch and generally found in storage tissues in tubers, cotyledons and endosperms. Reserve starch is laid down as large starch grains usually made up of a series of concentric layers successively deposited about a centre — the amyloplast membrane and accom- panying stroma becomes greatly distended and finally becomes so thin as to be scarcely perceptible as a pellicle enveloping the starch grain. Each leucoplast apparently produces only a single starch grain. These vary greatly in size (l-150)Li in diameter) and in number in different cells. Their shape is character- istic for a given species. 2. Elaioplasts These are plastids which develop a preponderance of oil which may appear in old chromoplasts which lose their pigment. Oil droplets are however formed independently of plastids. The oils are usually considered to be a reserve storage product. 5. Aleuroplasts These are colourless plastids which contain protein. The proteins are normally "globular" and are water soluble in contrast to fibrous proteins. They tend to crystallize if solvent is withdrawn. The size of the aleuroplast varies between 3 and 20/Lt. They are found in many fruits and seeds. The pro- tein bodies in developing wheat endosperm contained 68% protein, 23% lipid (most phospholipid), 6% nucleic acid present as ribosomes and 4% of phytic acid. The protein bodies are enclosed within a distinctive Hpo- protein membrane and the whole structure is considered to be an aleuro- plastid or proteoplast (Morton, Palk and Raison 1964) i.e., the plastids consist of a lipoprotein membrane enclosing the protein body and a region rich in ribosomes. The aleuroplast contains the enzymes and nucleic acid components necessary for the incorporation of amino acid into the protein. The protein body within the plastid is formed by the accumulation of storage protein within the plastid membrane. III. Methods for Isolation of Chloroplasts Chloroplasts may be isolated from leaves in aqueous or non-aqueous media. If the intracellular distribution of water-soluble enzymes is to be studied, it may be desirable to isolate the particles in non-aqueous solvents. However, chloroplasts isolated in this way may lack a number of the overall photochemical processes normally associated with chloroplasts which have been isolated in dilute salt or sugar solution. Protocols are given in Figs. 3, 6 and 7 below of methods which have been commonly used for the isolation of chloroplasts. 4. THE CHLOROPLAST 189 Grind leaves by hand with sand in 0-35 M NaCl, 002 m tris pH8, 001 M ascorbate Filter through muslin 200 g for one min Green supernatant 1000^ for 7 min Precipitate (cell debris — discarded) *ale green supernatant (broken chloroplasts, mitochondria, microsomes, cell sap) Precipitate ("whole" chloroplasts) Suspend in 0-35 m NaCl IOOO5 for 7 min Precipitate Supernatant ("whole" chloroplasts = Ci) (discard) I Suspend in 0035 m NaCl (=Cis) 5000^ for 10 min r Supernatant fluid (chloroplast extract = CE) Precipitate ("broken" chloroplasts = Cisi) Further washings Cis2, Cis3, etc. Fig. 3. Isolation of chloroplasts in aqueous medium (Whatley and Amon, 1963). A. ISOLATION IN AQUEOUS MEDIUM Most of the work which has been carried out on the enzymology of chloro- plasts have been done with chloroplasts isolated in 0-35m sodium chloride and frequently broken subsequently by suspension in much lower salt concentration. In the method described by Whatley and Arnon, 1963 (Fig. 3) spinach leaves were gently ground in buffered 0-35m sodium chloride with sand, using a pestle and mortar; other species have been used in a similar way. The resultant slurry was filtered through cheese cloth and submitted to a 190 D. O. HALL and F. R. WHATLEY brief centrifuging at slow speed (2005^) to remove coarse debris and nuclei. Higher speeds (lOOO^^) brought down the unbroken ("whole") chloroplasts, which were washed by suspending them in 0-35m sodium chloride and centrifuging again at 1000^. A suspension of "whole" chloroplasts (Ci) was obtained by taking up the green pellet in 0-35m sodium chloride. If the pellet was taken up in 0-035M sodium chloride the chloroplasts became "broken" (Cu) and a considerable amount of protein originally contained in the whole chloroplast was extracted (CE). Further washings of Cis by suspending in 0-035m sodium chloride and centrifuging down the chloroplasts to remove water-soluble components yielded suspensions of "broken" chloro- plasts labelled Cisi, Cis2, Cis3, etc., according to the number of washings. Kahn and von Wettstein (1961) have isolated chloroplasts in 0-35m NaCl (Ci) and examined them in the light and electron microscopes. Their electron micrographs shown in Figs. 4a and b represent those with smooth outlines and no clearly visible grana when viewed with the light microscope. These chloroplasts retain the normal structural features of those in the leaf and possess a lamellar system, stroma and surrounding membrane. Other chloro- plasts isolated in 0-35m NaCl lack the smooth outlines when viewed in the light microscope and in the electron microscope can be seen to consist solely of the lamellar system and lacking a surrounding membrane and stroma (Fig. 4c). Despite the absence of the confining structures the lamellar systems of such chloroplasts appear identical to those of the chloroplasts in the leaf. When these NaCl chloroplasts are treated with water (or dilute NaCl: fraction Cu) the surrounding membrane together with the stroma is lost and the regular arrangement of the lamellar discs is greatly distorted (Fig. 4d). These distorted, "broken" chloroplasts are however still capable of carrying out many biochemical reaction sequences. Other workers e.g. James and Das (1957), Park and Pon (1961), Greenwood, Leech and Williams (1963), and Leech (1964), have preferred the use of buffered 0-3-0-5M sucrose solutions for the isolation of chloroplasts, but otherwise follow similar procedures. Chloroplasts isolated in buffered sucrose have been examined under the electron microscope by Greenwood et al. (1963). An unwashed pellet (Ci) prepared from bean leaves contained chloroplasts together with some chloroplast fragments and mitochondria. These particles are all shown in Fig. 5a. The chloroplasts were of two easily distinguishable types, the first retaining the lamellar structure and stroma within a surrounding membrane (more opaque), the second lacking the membrane and stroma and corre- sponding to the naked lamellar structures (Fig. 4c) of Kahn and von Wett- stein in salt-isolated chloroplasts. If the unwashed chloroplast fraction (Ci) was subjected to a sucrose-glycerol gradient differential centrifuging (James and Das, 1957) the naked lamellar structures were selected without contami- nation— see Fig. 5c. On subjecting Ci to a sucrose gradient differential 4. THE CHLOROPLAST 191 Fig. 4. Sections of spinach chloroplasts isolated in salt media, fixed in 2% KMn04. For descriptive details see text. (Kahn and von Wettstein, 1961). 192 D. O. HALL and F. R. WHATLEY ^ ■ ^Xt\\H'' - *' %r ^ 4. THE CHLOROPLAST 193 centrifuging (Leech, 1964) intact chloroplasts were selected with minimal contamination — see Fig. 5b. B. ISOLATION IN NON-AQUEOUS MEDIUM Chloroplasts have also been prepared in non-aqueous solvents by several workers using essentially the methods of either Stocking, 1959 (Fig. 6) or Thalacker and Behrens, 1959 (Fig. 7). In Stocking's method spinach leaves Leaves frozen and lyophilized Ground in hexane/carbon tetrachloride (d= 1-32) Filter through muslin and glasswool 12,000^ for 15min Supernatant including upper surface deposit Sediment (discard) Add equal volume hexane 1000^ for 5 min Supernatant (discard) Green sediment (chloroplasts) Fig. 6. Preparation of chloroplasts in non-aqueous media (Stocking, 1959) were freeze-dried and ground in a mixture of hexane and carbon tetrachloride adjusted to give a density of 1-32. After filtering through cheesecloth the suspension was centrifuged at 12,000^ for 15 minutes and the precipitate (density greater than 1-32) was rejected. To the supernatant an equal volume Fig. 5. Sections of bean chloroplasts isolated in buffered sucrose media, fixed in Os04. For descriptive details see text. Magnification approximately (a) top: x8000 (b) bottom left: X 3200 (c) bottom right: x 5500. (Courtesy of A. D. Greenwood.) I 194 D, O. HALL and F. R. WHATLEY Frceze-dried Elodea Ground in petroleum ether Stand for 10 min Suspension Sediment (discard) 2000^ for 10 min Supernatant (discard) Precipitate Suspend in petroleum ether 800^ for 2-5 min (discard) Precipitate (chloroplasts) Further washings with petroleum ether Density gradient fractionation in carbon tetrachloride/petroleum ether id = 1-25-1 -40; centrifuging at 2000^ for 15 min Remove Fraction I (chloroplasts) and wash in petroleum ether Fig. 7. Isolation of chloroplasts in non-aqueous media (Thalacker and Behrens, 1959) of hexane was added and the mixture was centrifuged at 10(X)^ for 5 minutes. The green precipitate (density less than 1-32) consisted almost entirely of chloroplast material. In the method of Thalacker and Behrens (Fig. 7) leaves of Elodea were freeze-dried, ground in petroleum ether and allowed to stand for 10 minutes to allow the larger debris to settle out. The supernatant was then centrifuged for 10 minutes at 2000^ and the precipitate of chloroplast material collected. The chloroplasts were washed by further suspending them in petroleum ether and centrifuging at 800^. Finally a density gradient 4. THE CHLOROPLAST 195 fractionation between the density limits of 1-25 to 1-40 was carried out and fraction 1 (less than 1-32) was collected. The chloroplast fraction was finally washed once in the centrifuge with petroleum ether. Other investigations (e.g. Heber, Pon and Heber, 1963) with chloroplasts prepared in non- aqueous solvent systems have used essentially one or other of these methods. C. COMMENTS ON THE LOCALIZATION OF ENZYME ACTIVITY The difficulties of localizing the distribution of enzymes within chloroplasts have been discussed in some detail by Smillie (1963). When chloroplasts are isolated by grinding leaves in sodium chloride or sucrose solutions a common deficiency is that soluble proteins are lost. The loss of the protein may exceed 50 % of the total protein content of the chloroplast and has been shown to vary with such factors as the method of breaking the cells, the medium used in the isolation of the chloroplast, the plant tissue used and the condition and physiological age of the material. If the chloroplasts contain starch they are often disrupted during centrifuging since the starch is denser than the chloro- plast material. The loss of protein from the chloroplasts which occurs during the isolation has been repeatedly demonstrated and attention will be drawn to this phenomenon in subsequent discussions. Where the particular enzymic component under study is insoluble or relatively insoluble in water the chloroplasts isolated in aqueous media are often the most appropriate to use. Such chloroplasts exhibit the maximum range of activity of photochemical reactions, chloroplasts isolated in non-aqueous media being normally unable to catalyse such basic reactions as the Hill reaction (see below). The rates of reactions carried out in the light by chloroplasts, which depend on the presence of soluble proteins have, however, been found to be much lower than the rates of comparable preparations of leaf material. Two rather important examples of the loss of key proteins from chloroplasts during isolation in aqueous solvents include the loss of carboxydismutase from both "whole" and "broken" chloroplasts and the loss of the ferredoxin on breaking the chloroplast. Although very little of the cellular ferredoxin is found in chloroplasts isolated in dilute sodium chloride solutions ferredoxin is required for the reduction of NADP by enzymes which are bound to the chloroplasts and in the intact cell the ferredoxin is undoubtedly localized within the chloroplast. The loss of materials from isolated chloroplasts has also been demonstrated by electron microscopy (Kahn and von Wettstein, 1961). As mentioned above two types of plastids were found in the population of spinach chloroplasts isolated in a sodium chloride medium. One type retains an apparently continuous outer membrane and contains the interlamellar material similar to that seen in chloroplasts in these sections. The remainder of the plastids are damaged to a greater or lesser extent and their outer membranes, and 196 D. O. HALL and F. R. WHATLEY in more extreme cases interlamellar material, are missing. On treatment of the chloroplasts with dilute sodium chloride or water only the latter broken plastid type was observed. It is apparent that some tissues when treated by a procedure which gives a high percentage of intact chloroplasts from spinach yield in the main broken chloroplasts. This is an additional compli- cation which may be of importance in evaluating the results of experiments on chloroplasts isolated in aqueous medium. SmilUe (1963) goes on to point out that in cases where the isolation of the chloroplast in aqueous media causes the loss of much protein from their original site, fractionation in non-aqueous media may be a useful alternative. In this procedure described in Figs 6 and 7, freeze-dried tissues are used and the entire isolation is carried out in media in which proteins are insoluble. The proteins are then preserved in their original location and are not leached out of the subcellular structures (cf. isolation of nuclei — Chapter 2). It has been pointed out that the contamination of the chloroplasts by co-precipita- tion of proteins originating from other portions of the cell might take place during the grinding and subsequent isolation procedures. This has not so far turned out to be a serious problem in the isolation of chloroplasts. Most enzymes survive the isolation procedure well. Enzymes which can be demon- strated in aqueous extracts of cells can also be detected after non-aqueous isolation of the chloroplasts. Some difficulty may be experienced with the isolation of chloroplasts in non-aqueous media if they contain starch. The average density of the chloro- plast is increased when it has accumulated starch and the fractionation will not proceed in the same way in the case of chloroplasts containing starch as occurs with chloroplasts that do not contain starch. It has been particularly noted that in the gradient centrifuging method of the type described by Thalacker and Behrens (1959, Fig. 7) chloroplasts with high starch content are not adequately separated from other cell components. Because of the difficulty inherent in the fractionation of the leaf material by the non-aqueous method Smillie has chosen to use the unicellular alga Euglena gracilis. Although the organism contains storage polysaccharide granules these are located outside the chloroplasts. The organism can be grown under well controlled conditions, by contrast with the frequently variable conditions under which leaves can be grown. Leaves, however, have several types of cell instead of being a uniform material. Moreover Euglena can be lyophilized very rapidly and the lyophilized material can be broken down more readily than can leaf material. The fractionation and isolation of chloroplast material from Euglena has demonstrated very clearly that those fractions which contain chlorophyll, corresponding to chloroplast material, are characteristically rich in those enzymes which are confined to the chloroplasts in the intact cell, and a distri- bution of the enzyme activity identical with the distribution of chlorophyll 4. THE CHLOROPLAST 197 has been found to occur for a number of enzymes. Equally clearly other enzymes are not localized in the chloroplasts and are presumably hnked to glycolysis and respiratory processes. The basic method which is used is to carry out a fractionation as cleanly as possible and then to assay for some "endogenous marker" component, e.g. chlorophyll in the case of chloroplasts or succinic dehydrogenase and cytochrome oxidase in the case of the mitochondria. By comparison of the distribution of the enzymic activity which is under study and the distribution of the component characteristic of the particular organelle in the various fractions it is possible to arrive at a conclusion about which particular organelle is the original site of localization of the enzyme activity. This technique has been very successfully applied to animal tissues by de Duve, Wattiaux and Baudhuin, (1962) and to plant tissues by Smillie (1955, 1963) and by other workers. (See Chapter I for a general discussion of this ap- )roach.) IV. Enzymes of Chloroplasts A. ENZYMES FOR ASSIMILATORY POWER In order to reduce CO2 to the level of carbohydrates the chloroplast must first synthesize the components of the assimilatory power, viz. NADPH2 and ATP. These products are formed by the processes of cyclic and noncyclic photophosphorylation. Cyclic photophosphorylation which yields only ATP as a net product, can be represented by the equation : W ADP + orthophosphate .hi'ropiast "^ ^'^^ The process was first observed in isolated spinach chloroplasts by Arnon, Allen and Whatley (1954). Subsequent work showed that under appropriate experimental conditions this process is capable of high rates of ATP for- mation commensurate with the rates of photosynthesis in intact leaves (see review by Arnon, 1961). The process of noncyclic photophosphorylation on the other hand yields both ATP and NADPH2 according to the equation: NADP + ADP +orthophosphate +H2O chloroplast >NADPH2 + ATP+i02 The reaction was first observed with isolated chloroplasts by Arnon, Whatley and Allen (1958). 1. Noncyclic Photophosphorylation A scheme to represent the electron flow from H2O to NADP coupled with the formation of ATP is given in Fig. 8. It incorporates many of the 198 D. O. HALL and F. R. WHATLEY current ideas of the mechanism of the process (see, for example, Whatley and Losada, 1964 for references). In a light reaction catalysed by a pigment system in which chlorophyll b or other accessory pigment predominates, electrons are transferred from H2O (£"0 = + 0-81V) to plastoquinone (£"0 = caOV). Oxygen is released in this process as a waste product of the Reductase NADP iseX chl cytfi X^Pi 0 Light £"0. PH 7 -Fd -0-4 -0 2- 0- -cyt /?6 + 0-2- +0-4 +0-6- NADP -cytf OH Fig. 8. Scheme for noncyclic photophosphorylation. Details in text. Fd: Ferredoxin, chl a: chlorophyll a, chl b: chlorophyll h., PQ: plastoquinone. photo-oxidation of H2O. In a subsequent light reaction catalysed by a pigment system in which chlorophyll a predominates electrons are transferred from cytochrome / (E'o = +0-37V) to ferredoxin {E'o = -0-42V), This reduced ferredoxin then donates its electrons to NADP (£"0 = — 0-34V) in a dark reaction. The two photochemical reactions are joined by a series of dark reactions involving the transfer of electrons from plastoquinone to cytochrome/via plastocyanin in the direction of the thermochemical gradient. Part of the energy released during the passage of electrons is coupled to the formation of ATP. The participation and characterization of several of the enzymic intermediates is discussed below, (a) Photo-oxidation of water. The enzymic apparatus necessary for the photo-oxidation of water, with the accompanying evolution of oxygen as a waste product, is located in the water-insoluble portion of the chloroplast, i.e., washed chloroplast fragments (Cui, C152, C153— see Fig. 3). Photo- oxidation of water can also be carried out in the very small chloroplast sub-units representing a small number of quantasomes (Park and Pon, 1963; 4. THE CHLOROPLAST 199 Becker, Shefner and Gross, 1965). The ability of chloroplast fragments to carry out the photo-oxidation of water in the Hght can be determined by measuring the rate of oxygen evolution accompanying the photo- reduction of various artificial electron acceptors such as ferric oxalate, ferricyanide, quinones and various dyes. The continuing evolution of oxygen from water (the electron donor) by illuminated chloroplasts in the presence of artificial electron acceptors is called the Hill reaction after its discoverer. The detailed mechanism of that part of the Hill reaction which leads to the evolution of oxygen has not so far proved susceptible to experimental analysis. The photo-oxidation of water requires the presence of chloride as was first shown by Warburg in 1948. Chloride could be replaced by bromide but not by other anions and was considered to be essential for the evolution of oxygen. Later Bove, Bove, Whatley and Arnon (1963), confirmed Warburg's conclusion that chloride is essential only for those photosynthetic reactions in which oxygen is liberated. Chloride was not required for the cyclic photo- phosphorylation carried out by chloroplasts. A concentration of approxi- mately 0-01 M chloride is necessary to saturate the oxygen evolving reaction. Manganese is also shown to be related to oxygen evolution. Experiments summarized by Pirson (1960) and Kessler (1960) using the green alga Ankistrodesmus which could be adapted to use hydrogen as the reductant instead of water and which can be grown under manganese deficient con- ditions, localized the site of action of manganese at the oxygen evolving site in photosynthesis. Spencer and Possingham (1961) working with chloro- plasts isolated from manganese-deficient spinach leaves, came to the con- clusion that manganese was required for oxygen evolution and noncyclic photophosphorylation but was not required for the cyclic electron flow. Plastoquinone was shown to be necessary for the overall Hill reaction by Bishop (1959). The role of plastoquinone in the photoreduction of NADP by water, the physiological counterpart of the Hill reaction, has now been investigated . Since the noncyclic electron flow in chloroplasts has been shown to be composed of two partial reactions (i) the photo-oxidation of water leading to oxygen evolution and (ii) the subsequent photoreduction of NADP usually coupled with ATP formation, it has been possible to identify the site of action of plastiquinone with more certainty. Plastoquinone was found to be needed only for the photo-oxidation of water and not for the subsequent reduction of NADP. Plastoquinone may very well be the initial electron acceptor for the auxiliary light reaction catalysed by the accessory pigment (photoreaction B). However, from his work on fluorescent compounds, Duysens (1964) proposes that an unidentified substance termed "quencher" is the initial electron acceptor located between photoreaction B and plastoquinone. A number of substances have been found to exert a powerful inhibitory effect on the oxygen evolving reaction. These include phenylurethane and the 200 D. O. HALL and F. R. WHATLEY substituted phenyl ureas such as the herbicide CMU (3-(4-chlorophenyl)-l,l- dimethylurea), o-phenanthroHne, hydroxylamine and azide (Losada and Arnon, 1963). Duysens (1964) has suggested that CMU interrupts the electron flow immediately prior to the point at which plastoquinone accepts electrons. The site of action of the other inhibitors of oxygen evolution are quite unknown. The photo-oxidation of water (photoreaction B) is driven by light absorbed by the accessory pigment system, as indicated in Fig. 8. In this accessory pigment system, chlorophyll Z?650 is the primary light absorber and is ac- companied by small amount of chlorophyll a (chlorophyll ^673) which appears to act as the sink of the energy transfer chain. For the photo-reduction of NADP (photoreaction A) chlorophyll a with an absorption maximum at 683m/x (chlorophyll aesa) is the primary light absorber; it is accompanied by a pigment P700 (absorption maximum at or above TOOm/i) which acts as the energy sink for chlorophyll aess (French, 1964). {b) Plastocyanin. Katoh (1960) and Katoh, Suga, Shiratori and Takamiya, (1961) isolated a blue copper-containing protein called plastocyanin from the leaves of higher plants and from various algae. This protein has a molecular weight of approximately 21,000 and in the oxidized form possesses an ab- sorption spectra with a broad maximum at 597m)Li. The redox potential was found to be +0-370 volts. Plastocyanin contains two atoms of copper per mole and approximately 50 % of the copper contained in the chloroplast is present in plastocyanin. One mole of plastocyanin (2Cu2+ atoms) is present per 3-400 moles of chlorophyll in the spinach chloroplast (Katoh et al., 1961). Katoh and Takamiya (1963) found that added plastocyanin could become photoreduced on illumination with chloroplast fragments, i.e., it could act as a Hill reagent. In studying the photoreduction of NADP with water as the electron donor, Trebst (1963) found that the overall reaction was inhibited by the copper chelating agent salicylaldoxime. If ascorbate, in the presence of the dye dicWorophenolindophenol, was substituted for water as the electron donor lalicylaldoxime was no longer an effective inhibitor. It was suggested that plastocyanin might have been the copper-containing compound upon which salicylaldoxime acted. This was later substantiated by the experiments of Trebst and Pistorius (1965) who studied the role of plastocyanin in digitonin- extracted chloroplasts. On treatment with digitonin the plastocyanin was lost and at the same time the ability to carry out the Hill reaction was lost. The use of ascorbate as an alternative electron donor showed that electrons were readily donated to the electron transport chain through the mediation of the dye which could also be replaced by plastocyanin. This indicated that reduced plastocyanin is capable of reacting readily with the components of the chain and provides some evidence for the functional role of plastocyanin. These experiments are related to those of Nieman, Nakamura and Vennesland, 4. THE CHLOROPLAST 201 (1959) who used digitonin-treated chloroplasts and showed that they were capable of carrying out a photo-oxidation of reduced cytochrome c. On fractionation of the chloroplast preparation a supernatant factor was found which was necessary to permit the reaction of the cytochrome c with the electron transport chain. This was later shown by Katoh and Takamiya (1963) to be identical with plastocyanin. Further evidence of the role of copper-containing compounds in the electron transport chain comes from the work of Spencer and Possingham (1960) on copper deficient tomato plants. Chloroplasts isolated from leaves of copper deficient plants showed only a feeble Hill activity which they showed to be due to an effect on the reductive portion of the Hill reaction and not related to the oxygen evolving system, in contrast to manganese. The fact that purified plastocyanin is photo-reduced by chloroplasts and can also be photo-oxidized if the chloroplasts are pre-treated with digitonin has led Kok, Rurainski and Karmon (1964) to suggest that plastocyanin could mediate the transfer of electrons between the two light reactions of photosynthesis. Evidence for this functional role has been obtained by de Kouchkovsky and Fork (1964) who analysed the light induced absorption changes in the spectral region where the difference between the oxidized and reduced form of the copper protein is maximum, namely 591m/x. Another compound which markedly inhibits the Hill reaction at high concentrations is KCN. This probably functions in a manner similar to that of salicylald- oxime by reacting with the copper containing compound. (c) Cytochrome f. Special cytochrome components have been discovered in photosynthetic cells of green plants and several algae and appear to be concentrated in the chloroplasts (Hill and Scarisbrick, 1951 ; Davenport and Hill, 1952). Cytochrome / was isolated, as a haem protein of molecular weight 110,000, from photosynthetic tissues and shown to be present in chloroplasts in the ratio of 1 mole of cytochrome / to 400 moles of chloro- phyll. Cytochrome / has an unusually oxidizing redox potential (£'o, pH7 = + 0-365 V). When isolated from the chloroplasts it occurs in the reduced form, characterized by a very sharp a band at 554-5m/Li. Katoh (1959) has described the isolation and properties of a cytochrome of the c-type from various algae of the family Rhodophyceae, Phaeophyceae, Chlorophyceae, and Cyanophyceae. The cytochromes from these widely different algae had practically identical redox potentials and absorption spectra, with £"o, pH 7 = -f 0-30 to + 0-34 V and an a band at 553m/i. They appear to be the algal equivalent of cytochrome /, which itself is a cytochrome of the c-type. Cytochrome /has been isolated and purified from parsley, spinach and from the alga, Euglena. The presence of cytochrome / in chloroplasts can be shown directly since its spectrum is visible not only in leaves but also in isolated chloroplasts after acetone treatment to remove the chlorophyll. In the living state cytochrome 202 D. O. HALL and F. R. WHATLEY /may also be seen in pale varieties of plants or mutants of algae. Cytochrome /exists in the unilluminated leaf in the reduced form. It is not auto-oxidizable and no cytochrome /oxidase has ever been detected in plants. Cytochrome/ is not oxidized by cytochrome c oxidase and thus cytochrome / does not react with oxygen. However, on illumination of leaves or algae the reduced cytochrome / becomes oxidized (see Duysens, 1964, for discussion and references). A clear cut demonstration of the photo-oxidation of cytochrome /was obtained by Duysens (1955) with the red alga Porphyridium cruentum. In this case the a band of cytochrome / was not completely masked by the chlorophylls and spectral changes around 550m/x were clearly seen. Similar changes were also observed by Chance and Sager (1957) with a Chlamy- domonas mutant that had a low content of chlorophyll and carotenoids. In normal leaves the changes in spectrum at 550m)u. could not be observed because of masking by chlorophyll and evidence demonstrating the photo- oxidation of cytochrome / could only be inferred from changes at 420m/x. On turning off the light the cytochrome returned to the reduced level. Similar cytochromes have also been detected in photosynthetic bacteria. Smith and Baltscheffsky (1959) made the interesting observation that in chromatophores cytochromes could become photo-oxidized in the light but that this only occurred in the presence of a phosphate accepting system, adenosine di- phosphate + inorganic phosphate. The coupling of photo-oxidation of the cytochrome with phosphorylation was thus shown in the bacterial system and it is inferred by analogy that it takes place in the chloroplast. In Fig. 8 we represent the pigment system accepting an electron from cytochrome / and passing the electron on to ferredoxin in a reaction catalysed by the chlorophyll a system. The special pigment component of this system, P700 first observed by Kok in 1956 (see Kok, Cooper and Yang, 1963 for dis- cussion) is believed to participate as a catalyst within the system. {d) Ferredoxin. Spinach ferredoxin (Tagawa and Arnon, 1962) is a water- soluble protein of molecular weight of approximately 14,000, characterized by possessing two iron atoms per molecule in a non-haem combination and two labile sulphur atoms which are released on acidification as H2S. In addition the molecule contains six sulfhydryl groups in cysteine. The protein had been studied extensively under a series of different names before the identity of the various synonyms was recognized ; it was earlier studied as the methaemoglobin reducing factor, later as photosynthetic pyridine nucleotide reductase, as the NADP reducing factor, as the red enzyme, and finally as ferredoxin. One mole of ferredoxin is found per approximately 400 moles of chlorophyll both in leaves and in isolated whole chloroplasts. On breaking the chloroplasts the ferredoxin is released into the solution. The identity of the various substances mentioned above has been shown on the basis of complete amino acid analysis (Davenport and Hill 1960, Fry and San Pietro, 1963) by a comparison of the activity of the !1 4. THE CHLOROPLAST 203 purified compounds, and by a demonstration of their interchangeability in a number of reactions. The redox potential of spinach ferredoxin, (E'o pH 7-5) is — 0-430 V. The role assigned to ferredoxin in metabolism of the chloroplast is as an intermediate in electron transport at a very reduced potential. In the presence of illuminated chloroplasts ferredoxin will accept electrons by a photochemical reaction and can subsequently pass these electrons on to acceptors such as NADP in the presence of the appropriate enzyme, ferre- doxin-NADP reductase. Its role as a carrier of electrons can be readily studied by observing changes in the absorption spectrum and it has been shown to become reduced by accepting one electron per mole in a photo- chemical reaction and to donate this electron via the flavoprotein enzyme to NADP in a subsequent dark reaction (Whatley, Tagawa and Arnon, 1963). Ferredoxin also seems to serve as a branching point in the electron transport systems that result either in cyclic or noncyclic photophosphorylation. When the photoreduced ferredoxin is reoxidized by NADP (with the aid of ferredoxin-NADP reductase) noncyclic photophosphorylation results; when oxidized NADP is unavailable as an acceptor, the photoreduced ferredoxin is reoxidized by a bound component of the grana [possibly cytochrome be — see Section IV. a. 2. e.] and cyclic photophosphorylation results. The expected stoichiometry between the number of moles of ferredoxin reduced and of oxygen evolved in noncyclic photophosphorylation has also been observed (Arnon, Tsujimoto and McSwain, 1964). Other reactions in addition to the reduction of NADP or the catalysis of cyclic photo phosphorylation have also been identified. Reduced ferredoxin is involved for example in the reduction of nitrite, in the photoreduction of hydrogen and in the reduction of various dyes. Many of these reactions have been summarized recently by San Pietro and Black (1965). o-Phenanthroline, an inhibitor of photosynthesis, combines readily with ferredoxin, removing the iron atoms from the molecule and releasing H2S. This action is however not the basic reason for the inhibition of photo- synthesis by o-phenanthroline, which apparently acts directly on the oxygen evolving reaction. Mercury compounds also combine readily with ferredoxin (mercuric chloride and /7-chloromercuribenzoate have been studied in some detail) and the action of mercuric compounds on the inhibition of cyclic and noncyclic photophosphorylation in which ferredoxin is involved can be attributed directly to the reaction between these compounds and ferredoxin. (e) Ferredoxin-NADP reductase (1.6.99.4.). As already stated, ferredoxin- NADP reductase is a flavoprotein enzyme of the chloroplast which catalyses the transfer of the hydrogens from reduced ferredoxin to NADP. Shin, Tagawa and Arnon (1963) prepared the reductase in crystalline form and investigated a number of its properties. It has a much higher affinity for NADP than for NAD (400 times greater; Km for NADP ^ 9-78 x IO-^m; for NAD = 3-75 x IO-^m). Ogren and Krogmann (1963) showed that more K* 204 D. O. HALL and F. R. WHATLEY than 90% of the NADP but only 50% of the NAD in the leaf of spinach is located in the chloroplast. (These values were determined on chloroplast material isolated in non-aqueous solvent since chloroplasts isolated in aqueous solvents contain virtually no pyridine nucleotide). In view of the marked afiinity of the reductase for NADP it seems reasonable, therefore, to beUeve that NADP is of predominant biosynthetic significance in the chloroplast. In addition to its main physiological function as a ferredoxin-NADP reductase the enzyme was also found by Shin et al. (1963) to have other activities from which were derived an ability to catalyse the oxidation of NADPH2. They observed (i) transfer of hydrogens from NADPH2 to a number of hydrogen or electron acceptors such as FMN, FAD, ferricyanide and various dyes; (ii) NADP reductase was also found to have transhydro- genase activity. It catalysed a transfer of hydrogens from NADPH2 to NAD though not NADH2 to NADP; (iii) it acted as a NADPH2-cytochrome c reductase in the presence of one of several electron acceptors mentioned above such as vitamin K3, FMN, FAD or ferredoxin. In all these cases the role of the ferredoxin-NADP reductase was to transfer electrons from NADPH2 to the respective electron acceptor which could then reduce cyto- chrome c non-enzymically. These additional activities of the ferredoxin-NADP reductase, in addition to other evidence, indicate that the enzyme is the same substance as the chloroplast NADPH2 — diaphorase isolated and purified by Avron and Jagendorf (1956) and the chloroplast transhydrogenase isolated and purified by Keister, San Pietro and Stolzenbach (1960). The latter investigators have already discussed the similarities and possible identity of transhydrogenase (1.6.1.1.) and NADPH2 diaphorase. The spectrum of the enzyme which was crystallized by Shin et al. (1963) indicates that it contains flavin. Avron and Jagendorf, studying the enzyme under the name of diaphorase, found it to contain FAD. The enzyme is shown to be localized in the chloroplast since it is unnecessary to add the enzyme initially in studying the reduction of NADP by illuminated chloroplasts in the presence of ferredoxin. However, the extraction of chloroplasts by dilute tris buffer causes the solubilzation of the reductase, and treated chloroplasts cannot then reduce NADP via ferredoxin. On adding the purified enzyme or simply on adding the purified extract this ability reappears. Shin and Arnon (1964) have shown that the action of the enzyme is to become reversibly reduced by diaphorase and to be reoxidized by NADP, a phenomenon readily observed through changes in the absorption spectrum. Ferredoxin-NADP reductase is inhibited by adenosine-2'-monophosphate, a competitive inhibitor which was previously shown also to inhibit transhydrogenase and diaphorase activity. Mercuric chloride, /7-chloromercuribenzoate, and iodoacetate also inhibit. The dia- phorase activity was not inhibited by cyanide, o-phenanthroline, ethylene- 4. THE CHLOROPLAST 205 diamine tetraacetate (EDTA), 3-(4-chlorophenyl)-l,l-dimethylurea, (CMU) or azide. Since ferredoxin reacts with o-phenanthroline one cannot test the sensitivity of the ferredoxin-NADP reductase activity directly. The molecular weight of the protein appears to be approximately 35,000 and it contains 1 mole of FAD per mole enzyme. (f) Factors affecting coupled phosphorylation. The scheme for electron transport (Fig. 8) indicates that ATP formation is coupled to the electron flow. The overall reaction is represented by the equation : light NAD? + H2O + ATP + P — > NADPH2 + ATP + ^©2 This stoichiometry was first observed by Arnon et al. (1958) and has been confirmed by several investigators. Replacement of NADP by various arti- ficial electron acceptors, e.g. ferricyanide or quinones, still leads to the same ATP/2e~ ratio. Evidence on this point is summarized by Jagendorf (1962). In the absence of ADP and phosphate reduction of the acceptor is normally greatly diminished, provided no other component is rate limiting, but the rate can be restored to its original value by the addition of ADP and phos- phate. On the addition of a number of reagents which can act as uncouplers, e.g. NH4+, carbonyl cyanide w-chlorophenylhydrazone (see Losada and Amon, 1963 for further examples), the rate of electron transport remains either unchanged at the "ADP + P-stimulated" rate or may be slightly increased, whereas the ATP formation ceases. Compounds such as NH4"'', primary amines and several anions at high concentration, atebrine at 5 X 10~5m, and the very potent carbonyl cyanide phenylhydrazone inhibitors cause an uncoupling which can be reversed by simply washing them out of the chloroplasts. The electron transport can be irreversibly uncoupled by many fold dilution so as to reduce the cation concentration below a critical level. If chloroplast suspensions are frozen they usually become uncoupled to a considerable extent. Similar work on the uncoupling agents of oxidative phosphorylation by mitochondria have suggested the involvement of proteins in the mitochondria which couple phosphorylation and electron transport. The isolation of these coupling agents from mitochondria has been attempted with some success (Lehninger, 1964). Although it is not exactly clear how these protein com- pounds function, their participation in the coupling in oxidative phosphoryl- ation is indicated (see Chapter 3). In photosynthetic phosphorylation in chloroplasts the only experiments on isolation of the coupHng factors so far reported are those of Avron (1963) who found a 20% or somewhat greater stimulation on the re-addition of a heat labile, non-dialysable factor to depleted chloroplasts. Further experiments may demonstrate the obhgatory nature of protein coupling factors in photosynthetic phosphorylation which may function similarly to those isolated from mitochondria. 206 D. O. HALL and F. R. WHATLEY 2. Cyclic Photophosphorylation In this process the net product is ATP. It is thought to result from the passage of electrons from a photochemically produced reductant to a photo- chemically produced oxidant in an electron flow which is cyclic in nature (Arnon, 1959). In Figs. 9a,b schemes are suggested for two examples of this process. Figure 9a represents a system probably closely related to the system cyt Ag PQ) •Pi |chi a I D Light cyt /■ (a) Fig. 9. Schemes for cyclic photophosphorylation. Details in text. Fd: Ferredoxin, chl a: chlorophyll a, PQ : plastoquinone. occurring in vivo in which electrons are received by ferredoxin from the photochemical reaction and returned by a series of dark reactions via cyto- chrome b& and cytochrome/. The energy released by this transfer of electrons is coupled to ATP formation. The photo-oxidation of H2O, an integral part of noncyclic photophosphorylation, is not involved in the cyclic electron flow. Figure 9b represents a system in which washed chloroplast fragments depleted of ferredoxin (Cisi, see Fig. 3) catalyse the formation of ATP only on the addition of an artificial cofactor, e.g. phenazine methosulphate, vitamin K3 and flavin mononucleotide. These artificial cofactors substitute for ferredoxin and cytochrome b%, which are themselves components of the physiological electron flow system. Plastoquinone is included in Fig. 9b as an intermediate in the cyclic electron flow on the basis of the experiments of Krogmann and Oliver© (1962) and Whatley and Horton (1963), showing the depression of cyclic photophosphorylation when plastoquinone was removed and its restoration when plastoquinone was added back. There is no direct evidence available 4. THE CHLOROPLAST 207 on the participation of plastoquinone in the cyclic photophosphorylation catalysed by ferredoxin. The participation of several of the intermediates shown in Figures 9a and 9b is discussed below. (a) Plastocyanin. Direct evidence is lacking but Trebst (1963) has shown that salicylaldoxime at IO^^m inhibits cyclic photophosphorylation with vitamin K3 as the co-factor to about the same extent as it inhibits noncychc photophosphorylation. On the basis of this inhibition by salicylaldoxime, taken in conjunction with the removal and re-addition of plastocyanin to digitonin treated chloroplasts, Trebst concluded that plastocyanin partici- pates in the electron transport system of noncyclic photophosphorylation. It seems reasonable then to assume that plastocyanin also participates in the cycUc electron transport system. Details of plastocyanin are given in the section (IV. A. 1 .d) above. (6) Cytochrome f. No direct evidence for its role in cycHc photophos- phorylation is available. We place cytochrome / in the scheme adjacent to chlorophyll a by analogy with the position assigned to it in non-cyclic electron flow — see Section (IV.a.I.c) above. It should be pointed out that in green plants technical problems make it extremely difficult to observe any changes in the redox state of cytochrome / upon illumination. Although changes indicating the oxidation of cytochrome /have been seen with golden varieties of leaves or chlorophyll deficient mutants of Scenedesmus it has been pointed out by Hill and Bonner (1961) that these varieties may not necessarily reflect conditions in the normal plant. (c) Ferredoxin. Tagawa, Tsujimoto and Arnon (1963) have demonstrated a functional role for ferredoxin as a catalyst in cyclic photophosphorylation under anaerobic conditions in the presence of CMU to stop the photo- oxidation of water. Prior to this several investigators had detected ATP formation dependent upon ferredoxin ("photosynthetic pyridine nucleotide reductase"), under conditions where oxygen could reoxidize the reduced co-factor. Although this appeared to be a cyclic electron flow it actually closely resembled a non-cyclic system described above and was inhibited by CMU. This oxygen-dependent type of cyclic photophosphorylation has been termed pseudocyclic photophosphorylation. (See Arnon, 1965 for details and references). The cyclic photophosphorylation catalysed by ferredoxin and described by Tagawa et al. (1963) is sensitive to antimycin A, thereby differentiating it from cyclic photophosphorylation catalysed by artificial catalysts and non- cyclic photophosphorylation which are not sensitive to antimycin A. Reason- ing by analogy with the sensitivity of mitochondrial electron flow to antimycin A at the level of cytochrome b, the sensitivity of the ferredoxin catalysed photophosphorylation to antimycin A suggests that ferredoxin may react with the cytochrome b% found in chloroplasts and this in turn with cytochrome 208 D. O. HALL and F. R. WHATLEY /. Urbach and Simonis (1964) studying inhibition of the endogenous photo- phosphorylation in the alga Ankistrodesmus have found this to be sensitive to antimycin A, suggesting that in vivo the cyclic electron flow catalysed by ferredoxin is in fact operative. id) Ferredoxin-NADP reductase. There is no evidence to implicate the reductase in the electron transport of cyclic photophosphorylation catalysed by ferredoxin. Its role appears to be confined to the catalysis of NADP reduction in noncyclic photophosphorylation. However, the possibility can- not be excluded that the flavoprotein may act as a diaphorase in those artificial cyclic photophosphorylation systems which are catalysed by vitamin K3, FMN and various dyes. (e) Cytochrome be (General reference Losada and Arnon, 1964). In ad- dition to cytochrome/, Hill (1954) found another cytochrome (in the b group) which he called cytochrome b&, to be present in chloroplasts. It is auto oxidizable, has an E'o at pH 7 of —0-06 V and a sharply defined absorption band at 563mjLi. The molar ratio of fte to /was determined by Hill and Bonner (1961) to be 1-3. It is clear from the work of Hill and Bonner (1961) and of Lundegardh (1962) that chloroplasts of higher plants contain at least two characteristic cytochrome components, /and b^, and that these compounds are present in relatively high amounts to the extent of one mole of each cytochrome per 400 moles of chlorophyll. Cytochrome b% has not been isolated from plants since it appears to be bound firmly to the insoluble portion of the particle. It is however relatively easy to observe in acetone extracted chloroplasts. On the basis of its redox potential and the sensitivity of the ferredoxin catalysed cyclic photophosphorylation to antimycin A it seems reasonable to assign cytochrome b& to an electron transporting role between ferredoxin and cytochrome / There is no direct evidence to warrant the inclusion or exclusion of plastocyanin in the ferredoxin catalysed cyclic photophosphoryl- ation, even though there is, as mentioned above, evidence of the salicylald- oxime sensitivity of the vitamin K3 catalysed cyclic photophosphorylation. (/) Factors affecting coupled photophosphorylation. The same observations and comments apply to cyclic photophosphorylation as to the non-cyclic photophosphorylation described above (Section IV. a. 1./) The only direct evidence which bears on the subject comes from the work of Avron (1963) who has shown some degree of restoration of activity with protein compon- ents. B. ENZYMES FOR CO2 FIXATION CYCLE Isolated chloroplasts can fix CO2 and reduce it to the level of carbohydrate in the light as was first shown by Arnon, Allen and Whatley (1954) — see Allen, Arnon, Capindale, Whatley and Durham (1955) for further details. 4. THE CHLOROPLAST 209 The incorporation of CO2 in photosynthetic tissues takes place according to a scheme which may be termed the reductive pentose cycle or Calvin cycle. Evidence for the operation of the reductive pentose cycle in algae and leaves comes from the work summarized by Bassham et al. (1954). Subsequent work by Losada, Trebst and Arnon (1960) indicates that the reductive pentose cycle also operates in an isolated chloroplast system. In the condensed diagram (Fig. 10) the cycle is indicated as consisting of three phases. In the carboxylative phase ribulose diphosphate accepts a Carbohydrate synthesis by isolated chloroplasts. _^cn JO 10 Triose .^_fc.i Hexose fc.c««,/.k ■*■ 6 Ru-di- P 12 phosphat^-*^' phosphotr^ ^^"""^ Fig. 10. Condensed diagram of the reductive carbohydrate cycle in chloroplasts. The cycle consists of three phases. In the carboxylative phase (I), ribulose diphosphate (Ru-di-P) accepts a molecule of CO2 and is cleaved to 2 molecules of phosphoglyceric acid (PGA); in the reductive phase (II) PGA is reduced to triose phosphate; in the regenerative phase (III) triose phosphate is partly converted into Ru-di-P and partly into hexose phosphate and starch. All the reactions of the cycle occur in the dark. The reactions of the carboxyla- tive and reductive phases are driven by ATP and NADPH2 formed in the light. One complete turn of the cycle results in the assimilation of 1 mole of CO2 at the expense of 3 moles of ATP and 2 moles of NADPH2. (Losada et al, I960.) molecule of CO2 and yields two molecules of phosphoglyceric acid. In the reductive phase phosphogylceric acid is reduced to triosephosphate. In the regenerative phase triosephosphate is partly converted into ribulose diphos- phate and partly into hexose phosphate and starch. All of these reactions occur in the dark. The energy which they require is supplied by ATP and NADPH2 formed in the light (assimilatory power). One complete turn of the cycle results in the assimilation of one mole of CO2 at the expense of 3 moles of ATP and 2 moles of NADPH2. In Fig. 1 1 the carbon compounds occurring as intermediates in the cycle are summarized and the enzymes believed to be involved in their transformation are listed. These enzymes and their distri- bution within the cell are described in the following sections. I 210 D. O. HALL and F. R. WHATLEY C3 C3 C3 C3 C3 C3 PGA C, C3 C3 C, C3 C3 Triose P Ru-di-P 1. Carboxydismutase 2. PGA kinase and triose P dehydrogenase 3. Triose P isomerase 4. Aldolase 5. Phosphatase Hexose P Triose P 6. Transketoiase Triose P Tefrose P Pentose P 4. Aldolase 5. Phosphatose Triose P Heptose P 6. Transketoiase Pentose P 7. Phosphopentoisomerase 8. Phosphoketopentose epimerase 9. Phosphopentokinase Ru-di-P C3 Triose P [Sugors[ Fig. 1 1 . Summary of reactants and enzymes of the reductive pentose cycle. PGA : phos- phoglycerinc acid, Ru-di-P: Ribulose diphosphate. 1. Carboxylative Phase Ribulose- 1,5-diphosphate carboxylase (carboxydismutase: 4.1.1.39.,!). This enzyme catalyses the reaction: CO2 + D-ribulose- 1,5-diphosphate + H20->2 D-3-phosphoglycerate This enzyme has been isolated and purified from spinach leaves by Weissbach et al. (1956). The enzyme has also been identified in other leaves and in algae (see references in Thomas, 1960; Calvin and Bassham, 1962). Carboxydismutase is localized in the chloroplast. Water extracts (CE in Fig. 3) of chloroplasts isolated in 0-35m sodium chloride contain the enzyme 4. THE CHLOROPLAST 211 (Losada et al., 1960). Jacobi and Perner (1961) showed the loss of the carboxydismutase from whole chloroplasts on washing in 0-3 5m sodium chloride, indicating its original location in the chloroplasts. This loss of carboxydismutase may well acount for the loss of CO2 fixing ability during the isolation and washing of spinach chloroplasts (Smillie and Fuller, 1959). When chloroplasts were isolated in non-aqueous media Smillie and Fuller found that the activity of the carboxydismutase paralleled the chlorophyll content of the fractions of frozen dried leaves. Heber et al. (1963) further confirmed the location of carboxydismutase in chjproplasts from several species, isolating the chloroplasts in non-aqueous media. Smillie (1963) has also shown that carboxydismutase is localized in the chloroplast of light grown Euglena by carrying out the isolation procedure in non-aqueous media. The major component of the soluble leaf protein fraction from green leaves is a high molecular weight substance which sediments as a single protein in the ultra-centrifuge. It accounts for a remarkably high proportion (up to 40%-50%) of the total leaf protein (Corner, Kahn and Wildman, 1957). This protein, originally termed Fraction I protein (Wildman and Bonner, 1947) is localized in the chloroplast (Lyttleton and Ts'o, 1958) and has since been shown to exhibit carboxydismutase activity (van Noort and Wildman, 1964). Although the enzyme in green plants is confined to the chloroplasts it has also been shown by Trudinger (1956) and by Aubert, Milhaud and Millet (1957) to be present in the autotrophic, CO2 fixing, non-photosynthetic sulphur bacterium Thiobacillus denitrificans (in which, of course, no chloro plasts or chromatophores are present). The enzyme purified from leaves (Weisbach, Horecker and Hurwitz, 1956; Jakoby, Brummond and Ochoa, 1956) has a molecular weight of about 300,000, is unstable below pH6, is specific for ribulose-l,5-diphosphate and has a A'wi = approximately 2-3 x IO^'^m for ribulosediphosphate and about 1-5 X 10"2m for HC03~ at pH8. The latter is an unexpectedly high value for the principal CO2 fixing enzyme which in nature depends on an external CO2 concentration of 0-03 % CO2. In studying the stoichiometry of the reaction catalysed by the isolated enzyme it was found that in the presence of excess CO2, one mole of ribulose-l,5-diphosphate gave 2 moles of 3-phosphoglyceric acid. The reaction was apparently irreversible. The purified enzyme was inactive in the absence of divalent metal ions ; magnesium, nickel and cobalt are effective activators. The enzyme is also activated by sulfhydryl com- pounds (cysteine and glutathione) and is inhibited by sulfhydryl binding agents. Mercuric chloride at a concentration of 2 x 10~'*m inhibited the enzyme completely and /7-chloromercuribenzoate inhibited 50 % at this con- centration. Arsenite did not inhibit, indicating that dithiol groups were not involved. Cyanide was found by Trebst, Losada and Arnon (1960) to be an effective 212 D. O. HALL and F. R. WHATLEY inhibitor of the carboxylating enzyme, a concentration of 10-'*m giving 60% inhibition. For further discussion of the inhibitors of CO2 fixation in general see the review by Losada and Arnon (1963). 2. Reductive Phase According to the ideas expressed by Calvin in his description of the photo- synthetic CO2 fixation cycle the reductive phase involves the conversion of phosphoglyceric acid ii^to phosphoglyceraldehyde, a reaction sequence cata- lysed by two enzymes (i) phosphoglycerate kinase (2.7.2,3) and (ii) the NADP dependent triosephosphate dehydrogenase (1.2.1.13). The reactions catalysed by these enzymes are: (i) ATP + D-3-phosphoglycerate^ADP + D-l,3-diphosphoglycerate (ii) D-l,3-diphosphoglycerate + NADPH2-> NADP + D-glyceraldehyde-3-phosphate + orthophosphate. The action of the dehydrogenase has been measured both in the forward and reverse directions. Starting with glyceraldehyde-3-phosphate as a substrate, Jacobi and Perner (1961) added arsenate to render the reaction irreversible and were able to measure the activity of the dehydrogenase alone. Most other workers have chosen to assay the reactions starting with phosphoglyceric acid and have thereby obtained a composite measure of both the enzymes involved in the reductive phase. The enzymes have been isolated from the green tissues of several algae and higher plants (see Thomas, 1960 and Calvin and Bassham, 1962 for references). Both enzymes have been detected in water extracts of the chloro- plasts isolated in 0-35M sodium chloride by Losada et al. (1960) who claimed that this provided evidence that the enzymes are present in the chloroplasts of intact cells. However, Smillie (1963) has commented that since the per- centage of the total cellular activity was not stated he considered that the data were insufficient to justify this claim. The activity found in the chloroplast extract was low compared with the values for the intact leaves indicating that the bulk of the activity was present in non-chloroplast fractions. Jacobi and Perner (1961) have already observed that "whole" chloroplasts isolated in 0-35m sodium chloride lose triosephosphate dehydrogenase ac- tivity during washing at the same time as they lose their water soluble proteins by leakage. This implies an original localization for the NADP-dependent dehydrogenase in the chloroplasts. The ready leakage of chloroplast proteins probably accounts for the low activity reported by Losada et al. (1960) since the "whole" chloroplasts from which the cytoplasmic extract was made had already undergone preliminary washing. A direct demonstration of the original distribution of the enzyme within the cell has been obtained by Smillie (1963) for pea leaves and for Euglena, and by Heber et al. (1963) 4. THE CHLOROPLAST 213 for spinach and other leaves. Using chloroplasts isolated in non-aqueous media they showed that all the NADP-dependent triosephosphate dehydro- genase was localized within the chloroplast. The phosphoglyceric acid kinase is however distributed between the chloroplast and the rest of the cytoplasm an observation consistent with its participation both in photo- synthesis and in glycolysis. In addition to the NADP-dependent dehydrogenase which is closely as- sociated with photosynthetic reactions, green leaves also contain a NAD- dependent dehydrogenase (Gibbs, 1952; Arnon, 1952). This has been detected in water extracts of isolated chloroplasts by Losada et al. (1960) and by Smillie and Fuller (1960) and is also found in non-green tissues of several plants where it presumably participates in glycolysis. Further experiments using chloroplasts prepared in non-aqueous solvents have shown that the enzyme is found in both chloroplast and non-chloroplast fractions, 20%- 40 % of the total amount of the NAD-dependent triosephosphate dehydro- genase activity being found in the chloroplast. It is worth noting that Rosenberg and Arnon (1955) have found a third enzyme which oxidizes triosephosphate but which acts in the absence of phosphate or arsenate and catalyses a reversible conversion of phosphoglycer- aldehyde to phosphoglyceric acid. It requires NADP but does not require cooperation of the phosphoglyerate kinase. The dehydrogenase is present only in green tissue but its role in photosynthetic reactions is unknown. The triosephosphate dehydrogenases are sensitive to iodoacetate and addition of this inhibitor would be expected to result in the prevention of the reductive steps of photosynthetic CO2 fixation. Work on the inhibition of photosynthesis by iodoacetate using intact Elodea leaves (Simonis and Weichart, 1958), and with Chlorella (Kandler, Liesenkotter and Oaks, 1961), has shown the accumulation of phosphoglyceric acid and phosphoenolpyru- vate and a decrease in sugar phosphates, a result in agreement with this expectation. However, other workers have found that in isolated systems the enzyme phosphoribulokinase (see below) is sensitive to iodoacetate and this is probably the site of action of iodoacetate with isolated chloroplasts. Investigations have also been made on the possible competitive inhibition by various sugar phosphates of the triosephosphate dehydrogenases, d- sedoheptulose-7-phosphate was shown by Gibbs (1963) to inhibit CO2 fixa- tion by isolated chloroplasts and in addition to inhibit both the NADP- and NAD-linked dehydrogenases. Work with threose-2,4-diphosphate (a specific inhibitor of muscle and yeast triosephosphate dehydrogenase) has however, given equivocal results and the use of this inhibitor has so far proved to have been of limited value (see Gibbs, 1963). Gibbs and Turner (1964) have summarized additional details of all three dehydrogenases and of the phosphoglycerate kinase from leaf material. 214 D. O. HALL and F. R. WHATLEY 3. Regeneration of the CO2 Acceptors (a) Triosephosphate isomerase (5.3.1.1.). This enzyme catalyses the re- action: D-Glyceraldehyde-3-phosphate-> Dihydroxyacetone phosphate. The isomerase has been found in pea seeds (Tewfik and Stumpf, 1951), in spinach leaves (Peterkovsky and Racker, 1961) and in algae (Richter, 1959). On the basis of i^C labelling experiments with spinach chloroplasts, Trebst and Fiedler (1962) and Losada et al. (1960) have concluded that the photo- synthetic scheme according to Calvin operates in the isolated chloroplast system. This implies the presence of the enzyme triosephosphate isomerase in the systems. Recently Smillie (1963) has reported on the intracellular distribution of this enzyme in both pea leaves and Euglena; by isolating sub- cellular fractions in non-aqueous media about 40% of the total isomerase activity was shown to be in the chloroplast fraction in both organisms. This is in accordance with the expected participation of triosephosphate and isomerase in both photosynthesis and glycolysis. Gibbs and Turner (1964) have summarized additional information on this enzyme. (b) Aldolase (4.1.2.7.). Aldolase catalyses the reaction: Dihydroxyacetone phosphate + D-Glyceraldehyde-3-phosphate -»D-Fructose-l ,6-phosphate. and is believed also to catalyse the second similar aldolase type conversion: Dihydroxyacetone phosphate + D-Erythrose-4-phosphate ->D-Sedoheptulose-l,7-diphosphate. The enzyme has been demonstrated in leaves and algae (references are given in Thomas, 1960). The enzyme has been shown to be present in water extracts of isolated spinach chloroplasts (Losada et al., 1960) as well as "whole" chloroplasts by Jacobi and Perner (1961) who also showed that the enzyme leaks out of the chloroplasts during washing. After these results had indicated a partial localization of aldolase within the chloroplast, the work of Smillie (1963) with subcellular fractions of Euglena prepared in non-aqueous media showed that some 60% of the aldolase activity within the cell was present in the chloroplast fraction. These data are in agreement with the participation of aldolase in both photosynthetic reactions and glycolysis. Further details of this enzyme are given in the review of Gibbs and Turner (1964). (c) D-fructose-\,6-diphosphatase (3.1.3.11.). This enzyme catalyses the reaction : D-Fructose-l,6-diphosphate + H20-> D-Fructose-6-phosphate + orthophosphate 4. THE CHLOROPLAST 215 The enzyme was shown to be present in leaves (see Thomas, 1960 and Calvin and Bassham, 1962 for references) and in algae (Peterkovsky and Racker, 1961, SmiUie, 1964). The enzyme has been shown to be present in the aqueous extract of isolated spinach chloroplasts (Losada et ai, 1960). Smillie (1964) has clearly distinguished between the distribution of alkaline and acid phosphatases within different parts of the pea plant and has shown that the alkaline phosphatase is confined to the green tissues. In Euglena it has been shown to be completely confined to the chloroplasts by fractionation in non- aqueous media, whereas the acid phosphatases are associated with the non- chloroplast fractions. This indicates the importance of the alkaline fructose- diphosphatase in the photosynthetic organelle. The enzyme is highly specific for D-fructose-l,6-diphosphate; it requires Mg2+ for its activity and has an optimum pH between 8-5 and 9-5 (see Gibbs and Turner, 1964 for additional details). {d) Transketolase (2.2.1.1.). This enzyme catalyses the reactions: D-Glyceraldehyde-3-phosphate + D-Fructose-6-phosphate -> D-Erythrose-4-phosphate + D-Xylulose-5-phosphate and D-Glyceraldehyde-3-phosphate + D-SedoheptuIose-7-phosphate -> D-Ribose-5-phosphate + D-Xylulose-5-phosphate. Transketolase has been found in green leaves by numerous investigators (see Thomas, 1960 and Calvin and Bassham, 1962 for references). It has also been found in algae by Peterkovsky and Racker (1961) and Smillie (1963). On the basis of experiments involving the fractionation of subcellular par- ticles in non-aqueous media, Smillie (1963) has shown that transketolase is present in the chloroplasts of Euglena but is also found in other parts of the cell indicating a dual metabolic role for this enzyme. The evidence for the occurrence of the enzyme in isolated chloroplasts rests upon i^C labelling experiments which indicate that the complete photosynthetic reductive cycle operates in vitro (Losada et al., 1960; Trebst and Fiedler, 1962). The presence of transketolase may be inferred from these experiments. This enzyme is described in further detail in the review of Waygood and Rohringer (1964). (e) Ribosephosphate isomerase (phosphopento-isomerase) (5.3.1.6.). This enzyme catalyses the reaction : D-Ribose-5-phosphate -> D-Ribulose-5-phosphate The enzyme has been isolated and partially purified from spinach leaves by Weissbach, Smyrniotis and Horecker (1954). Peterkovsky and Racker (1961) have confirmed the presence of ribosephosphate isomerase in spinach leaves and shown it also to be present in Euglena and Chlorella. Smillie (1963) has I 216 D. O. HALL and F. R. WHATLEY shown that the enzyme is present in the chloroplasts of Euglena but is also found in other subcellular fractions. The i^c labelling experiments of Losada et al. (1960) and Trebst and Fiedler (1962) indicate the presence of this enzyme in isolated spinach chloroplasts. Further details of the enzyme are given in the review of Waygood and Rohringer (1964). (/) Ribulosephosphate-3-epimerase (phosphoketopentose epimerase) (5.1. 3.1.). This enzyme catalyses the reaction: D-Xylulose-5-phosphate -> D-Ribulose-5-phosphate The enzyme has been demonstrated to occur in spinach leaves (Horecker, Hurwitz and Smyrniotis, 1956; Peterkovsky and Racker, 1961) and it is present also in Euglena and Chlorella (Peterkovsky and Racker, 1961). Its presence in the isolated chloroplasts is inferred from the ^^C labelling experi- ments. Waygood and Rohringer (1964) have summarized further details of the enzyme. ig) Phosphoribulokinase (phosphopentokinase) (2.7.1.19.). This enzyme catalyses the reaction: ATP + D-Ribulose-5-phosphate -> ADP + D-Ribulose-l,5-diphosphate The enzyme has been purified from spinach leaves by Hurwitz, Weissbach, Horecker and Smyrniotis (1956) (see Thomas, 1960 for other references). Losada et al. (1960) indicated that the enzyme is present in water extracts of isolated spinach chloroplasts. Peterkovsky and Racker (1961) showed that the enzyme was present in cell free extracts of Euglena and Chlorella, while Smillie (1963) localized the phosphoribulokinase as occurring solely in the chloroplasts oi Euglena, after isolating the chloroplasts in non-aqueous media. The properties of the purified enzyme were studied by Hurwitz et al. (1956) who found that it was specific for ribulose-5-phosphate and ATP. The Michaelis constants were 2-5 X 10"% for ribulose-5-phosphate and 2-8 x 10~^M for ATP. The phosphorylation reaction proceeds at a maximum velocity at pH 7-9 and requires Mg2+ at a concentration of 5 x 10~3m for maximum velocity. Phosphoribulokinase appears to be a sulphydryl enzyme. It is inhibited by the addition of the heavy metals Cu2+ and Hg2+ and by /j-chloromercuribenzoate. The inhibition by metal and by /?-chloromercuri- benzoate was completely reversed by the addition of cysteine. Trebst et al. (1960) and Calo and Gibbs (1960) found that iodoacetate inhibited the carbo- xylative phase of photosynthetic CO2 fixation in isolated chloroplasts, and the latter workers showed in addition that iodoacetamide inhibits the phos- phoribulokinase, thus establishing the site of action of this inhibitor in photosynthesis. Table I shows a summary of the known intracellular distribution of the enzymes involved in photosynthetic CO2 fixation. The enzymes have been J 4. THE CHLOROPLAST 217 Table I. Distribution of Enzymes of the Reductive Pentose Cycle in Several Plants. Numbers refer to listing of enzymes in Fig. 1 1 . (For references, see text). Spinach Pea Euglena 1. Carboxydismutase + + + + + + 2. Triose phosphate dehydrogenase (NADP) + + + + + + 2. Phosphogylcerate kinase + + + 3. Triose phosphate isomerase + + ■ + 4. Aldolase + — + 5. FDP phosphatase (alkaline) + + + + + 6. Transketolase + — + 7. Phosphopentoisomerase + —■ + 8. Phosphoketopentoepimerase + — + 9. Phosphopentokinase + ~ + + + = Present in chloroplast + + = Confined to chloroplast — = Unknown. discussed individually in the preceding sections where the evidence to support their placing in the table is given. C. ADDITIONAL ENZYME REACTIONS 1. Carbohydrate Metabolism (a) Formation of glucose-l-phosphate from fructose-6-phosphate. The con- version of fructose-6-phosphate (a nett product of the reductive CO2 fixation cycle) to glucose-l-phosphate (precursor of starch) requires the presence of two enzymes. These are (i) Glucosephosphate isomerase (5.3.1.9.) which catalyses the reaction: D-Fructose-6-phosphate -> D-Glucose-6-phosphate and (ii) Phosphoglucomutase (2.7.5.1.) which catalyses the reaction: D-Glucose-6-phosphate + D-Glucose-l,6-diphosphate -> D-Glucose-l,6-diphosphate + D-Glucose-1 -phosphate The conversion of P^CJ-glucose-l -phosphate into PC]-fructose-6-phosphate has been shown in intact leaves and leaf homogenates of sugar beet by Burma and Mortimer (1956) indicating the presence of both enzymes in leaf tissues. The glucosephosphate isomerase has been isolated and partially purified from seeds of Phaseolus radiatus by Ramasamna and Giri (1956). The isomerase is widely distributed in plants and in animals. The phospho- glucomutase has been reported by Sissakian (1958) to be localized in the chloroplast. Recent evidence by Bird, Porter and Stocking (1965) using 218 D. O. HALL and F. R. WHATLEY chloroplasts from tobacco leaves isolated in non-aqueous media has con- firmed the presence of both glucose phosphate isomerase and phosphogluco- mutase within the chloroplast. In the special case of Euglena which forms starch outside the chloroplast, the isomerase and the mutase are found in the non-chloroplast fraction (Smillie 1963). Photosynthetic experiments using 1^002 in vivo (see Bassham and Calvin, 1960 for references) have shown the occurrence of the intermediates fructose- 6-phosphate and glucose- 1 -phosphate as early products of CO2 fixation, suggesting that glucose- 1 -phosphate is formed from fructose-6-phosphate by the action of the isomerase and the mutase. Since sucrose has been shown by Stocking, WiUiams and Ongun (1963) to be formed within the chloroplast and presumably requires the prior formation of glucose- 1 -phosphate as an intermediate, the presence of the isomerase and mutase in the chloroplast had already been presumed. {b) Starch synthesis. Starch consists of two components, amylose and amylopectin. In amylose glucose molecules are joined by a-l,4-linkages in an unbranched chain. In amylopectin there is a high degree of branching with relatively short a-l,4-Hnked glucose chains connected by a-l,6-linkages to other a-l,4-linked glucose chains (Feingold, Neufeld and Hassid, 1964). Two enzymes are needed to synthesize starch. These are phosphorylase and the Q enzyme or branching enzyme. (i) Phosphorylase (2.4.1.1.) catalyses the reaction: a-D-Glucose-1 -phosphate + (a-l,4-glucosyl)ra -> (a-l,4-glucosyl)n+i + Orthophosphate The enzyme is widely distributed in plants both in green and non-green parts. In leaves of algae it has been found in both the chloroplasts and in the cytoplasm (see Sissakian, 1958 and Thomas, 1960 for references). There is no absolute agreement between various workers concerning the distribution of phosphorylase. Some workers emphasize that it is predominantly localized in the chloroplasts and others believe in an equal distribution of the enzyme between chloroplast and cytoplasm. Stocking (1959) isolated chloroplasts from tobacco leaves in non-aqueous media and detected at least 54 % of the total phosphorylase activity in the chloroplast fraction. Since starch is in any case synthesized in higher plants only within the chloroplasts or leuco- plasts and is an insoluble product, it seems reasonable to expect that the enzymes required for its synthesis will be localized within the chloroplast or leucoplast. There is an alternative method known for the synthesis of a-l,4-linkages; it utilizes UDP-D-glucose as the substrate (see Section IV.c.l.^). This enzyme system has been found in bean cotyledons by Rongine de Fekete, Leloir and Cardini (1960). An interconversion of sucrose to starch via UDP-glucose was 4. THE CHLOROPLAST 219 also envisaged by them. The pathway via UDP-glucose would be energetically more favourable than the phosphorylase pathway. Unfortunately, however, no information is available concerning the distribution of this synthetase enzyme within the cell. (i) The Q (or branching) enzyme (a glucan-branching glycosyl transferase : 2.4.1.18.) catalyses the formation of a-l,6-linkages in amylopectin. The re- action occurs by the transfer of a terminal segment of an a-l,4-linked glucose chain to the primary hydroxyl of a non-terminal D-glucose residue, i.e., to position 6. We know nothing of its intracellular location but its presence in chloroplasts and in leucoplasts must be inferred from the ability of these organelles to deposit starch internally. (c) Starch breakdown. In view of the equilibrium constant of the synthesis of starch from glucose- 1 -phosphate (which is directly and markedly affected by the pH) it has been postulated that the action of phosphorylase is the ^imary mechanism responsible for the breakdown of starch in plants. An alternative enzyme system responsible for starch breakdown has been provided by the enzyme a-amylase (3.2.1.1.) which hydrolyses the a-1,4- linkages of starch in a random fashion. A similar enzyme jS-amylase (3.2.1.2.) removes maltose units (glucosyl-l,4-a-glucose) hydrolytically from starch. Although the enzymes are widely distributed they are especially active in germinating seeds where rapid starch breakdown is required. The amylases are presumably located in the plastids, as this is where starch is stored. The enzymes have been detected in leaves and algae (see Sissakian, 1958 and Thomas, 1960 for references). ** {d) Sucrose synthesis. Two related mechanisms have been identified for sucrose synthesis in plants. These are catalysed by the enzymes: (i) UDP glucose-fructose glucosyltransferase (2.4.1.13.) which catalyses the reaction: UDP-D-Glucose + D-Fructose ->'UDP + Sucrose (ii) UDPglucose-fructose-phosphate glucosyltransferase (2.4.1.14.) which catalyses the reaction : UDP-D-Glucose + D-Fructose-6-P -> UDP + Sucrose-6-phosphate The involvement of a phosphatase to split off the phosphate from the sucrose- 6-phosphate is presumed since no sucrose phosphate accumulates in the plant. The operation of such a phosphatase, which has been identified in some tissues, would render the second reaction irreversible. Bird et al. (1965) have shown sucrose-6-phosphatase to be present in tobacco chloroplasts isolated in non-aqueous media. The substrate for the transferases, UDPG, is synthesized by the enzyme: (iii) UDPG pyrophosphorylase (2.7.7.9.) which catalyses the reaction : D-Glucose-1 -phosphate + UTP -^ UDPG + Pyrophosphate 220 D. O. HALL and F. R. WHATLEY This enzyme was also found in tobacco chloroplasts isolated in non-aqueous media by Bird et al. (1965). The enzyme is absent from the cytoplasm. The two transferases were first isolated from wheat germ by Leloir and Cardini (1955). Burma and Mortimer (1956) have reported the synthesis of sucrose by such mechanisms using excised sugar beet leaves and leaf homo- genate, indicating the presence of the two enzymes in leaf material. Evidence indicating the occurrence of both transferases in chloroplasts has been given by Bird et al. (1965), as noted above. Radioactive sucrose is formed during the incorporation of i4C02 in the light by Chlorella, Scenedesmus and soyabean leaves and is the first free sugar to become labelled to any extent (see Calvin and Bassham, 1962). The intracellular distribution of the [i'*C]-sucrose formed in photosynthesis was studied by Stocking et al. (1963) in tobacco leaves. After treatment with i'*C02, leaves were freeze-dried and separated in non-aqueous media to isolate various subcellular fractions. After a 20 sec treatment with i'^C02, sucrose appears in the chloroplast fraction only. Appreciable sucrose did not appear in the non-cellular fractions up to 1 to 2 min after i4C02 feeding had taken place. This suggests that the primary site of sucrose synthesis in tobacco leaves is in the chloroplast and implies therefore that there is glucosyl trans- ferase in the chloroplast. This is in agreement with the identification in isolated chloroplasts of the transferases, pyrophosphorylase and phosphatase, required for the synthesis of sucrose. The properties of these enzymes are described in detail in the review by Feingold et al. (1964). 2. Carboxylic Acids (a) Entry of PGA into the Krebs cycle. The enzymes needed to transform phosphoglyceric acid to pyruvate include : (i) Phosphoglycerate mutase (5.4.2.1.) [possibly (2.7.5.3.)] which catalyses the reaction : 3-Phospho-D-glycerate -> 2-Phospho-D-glycerate (ii) 2-phospho-D-glycerate hydrolyase (enolase) (4.2.1.11.) which catalyses the reaction : D-2-Phosphoglycerate -> Phosphoenolpyruvate + H2O (iii) Pyruvate kinase (2.7.1.40) which catalyses the reaction: Phosphoenolpyruvate + ADP -> Pyruvate -f ATP These three enzymes have been found in the leaves of various plants (see Gibbs and Turner, 1964 for references). Evidence for the occurrence of the enzymes in spinach chloroplasts comes from experiments on aqueous chloro- plast extracts (Rosenberg, Capindale and Whatley, 1958). Although the 4. THE CHLOROPLAST 221 evidence is insufficient to determine the extent to which the enzymes are locahzed within the chloroplast of spinach, SmilUe (1963) found approxi- mately 10% of the enolase activity to be concentrated in the chloroplasts in pea leaf fractions isolated in non-aqueous media. Pyruvate would normally be expected to enter the Krebs cycle via CoA formed by way of the pyruvate oxidase system (i.e. pyruvate dehydro- genase, 1.2.4.1; lipoate acetyl transferase, 2.3.1.12; and lipoamide dehydro- genase, 1.6.4.3.). Although the system has been characterized in detail in bacterial and animal cells, little is known about it in plant systems, apart from the observation that certain plant mitochondrial systems can oxidize pyruvate. In all probability the pyruvate oxidase system is not present in chloroplasts. The enzymes which convert phosphoenolpyruvate to malate, aspartate and oxaloacetate have been detected both in green leaves and in water extracts of isolated spinach chloroplasts (see Rosenberg et al., 1958 for references; also Zelitch and Barber, 1960b for malate dehydrogenase in washed spinach chloroplasts). These enzymes include : (iv) Phosphopyruvate carboxylase (4.1.1.31.) which catalyses the reaction: phosphoenolpyruvate + CO2 + H2O -> oxalacetate + orthophosphate (v) Malate dehydrogenase (1.1.1.37.) which catalyses the reaction: Oxalacetate + NADH2 -> L-Malate + NAD (vi) Aspartate aminotransferase (2.6.1.1) which catalyses the transforma- tion: Oxalacetate + L-Glutamate -> L-Aspartate + 2-Oxoglutarate For further details of phosphoenolpyruvate carboxylase and malate de- hydrogenase the review by Davies and ElHs (1964) may be consulted. Sanwal, Zink and Din, (1964) have given further details of the enzyme aspartate transaminase in their review article. (b) Krebs cycle enzymes. Losada and Arnon (1964) have drawn attention to the occurrence of a number of enzymes participating in the Krebs cycle which were found in water extracts of isolated spinach chloroplasts. Tests for individual enzymes have demonstrated the requisite enzymic equipment for the conversion of pyruvate to acetate and oxoglutarate via citrate and isocitrate, the continued formation of oxalacetate necessary for the operation of the condensing enzyme presumably coming via the carboxylation of phosphoenol-pyruvate to oxalacetate. The closely related malate and fumarjite presumably arise when excess reduced pyridine nucleotide is available (the phosphoenolpyruvate carboxylase and the malate dehydrogenase are discussed above in section (IV c. 2.a)). Those enzymes normally associated with the Kreb's cycle which have been identified in chloroplast extract (CE) are the acetate activating enzyme 222 D. O. HALL and F. R. WHATLEY (acetyl CoA synthetase, 6,2.1.1), condensing enzyme (citrate synthase, 4.1.3.7.), aconitate hydratase (4.2.1.3.), isocitrate dehydrogenase (1.1.1.42.), malate dehydrogenase (1.1.1.37.) and fumarase (fumarate hydratase, 4.2.1.2.) — these enzymes catalyse the conversion of oxalacetate to fumarate. The enzyme succinate dehydrogenase (1.3.99.1.) has not been reported in isolated chloroplasts. The available data are insufficient to admit us to claim the localization of these enzymes within the chloroplasts or to specify the extent to which their distribution indicates an original association with the chloroplasts. It is of interest to point out that Zelitch and Barber (1960a) have obtained mito- chondrial preparations from spinach leaves which can carry out the oxi- dation of succinate, citrate, isocitrate, oxoglutarate, pyruvate and NADH2 accompanied by ATP formation (oxidative phosphorylation). The enzymes required for this complex of oxidations are obviously located within the mitochondria, and might conceivably represent a source of contamination in the chloroplast extract. (c) Two carbon acids. The enzyme glycoUate oxidase (1.1.3.1.) catalyses the reaction : GlycoUate + O2 -> Glyoxylate + H2O2 The enzyme has been identified in chloroplasts of various leaves (see Zelitch, 1964 and Da vies and Ellis, 1964, for references and details of the properties of the enzyme). Pierpoint (1962) using a sucrose density gradient centrifuging method to fractionate tobacco leaf homogenates, states that very little of the glycoUate oxidase appeared to be associated with the chloroplast fraction but is instead principally a soluble enzyme. However some fraction of the glycoUate oxidase activity always remains associated with chloroplasts. The function of the enzyme in chloroplasts is unclear although it has been suggested that it forms part of the terminal oxidase system in conjunction with gly oxalate reductase (Zelitch, 1964). The glycoUate oxidase activity is weak or absent in extracts of etiolated plants but on illumination the activity increases markedly. Tolbert and Cohan (1953) have shown that this response to light is indirect. Etiolated plants sprayed with glycoUate in the dark showed an increase in the glycoUate oxidase activity and it is suggested that glycoUate oxidase is an adaptive enzyme, appearing in response to the formation of glycoUate produced during photosynthesis. It is also interesting that the oxidase activity is induced in cell free extracts of etiolated plants on the addition of glycoUate. Although at first sight the involvement of light in the inducing of glycoUate oxidase appears to indicate the localization of the enzyme within the chloroplast, the fact that the effect is indirect, depending eventually on the production of glycoUate upon greening, make such a conclusion unreliable. 4. THE CHLOROPLAST 223 The glycollate pathway in photosynthesizing cells is further discussed in the paper of Tolbert (1963). The enzyme glyoxylate reductase (1.1.1.26) catalyses the reaction: Glyoxylate + NADH2 -> Glycollate + NAD The enzyme is present in spinach leaves and occurs in washed suspensions of spinach chloroplasts (Zelitch and Barber, 1960b). The enzyme has been crystallized from tobacco leaves (see Davies and Ellis 1964, for details of the enzyme). Crude extracts of tobacco and spinach leaves have activity with NADH2 for glyoxylate reduction. Zelitch and Gotto (1962) have partially separated the NADP-and NAD-dependent enzymes and shown them to be distinct entities. It has been suggested that the function of the enzyme is as a terminal oxidase system in conjunction with glycollate oxidase. Butt and Peel (1963) have suggested that the glycollate-glyoxylate cycle participates in the re- generation of ATP in illuminated Chlorella by removing excess NADH2. There is no reported evidence to indicate the intracellular localization of the NADP dependent glyoxylate reductase. In tissues rich in fats, particularly storage tissues, e.g. castor bean seeds, glyoxylate is involved in the operation of the glyoxylate cycle, whereby acetate is converted to malate and other intermediates of the Krebs cycle. Of the two special enzymes involved, malate synthase (4.1.3.2.) and iso- citratase (isocitrate lyase) (4.1.3.1.), only the former has been detected in leaf tissue (see Davies and Ellis, 1964 for further discussion of the glyoxylate cycle in plants). , 3. Amino Acid Synthesis A number of enzymes concerned with amino acid synthesis have been identified both in leaves and algae and in water extracts of isolated chloro- plasts. These enzymes are (a) glutamate dehydrogenase, catalysing the con- version of ammonia to a-amino groups and (b) transaminases, catalysing the transfer of a-amino groups to newly synthesized a-keto acids. (a) Glutamate dehydrogenase ( 1 .4. 1 .2). This enzyme catalyses the reaction : 2-Oxoglutarate + NH3 + NADH2 -> L-Glutamate + H2O + NAD Glutamate dehydrogenase has been found in leaves (see Thomas, 1960 for references). Although the enzyme appears to be principally localized in the mitochondria (Sanwal and Lota, 1964) it has been detected in water extracts of isolated chloroplasts (see Losada and Arnon, 1964). (b) Transaminases (2.6.1.-.). These enzymes catalyse the general reaction: a-Keto-acid A + a-Amino-acid B -> ' a-Amino-acid A + a-Keto-acid B 224 D. O. HALL and F. R. WHATLEY Transaminases specific for glutamate, aspartate and alanine have been identified in leaves and seedlings of a number of plants. Aspartate transaminase was already mentioned in section (IV.c.2.a) as being present in aqueous extracts of chloroplasts isolated from spinach leaves. It is also present in extracts of castor bean endosperm and wheat germ, and in mitochondria, indicating a partial localization in both chloroplasts and mitochondria. In experiments with whole Chlorella cells a high proportion of the ^^CO^ fixed in the light may appear very rapidly in amino acids, especially alanine, glutamate, aspartate and serine. The synthesis of these amino acids can account for 60% of the i'*C fixed by the algae. The available evidence indicates that there are at least two pools of these predominant amino acids and that one of them becomes labelled with such rapidity as to indicate "that the site of their synthesis must be freely accessible to their photosynthetically formed precursors, viz., phosphoenolpyruvate and phosphoglyceric acid" (Calvin and Bassham, 1962). These authors concluded that in Chlorella the more rapidly labelled pools of the amino acids are located at the site of photo- synthetic crabon reduction, probably in the chloroplast. This supports the conclusion that the transaminases (e.g. aspartate transaminase, (2.6.1.1.) and alanine transaminase, (2.6.1.2 and 2.6.1.12.) are located within the chloro- plast. 4. Protein Synthesis App and Jagendorf (1964) have pointed out the difficulties in obtaining substantive evidence for protein synthesis in isolated chloroplasts. However, recent work of Spencer and Wildman (1964), Eisenstadt and Brawerman (1964) and Goffeau and Brachet (1965) indicates that protein synthesis (the incorporation of the amino acids into proteins) may occur in isolated chloroplasts of tobacco, Euglena and Acetabularia. Chloroplasts contain ribosomes which when extracted from the chloroplasts are able to incorporate amino acids into proteins (see Goffeau and Brachet, 1965, for references). Isolated chloroplasts may contain DNA (see Gibor and Granick, 1964 and Kirk, 1963 for references) and also RNA (Eisenstadt and Brawerman, 1964). If the chloroplasts are to synthesize proteins they must be capable of activating the amino acids. Amino acid activating enzymes have been demon- strated in isolated chloroplasts (Bove and Raacke, 1959; Marcus, 1959). These enzymes (6. 1 . 1 .-) catalyse the general reaction : Amino acid + sRNA + ATP -> Amino acyl-sRNA + AMP + Pyrophosphate Bove and Raacke (1959) found that water extracts of isolated chloroplasts contained the enzymes needed to activate tyrosine and to a lesser extent 4. THE CHLOROPLAST 225 methionine and leucine. Washed "whole" chloroplasts were found by Marcus (1959) to activate leucine, isoleucine and valine and to a lesser extent histidine, tyrosine, asparagine, cysteine, proline and alanine. Amino acid activation was also detectable in the cytoplasm, but when the amino acids listed above were tested with cytoplasmic extracts the relative rates of their activation were different from those found with the chloroplast extract. This indicates that the amino acid activation observed in the chloroplasts was probably originally associated with and located in the chloroplast and did not represent simply contamination from extra-chloroplastic sources. In the experiments of Spencer and Wildman (1964) with tobacco leaf fractions the product of amino acid activation, viz. aminoacyl-sRNA, as well as the enzymes themselves, were found in the chloroplast fraction. In experiments on the incorporation of ^^C labelled amino acid, 67 % of the i^C incorporated in one min. was found in the fraction containing amino acyl-sRNA, but the incorporation into this fraction dropped to 25 % of the total i^c incorporated after 30 min. This indicates that the amino acyl-sRNA is an early intermediate of amino acid incorporation, the fraction containing it attaining a maximum radioactivity after 5 min. This labelling was main- tained during the subsequent period of amino acid incorporation, so that the percentage of ^^C in the amino acyl-sRNA fraction decreases with time after 5 min. 5. Lipid Synthesis The ability of isolated spinach and lettuce chloroplasts to incorporate acetate in the light into lipids has been shown by a number of investigators (see Stumpf e^ al., 1963, for references). Stumpf and James (1962) and Stumpf, Bove and Goffeau (1963) found that the system for the light-stimulated incorporation of acetate into oleate and palmitate required the presence of CoA, HCO3-, Mg2+ and Mn2+, and was stimulated when NADP, ADP and orthophosphate were present. They interpreted their results to show that the acetate incorporation into fatty acid in the chloroplast depended upon the occurrence of noncyclic photophosphorylation (see Fig. 8) which results in the formation of NADPH2, ATP and oxygen. They also concluded that in fatty acid synthesis by illuminated chloroplasts light has another, as yet unknown, role besides simply promoting the formation of NADPH2, ATP and O2 during noncyclic photophosphorylation. The individual enzymes concerned with the biosynthesis of long-chained fatty acids from acetate have not been studied in isolated chloroplasts and one can only assume that all the enzymes required for the biosynthesis of these fatty acids are present in the chloroplasts. 6. Pigment Metabolism (a) Chlorophyll. The steps believed to be responsible for the biosynthesis of chlorophyll have been well outlined in the review by Smith and French 226 D. O. HALL and F. R. WHATLEY (1963). The initial steps include the condensation of succinyl CoA and glycine to S-amino-laevulinic acid (S-ALA), the condensation of which leads to the tetrapyrrole structure which is subsequently transformed via Mg-protopor- phyrin to chlorophyll a or chlorophyll b. The enzymes required for all the transformation steps have not been identified in plant material, although it is clear that chlorophyll is accumulated and presumably synthesized within the grana of the chloroplasts. The enzyme chlorophyllase (3.1.1.14) which may catalyse the phytylation of chlorophyllide has, however, been identified in chloroplasts (see Sissakian, 1958). A few of the other enzymes needed in chlorophyll synthesis have been identified in photosynthetic bacteria and in Chlorella. The study of chlorophyll synthesis, using labelled intermediates and mutants of Chlorella has established the probable biosynthetic pathway in the algae. The early precursors, succinyl-CoA and glycine, are probably both syn- thesized in the chloroplasts. Glycine can be formed by transamination from glyoxylate, which is itself an early product of CO2 fixation within the chloro- plast (Zelitch, 1964). The enzymes required for the formation of a-oxo- glutarate from phosphoenolpyruvate are present in chloroplasts. (see Section YW.c.l.b). The oxidation of a-oxoglutarate (a reaction not thus far demonstrated in chloroplasts) would then be expected to yield succinyl- CoA, which could be fed directly into the reaction sequence leading to the synthesis of chlorophyll. It has also been shown that succinic dehydrogenase is significantly absent from the chloroplast, thus preventing the operation of the Krebs cycle. {b) Carotenoids. These pigments (carotenes and xanthophylls) are found to vary in the extent of their occurrence in plastids, depending on the physio- logical state of the plastid. In experiments with 14CO2 there was rapid incor- poration of i^C into j8-carotene in illuminated seedlings and leaves. Illumi- nation of etiolated plants (from which carotene is absent) resulted in the formation of functional chloroplasts in which the carotenoids and xantho- phylls were synthesized together (see Goodwin, 1960 for references). It has been deduced that j3-carotene produced on illumination is formed de novo in newly formed chloroplasts from photosynthetically fixed CO2. Biosynthesis of the carotenoids (tetra-terpenes) is believed to involve the synthesis of mevalonic acid from acetate or leucine and its transformation to isopentenylpyrophosphate, which is the unit which becomes polymerized to form the terpene unit. jS-Carotene and the xanthophylls would appear to be synthesized in the chloroplast. However, not all of the carotenoids present in the leaf are found within the chloroplast, some being localized in chromoplasts and being characteristic of non-green tissues. 7. Nitrate Metabolism The reduction of nitrate to ammonia by leaf homogenates can be accom- 4. THE CHLOROPLAST 227 plished in the dark using reduced pyridine nucleotides or in the hght photo- chemically. Non-green parts of the plant (and leaves in the dark) may carry out nitrate reduction using the pyridine nucleotide dependent nitrate reduc- tase (1.6.6.2) and subsequent enzymes to the level of ammonia (NH4+)via nitrite reductase (1.6.6.4), but in the chloroplast it appears that a somewhat different mechanism may occur. Ramirez, Del Campo, Paneque and Losada (1964) have shown that flavin nucleotides can be photochemically reduced by isolated chloroplasts and subsequently used for the reduction of nitrate to nitrite using the enzyme nitrate reductase, which they isolated from spinach leaves. The further reduction of nitrite to NH4+ was shown by Paneque, Ramirez, Del Campo and Losada (1964) to proceed by way of photoreduced ferredoxin as the electron carrier. The enzyme complex nitrite reductase needed for this last reaction sequence was also isolated from spinach leaves. It is presumed that in the photochemical nitrate and nitrite reducing systems, in contrast to the dark system where pyridine nucleotides are the reducing agents, the reductase enzymes are present inside the chloroplasts. This is rendered more probable since the protein electron carrier, ferredoxin, is known to be localized within the chloroplast. A number of the enzymes involved in the dark and photochemical reduc- tion of nitrate to ammonia have been identified and partly purified from Jiigher plants, but no attempt has been made to localize them within a parti- I^Hilar cell organelle (see Kessler, 1964 for references). 8. Adenosine Triphosphatases (ATPases) (3.6.1.-) These enzymes catalyse the reaction: ATP + H2O -> ADP + Orthophosphate Three types of ATPases have been reported in isolated chloroplasts, namely (i) dark, (ii) light-dependent, and (iii) light-triggered ATPases (see Bennun and Avron, 1964 for references). In the dark, chloroplasts hydrolyse ATP at a very low rate. This rate is stimulated several-fold in the light due to the "triggering" of an ATPase, which is detectable only in the presence of Mg2+ and a large quantity of cysteine. By contrast the light-dependent ATPase is stimulated by Ca2+; in the dark no ATPase is detectable in the presence of Ca2+. It should be noted that the maximum rates of ATPase activity associated with the chloroplasts are 1/50 of the maximum rates of photophosphorylation (Avron, 1962), indicating that the ATPases probably do not contribute markedly to the loss of ATP during photophosphorylation. V. Adenylate Kinase (2.7.4.3) This enzyme catalyses the reaction : ^ ATP + AMP -> ADP + ADP (frhe enzyme has been shown to be present in isolated spinach chloroplasts (Mazelis, 1956) and is easily removable by washing "whole" chloroplasts. 228 D. O. HALL and F. R. WHATLEY When this enzyme is present AMP can act as the phosphate acceptor in a phosphorylation experiment, and under these conditions is as effective in photosynthetic phosphorylation as ADP (Allen, Whatley and Arnon, 1958). Smillie (1963), using a non-aqueous solvent technique, has shown that the enzyme is present both in the chloroplast and in the non-chloroplast fractions isolated from Euglena. 10. Various Redox Enzymes (a) Glucose-6-phosphate dehydrogenase (1.1.1.49). This enzyme catalyses the reaction: Glucose-6-phosphate + NAD? -> Glucono-j8-lactone-6-phosphate + NADPH2 This dehydrogenase has been extracted and purified from acetone treated spinach, wheat and tobacco leaves and from algae (see Thomas, 1960 and Waygood and Rohringer, 1964 for references). Evidence for the occurrence of the enzyme in chloroplasts comes from unpublished observations of Whatley with water extracts of isolated spinach chloroplasts, where the reduction of NADP was followed with glucose-6- phosphate as the substrate. Losada et al. (1960) have also shown that glucose-6- phosphate greatly stimulates i^C02 uptake in an isolated chloroplast system. It has been shown by Smillie (1963) that the enzyme is largely concentrated outside the chloroplast in Euglena. {b) Phosphogluconate dehydrogenase (1.1.1.44). The reaction catalysed by this enzyme is : 6-Phospho-D-gluconate + NADP -> D-Ribulose-5-phosphate + CO2 + NADPH2 The enzyme is widely distributed in all parts of the plant, including the leaves (Waygood and Rohringer, 1964). Evidence for its occurrence in chloroplasts comes from the experiments of Losada et al. (1960) where 6-phospho- gluconate stimulated the photosynthetic i''C02 uptake of isolated spinach chloroplasts some fifteen-fold. The enzyme is largely concentrated outside the chloroplasts in Euglena (Smillie, 1963). (c) Catalase (1.11.1.6). This enzyme catalyses the reaction: H2O2 + H20a > O2 + ^20 Krogmann (1960) has shown that about 30% of the catalase activity of the whole leaf homogenate was associated with the unwashed chloroplast, which retained this activity for one washing and gradually lost the catalase activity on subsequent washing. The enzyme was also found in broken chloroplasts isolated from spinach leaves by Trebst and Eck (1961) who studied the effect of various catalase inhibitors on H2O2 accumulation in photosynthetic phosphorylation. 4. THE CHLOROPLAST 229 (d) Polyphenol oxidase (o-diphenol oxidase) (1.10.3.1). This enzyme catalyses the reaction : o-Diphenol + IO2 ->■ o-Quinone + H2O Polyphenol oxidase is present in many leaves, but is not universally distri- buted (see Thomas, 1960, for references). Arnon (1949) demonstrated that in sugar beet the enzyme was associated with broken, washed chloroplasts. Trebst and Wagner (1962) found that freshly isolated spinach chloroplasts showed little if any polyphenol oxidase activity but on breaking the lipid structure of the chloroplasts the latent polyphenol oxidase activity was revealed. (e) Diaphorase (or transhydrogenase). As noted in section (IV.A.l.e), diaphorase, transhydrogenase, and ferredoxin-NADP reductase are probably one and the same enzyme and occur in isolated chloroplasts. The diaphorase is further discussed in section (IV.A.l.e) under the name ferredoxin-NADP reductase. (/) Cytochrome c photo-oxidase system. No cytochrome c oxidase (1.9.3.1) has been found in chloroplasts although it is present in plant mitochondria (Hackett, 1955). However, in the light reduced cytochrome c is oxidized by oxygen or NADP using isolated chloroplasts which have been pretreated with digitonin. This cytochrome c photo-oxidase activity, which is cyanide in- sensitive, is a manifestation of the activity of a terminal portion of the photo- synthetic electron transport chain. It should be noted that cytochrome /, which occurs naturally within the chloroplast in the reduced form, becomes oxidized on illumination and reduced again in the dark (see review by Duysens, 1964), another manifestation of this type of activity. Miscellaneous Enzymes r^jX:i^^ Imj (a) Hexokinase (2.7.1.1.) Catalyses the reaction: ATP + D-Hexose -> ADP + D-Hexose-6-phosphate le enzyme is widespread in the plant kingdom (see Waygood and Rohringer, 1964, for references). However, hexokinase has not been directly demonstrated in isolated chloroplasts. The only evidence suggesting its occurrence in chloroplasts comes from the experiments of Losada et al. (1960), where the addition of glucose or fructose to the isolated chloroplast system assimilating i^C in the light resulted in a 10-15 fold increase in CO2 uptake. It seems justifiable to conclude that the "chloroplasts have the enzyme systems necess- ary for catalysing the rearrangements of the added primary substances to yield the pentosemonophosphate which is needed for the carboxylation phase of CO2 assimilation." The enzymes would include hexokinase. ib) Phospholipase C (3.1.4.3) and Phospholipase D (3.1.4.4). Both these enzymes hydrolyse phospholipids, and have been detected in plastids isolated 230 D. O. HALL and F. R. WHATLEY from a number of leaves, although the enzymes are also present in the non- chloroplast fractions of leaves (see Barron, 1964 for references). D. INDUCED ENZYME FORMATION The localization of some enzymes within the chloroplast may be deduced from the behaviour of etiolated plants on illumination. The rapid greening which occurs in the light is accompanied by the development of the lamellar structure of the chloroplast and at the same time there is the development of a number of chloroplast enzymes. Increases in the total protein content of the plastid fraction have been noted and the development of a particular enzyme has been studied. On illumination, increases occur in the activity of the NADP-dependent triosephosphate dehydrogenase, ferredoxin, ferredoxin- NADP reductase (transhydrogenase), ribulose-1, 5-diphosphate carboxylase, alkaline fructose- 1, 6-diphosphatase and glycollate oxidase. It has been shown that the glycollate oxidase is induced by the prior formation of gly- collate in the light as an early product of photosynthesis (see Section IV.c.2c). These enzymes appear to be quite definitely located within the chloroplast. In addition Smillie (1962) has pointed out that certain enzyme activities in cells containing developing chloroplasts undergo changes which are more closely connected with the changes in photosynthetic rates (initially low and increasing) than with the changes in respiration rates (initially high and de- creasing). This was true of ribulose-1, 5-diphosphate carboxylase and of ferredoxin. However, other enzymes e.g. enolase and 6-phosphogluconate dehydrogenase, showed changes related more closely to the changes in the respiration rate and were presumably associated with the non-chloroplast fraction. The changes in the activity of the enzyme transketolase were inter- mediate, suggesting its participation in both photosynthesis and respiration and therefore its partial localization in the chloroplast in addition to its occurrence in the cytoplasm. Both of these approaches afford useful tech- niques for studying the intracellular localization of enzymes. V. Comparison of Oxidative and Photosynthetic Phosphorylation I The electron transport and accompanying ATP formation carried out by mitochondria in oxidative phosphorylation and by chloroplasts in noncyclic photophosphorylation are shown in simplified form in Fig. 12. In mito- chondria the starting point is NADH2, electrons being transferred in a series of identifiable steps through flavoprotein and ubiquinone to cytochrome c and eventually to molecular oxygen yielding water as the end product (see Chapter 3). Each of the steps represents the passage of an electron from a i 4. THE CHLOROPLAST 231 more reduced to a more oxidized potential, and each step is coupled to the formation of a molecule of ATP. For purposes of comparison the second step, ubiquinone to cytochrome c, is emphasized in the figure. In chloroplasts the starting point is water, electrons being transferred in a series of identifiable steps through plastoquinone and cytochrome /to NADP, yielding NADPH2 as the end product. The step plastoquinone to cytochrome / represents the "fall" of an electron to a more oxidized state and is accompanied by the formation of a molecule of ATP. Although the overall electron flow pathways of oxidative and photo- ^nthetic phosphorylation appear to be direct opposites the figure indicates that this is not true of all the component partial reactions. The actual steps resulting in ATP formation, a product common to both reactions, accompany the release of chemical energy during a redox reaction (compare especially le heavy lines in Fig. 12). The electrons are, however, brought to the more Photosynthetic phosphorylation PN PNH2 Oxidative phosphorylation £0 (volts) ^-0-4 PNH2 PN V PQ- ATP hv ATP -UO ^ATP 04 cyt ■ V ATP H2O O2 ^0-8 (b) O2 H2O 'lo. 12. Diagramatic comparison of electron transport and ATP formation in photo- synthetic and oxidative phosphorylation. PN, PNH2: oxidized and reduced pyridine nucleotides, PQ : plastoquinone, UQ : ubiquinone, cyt : cytochrome. reducing potential by very different means in the two processes. In oxidative phosphorylation the reducing electrons in NADH2 arise by a thermochemical reaction from substrate molecules e.g. pyruvate; by contrast, in noncyclic photophosphorylation the reducing electrons result from a photochemical reaction in which water acts as the electron donor, and plastoquinone is the initial electron acceptor. Following the reaction sequence leading to ATP formation (heavy line in Fig. 12) a second photochemical reaction leads to the reduction of NADP. Neither photochemical reaction is accompanied by ATP formation. In noncyclic photophosphorylation the ATP formation is a dark I 232 D. O. HALL and F. R. WHATLEY reaction only secondarily dependent on the photochemical reactions, and closely resembles the dark production of ATP in the mitochondrial system. In cyclic photophosphorylation (Fig, 9) the ATP formation is also a dark reaction dependent on a prior photochemical step. ATP formation is sensitive to a number of inhibitors which act either as inhibitors of electron transport or as uncoupling agents. The same inhibitor does not always produce the same effect in both mitochondrial and chloro- plast systems (Avron and Shavit, 1963; Losada and Arnon, 1964). Although the formation of ATP is in fact a similar thermochemical reaction in both oxidative and photosynthetic phosphorylation, the differential effects of inhibitors indicate that the details of the two processes may be appreciably different. This is true of the intermediates in the two electron transport sequences and also of the ATP formation with its associated couphng factors. The metabolism of mitochondria and chloroplasts may be contrasted by emphasizing that mitochondria appear to be organelles adapted to yield ATP, using respiratory substrates as the energy source and oxygen as terminal electron acceptor. They contain the necessary complement of Krebs' cycle enzymes as ancillary equipment to enable them to degrade the substrates by way of reduced pyridine nucleotide. Mitochondria are thus largely concerned with the oxidation of the substrates, and any synthetic reducing activities they show are associated with the NADH2 and NADPH2 formed from the substrates. By contrast chloroplasts yield ATP together with NADPH2 discarding oxygen as a waste product. They are largely concerned with reactions needing a large supply of reductants. The principal synthetic reactions characteristic of the chloroplasts are CO2 fixation leading to sucrose and starch synthesis, and the synthesis of fats, pigments and proteins. References Allen, M. B., Amon, D. I., Capindale, J. 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Origin and Formation of Lysosomes and Lysosomal Enzymes . . 258 V. Lysosomes and Ingestion Processes (Phagosomes and Phago-Lyso- somes) 261 A. Microscopic and Sub-Microscopic Aspects of Ingestion Processes 262 B. Uptake of Proteins 267 C. Uptake of Lipids 271 D. Uptake of Nucleic Acids and Viruses 273 E. Uptake of Other Macromolecular and Colloidal Material . 274 F. Uptake of Vital Dyes 275 G. Factors Influencing Permeability, Uptake, and Transport . 277 H. Metabolism of Uptake Processes 281 I. Secretion as Reversed Pinocytosis 282 J. Degradation of Ingested Macromolecular Material, Especially of Proteins ........ . 283 K. Accumulation of Poorly-digestible Material in Phago-Lyso- somes (Residual Bodies) ....... 285 VI. Autolysis and Autophagy 286 A. Physiological Autolysis 286 B. Autophagy 288 VII. Relationship of Lysosomes and Phagosomes to Immune Processes . 289 A. Antibody Formation ....... 289 B. Uptake of Antigen- Antibody Complexes . . . .291 ^ This work was supported by research grants from the U.S. Public Health Service (No. GM 12123) and from the American Heart Association. 239 240 W. STRAUS C. Antigen and Antibody Reactions at the Cell Surface . D. Recognition of "Self" ...... VIII. Pathological Changes Related to Lysosomes and Phagosomes . A. Pathological Enlargement of Lysosomes and Phagosomes B. Release of Lysosomal Enzymes In Vivo C. Extrusion of Lysosomes and Lysosomal Enzymes D. Possible Role of Lysosomes in Inflammation, Necrosis, Shock and Virus Infection ....... IX. Appendix. Methods for the Study of Lysosomes and Phagosomes A. Isolation of Lysosomes ...... B. Cytochemical Reactions for Lysosomal Enzymes C. Markers for Phagocytic Vacuoles .... X. Concluding Comments ....... Acknowledgement References ......... 293 293 294 294 296 296 297 298 298 301 303 305 306 306 I. Introduction The history of the study of lysosomes began with the discovery by de Duve, Pressman, Gianetto, Wattiaux and Applemans (1955) that a group of acid hydrolytic enzymes was concentrated in the "light" mitochondrial fraction isolated from liver homogenates of rats by diiferential centrifuging. The enzymes were thought to belong to a special type of cytoplasmic granule, 0-2 to 0-4 fx diameter, and to function in the degradation of various biological substrates; therefore, the name "lysosomes" was given to these granules (de Duve et al., 1955).. De Duve and co-workers also observed that the hydrolytic enzymes oi the isolated lysosome fraction were active only if the granules were damaged by osmotic shock, freezing and thawing, or by deter- gents (de Duve, 1959a). De Duve concluded that the lysosomes were sur- rounded by a membrane of lipoprotein nature restricting the accessibility of external substrates to the internal enzymes. Soon after the separation of lysosomes from liver cells by de Duve and coworkers, granules containing the same types of acid hydrolytic enzymes were isolated from kidney homogenates of normal rats (Straus, 1954, 1956). The size of the kidney lysosomes varied within a wide range, from 0-1 to 3 /* diameter. Figure 1 shows a purified kidney fraction with granules of intermediate size (0-5 to 1-5 ju diameter). The most interesting property of the kidney lysosomes was their ability to concentrate injected foreign pro- teins. After injection of egg white or horse-radish peroxidase, for example, the size of many granules increased, and the foreign proteins were con- centrated together with the hydrolytic enzymes in the isolated lysosome fractions (Straus and Oliver, 1955; Straus, 1957a, b). These experiments {suggested for the first time that the lysosomes may function in the digestion or detoxification of foreign material ingested by pinocytosis or phagocytosis, I and that the well-known property of the reticulo-endothelial system to 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 241 segregate ingested foreign material in intracellular granules may be closely related to the activity of lysosomes. Progress during the early phase of lysosome research was based mainly on the biochemical analysis of isolated fractions. In recent years, however, cytochemical methods and electron microscopy have contributed much to Fig. 1 . Lysosomes of intermediate size (0-5-1 -5 /x diameter) isolated from rat kidney, x 400. (Reproduced with permission from the J. biol. Chem. 207, 750 (1954.) the advance of knowledge in this field. In particular, the cytochemical reac- tion for one lysosomal enzyme, acid phosphatase (3.1.3.2.), was very useful in identifying lysosomes in various tissues. Several years after the early cyto- chemical detection of acid phosphatase in kidney droplets (Holt, 1954), this cytochemical method was extended to the identification of lysosomes in other tissues (NovikoflF, 1961; and others). Of special interest was the demonstration by Barka, Schaff'ner and Popper (1961) that acid-phosphatase- positive granules were numerous in the cells of the reticulo-endothelial system. Early observations by electron microscopy led to a misunderstanding of the relationship between lysosomes and mitochondria. Several electron micro- scopists had come to the conclusion that the lysosomes of kidney and certain other cells were derived from mitochondria. This error, which can now be understood by the existence of "autolytic vacuoles" containing mitochondria (Section VI) was corrected only in 1960 by Miller in his work on the re- absorption of injected haemoglobin by the tubule cells of the kidney (Miller 1960). In th? case of the liver, fortunately, no such confusion between ly- 242 W. STRAUS sosomes and mitochondria occurred, and the tentative identification by electron microscopy of lysosomes with pericanaUcular bodies (Noviicoff, Beaufay, and de Duve, 1956) was confirmed by later work. The identifica- tion of polymorphous bodies as lysosomes was much facilitated during recent years by the application of the cytochemical acid phosphatase method of Gomori (1941, 1952), at the electron microscope level (Holt and Hicks, 1961b; Daems, 1962; Miller, 1962; Novikoff, 1963; Miller and Palade, 1964; Ericsson, 1964). As may be seen from this brief review on the development of lysosome research, the study of these granules is now accessible to three main ap- proaches: biochemical analysis of lysosomal enzymes in isolated fractions combined with tests for "latency", cytochemical staining reactions for lysosomal enzymes, and histochemical electron microscopy for acid phos- phatase. In view of the changing properties of lysosomes during their life cycle, a correlation of biochemical with cytochemical methods seems to be ^„ essential for the study of these cell organelles. Since excellent reviews on lysosomes have been written in recent years by de Duve (1959a) and by Novikoff (1961), and since various aspects of the problem were discussed at a recent symposium on lysosomes (De Reuck and Cameron, 1963), it is not appropriate to repeat in detail points already covered there.^ In the following pages the main emphasis will be on the relation- ship between lysosomes and pinocytosis and phagocytosis, a subject with which the writer has some experience from his own work. A comment on terminology: The reading of the literature on lysosomes is made more difficult by the proliferation of new terms for the same type of particles. In part, new names were given to bodies before their function and origin were well understood. In order to avoid confusion of terms in this review, it seems appropriate to comment on terminology at this stage. "Lysosomes" were defined by de Duve and co-workers (1955) as cyto- plasmic granules containing high concentrations of several acid hydrolytic enzymes and showing "latency" of these enzymes. On the basis of more Tecent knowledge of lysosomes, de Duve (1963a) distinguished four sub- types of lysosomes : a storage granule (for lysosomal enzymes), a digestive vacuole, an autophagic vacuole, and a residual body. The properties on which these terms are based will be discussed in detail in the following Sections. The term "phagosome" was suggested originally to indicate the segregating ability of granules which also contained the lysosomal enzymes (Straus, 1958, 1959). Later, when the relationship between lysosomes and phago- 1 While this article was in press, several comprehensive reviews on lysosomes appeared. Berthet (1965), Weissmann (1965), and Wattiaux and de Duve (1966) discussed the genera! properties and function of lysosomes, and Woessner (1965a) reported on lysosomes and lysosomal enzymes with special reference to connective tissue. I 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 243 somes was understood better, the term "phagosome" was suggested to designate phagocytic or pinocytic vacuoles or vesicles alone, before their fusion with lysosomes (de Duve, 1963a; Straus, 1963, 1964a, b). The term "phago-lysosome" or "lyso-phagosome" was used for the combined granules (Straus, 1963, 1964a, b). This terminology simplified the description of experiments in which the fusion of phagosomes with lysosomes was deduced from double staining for acid phosphatase and injected plant peroxidase in the same tissue sections (Straus, 1963, 1964a, b). Enlarged lysosomes which also contained mitochondria or other cell components were termed "cytolysomes" by Novikoff (1960). Since the original idea that these bodies function in cytolysis was not confirmed, the terms "areas of focal degradation" (Hruban, Spargo, Swift, Wissler and Kleinfeld 1963) or "autophagic vacuoles" (de Duve, 1963b) seem to be more appropriate. The term "segresome" was proposed by Tanaka (1962) for granules in which vital stains were segregated. The term "cytosegresomes" was used by Ericsson and Trump (1964) for areas of focal degradation. The neutral term "composite bodies" was preferred by Straus (1961) for much enlarged phago-lysosomes which seemed to contain also cell fragments (mitochondria, nuclei, brush border material). Thus, the terms cytolysome, area of focal degradation, autophagic (autolytic) vacuole, cytosegresome, or composite body all refer to similar types of structures. Gordon, Miller and Bensch (1965) recently suggested a unifying terminology. The terms "proto-lysosomes" were introduced for "young" lysosomes, "telo-lysosomes" for "old" lysosomes (phago-lysosomes), and "auto-lysosomes" for bodies functioning in the digestion of endogenous cell material (autophagic vac- uoles). The diagram from the paper of Gordon, Miller, and Bensch (1965) showing the interrelationship of these particles, is reproduced in Fig. 2. The term "cytosome" was used by Lindner (1957) and by Schulz (1958) and latefr by other electron microscopists to designate dense and polymor- phous bodies in electron micrographs which probably were lysosomes. "Cytosis" was suggested by Novikoff (1961) as a general term for ingestion processes by pinocytosis or phagocytosis. "Endocytosis" for intake and "exocytosis" for extrusion were proposed by de Duve (see, de Reuck and Cameron, 1963, p. 126). Zucker-Franklin and Hirsch (1964) suggested the term "exoplasmosis" for the release of enzymes from cells or into phagocytic vacuoles. The terms used by earlier investigators in relation to ingestion processes were reviewed by Chapman- Andresen (1962). h. II. Properties of Lysosomes A. ENZYMES /. Types of Lysosomal Enzymes As mentioned above, the high concentration of certain hydrolytic enzymes and their "latency" in the intact granules were considered by de 244 W. STRAUS Telo-lysosome (Dense body) Telo-lysosome (Dense body) Residuol body (Lipofuscin, etc.) Fig. 2. Interrelationship of cytoplasmic granules, as suggested by Gordon, Miller and Bensch (1965), functioning in intracellular digestion in strain L cells. (Reproduced with the permission of/. Cell Biol.) Duve (1963a) to be the most characteristic properties of lysosomes. The fojlovving five enzymes were found to be concentrated in rat-liver lysosomal fractions by de Duve and co-workers (1955) and de Duve (1959a): acid I i 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 245 d ^. thospiiatase, acid ribonuclease (2.7.7.16), acid deoxyribonuclease (3.1.4.6), '^-glucuronidase (3.2.1.31), and cathepsin (3.4.4.9). The same enzymes were so found to be highly concentrated in "droplet" fractions isolated from at kidney (Straus, 1954, 1956). More recently, . the same acid hydrolases were demonstrated in partially purified fractions isolated from polymorpho- nuclear leucocytes (Cohn and Hirsch, 1960a), eosinophil leucocytes (Archer nd Hirsch, 1963), macrophages (Cohn and Wiener, 1963a), and in fractions isolated from spleen (Bowers, 1964), intestine (Hsu and Tappel, 1964) and brain (Koenig, Gaines, McDonald, Gray and Scott, 1964). Certain other^ hydrolytic enzymes were reported to be jconcentrated in lysosome fractions of liver: arylsulphatase (3.1.6.1) (Viala and Gianetto, 19^5; Roy, 1958), ^S-galactosidase (3.2.1.23), jS-N-acetylglucosamine hydro- lase (3.2.1. -or 3.2.1.29), a-mannosidase (3.2.1.24) (Sellinger, Beaufay, Jacques, Doyen andde Duve, 1960), collagenase (3.4.4.1 9) (Frankland and Wynn, 1962; Schaub, 1964), a-glucosidase (3.2.1.20) (Lejeune, Thines-Sempoux, and Hers, 1963), hyaluronidase (4.2.99.1) (Aronson and Davidson, 1965) and phos- phatidate phosphatase (3.1.3.4) (Wilgram and Kennedy, 1963; Sedgwick and Hiibscher, 1965). The presence of phosphoprotein phosphatase (3.1.3.16) in lysosomes, reported by Paigen and Griffiths (1959) was not confirmed by Tappel, Sawant and Shibko (1963) by analysis of highly purified preparations. Jhc possibiHty of a high concentration of alkaline phosphatase (3.1.3.1) in lysosomes of leucocytes (Cohn and Hirsch, 1960a), and of lipase (3.1.1.3) in the lysosomes of macrophages (Cohn and Wiener, 1963a) requires confirma- tion since these enzymes were not present in high concentrations in lysosomes of other cells. Uncertainties arose concerning the concentration of esterase (3.1.1.1) lysosomes. The lysosomes show strong staining with cytochemical reactions r esterase (Holt and Withers, 1952; Shnitka and Seligman, 1961) but iochemical determination of esterase in tissue fractions indicated that the main esterase activity was in the microsomal fraction (Underhay, Holt, Beaufay and de Duve, 1956). However, the two enzymes seem to be different since the microsomal esterase was sensitive and the lysosomal esterase was resistant to inhibition by organo-phosphorous compounds (Shnitka and Seligman, 1961 ; Holt, 1963). Hess and Pearse (1958) suggested that the cyto- chemical esterase reaction might be due to cathepsin. This question was clarified to a certain extent by the work of Shibko and Tappel (1964) who observed esterase activity in highly purified lysosomes from rat liver. The activity was lower than that of the other hydrolytic enzymes and the enzyme was fully active in the intact granules. Shibko and Tappel suggested, there- fore, that esterase was bound to the lysosomal membrane. It is of considerable interest that lysosomes seem to contain an oxido- reductase. A NADH2 cytochrome c reductase (1.6.99.3) was detected by Cagan and Karnovsky (1964) and by Rossi and Zatti (1964a) in the granules I 246 W. STRAUS of leucocytes. The presence of the same enzyme was also reported in highly purified liver lysosomes by Tappel and Ragab (1964). It should be noted that most of the hydrolytic enzymes mentioned were much more concentrated in the isolated lysosome fractions than in the ori- '^nal homogenates. However, lysosomes may contain several other enzymes in low concentrations. It is technically difficult to determine whether low concentrations of enzymes in particulate fractions belong to cell organelles in vivo, or whether they were adsorbed on to granules during their isolation. This complication is especially difficult to surmount in the case of lysosomes. As will be mentioned later, enzymes characteristic of other cell organelles may be incorporated into lysosomes by processes of fusion. For example, the fusion of lysosomes with phagosomes will introduce phagosomal enzymes, and ATPase activity (3.6.1.3) present in highly purified kidney lysosomes (Straus, 1954) may be derived from the phagosomes. Low concentrations of alkaline phosphatase observed bv Straus n9S4^ and by Shibko and Tappel (1965) in highly purified lysosomal fractions of rat kidney may belong to the phagolysosomes, since contamination of the purified fractions by microsomes (rich in alkaline phosphatase) was very low in these fractions. The sequestra- tion of lysosomes together with mitochondria and cell fragments (Sections VI. and VIII. a) may introduce low concentrations of non-lysosomal enzymes into these "composite bodies." These considerations show that biochemical and cytochemical observations have to be correlated in the analysis of ly- sosomal enzymes. / 2. Heterogeneity of Lysosomes and Lysosomal Enzymes t^Kj It has already been mentioned that lysosomes vary greatly in size and \ I density. A simple cytochemical staining reaction for acid phosphatase often I shows at first sight striking differences in the size, number, and location of V acid phosphatase-positive granules in the same tissue section. This difference can be seen between the proximal and distal tubule cells in kidney sections (Straus, 1964b) and between parenchymal and Kupffer cells in liver sections. The ultrastructure of the lysosomes as observed by electron microscopy, also reveals the difference in size and the pleomorphism of the bodies (dense bodies, vacuolated bodies, multivesicular bodies, lamellated bodies (Farquhar and Palade, 1962; Miller and Palade, 1964). The heterogeneity of lysosomes is related to the dynamic changes which take place in the granules during their life cycle. As will be reviewed in greater detail in Section V.A.2, lysosomes often fuse with pinocytic or phagocytic vacuoles in which various ingested material is concentrated and gradually digested. Small areas of the cytoplasm may be sequestered together with lysosomes and may form "autolytic vacuoles" (Sections VI. and VIII.a). Since the changes connected with fusion or degradation are probably more 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 247 pronounced in some lysosomes than in others of the same cell or tissue, the heterogeneity of the granules is accentuated. It may be expected that the hydrolytic enzymes also undergo changes! during the life cycle of the lysosomes. The concentration of hvdrolytir: p.nyymfis probably is greater in "young" lysosomes than in older ones which have fused ) with phagosomes. If lysosomes are sequestered together with larger cell components (Sections VI. and VIII.a), the original enzyme concentration may be further diluted. Another cause of heterogeneity would be introduced if differences existed in the rates of synthesis or decay between the various lysosomal enzymes. Nothing definite is known about this point. It also is not known to what extent enzymes which are highly concentrated in most lyso- somes, may be present in only low concentration in others, or may be com- pletely lacking. In order to answer this question, it would be necessary to isolate "pure" lysosomes from homogenous cell types, and to correlate bio- chemical, cytochemical and electron microscopic observations. Lutzner(1964) attempted to obtain such a correlation on different types of blood cells and observed considerable heterogeneity. Shibko, Caldwell, Sawant and Tappel (1963) reported that muscle tissue showed a high concentration of cathepsin and acid RNase but a low concentration of acid phosphatase, aryl-sulphat- ase, ^-glucuronidase, and /S-galactosidase. It would be interesting to test whether purified lysosomes isolated from muscle tissue have the same pro- perties and whether the enzymes were derived from lysosomes of muscle cells or blood cells. In spite of these variations, the similarities in the enzyme content of different lysosomes are probably more marked than the differences. As was mentioned, the same hydrolytic enzymes were found to be concentrated in the lysosomes oTtiver, kidney, leucocytes, macrophages, spleen, intestine, and brain. It therefore seems justified to treat the lysosomes as a distinct group of cytoplasmic particles, and to sub-divide them into certain types as proposed by de Duve (1963a). 3. Distribution Patterns of Lysosomal Enzymes The difference in size and density of the lysosomes makes it difficult ori impossible to isolate a large proportion of these granules from a complex! tissue in a highly purified state (Section IX. a.). Most investigators found it easier, therefore, to compare the distribution patterns of lysosomal en- zymes in partially purified fractions obtained from tissue homogenates by differential centrifuging or by density gradient centrifuging. Certain factors which alter the distribution of lysosomal enzymes in vivo and in vitro were not well understood until recently. With fractions isolated from kidney homogenates, for example, it was observed that the distribution pattern of lysosomal enzymes was altered after intraperitoneal injection of egg white (Straus, 1957a). Similar changes in the distribution of lysosomal 248 W. STRAUS enzymes were observed by Cohn and Hirsch (1960b) and by Cohn and Wiener (1963b) in granule fractions isolated from leucocytes and macro- phages after phagocytosis of bacteria. In both cases, fusion of lysospmes with phagosomes (Section V.a.2) was responsible for the altered distribution pattern of the enzymes. Similar changes may be expected if lysosomes fuse with phagosomes containing unknown, endogenous material in untreated animals and during the subsequent degradation of this material. In view of \\ these continuous changes during the life cycle of the granules, no constancy of their centrifugal behaviour can be expected. The risk of artifacts of diffusion or adsorption of enzymes occurring during ^ / the fractionation of tissue homogenates has been pointed out by several l^- investigators (see, for example, Schneider and Hogeboom, 1951). In the case of lysosomes, it is important to consider the variation in the fragihty of the lysosomal membranes (Section II. c) in relation to the functional state of the granules. Artifacts of diffusion, therefore, occur with some types of lysosomes more readily than with others. Thus, "phago-lysosomes" newly formed after ingestion of bacteria or proteins (Cohn and TTirsch, 1960b, Straus, 1957a) may be especially fragile, and they may release their hydrolytic enzymes, in part, into the supernatant fluid during the horao- genization and fractionation of the tissue. The study of distribution patterns of lysosomal enzymes is further com- yljj plicated by the existence in the same tissue of different enzymes which ^y^ I react with the same substrates. These enzjmies may have similar or different intracellular locahzation. The possibility of different enzymes in lysosomal and supernatant fractions from rat liver was raised by Reid and Nodes (1963) in the case of acid ribonucleases, and by Shibko and Tappel (1963) in the case of acid phosphatases. The most comprehensive study of the multiplicity of hydrolases which react with the same substrate in guinea pig \ and mouse liver was made by Neil and Horner (1964a, b). These investigators ,', 'r?^' r^- • • Fig. 3. Phago-lysosomes in kidney cells, 48 hr after intravenous injection of horse-radish peroxidase; stained with benzidine. Note that a large phagosome (phago-lysosome) in lower part of picture contains a crystal, and that most of the peroxidase in other phagosomes of this nephron was already digested. x660. Fig. 4. Two phago-lysosomes in kidney cells showing crystals, 48 hr after intravenous injection of horse- radish peroxidase; stained for acid phosphatase by an azo-dye procedure, x 660. Facing page 250 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 251 in the lysosomes of the kidney medulla during magnesium deprivation (Schneeberger and Morrison, 1965; Battifora, Eisenstein, Laing, and McCreary, 1966). C. LYSOSOMAL MEMBRANE AND ACTIVATION OF ENZYMES The importance of the lysosomal membrane for the "activation" of the hydrolytic enzymes in vitro and in vivo was recognized by de Duve and co-workers early in their work. These investigators systematically studied various treatments which injured the membrane (freezing and thawing, mechanical disruption, osmotic shock, surface-active agents, proteolytic agents, and "autolysis") (Gianetto and de Duve, 1955; Wattiaux and de Duve, 1956; Beaufay and de Duve, 1959). In general, each treatment caused the release of the various hydrolytic enzymes in a similar fashion, although slight variations from one enzyme to the other were observed. These pro- perties supported the hypothesis that the different enzymes belong to the same type of organelle. The "latency " of the enzymes could be explained, according to de Duve and co-workers, by envisaging a model in which the enzymes were contained in a sac of fluid enclosed by a semipermeable membrane. A different model was proposed by Koenig and Jibril (1962). According to these in- vestigators, the hydrolytic enzymes are linked within the granules by ionic bonds to a matrix of a glycolipid nature. This hypothesis was based on the observation that cationic substances caused the release of the enzymes. Jacques, Ennis and de Duve (1964) did not confirm the releasing effect of some of the cationic compounds tested by Koenig and Jibril. Sawant, Desai and Tappel (1964a) discussed the possibility that lysosomal enzymes] might be bound to the membrane of the granules by electrostatic bonds. During recent years, the effects of the following agents which cause release of lysosomal enzymes in vitro have been studied : Vitamin A (Lucy, Dingle, and Fell, 1961 ; de Duve, Wattiaux and Wibo, 1962; Dingle, 1963 ; Weissmann and Thomas, 1963; Dingle, Sharman, and Moore, 1966), vitamin D, vitamin E, vitamin K, and sterols (de Duve et al., 1962), endotoxin (Weissmann and Thomas, 1962), streptolysin (Weissmann, Keiser and Bernheimer, 1963; Hirsch, Bernheimer and Weissmarm, 1963), ultraviolet irradiation (Weiss- mann and Dingle, 1961); Pesai, Sawant and Tappel, 1964) and cations (Koenig and Jibril, 1962; Sawant, Desai and Tappel, 1964a; Jacques, Ennis and de Duve, 1964). Of practical interest is the observation by Sawant et al. (1964a) that the enzymes were released more rapidly in 0-25 m sucrose, often used for fractionation experiments, than in 0-7 m sucrose. Kidney lysosomes were found by Shibko and Tappel (1965) to be much less affected by changes in the osmolarity of the suspending medium than liver lysosomes. Shibko, Pangborn and Tappel (1965) observed that the enzymes were released from kidney lysosomes in two stages during incubation at 37°. In 252 W. STRAUS the first stage, increasing amounts of the enzymes became available to the external substrates but the granules did not release their enzymes and did not show any change in morphology. In the second stage, the enzymes were released at the same time as the granules disintegrated. Several of the agents which cause the release of lysosomal enzymes also labilize the mem- branes of mitochondria and erythrocytes (Dingle, 1963; Keiser, Weissmann and Bernheimer, 1964). It may be of interest to mention in this context that in kidney homogenates in 0-88 m sucrose at room temperature, swelling of mitochondria occurred much more readily, than swelling of lysomes (Straus, 1959). Only one agent, cortisone, seemed to make the lysosomal membrane more resistant toward the release of enzymes (Weissmann and Dingle, 1961 ; de Duve, et al, 1962; Weissmann and Thomas, 1962, 1963). It would be of considerable interest if cortisone specifically affected the lysosomal membrane. y In all these studies, the possibility does not seem to have been considered i sufficiently that the membrane properties may differ with the physiological I state of the cytoplasmic granules. It Nvould not be surprising if the membranes of "young" lysosomes, "old" lysosomes, lysosomes soon after their fusion with phagosomes, and lysosomes after pathological enlargement (Section VIII. A.) behave quite differently. Reports in the literature also indicate that the properties of lysosomal membranes are not uniform. Rahman (1964) showed that the membrane properties of lysosomes (as studied by release of acid phosphatase during incubation at pH 5-2 at 37°, or after treatment with phospholipase at pH 7-0) were quite different in homogenates of liver, spleen, and thymus. Bitensky (1963), by u^ing Gomori's acid phosphatase reaction in a controlled temperature freezing and sectioning procedure, noted that the -fragility of the lysosomes_v;aried with the physiological state of the granules. The factors causing the release of enzymes from lysosomes are little understood. Whereas de Duve and co-workers, in general, found that the release proceeded in a parallel fashion for different enzymes, Sawant, Desai and Tappel (1964a) noted that the various enzymes were released to a different extent. As was pointed out by de Duve et al. (1962), the releasing effect by biologically active agents in vitro does not prove that these agents also have this effect in vivo. Before the effect of an agent on the lysosomal membranes in vivo can be evaluated, it should be determined by light micro- scopy or electron microscopy whether or not the agent caused the trans- formation of one type of lysosome into another. This question of the release of lysosomal enzyme in vivo will also be discussed in Section VIII. The fusion of lysosomes with phagosomes, to be discussed later (Section V. A. 2.), is probably closely related to properties of the membranes. This was foreseen in the hypothesis by Bennett (1956) on "membrane flow". The factors causing the fusion of membranes between lysosomes, phagosomes, and plasmalemma, are unknown. -I 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 253 III. Occurrence of Lysosomes A quick estimation of the number and location of lysosomes in different types of cells can be obtained in many cases by the cj^ochemical staining for jS-glycerophosphatase in formaUn-fixed, frozen sections. Such a_ survey shows that lysosomes are numerous in a number of epithelial cells of organs of absorption, secretion and excretion (kidney, liver, prostate, epi- didymis, for example). Considerable numbers of lysosomes are also present in epithelial cells of the intestine, lung, uterus, and in nerve cells. Relatively few acid phosphatase-positive granules were seen in muscle cells and in the acinar cells of the pancreas. On the other hand, all phagocytic cells or reticulo- endothelial cells (macrophages) contain large numbers of lysosomes: for example, the phagocytic cells of the spleen, bone marrow, liver, and connec- tive tissue. It should be noted that acid phosphatase may not always be a reliable marker for lysosomes and that a cytochemical reaction for acid phosphatase of a cell particle only points to its possible lysosomal nature but is not sufficient proof. This has to be emphasized since many investigators seem to rely on this criterion alone for the detection of lysosomes. In order to establish the lysosomal nature of a cell particle, it has to be shown that several acid hydrolytic enzymes are present in relatively high concentra- tions and that the enzymes show "latency" (see also de Duve, 1963a). In the following paragraphs, a few representative studies from the literature have been selected in which pertinent data on lysosomes in various tissues may be found. A. LYSOSOMES IN EPITHELIAL CELLS 1. Liver The fundamental biochemical work which established the presence of acid | -n hydrolytic enzymes in a distinct class of cytoplasmic granules was done on liver by de Duve and co-workers (1955) and de Duve (1959a). Sawant, Shibko, Kumta and Tappel (1964) succeeded in improving considerably the procedure for the isolation of liver lysosomes. By electron microscopy, Holt and Hicks (1961b) demonstrated the re- action product of acid phosphatase in liver lysosomes. This was also achieved by Daems (1962) who investigated the relationship between liver lysosomes and the vacuoles formed after injection of dextran and iron-dextran. Kent, Volini, Orfei, Minick and de la Huerga (1963), Kent, Minick, Volini, Orfei and de la Huerga (1963) in similar work, studied the uptake of iron-dextran by liver cells and its appearance in lysosomes; they noticed, by electron micro- scopy, increased uptake after injury associated with the fusion of granules. Increased uptake of a protein after injury and enlargement of phago-lyso-i^ somes in liver cells, were also reported by Straus (1963, 1964a) by double cytochemical staining for acid phosphatase and injected plant peroxidase (1.11.1.7). 254 W. STRAUS 2. Kidney Biochemical analysis of 5 lysosomal enzymes in highly purified "droplet" fractions from kidney homogenates was reported by Straus (1954, 1956). Maunsbach (1964a) and Shibko and Tappel (1965) also obtained highly purified kidney lysosomes and studied some of their properties (see Section IX. A.). In cytochemical work, Shnitka and Seligman (1961) stained kidney lyso- somes by the reaction for esterase, and differentiated inhibitor resistant esterase in the lysosomes from an inhibitor-sensitive enzyme in the cyto- plasm. Wachstein, Meisel and Ortiz (1962) compared by cytochemical staining for acid phosphatase lysosomes in kidneys of rat, mouse, guinea pig, and rabbit. Straus (1964a, b) investigated phago-lysosomes in tubule cells of rat kidney by double staining for acid phosphatase and injected peroxidase. Acid phosphatase-positive granules in cells of the collecting tubules in potassium deficiency were investigated by histochemical electron microscopy by Morrison and Panner (1964). Ericsson and Trump (1964) and Ericsson (1964) studied by histochemical electron microscopy acid phosphatase- positive granules in the proximal tubule cells. Fisher (1964) using the same method, investigated the lysosomes during increased protein absorption induced by nucleoside nephrosis. Miller and Palade (1964) by histochemical electron microscopy, demonstrated the presence of acid phosphatase and re- absorbed haemoglobin or ferritin in the same granules. 3. Intestine Biochemical studies on the intracellular distribution of lysosomal enzymes were reported by Hsu and Tappel (1964). Gitzelmann, Davidson and Osin- shak (1964) also fractionated intestinal homogenates and studied the acid phosphatase reaction of the isolated fractions biochemically and in the elec- tron microscope. Sheldon, Zetterquist and Brandes (1955) were the first to demonstrate acid phosphatase-positive granules at the level of the electron microscope. More recently, Behnke (1963) observed enlarged lysosomes, by histochemical electron microscopy for acid phosphatase, in foetal rat duodenum during certain stages of differentiation, and Behnke and Moe (1964) investigated Paneth cells by the same method. Barka (1964) applied the Gomori reaction for acid phosphatase at the electron microscope level to identify lysosomes in intestinal epithelial cells and possible changes during fat absorption. 4. Brain In biochemical work, Koenig et al. (1964) fractionated mitochondrial and microsomal fractions from rat brain into several sub-fractions by dis- continuous density gradient centrifuging. They showed that 5 lysosomal 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 255 enzymes were concentrated in some of the sub-fractions and also presented electron micrographs of the sub-fractions. By cytochemical staining for acid phosphatase, Becker, Goldfischer, Shin and Novikoff (1960), Barron and Sklar (1961), and Anderson and Song (1962) showed the presence of numerous lysosomes in nerve cells. Chouinard (1964) correlated the cytochemical reactions for several lysosomal enzymes with the periodic acid-Schiff re- action, in Purkinje cells. Histochemical electron microscopy was applied to demonstrate the acid phosphatase reaction product in lysosomes of brain stem by Torack and Barrnett (1962) and in neurosecretory cells by Osinshak (1964). 5. Prostate Bertini and Brandes (1964) reported (in an abstract) the biochemical analysis of acid phosphatase and cathepsin in fractions isolated from prostate homogenates by differential centrifuging. Brandes, Groth and Gyorkey (1962) and Brandes (1963, 1965) investigated lysosomes in prostatic epi- thelium by electron microscopy, and by cytochemical staining for acid phosphatase. 6. Epididymis In biochemical work, Schneider and Kuff (1954) observed concentra- tion of acid phosphatase activity in isolated Golgi material. By cytochemical staining for acid phosphatase, Allen and Slater (1958) demonstrated the presence of many lysosomes in the epithelial cells of the epididymis. 7. Urinary Bladder Kanczak, Krall, Hayes, and Elliot (1965) isolated lysosomal fractions rich in acid phosphatase and jS-glucuronidase from canine and bovine transitional epithelium. The granules were also identified in situ by their cytochemical reaction for acid phosphatase. 8. Uterus, Ovaries and Endometrium Nilsson (1962c) and Fuxe and Nilsson (1963) observed lysosomes in the uterus of spayed mice by cytochemical staining for acid phosphatase and esterase, and noted the effect of oestrogen. Nilsson (1962a, b) investigated lysosomes by electron microscopy in the human uterus during the folicular and luteal phases. Banon, Brandes and Frost (1964) made a cytochemical study of lysosomes in rat ovaries and endometrium during the oestrus cycle. Woessner (1965b) analysed changes in the concentration of several lysosomal enzymes in homogenates and fractions from rat uterus in relation to preg- nancy, post-partum involution, and collagen breakdown. P. Thyroid, Adrenals Hosoya (1963) fractionated thyroid tissue and found that acid phosphatase was concentrated in a fraction which also showed dense bodies under the 256 W. STRAUS electron microscope. Wollman, Spicer and Burstone (1964) demonstrated lysosomes in thyroid epithelial cells by cytochemical staining for acid phos- phatase and esterase and studied the relationship to colloid droplets. Smith and Winkler (1966) fractionated homogenates from the bovine adrenal medulla by centrifuging in a discontinuous sucrose gradient. They separated the lysosomal fraction (in which 6 acid hydrolytic enzymes were concentrated) from the mitochondrial fraction and the chromaffine granule fraction. B. LYSOSOMES IN RETICULO-ENDOTHELIAL CELLS AND BLOOD CELLS The isolation of lysosomes and the biochemical analysis of their hydrolytic enzymes from polymorphonuclear leucocytes was reported by Cohn and Hirsch (1960a), from eosinophil leucocytes by Archer and Hirsch (1963), and from macrophages by Cohn and Wiener (1963a). Lutzner (1964) briefly reported the isolation, biochemical analysis and electron microscopy of lysosomes Jrom various types of blood cells. Marcus, Zucker-Franklin, Safier, and Ullman (1966) fractionated homogenates from human blood platelets and obtained fractions rich in acid phosphatase, cathepsin, and jS-glucuronidase. In cytochemical work, acid phosphatase in lysosomes was demonstrated in reticulo-endothelial cells of spleen, liver, thymus, lymph nodes, and other organs, by Barka and co-workers (1961). Thorbecke, Old, Benacerraf and Clarke (1961) observed acid phosphatase-positive granules in Kupffer cells of liver during various treatments modifying the activity of the reticulo- ) endothelial system. Acid phosphatase- and esterase-positive granules in alve- olar and peritoneal macrophages and in polymorphonuclear leucocytes were demonstrated by Dannenberg, Burstone, Walter and Kinsley (1963). Acid phosphatase-positive vacuoles in blood platelets were detected by histo- chemical electron microscopy by White, Krivit and Vernier (1964). The presence of lysosomes in lymphocytes and their increase after phytohaemag- glutinin treatment was reported by Hirschhorn and co-workers (1965), Allison and Malucci (1964a), Diengdoh and Turk (1965), and Parker, Wakasa, and Lukes (1965) (see also Chapter 7). Straus (1964a, Fig. 8 and 1964d, Fig. 10) showed "phago-lysosomes" in Kupffer cells of liver and in macro- phages of spleen by double staining for acid phosphatase and injected peroxidase in the same tissue sections. In experiments, illustrated in Figs. 5-7 of the present article, this procedure was applied to demonstrate phago- lysosomes in macrophages of thymus, lung, and prostate. Rahman (1962a, b) purified "dense bodies" probably derived from phago- cytic cells of the thymus after X-ray irradiation and measured acid phospha- tase and j3-glucuronidase activities of the isolated fractions. Ruyter (1964) showed acid phosphatase-positive granules in osteoclasts cytochemically. Vaes and Jacques (1965) fractionated bone tissue homogenates and demon- Fig. 5. Phago-lysosomes in macrophages of lung, 3 hr after intravenous injection of horse-radish peroxidase. Note purple (blue-red) color of phago-lysosomes in macrophages after double staining for acid phos- phatase and peroxidase in the same section, x 660. 6. Phago-lysosomes in macrophages of connective Lie of prostate, 2 hr after intravenous injection of se-radish peroxidase. Note purple (blue-red) color arge phago-lysosomes in macrophages after double ning for acid phosphatase and peroxidase, and acid ■sphatase reaction (without peroxidase) in lyso- les of prostatic epithelial cells. ■ 660. % Fig. 7. Phago-lysosomes in macrophages of thymus, 24 hr after intravenous injection of horse-radish per- oxidase; double staining for acid phosphatase and peroxidase. Note that the peroxidase in some phago- lysosomes seems to have been digested already, x 660. mm • ' 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 257 strated the concentration of 8 lysosomal enzymes in the "light" mitochondrial (lysosomal) fraction. C. LYSOSOMES IN DIFFERENT SPECIES Acid phosphatase activity in fractions isolated from amoebae was studied in relation to metachromatic vacuoles by Quertier and Brachet (1959). LagunoflF(1964) observed no significant change in the acid protease and acid phosphatase activities of amoebae after pinocytosis of proteins. Biochemical investigation of 6 lysosomal enzymes in muscle, spleen, and hver of various animals (marine invertebrates, bees, frogs, turtles, pigeons, ox, sheep, and hogs) was carried out by Shibko, Caldwell, Sawant and Tappel (1963). Zeidenberg and Janoff (1964) analysed 3 lysosomal enzymes in the particulate fractions of kidneys from fishes. Cytochemical staining for acid phosphatase and esterase in food vacuoles of protozoa (paramecium) was applied by Miiller (1962); Miiller and Toro (1962); MuUer, Toth and Toro (1962); Muller, Rohlich, Toth and Tor6 (1963); Miiller, Toro, Polgar and Druga (1963) and by Rosenbaumand Witt- ner (1962). Acid phosphatase-positive granules in amoeba were reported by Birns (1960), in cihates by Seaman (1961) and by Klamer and Fennell (1963), and in planarians by Rosenbaum and Rolon (1960a). Rosenbaum and Ditzion (1963) made cytochemical observations on lysosomal enzymes in the digestive glands of a snail. Brandes, Buetow, Bertini and Malkoff (1964) studied the development of autophagic vacuoles after carbon starvation in Euglena, by histochemical electron microscopy for acid phosphatase. Wachtler and Pearse (1966) demonstrated lysosomes in the pars distalis of the amphibian pituitary by cytochemical staining for acid phosphatase, /S-glucosaminidase, j8-glucuro- ^^^dase, sulphatase, and esterase. ^^P D. LYSOSOMES IN TISSUE CULTURE CELLS Wattiaux (1962) separated fractions enriched in several lysosomal en- zymes, from HeLa cells, and Munro, Daniel and Dingle (1964) analysed fractions isolated from cultured fibroblasts. Cohnand Benson (1965b) measured the increase in 3 lysosomal enzymes in monocytes during differentiation in tissue culture. Acid phosphatase-positive granules were demonstrated in monocytes and macrophages in tissue culture by Weiss and Fawcett (1953), in HeLa cells by Green and Verney (1956), in fibroblasts by Ogawa, Mizuno and Okamoto (1961), in cultures of chick cells by Mulnard (1961) and Flaxman and Mulnard (1961). Hirschhorn, Kaplan, Goldberg, Hirschhorn and Weiss- man (1965) observed a striking increase in acid phosphatase-positive granules in lymphocytes in tissue culture in the presence of haemagglutinin, and made correlated biochemical measurements of enzyme activities. I 258 W. STRAUS E. LYSOSOME-LIKE GRANULES IN PLANT CELLS Lysosome-like granules also seem to exist in plant cells. Walek-Czernecka (1962, 1963, 1965) observed that the spherosomes of plant cells showed cytochemical reactions for acid phosphatase, esterase, j8-glucuronidase, j3- galactosidase, j8-glucosidase, lipase, arylsulphatase, and desoxyribonuclease, and was the first to suggest that these granules might be analogous to animal cell lysosomes. Olszewska and Gabara (1964) reported that granules in plant cells showing the cytochemical reactions for esterase, acid and alkaline phosphatase, arylsulphatase, desoxyribonuclease and jS-glucuronidase seemed to be involved in the formation of the cell plate after cell division, and pointed out the analogies between spherosomes and animal cell lysosomes. Harrington and Altschul (1963) measured acid phosphatase activities in particulate frac- tions isolated from the embryo and endosperm of germinating onion seed and concluded that lysosome-like particles were present. Matile, Balz, and Semadeni (1965) purified spherosomes from corn and tobacco seedlings by continuous density gradient centrifugation. Acid proteinase, acid phospha- tase, esterase, and acid ribonuclease were concentrated in fractions which, by electron microscopy, showed the presence of spherosomes. The authors believe that the spherosomes of plant cells, in analogy with animal cell lysosomes, function in the autolytic degradation of cell material. IV. Origin and Formation of Lysosomes and of Lysosomal Enzymes The formation of lysosomes and of their enzymes is not yet understood completely. It was suggested that "young" lysosomes might be a type of secretory granule comparable to the zymogen granules of the pancreas (Straus, 1957a). The main difference between both types of secretory granules would be in the pH optima of their hydrolytic enzymes, and in the fact that secretion of enzymes from the cells into ducts occurs in the case of zymogen granules, and that utilization of enzymes for intracellular digestion in the same cells in which they are produced occurs in the case of the lysosomes. It may be expected that the synthesis of the lysosomal enzymes takes place on the ribosomes of the endoplasmic reticulum where the synthesis of other proteins is known to occur. One lysosomal enzyme, acid phosphatase, appeared initially in the cisternae of the endoplasmic reticulum in ciliae, as demonstrated by Carasso, Favard and Goldfischer (1964) by electron micro- scopy. Later, the acid phosphatase in these cells was seen in small pinocytic vesicles which seemed to bud off from the food vacuole. Brandes (1965) demonstrated by electron microsopy that acid phosphatase-positive bodies were formed in the rough-surfaced endoplasmic reticulum of prostatic and seminal vesicle epithelial cells. 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 259 As shown by Caro and Palade (1964), secretory enzymes formed in the endoplasmic reticulum of the pancreas are assembled in the Golgi region and later discharged at the periphery of the cells. The suggestion of NovikoflF (1961) that the lysosomal enzymes are also assembled in the Golgi zone therefore seems plausible. Novikoff observed a positive acid phosphatase reaction, by electron microscopy, in Golgi cisternae and in small vesicles close to them, in several types of cells. Smith (1963) also reported acid phosphatase activity within the inner Golgi cisternae and in small vesicles associated with them, in the adenohypophysis, and increased activity at both sites during periods of heightened secretion. Osinshak (1964) in a careful study by electron microscopy of neurosecretory cells, observed that some Golgi cisternae in these cells showed the reaction product for acid phosphatase. The Activity was also seen in small vesicles, probably young secretory granules, which formed from the Golgi cisternae. Osinshak sug- gested that acid phosphatase was "carried over" and was transient, and was not related to the neurosecretory process. No evidence could be found by Osinshak for the origin of acid phosphatase in the large lysosomes of the neurosecretory cells. Gordon, Miller and Bensch (1965) clearly demonstrated a relationship between Golgi vesicles, probably containing acid phosphatase, and phago-lysosomes. According to the observations of these authors, the small vesicles of "multivesicular bodies" are enzyme carriers derived from the Golgi apparatus. Figure 8 reproduced from the article by Gordon, Miller, and Bensch (1965), shows such small vesicles together with exogenous gold-labelled DNA-protein complexes in a multivesicular body of a fibro- blast in tissue culture. Sainton and Farquhar (1966) also demonstrated clearly that lysosomes are derived from the Golgi complex. These investi- gators examined developing leucocytes from rabbit bone marrow by electron microscopy and observed that two types of lysosomes, the "specific granules" and the "azurophil" granules, originated from different faces of the Golgi complex during different stages of granulocyte development. Interesting observations on the relationship between the Golgi zone and acid phos- phatase-positive granules were reported by Mulnard (1961) and by Flaxman and Mulnard (1961) in cultured chick cells, using light microscopy, and by Brandes et al. (1964) on carbon-starved Euglena, using electron microscopy. Other investigators, however, did not find any acid phosphatase activity, by electron microscopy, in the Golgi zone. Barka (1964) looked for this in vain in the intestinal epithelial cells, and Behnke and Moe (1964) in the Paneth cells (secretory cells of the intestinal epithelium). Miller and Palade (1964) observed a positive acid phosphatase reaction in the Golgi cisternae only in 7 out of 170 sections of kidney cells using electron microscopy. Thus, the question of the acid phosphatase activity in the Golgi apparatus is still controversial. Perhaps, acid phosphatase is only detected in the Golgi zone in cells which do not contain many "old" lysosomes and in which 260 W. STRAUS Fig. 8. Multivesicular body, reproduced from ihe paper by Gordon, Miller, and Bensch (1965), showing relationship of phagosome, containing ingested gold-DNA-protein com- plex, to vesicles derived from the Golgi apparatus, x 32,000. (Reproduced with the permission of /. Cell Biol.) lysosomal enzymes (or lysosomes) are newly formed after stimulation of the enzyme-forming system by increased pinocytosis or autophagy. Several investigators observed that lysosomal enzymes increased in activity after various treatments. However, in most cases, it was not certain whether there was a real increase of lysosomal enzymes in one type of cell, or whether an infiltration of macrophages, rich in lysosomes, into the tissue had taken place. Recently, however, several workers have demonstrated a real increase of lysosomes or of lysosomal enzymes in one type of cell: in the cells of the collecting tubules after K+ deficiency (Morrison and Panner, 1964), in macrophages differentiating from monocytes in tissue culture (Cohn and Benson, 1965a), and in lymphocytes in tissue culture in the 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 261 presence of hemagglutinin (Hirschhorn et al., 1965). Cohn and Benson (1965b) observed that the formation of lysosomes and of hydrolytic en- zymes in macrophages, cultured in vitro, was directly related to the uptake of proteins by pinocytosis. Some investigators have expressed quite different ideas about the origin and development of lysosomes to those mentioned above. According to van Lancker (1964), acid hydrolytic enzymes, probably synthesized in the endoplasmic reticulum, would appear in areas of focal degradation (Sections VI. and VIII.). The hydrolytic enzymes, due to their greater resistance towards autolytic degradation, would accumulate there while the other portions of the sequestered areas undergo autolysis. In this way, enlarged areas of focal degradation would be transformed into smaller lysosomes of normal size. Most investigators who expressed these, or similar ideas (Ashford and Porter, 1962; van Lancker, 1964; Confer and Stenger, 1964) have mainly studied pathologically altered lysosomes. The hypothesis does not explain how the hydrolytic enzymes arrive at the areas of focal degrada- tion in the many cases where the sequestered areas do not contain endoplasmic reticulum. It also ignores processes of fusion between lysosomes and phago- somes which often seem to be related to the development of areas of focal degradation (Straus, 1964a, b; see also Section VIII. a.). Another observa- tion which speaks against the derivation of normal lysosomes from areas of focal degradation is the high concentration of hydrolytic enzymes in highly purified lysosomes from kidney and liver (Straus, 1956; Sawant etal., 1964c). Although a moderate degree of concentration of enzymes is explicable by van Lancker's hypothesis of passive accumulation, the high concentra- tions actually found are similar to the concentrations of hydrolytic enzymes in zymogen granules. Recent observations by Locke and Collins (1965) suggest a way by which Golgi vesicles may bring lysosomal enzymes to sequestered regions of the cytoplasm. In the fat body of a butterfly larva studied by Locke and Collins, small regions of the endoplasmic reticulum, mitochondria, or other cytoplasmic areas, were surrounded by numerous Golgi vesicles. The vesicles fused, thus isolating these areas from the rest of the cytoplasm. Subsequently, the inner of the two "isolation membranes" was resorbed, and processes of coalescence and lysis within the "isolation bodies" were observed. The process described by Locke and Collins also seems to explain why "autophagic vacuoles" are surrounded sometimes by one and sometimes by two membranes. V. Lysosomes and Ingestion Processes (Phagosomes and Phago-Lysosomes) For reasons discussed by de Duve (1959b), lysosomal enzymes are prob- ably concerned with the degradation of metabolites, rather than with transfer 262 W. STRAUS or synthetic reactions. This idea is confirmed by the relationship between lysosomes and the reticulo-endothehal system. As will be discussed in the following paragraphs, the material broken down, in many cases, had been taken up from the environment by pinocytosis or phagocytosis. Even in the case of physiological breakdown of cell components, a type of intracellular pinocytosis or phagocytosis might be involved ("autophagy", de Duve, 1963a, b). Thus the problem of the lysosomes is closely related to fields of inquiry which have been known to cytologists and physiologists for many years under the terms of pinocytosis, phagocytosis, and ''Speichemng". Many investiga- tions dating back over 50 years showed that macrophages and endothelial cells in spleen, lymph nodes, bone marrow, thymus, liver, connective tissue and some other organs could take up such diverse materials as bacteria, colloidal carbon, metal oxides, dyes, and proteins, and segregate them in intracellular granules or vacuoles. Granules in the epithelial cells of the kidney and intestine were found to take part in the " Speicherung" of ingested material (Metchnikoff, 1892; Aschoff, 1924; MoUendorff, 1920, 1925; Gerard and Cordier, 1934; Kedrowski, 1933a, b). These properties were related to the defence of the organism against harmful agents. Whereas phagocytosis, i.e., the ingestion of solid particles, was studied mainly in mammalian macrophages and leucocytes, pinocytosis, i.e., the ingestion of fluid droplets, was first observed in tissue culture cells by Lewis (1931, 1937) and in amoebae by Mast and Doyle (1934). During the last 10 years better understanding of pinocytosis is mainly due to pioneering investigations on amoebae by Holter (I960, 1961) and by Chapman- Andresen. Chapman- Andresen (1962), in her recent studies on pinocytosis in amoebae has also briefly reviewed ingestion processes in other types of cells. The submicroscopic aspects underlying pinocytosis and phagocytosis are essen- tially similar. Therefore, a general term, "phagosome" (Straus, 1958, 1959) may be used to designate granules in which material ingested by pinocytosis or phagocytosis is segregated. A. MICROSCOPIC AND SUBMICROSCOPIC ASPECTS OF INGESTION PROCESSES During the last few years, a better understanding of ingestion processes has been obtained, mainly by electron microscopy. Application of fluorescence microscopy and cytochemistry have also led to useful new information. On the •basis of these observations, the following stages of ingestion processes could be distinguished : (a) adsorption of macromolecular or colloidal material on to the cell surface and formation of pinocytic or phagocytic vesicles (phago- somes) containing this material, (b) fusion of phagosomes with lysosomes, (c) intracellular transport or transport across cells by small phagosomes. 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 263 1. Adsorption Phase and Formation of Micropinocytic Vesicles Observations by light microscopy of cells in tissue culture or of amoebae show the appearance of funnels or invaginations of the cell surface, or the engulfment of droplets of the surrounding medium by thin extensions of the cell surface. By electron microscopy, pinocytic vesicles of submicroscopic size (Clark, 1957, 1959; Miller, 1960; Farquhar and Palade, 1961) or larger vacuoles of microscopic size (Ericsson, 1964) could be distinguished close to the cell surface. It is now generally recognized that macromolecular material, prior to its ingestion, is adsorbed onto a layer of the cell surface. Thus, Brandt and Pappas (1960, 1962) and Nachmias and Marshall (1962) demon- strated by electron microscopy the adsorption of colloidal particles of Th02 and ferritin onto the surface layer of amoeba, and Brandt (1958) and Chap- man-Andresen (1962) described adsorption of fluorescent-labelled proteins onto this layer. Jansco (1955) demonstrated adsorption of colloidal gold onto the surface of Kupffer cells of liver. The adsorption of proteins onto the cell surface of mammalian cells was reported by Ryser, Aub and Caulfield (1962), Easton, Goldberg and Green (1962a), Cormack and Ambrose (1962), Straus (1962a, 1963) and Aronow, Danon, Shahar and Aronson (1964). It would be of considerable interest to determine the chemical nature of the surface layer of absorbing cells. Mucopolysaccharides are often considered to be characteristic constituents of this layer (Bairati and Leh- mann, 1953; Brandt, 1962; Bennett, 1963, Rambourg, Neutra and Leblond, 1966). O'Neill (1964) succeeded in isolating the surface coat of Amoeba proteus, after shrinking the cells by concentrated sucrose solution. The surface preparation contained 35% lipid, 26% protein, and 16% polysaccharide. No sialic acid, which is present in the surface membranes of mammalian cells, was found (O'Neill, 1964). Following the adsorption of material onto the cell surface, small pinocytic vesicles develop which transport the adsorbed materials into the cells. This process may be related to "membrane vesiculation" as suggested by Bennett (1956). The vesicles often coalesce into larger vacuoles in which the ingested material accumulates. In some cases, phagocytic vacuoles (phagosomes) of relatively large size seem to develop directly at the cell surface (Straus, 1962a, 1964a; Ericsson, 1964). Interesting details of structures forming at the cell surface during the uptake of Th02 by the endothelial cells of the liver may be seen in electron micrographs of Toro, Rusza and Rohlich (1962), during the uptake of blood proteins by oocytes in electron micrographs by Roth and Porter (1964) and during the ingestion of ferritin in spinal ganglion cells of toads in electron micrographs of Rosenbluth and Wissig (1964). In recent work, when glutaraldehyde instead of OSO4 was used for the fixation of tissues for electron microscopy, microtubules were detected in many cells. According to Sandborn, Koen, McNabb and Moore (1964), these structures may serve in the transport of fluids or of suspended solids, and M 264 W. STRAUS micropinocytic vesicles may represent dilatations of this microtubular system. It is interesting to note that microtubules were also seen in lysosomes and phagosomes. Journey (1964) demonstrated their presence by electron microscopy in lysosomes and phagocytic vacuoles of peritoneal macro- phages, and Boler and Arhelger (1966) found them in lysosomes of rabbit kidney cells but not in the kidneys of rats and humans. (A diagram showing the relationships of the various structures is given in Fig. 2.) 2. Fusion of Phagosomes with Lysosomes The first observations on the fusion of pinocytized fluid droplets with structures, probably lysosomes, were made by Robineaux and Frederic (1955) on leucocytes, and by Rose (1957) on HeLa cells in tissue cuhure, using phase contrast cinematography. Hirsch (1962) who identified the specific granules of leucocytes as lysosomes (Cohn and Hirsch, 1960a), also observed by phase contrast cinematography the fusion of phagocytic vacuoles with lysosomes after ingestion of bacteria into these cells. The relationship between lysosomes and phagosomes in the tubule cells of the kidney and in Kupffer cells of the liver was studied by the writer (Straus, 1963, 1964a, b), and the fusion of both types of granules was reported. This could be demonstrated by staining phagosomes and lysosomes in contrasting colours in the same cells, after injection of plant peroxidase (Section IX. c). It should be noted that the size of phagosomes and lysosomes which fuse may vary widely from sub-microscopic to microscopic dimensions. Thus, small or large phagosomes may fuse with small or large lysosomes. Recently, the fusion of phagosomes with lysosomes in the tubular cells of the kidney was also demon- strated by electron microscopy by Ericsson (1964) after intravenous injection of homologous haemoglobin into male rats. Figures 9 and 10 are electron micrographs made by Dr. J. E. L. Ericsson (Ericsson, 1964, figures 101 and 102) showing kidney cells of animals injected intravenously with homologous haemoglobin. In Fig. 9, a lysosome (acid phosphatase reaction product) lies close to a phagosome, and in Fig. 10, fusion of a lysosome with a phagosome seems to occur. Miller and Palade (1964) reported the simultaneous appear- ance of intraperitoneally injected haemoglobin together with the acid phos- phatase reaction product in granules of kidney cells in a study by electron microscopy. They did not detect fusion of large phagosomes with pre-existing lysosomes. It may be suggested that in the experiments of Miller and Palade, haemoglobin after intraperitoneal injection into female mice was reabsorbed gradually over an extended period of time. Under these conditions, mainly small phagosomes (micropinocytic vesicles) probably fused with pre-existing lysosomes, as was also suggested by Miller and Palade. The fusion of large phagosomes (phagocytic vacuoles) with lysosomes probably occurred in- frequently or not at all. After intravenous injection of proteins, on the other hand, the initial load of the injected protein is much higher than after 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 265 Fig. 9. Two hr after injection of homologous haemoglobin. The lysosome (cytosome) (C), incubated in the Gomori medium for acid phosphatase appears Hght after post-fixation in 2% osmium tetroxide buffered with s-collidine (Ericsson, 1964). The phagosome (Dr) containing reabsorbed haemoglobin appears dark, x 27,000. (Reprinted with permission from Acta path. Microbiol. Scand.; cf. Ericsson, 1964.) Fig. 10. Four hr after injection of homologous haemoglobin. The lysosome (C) appears light and the phagosome (Dr) appears dark. Some of the dense material (Dm) from the droplet seems to have been delivered to the lysosome. x 14,700. (Reprinted from Acta path. Microbiol. Scand.; cf. Ericsson, 1964.) 266 W. STRAUS intraperitoneal injection, and many large phagosomes fuse with pre-existing lysosomes at about the same time (20 to 40 minutes after intravenous injec- tion of peroxidase (Straus, 1963, 1964a, b) thus facilitating detection of fusion by electron microscopy (Ericsson, 1964). In addition, the glomerulus of male rats may be more permeable to proteins than the glomerulus of female mice. It should also be noted that the molecular weight of peroxidase (44,000) is lower than that of haemoglobin (68,000), and that therefore more peroxidase than haemoglobin may pass the glomerulus, and arrives at the absorbing surfaces of the epithelial cells. Fusion of phagosomes containing ingested bacteria with lysosomes was also observed by electron microscopy in macro- phages and in leucocytes (North and Mackaness, 1963a, b; Lockwood and AlHson, 1964a, b; Zucker- Franklin and Hirsch, 1964; Aronow et al., 1964; Horn, Spicer and Wetzel, 1964). Gordon, Miller and Bensch (1965) showed that newly formed phagosomes, labelled with gold-DNA-protein complexes, fused with late phago-lysosomes, pre-labelled with colloidal iron, in fibro- blasts in culture. In studying the fusion of cytoplasmic granules, it has to be considered that this probably is a rapid process (see Hirsch, 1962) and there- fore may be difficult to detect in fixed tissue. Section VIII. A. will discuss the fusion of lysosomes and phagosomes in pathologically altered tissue to form enlarged phagolysosomes. Food vacuoles of protozoa may be considered to be types of phago- lysosomes. Rosenbaum and Rolon (1960a) and Muller, Rohlich, Toth and Toro (1963) investigated the appearance of lysosomal enzymes in food vacuoles by cytochemical methods. Although the transfer of the enzymes into the vacuoles has not yet been studied in detail, a close analogy with the fusion of lysosomes and phagosomes in mammahan cells may be expected. An important new aspect of the fusion of lysosomes with phagosomes was revealed by the work of Wollman et al. (1964) on thyroid cells. Whereas the phago-lysosomes mentioned in the preceding paragraphs functioned in the segregation and degradation of exogenous material, the phago-lysosomes in the thyroid cells seem to function in the degradation of an endogenous protein containing the precursor of a hormone. As was shown by Wollman et al. (1964) and by Nadler, Sarkar and Leblond (1962), colloid material containing thyroglobulin is ingested from the lumen into the thyroid cells by pinocytosis and accumulates in apically located droplets. These may be considered to be types of phagosomes. Wollman and co-workers induced the new formation of colloid droplets by injection of thyroid stimulating hormone (TSH) into hypophysectomized animals, and they identified lysosomes by the cytochemical reaction for acid phosphatase and esterase. Characteristic changes in the location of granules suggested a fusion of lysosomes with phagosomes. The investigators discussed the possibility that thyroxine is split from thyroglobulin within the phago-lysosomes, and is then released. The relationship between colloid droplets and lysosomes in the thyroid was 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 267 investigated with the electron microscope by Wetzel, Spicer and WoUman (1965). Since lysosomes are numerous in the epithelial cells of endocrine glands, the liberation of active portions of hormones, after partial degrada- tion in phago-lysosomes, may take place in other endocrine organs. This possibility was also discussed by Jung and Niisslein (1962) when they observed a high concentration of proteolytic enzymes in endocrine tissues. 3. Intracellular Transport and Transport across Cells by small Phagosomes Several investigators have observed that proteins and colloidal particles can be transported by micropinocytic vesicles across endothelial cells of blood vessels (Alksne, 1959; Farquhar and Palade, 1961, 1962; Staubesand, 1963; Fernando and Movat, 1964; Palade and Bruns, 1964; Casley-Smith, 1965, see also Majno, 1965). Moore and Ruska (1957) proposed the term "cytopempsis" for transport across cells. Transport of fat (Palay and Karlin 1959) and of proteins (Clark, 1959) in small vesicles across the intestinal epithelial cells was observed by electron microscopy. The transport of injected plant peroxidase across the epithelial cells of the liver, probably by small pinocytic vesicles, was suggested from cytochemical observations (Straus, 1962a, 1963, 1964a). In the tubule cells of the kidney, small pinocytic vesicles containing injected peroxidase or haemoglobin probably originate at the base of the brush border and fuse into large apical phagosomes (Miller, 1960; Straus, 1961, 1962a, Ericsson, 1964). In addition small phagosomes may originate from the large apical phagosomes themselves (Straus, 1964b). This latter process may be similar to the formation of small vesicles from food vacuoles as observed in protozoa by Roth (1960) and by other investigators. It was suggested that the injected peroxidase was transported by these small phagosomes either to pre-existing lysosomes, or that it was transported across the cells to the infoldings of the basal cell membranes and was brought back to the peritubular capillaries or spaces. (Straus, 1961, 1962a, 1964b). Menefee, Mueller, Miller, Myers and Bell (1964b) made observations that suggested transport of injected globin by small pinocytic vesicles across the tubular epithelial cells of the kidney. It is of interest that Marchesi and Barrnett (1963, 1964) demonstrated nucleoside phosphatase activity in micropinocytic vesicles of some but not all endothelial cells. These investigators discussed the possible relationship of this enzyme to transport processes. Pappas and Kaye (1964) reported ATPase activity on the lateral membranes and in intercellular spaces, but not in the micropinocytic vesicles, of the corneal endothelium. Adenosine triphospha- tase was also present in the apical infolding of the epithelium in the ciliary processes of the eye (Pappas and Kaye, 1964). B. UPTAKE OF PROTEINS Although it is now accepted by most investigators that ingested proteins are segregated in phagosomes and phago-lysosomes, the mitochondria were 268 W. STRAUS thought for several years to be sites of protein uptake (Zollinger, 1950; Oliver, McDowell and Lee, 1954; Rhodin, 1954; Straus and Oliver, 1955; Haurowitz, Reller and Walter, 1955; Gansler and Rouiller, 1956). This opinion is still encountered in recent publications (Fisher, 1964; Menefee, Mueller, Miller, Myers and Bell, 1964), However, the hypothesis that mito- chondria take up proteins was questioned by Gitlin, Landing and Whipple (1951), Straus (1954, 1956, 1957a, b, 1959), Ingraham (1955), and Jansco (1955). The sequestration of mitochondria together with phagolysosomes in "composite bodies" in some cases (Section VL and VIIL) may explain, in part, the earlier confusion. The problem of protein uptake, specially in kidney cells and amoebae, was clarified to a large extent by electron microscopy. As was mentioned in Section V. a. 1 , proteins are adsorbed onto the surface layers of pinocytizing cells, and small vesicles which seem to pinch off from infoldings of the surface membranes carry the proteins to apical vacuoles. The electron micrographs by Miller (1960) and Ericsson (1964) showing absorption of haemoglobin in the tubule cells of the kidney, and those by Roth and Porter (1964) showing uptake of proteins by oocytes, illustrate in a striking way the sub- microscopic aspects of this process. Small and large phagosomes probably are related to the smooth-surfaced endoplasmic reticulum seen in electron micrographs. It is not known whether or not ingested proteins can also penetrate to the rough-surfaced endoplasmic reticulum. If antigens can penetrate into cell nuclei as reported by Coons (1956) and Wellensiek and Coons (1964), the passage of proteins through the rough-surfaced endo- plasmic reticulum is possible. As was pointed out by Holtzer and Holtzer (1960) and by Straus (1964d), nuclei may adsorb proteins during fixation or isolation, and hence the apparent locahzation of injected proteins (antigens) in cell nuclei may be an artifact. In the following paragraphs, the uptake of proteins by various tissues is reviewed briefly. 1. Cells of the Reticulo-Endothelial System Kedrowski (1933b) in his investigations on the "segregation apparatus" in cells of the reticulo-endothelial system also demonstrated the uptake of haemoglobin in granules of macrophages and histiocytes of the connective tissue. Sabin (1939), Kruse and McMaster (1949), and Latta, Gitlin and Janeway (1951) noted uptake of dye-protein complexes in granules of the reticulo-endothelial cells throughout the organism. Schiller, Schayer and Hess (1953), and Mancini et al. (1961) using fluorescent-labeled proteins observed their deposition in the cells of the reticulo-endothelial system. Jansco and Jansco-Gabor (1952, 1954) made interesting observations on the uptake of proteins by macrophages and histiocytes and the effect of hista- mine on it. Straus (1964a, Fig. 8 and 1964d, Fig. 10) demonstrated the I 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 269 uptake of injected plant peroxidase by "phago-lysosomes" of macrophages in liver (Kupffer cells) and spleen. In Figs. 5-7 of the present article the uptake of peroxidase by phago-lysosomes in macrophages of thymus, lung, and prostate (connective tissue) is illustrated. In this context, it may be of interest to mention the probable existence of phagosomes (peroxidase uptake) in mast cells (Fig. 11 ; see legend to Fig. 1 1 for further comment, and Section IX. c. for more details on the peroxidase technique). 2. Kidney Cells The ability of kidney cells of amphibians to segregate proteins in intracel- lular granules was investigated by Lambert (1936). Randerath (1947) discussed the uptake of abnormal proteins (Bence-Jones) by human kidney. The early literature on "hyaline droplets" in relation to protein uptake was reviewed by Rather (1952) who himself investigated the re-absorption of haemoglobin and its effects on droplet formation (Rather, 1948). Smetana (1947) and Kruse and McMaster (1949) observed deeply-stained droplets in kidney cells after injection of dye-protein complexes. Oliver et al. (1954) investigated the appearance of protein absorption droplets by staining with iron-haema- toxylin and the Gram method. Straus (1957b, 1959, 1961, 1962a, 1964a, b) studied the uptake of plant peroxidase in "phagosomes" of the tubule cells and their differentiation from mitochondria. Niemi and Pearse (1960) demon- strated the appearance of injected egg white conjugated with a fluorescent dye in kidney droplets and the different localization of protein absorption droplets and mitochondria. Miller (1960) and Miller and Palade (1964) investigated the reabsorption of heterologous haemoglobin, and Ericsson (1964) studied the uptake of injected homologous haemoglobin and the de- velopment of phagosomes and phago-lysosomes. The formation of protein absorption droplets in the cells of the glomerulus was investigated by electron microscopy after injection of ferritin by Far- quhar and Palade (1960, 1961, 1962), and by the fluorescent antibody tech- nique by Kurtz and Feldman (1962). Aggregates of injected globin were observed in the glomerulus during passage through the basement membrane, by Menefee and co-workers (1964a). The uptake of fluorescent-labelled proteins by kidney slices was reported by Holtzer and Holtzer (1960), and the uptake of plant peroxidase by Miller et al. (1965). 5. Liver Parenchymal Cells In contrast to the Kupffer cells, most investigators have found that liver parenchymal cells do not take up proteins in significant amounts. Mayers- bach (1957), however, concluded that fluorescent-labelled proteins entered the liver cells. Straus (1962a, 1963, 1964a) observed that only small amounts of injected plant peroxidase entered the normal liver cells, especially in the 270 W. STRAUS peri-portal areas, and appeared in phago-lysosomes close to the bile capil- laries (see also Section VIII. a.). 4. Intestinal Epithelial Cells of New-born Animals As was shown by Brambell (1958), antibodies from the milk of the mother are absorbed through the mucosa of the intestine of new-born animals, and this process ceases after 10-20 days. Clark (1959) and Payne and Marsh (1962) fed fluorescent-labelled proteins to new-born rats, mice and pigs and demonstrated their locaHzation in large vacuoles of the intestinal epithelial cells. Figures 12 and 13 show that plant peroxidase, fed to new-born rats appears in apical phagosomes and, later, in large phago-lysosomes. 5. Nerve Cells Rosenbluth and Wissig (1964) observed by electron microscopy that nerve cells of the toad take up ferritin by pinocytosis. It will be interesting to see whether the capacity for pinocytosis of proteins is a general property of nerve cells. 6. Erythroblasts Bessis (1963) showed that erythroblasts take up ferritin by pinocytosis from the surrounding reticulo-endothelial cells of the bone marrow. 7. Embryonic Cells The uptake of proteins by embryonic cells was reviewed by Schechtman (1956) and by Brambell (1958). Telfer (1961) studied the uptake of blood proteins by oocytes of saturniid moths by staining with fluorescent-labelled antibodies. Roth and Porter (1964) investigated by electron microscopy the uptake of protein by oocytes of a mosquito. In these species, proteins formed outside the egg are absorbed into the egg from the blood; in other species, yolk protein is synthesized in the egg itself. Dalq (1963), on the basis of his own work and that of Pasteels and Mulnard, presented an interesting dis- cussion on the formation of yolk bodies in relation to phagosomes and lyso- somes. 8. Tumour Cells In vivo Easty (1964) tested the uptake of two fluorescent-labelled proteins by 12 different tumours and did not observe a significant uptake by most of them. Easty reported uptake mainly by the cells of the reticulo-endothelial system and by the tubule cells of the kidney. However, Raimondi (1964) observed by electron microscopy that radio-iodinated serum albumin, injected into two patients with brain tumour (astrocytoma), appeared in pinocytic vesicles of the neoplastic and endothelial cells. ca ^j- u 2^ 5i 4) n 2 "5 £ P C O — X> C ^ g 'g a op 5P== -o '5 . TO 4) *- fN X) 2 O , «i O U _c 60 T3 es ^■ (1> ^ O J= a-o ■" .E 3 1) E -S'o So o ^ >» 1> 60 <> « X (U JO <^« j: U (1> o "" 60 U ^'o .5 i u « «; o 6 .2 4> CO W T2 ^ x: =^ r<^ f^ "m « I- v (» .b: — TO IJ 3 >< a -! «« CO 2| S 60 O ■>< -E « o « 5 C "J « x: 5 •- ^ 3 CO T3 2 -o & CO (« CO • — O V S ^ h i2 6 > « O O O CO 3 «^ O 73 ■^ c « 5J 2 .E O CO 1) JZ 60 60 S =3 CO o 60 •§.E S ? 52 T3 . .— (/) u? '^ CJ '■^ CO c-2 ^ §^-§ • - x> <-> O 3 (U ^.E i- C X U O •- C X < ■— .E o CO CO a> Ji E o. CO .2 V 3 c ,'ff fractions. Loss of enzymes during the isolation procedure was not excluded by these investigators. The small fraction of phago-lysosomes, observed by Straus (1959, Fig. 19D) to float after high-speed centrifugation on the surface of liver homogenates, obtained from rats injected with horse-radish peroxidase, may have corresponded to lipofuscin granules. In addition to peroxidase-positive granules, this layer sometimes contained higher concen- trations of acid phosphatase than the homogenate (W. Straus, unpublished *^--t)bservation). r — Baudhuin, Hers and Loeb (1964) observed by electron microscopy, that / glycogen accumulated in large vacuoles of liver cells in certain types of glyco- ' gen disease. They suggested that this process might be related to the lack of glucosidase, normally present in lysosomes of liver. VI. Autolysis and Autophagy A. PHYSIOLOGICAL AUTOLYSIS Involvement of lysosomes in the breakdown of tissues and cells has been observed in certain cases of physiological regression. These processes are of special interest since they seem to be under hormonal control. In these studies the question arose whether the breakdown of tissue was caused by lysosomes in regressing epithelial cells or in macrophages, or in both. /. Regression of the Mullerian Duct These embryonic organs regress in the male between the ninth and thir- teenth day of development. Brachet, Decroly-Briers and Hoyez (1958) were the first to show that the total and free activities of 3 lysosomal enzymes (acid phosphatase, cathepsin and ribonuclease) increased in chick embryos during the regression of the Mullerian duct. This was confirmed by Scheib (1963). Scheib (1964), in cytochemical studies, observed an increase in the number and size of the lysosomes in the regressing epithelial cells. In addition, there was an increase in the number of macrophages in the conneciive tissue. Scheib suggested that the increase in size of the lysosomes was related to the release of lytic enzymes in vivo. 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 287 2. Regression of the Tail in Xenopus Larvae This process, which is under the control of the thyroid hormone, was investigated by Weber and co-workers. Weber (1963) reported that the amount of cathepsin, a lysosomal enzyme, markedly increased in the regres- sing tail. Salzmann and Weber (1963) demonstrated cytochemically that most of the lysosomes in the regressing tail were in macrophages. A marked increase of several lysosomal enzymes during the regression of Xenopus tails was also observed by Eekhout (1964), Recent electron microscope studies by Weber (1964) indicated that changes in muscle fibres and mito- chondria of the regressing tissue occurred independently of the lysosomes. Thus, the lysosomes are not the primary cause of autolysis during the regres- sion process in Xenopus tail. However, the lysosomes of the macrophages in the regressing tail function in cleaning up injured or dead cells. 3. Muscular Dystrophy Invading macrophages and leucocytes seem to be the main source for the increase of lysosomal enzymes in dystrophic muscle (Tappel et al., 1963). The lysosomes of macrophages participate in the breakdown of injured or dying cells. There is no indication so far that lysosomes in muscle cells are involved in the regression of the tissue. 4. Post-Partum Involution of the Uterus and Regressive Changes in the Ovaries and the Endometrium during the Oestrous Cycle Post-partum involution of the uterus was investigated by Lobel and Deane (1962) using acid phosphatase as a cytochemical marker for lysosomes. Although little enzyme activity was observed in the uterus of virgin or pregnant rats, marked activity developed immediately post-partum in placental detachment sites, epithelial cells, in macrophages, and stromal cells of all uterine layers. Lobel and Dean suggested that the lysosomes function in the resorption and reorganization of the tissue in post-partum involution. Banon, Brandes and Frost (1964) investigated the changes in lysosomes in the ovaries and endometrium during the oestrous cycle. In the corpora lutea undergoing involution, the lysosomal activity was found to be concentrated in surrounding macrophages rather than in the lutein cells themselves. However, during folicular atresia and also in the endometrium during the oestrous cycle, the lysosomes of the regressing cells themselves increased in size and seemed to take part in the lytic process. 5. Holocrine Secretion in Sebaceous Glands Brandes, Bertini and Smith (1965) made an interesting study of this process by histochemical electron microscopy. With the maturation of the cells, the number of lysosomes increased greatly. They seemed to be derived from small vesicles of the Golgi apparatus. During the disintegration of the mature cells, the lysosomes released their enzymes into the general cytoplasm. 288 W. STRAUS 6. Bone Resorption Electron micrographs by Scott and Pease (1956), Gonzales and Karnovsky (1961) and Hancox and Boothroyd (1961) indicated that osteoclasts were very active in phagocytosis (ruffled border) and contained numerous phago- cytic vacuoles. Schajowicz and Cabrini (1958) observed that acid phosphatase activity was marked in osteoclasts and chondrioclasts. Handelman, Morse, and Irving (1964) found acid phosphatase activity concentrated in a junctional band between osteoclasts and bone, suggesting that hydrolytic enzymes may be active at the surface of the osteoclasts. Vaes (1965) reported that lysosomal enzymes were released into the external medium during resorption of bone cells in tissue culture in the presence of parathyroid hormone. Woods and Nichols (1965a, b) found that particulate fractions isolated from rat bone were rich in collagenase activity. All these observations seem to indicate that the lysosomes of osteoclasts take part in bone resorption and that the degradation of bone material may take place either in phagolysosomes within the cells, or by lysosomal enzymes secreted at the cell surface, or at both sites. 7. Autolysis In vitro The autolytic effect of purified lysosomes on isolated nuclei, mitochondria and microsomes was investigated by Sawant, Desai and Tappel (1964b), and the release of lysosomal enzymes during autolysis in vitro was investigated by Van Lancker and Holtzer (1959a, 1963). B. AUTOPHAGY Enlargement of lysosomes has been observed in many types of cells under various physiological and pathological conditions. The formation and func- tion of these enlarged bodies is not yet completely understood. In electron micrographs, areas of the cytoplasm containing lysosomes (cytosomes), mitochondria, endoplasmic reticulum, or other cell components have been seen to be sequestered from the rest of the cytoplasm by a surrounding mem- brane (Ashford and Porter, 1962; Novikoflf and Essner, 1962; Moe and Behnke, 1962; Hruban et al, 1963). Acid phosphatase activity has often been detected in these areas. Similar bodies containing residual mitochondrial structures had been described previously in kidney cells by Rhodin (1954), Clark (1957), Novikoff (1959), and Miller (1960). These enlarged bodies were called "cytolysomes" by Novikoff and Essner (1962), "areas of focal degradation" by Hruban et al. (1963), and "autophagic vacuoles" by de Duve (1963b). Novikoff and Essner (1962) and Napolitano (1963) discussed whether these bodies have a function in the regulation of normal cell meta- bolism or only in the autolysis of abnormal cell components. The presence of such enlarged bodies in normal liver cells (Daems, 1962; Novikoff and Shin, 1964), in the regressing Mullerian duct (Scheib, 1964), in the foetal rat duodenum during certain stages of differentiation (Behnke, 1963), and 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 289 in brown adipose tissue during rapid mobilization of lipid (Napolitano, 1963), indicate that "autophagic vacuoles" may develop in normal cells during marked alterations of metabolism. An interesting example of such a process was demonstrated by Elliot and Bak (1964) in Tetrahymena. During ageing of these cells, a proportion of the mitochondria was broken down in autophagic vacuoles showing acid phosphatase activity. The electron micro- graph from the paper of Miller and Palade, reproduced in Fig. 14, shows such enlarged bodies in kidney cells. The reaction product for acid phos- phatase, remnants of mitochondria, and increased density, probably indi- cating ingested haemoglobin (see Section VIII. a.), may be recognized in the bodies. In other cases in which enlargement of lysosomes has been reported, drastic treatments which might be expected to induce pathological changes were used. The formation of many of these bodies, therefore, may be related to pathological alterations (see Section VIII.). VII. Relationship of Lysosomes and Phagosomes TO Immune Processes A. ANTIBODY FORMATION It is known from the work of Sabin (1939), Coons, Leduc and Kaplan (1951) and others, that foreign proteins are taken up by the cells of the reticulo-endothelial system by pinocytosis. Although the cells of the reticulo- endothelial system themselves are not thought by most investigators to produce antibodies, an important role of macrophages in antibody formation was suggested by Fishman. In Fishman's experiments, antibody formation against T2 bacteriophage occurred in cultures of lymph node cells to which cell-free filtrates from homogenized macrophages had been added. The macro- phages had been incubated previously with T2 bacteriophage (Fishman, 1961). The antibody-inducing material from the macrophages was ribo- nuclease-sensitive (Fishman and Adler, 1963). The investigators suggested that the antigen taken up by the macrophages was partially degraded there, and that complexes of antigen fragments and ribonucleic acid were trans- ferred to antibody-producing cells. A similar theory has been put forward by Campbell and Garvey (1961, 1963), If partial degradation of antigens precedes antibody formation, this may well take place in the phago-lysosomes of the reticulo-endothelial cells. It is of interest in this context that Lapresle and Durieux (1957) found that antibodies against serum albumin react with split products of serum albumin degraded by spleen extracts. After partial degradation, the antigen fragments, according to this hypothesis, leave the phagolysosomes and reach the endo- plasmic reticulum or the nucleus. It is of interest, therefore, that according to Coons (1956) and Wellensiek and Coons (1964), antigens often penetrate 290 W. STRAUS Fig. 14. Kidney cell of female mouse injected with haemoglobin intraperitoneally two hours previously. Note the reaction product for acid phosphatase around the bodies and rem- nants of mitochondria in the interior. The increased density suggests segregation of injected haemoglobin. Magnification x 45,000; inset x 30,000. (Electron micrograph from the paper of Miller and Palade (1964), reproduced with the permission of 7. Cell Biol.) 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 291 into the nuclei of cells. Fishman's theory is also supported by Nossal, Ada and Austin (1964) who used a sensitive autoradiographic method to detect 1311-labelled antigen in the cells of lymph nodes during antibody formation. They observed that radioactivity was present only in the macrophages but not in the surrounding plasma cells. Lymphocytes, now generally considered to be precursors of antibody-producing cells, normally contain few lyso- somes. However, the number of lysosomes in lymphocytes increased greatly after sensitization with dead tubercle bacilli or exposure to haemagglutinin (Hirschhorn et al, 1965; Allison and Malucci, 1964b; Diengdoh and Turk, 1965). The transfer of antigenic material from macrophages to plasma cells might take place via cytoplasmic bridges. Schoenberg, Mumaw, Moore and Weissberger (1964) demonstrated that such bridges exist, especially in immunized animals. They occur between phagocytic and antibody-producing cells in lymph nodes and spleen. Deane (1964) also noted by electron micro- scopy that there was a close association between plasma cells and macro- phages in the lamina propria of the gut, suggesting exchange of material between both types of cells. The question of the persistence of antigens or antigen fragments is often raised in relation to the theories of antibody formation. Garvey and Campbell (1957) concluded that small amounts of S^s which persisted in the liver for several years, belonged to antigenic fragments bound to ribonucleic acid, of the original S^^ labelled protein (see also Campbell and Garvey, 1963). Speirs (1963) found radioactivity derived from tritiated tetanus toxin in lymphocytes and macrophages as long as 9 months after injection into immunized mice. If there is persistence of such antigenic fragments, this may be at cell-sites far from lysosomes, or the fragments may be resistant to lytic enzymes. B. UPTAKE OF ANTIGEN-ANTIBODY COMPLEXES Sabesin (1963) showed by electron microscopy that eosinophils were especially active in the uptake of antigen (ferritin)-antibody complexes. This calls to mind the well-known eosinophilia of allergic states. The uptake of 1131-labelled antigen-antibody complexes by spleen cells in vitro was investi- gated by Patterson et al. (1962). These authors made the interesting observa- tion that the larger complexes formed from equivalent amounts of antigen and antibody were pinocytized more readily than the smaller complexes formed in antigen excess. Tahaba, Kinuwaki and Hayashi (1964) studied the uptake of soluble complexes of bovine-serum-albumin and rabbit-anti-bovine-serum- albumin by tissue culture cells using phase contrast microscopy. They observed cell damage and greatly increased pinocytosis. In some of the above cases the antigen-antibody complexes probably were •N 292 W. STRAUS digested in the phago-lysosomes of the phagocytic cells, and then did not cause further injury. However, antigen-antibody complexes may also be deposited at tissue sites where they are inaccessible to lysosomal enzymes. In such cases they may cause various kinds of cellular injury. McCluskey and Benacerraf (1959) produced acute glomerulo-nephritis, necrotizing arteritis, and acute endocarditis by injection of soluble antigen-antibody complexes (ovalbumin and bovine serum albumin) into mice, and they demon- strated the deposition of the complexes at the sites of tissue damage. Andres, Seegal, Hsu, Rothenberg and Chapeau (1963) produced glomerular nephritis in rabbits by repeated injection of serum albumin. With the aid of ferritin- labelled antibody the investigators showed that ferritin aggregates, probably representing antigen-antibody complexes, were trapped between the basement membrane and the epithelial cells of the glomerulus. The ratio of antigen to antibody (particle size of the complex) was an important factor for the development of glomerulo-nephritis. Dixon (1963) discussed the possible involvement of antigen-antibody complexes in the causation of glomerulo- nephritis, rheumatoid arthritis, lupus erythematosus, and polyarteritis nodosa. He pointed out that weak antigens, which produce soluble antigen- antibody complexes, are most likely to cause chronic injury. The essential factor, according to Dixon, is not the antigenic specificity, but the formation of aggregates which may form insoluble deposits at inaccessible sites (close to the basement membranes, for example). The possible involvement of humoral factors was also discussed by Dixon. The so-called Arthus reaction and reversed Arthus reaction, associated with focal vascular necrosis, is related to the deposition of antigen-antibody complexes in the vessel walls and the uptake of the complexes by phagocytic cells. Daems and Oort (1962) studied this process by electron microscopy and noticed degranulation of leucocytes and the formation of phagocytic vacuoles containing the immune complex. Movat, Urinhara, MacMorine and Burke (1964) and Urinhara and Movat (1964) demonstrated by electron microscopy that the Arthus reaction was initiated by the precipitation of antigen-antibody complexes in the lumen and walls of blood vessels. They showed that during the uptake of antigen-antibody complexes by leucocytes and the subsequent fusion of lysosomes with phagosomes, a permeability- increasing factor was released. Those antigens which are good antibody producers may give rise to anaphylactic shock when the antigen combines with antibody. This may be associated with the release of histamine, probably from mast cells (Cochrane, 1963a, b). Movat, Mustard, Taichman and Urinhara (1965) noted that antigen- antibody complexes were phagocytized by blood platelets, and that during their subsequent aggregation histamine, serotonin, and lysosomal enzymes were released. Fennell and Santamaria (1962) reported that anaphylactic shock in rats was associated with liver cell necrosis in the mid-zonal and 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 293 peri-portal areas and with alterations in the size and location of the lyso- somes. Blackwell (1965) also observed that after injection of antigen into sensitized rats, the liver contained large eosinophilic globules, positive for acid phosphatase and esterase, and related them to the optically clear vacuoles of the same cells. Treadwell (1965) found a 6-7 fold increase of acid phosphatase activity in blood serum soon after induction of anaphylactic shock and a concomitant decrease of 3 lysosomal enzymes in liver homo- genates. C. ANTIGEN AND ANTIBODY REACTIONS AT THE CELL SURFACE As mentioned in Section V. a. L, foreign proteins (antigens) may become adsorbed onto the cell surface during the early phase of pinocytosis. Greater adsorption onto membranes occurs in injured cells (Holtzer and Holtzer, 1960; Straus, 1964d). Easton, Goldberg and Green (1962b) noted that anti- body and complement reacted with antigen at the cell surface causing injury to the cell membrane and leakage of material ("immune cytolysis"). The fragility of lysosomes was increased when ascites tumour cells in tissue culture were exposed to immune serum and complement (Bitensky, 1963). Dumonde, Roodyn, and Prose (1965) observed that immune ferritin-labelled antibodies from rabbits were bound at the surface of isolated lysosomes and mito- chondria from rat liver together with large amounts of complement. No release of acid phosphatase from lysosomes and no change of oxidative phosphorylation in mitochondria was noted during the complement fixation reaction. Boyden and Sorkin (1960) and Boyden, Sorkin and Spark (1960) reported that immune cells were coated at their surface by a cytophilic, non-precipitating antibody which cause increased uptake of antigen. These diverse observations show that our understanding of the processes taking place during immune reactions at the cell surface are far from clear. D. RECOGNITION OF SELF The distinction of "self" and "non-self" is one of the least understood properties of the immune process. Boyden (1962) pointed out that phagocytic cells, too, have the ability of self recognition. They do not take up cells of the same organism, except if the cells are injured. Boyden suggested that humoral factors might be involved and might be similar to those involved in chemotaxis. Perkins and Leonard (1962) observed that, in the absence of type-specific antisera, erythrocytes from more distantly related animals were phagocytized preferentially by mouse peritoneal mononuclear cells in vitro, and that this selectivity could be modified by the addition of specific erythro- cyte antibodies. Nelson and Buras (1963) also noted that homologous and heterologous erythrocytes were phagocytized at different rates, and that 294 W. STRAUS Spleen cells preferentially took up homologous, and liver cells heterologous erythrocytes. The most interesting observations on this problem were made by Ada, Nossal and Austin (1964), who injected pil]-labelled antigenic, minimally antigenic, and non-antigenic (homologous) proteins and studied their distri- bution in the polipleal lymph nodes by autoradiography. They observed that the macrophages in the medulla took up antigenic and non-antigenic proteins to an equal extent but that the macrophages in the lymphoid follicles only took up antigenic proteins (with the exception of autologous gamma- globuHn). They concluded that this selectivity was due to opsonins (see Section V. g. 2. above) rather than to inherent properties of the phagocytic cells themselves. VIII. Pathological Changes Related to Lysosomes and Phagosomes a. pathological enlargement of lysosomes and phagosomes The possibility has already been discussed (Section VI.) that enlarged lysosomes ("areas of focal degradation", "autophagic vacuoles") may develop during sudden drastic changes in metabolism under relatively physiological conditions. However, many of the reports of enlargement of lysosomes were after treatment which must have caused cell injury. Hruban et al. (1963) observed areas of focal degradation in kidney, liver, and pancreas after potassium deficiency, after partial occlusion of the renal vein, or after injection of toxic agents. Novikoff (1959) reported enlargement of lysosomes in kidney cells after ligation of the ureter. Enlargement of lysosomes in nerve cells after axon section has also been observed (Barron and Sklar, 1961). The question has to be raised in each case, therefore, whether injury to cell components preceded the development of autophagic vacuoles and was the cause of it, or whether autophagic vacuoles developed without cell injury and functioned in the autolytic degradation or turnover of normal cell components. Another point does not seem to have been considered sufficiently in the interpretation of areas of focal degradation. This is the possibility that 1 materials ingested from the blood stream may have participated in the ■ enlargement of bodies resulting from the fusion of phagosomes with lyso- somes. Lysosomes and phagosomes are enlarged after fusion. A further enlargement of the bodies occurred in kidney and liver cells after injection of relatively high doses, or toxic preparations of horse-radish peroxidase (Straus, 1961, 1963, 1964a, b and Figs. 15 and 16 of the present article). The enlarged bodies in kidney cells often seemed to include cell fragments, mitochondria, or nuclei, in addition to lysosomes and phagosomes ("com- posite bodies", Straus, 1961). In a similar way, uptake of injected material Fig. 15. Pathologically enlarged phagosomes in liver cells, 3 hr after intravenous injection of a high dose of plant peroxidase; staining with benzidine. The same large granules also showed acid phosphatase activity (not demonstrated here), x 660. Fig. 16. Pathologically enlarged lysosomes (phago- lysosomes) in kidney cells, 48 hr after intravenous injection of plant peroxidase ; stained for acid phos- phatase. The animal had pyelo-nephritis. x 660. Facing page 294 I 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 295 in phagosomes and its transport to areas of focal degradation may have occurred in the experiments of Ashford and Porter (1962) with glucagon, in the experiments of Hruban et al. (1963) with toxic substances, and in the experiments of Novikoff and Essner (1962) with the neutral detergent Triton WR-1339. Even when cell injury was induced by treatments mentioned on the preceding page not involving injection of foreign compounds (K+ deficiency, partial occlusion of the renal vein, ligation of the ureter, axon section), the treatments may have caused the entry of increased amounts of serum proteins, and these, segregated in phagosomes, may have been trans- ported to the areas of focal degradation. Enlarged phago-lysosomes probably are related to the so-called "watery"! vacuoles" of liver cells. According to Trowell (1946), increase of sinusoidal /^ pressure and anoxia are main factors in the development of these vacuoles./ Doniach and Weinbren (1952), Aterman (1958), Nairn, Chadwick and McEntegart (1958), Anderson, Cohen and Barka (1961) and Schlicht (1963), demonstrated the presence of injected substances or of serum proteins in such vacuoles of liver cells. In recent years, several investigators have dis- cussed the formation of enlarged vacuoles in relation to lysosomes and in- ingestion processes. Vacuoles of this type in liver cells have been described after injection of dextran and iron-dextran (Daems, 1962; Kent et al. (1963a, b), mercury sulphide (Oudea, 1963), and sucrose (Wattiaux, Wattiaux- DeKoninck, Rutgeerts and Tulkens, 1964; Brewer and Heath, 1963). Similar large vacuoles have been observed in kidney cells after the injection of sucrose (Janigan and Santamaria, 1961; Trump and Janigan, 1962; Mauns- bach, Madden and Latta, 1962). It is possible that in all these cases, cell injury was associated with the increased uptake of material from the blood and with the fusion of numerous lysosomes with phagosomes. How are enlarged phago-lysosomes, which appear round and relatively empty in electron micrographs, related to the irregularly-shaped areas of focal degradation, which appear relatively dense in electron micrographs? This may depend on the size and number of lysosomes and phagosomes that fuse and on whether other cell components (such as mitochondria, endo- plasmic reticulum or microbodies) are sequestered together with phagosomes and lysosomes. The following hypothesis was suggested for the formation of enlarged phago-lysosomes (Straus, 1964b). During the pathological fusion of several lysosomes with several phagosomes some of the hydrolytic enzymes may be released into the cytoplasm in vivo. The cells might react against this irritation by sequestering these areas from the rest of the cytoplasm and sur- rounding them with a membrane. Although acid phosphatase activity can often be demonstrated in these areas by histochemical electron microscopy, no distinct cytosomes (lysosomes) can be seen in electron micrographs of many of these structures. This may indicate that the hydrolytic enzymes have been released from the lysosomes within these areas. 296 W. STRAUS Although phagosomes may participate in many cases in the formation of areas of focal degradation, it is possible, of course, that other autophagic vacuoles may develop independently of phagosomes. 'B. RELEASE OF LYSOSOMAL ENZYMES IN VIVO In his earlier work on lysosomes, de Duve suggested that lysosomes might be a frequent cause of cell injury and necrosis ("suicide bags"). However, this opinion has not yet been confirmed (de Duve 1963a) and a general release of lysosomal enzymes into the cytoplasm in vivo does not occur frequently. It is not difficult to detect the release of lysosomal enzymes from isolated cell fractions, and several agents causing release have been mentioned in Section II. c. It is difficult, however, to determine whether lysosomal enzymes have been released in vivo. The membranes of pathologically enlarged lyso- somes are probably very fragile so that enzymes are released during homo- genization or fractionation of the tissue (see Sections II. A., II. c). Another source of error arises if pathological tissues contain areas of necrosis sur- rounded by viable tissue. The lysosomal enzymes from the necrotic areas only may be released in vivo (van Lancker and Holtzer, 1959a) and may then appear in the supernatant fluid after fractionation of the tissue. Rigorous correlation of cytological (or cytochemical) and biochemical observations is therefore essential for the study of lysosomes in pathological tissues. The occurrence of a limited release of lysosomal enzymes in sequestered areas of the cytoplasm in vivo was suggested in Section VIII. a. (see the discussion on "composite bodies"). A similar effect in shock and in virus infection will be mentioned below (Section VIII. d.). General release of enzymes from the lysosomes into the cytoplasm is known to occur in leucocytes after treatment with streptolysin (Hirsch et al, 1963). C. EXTRUSION OF LYSOSOMES AND OF LYSOSOMAL ENZYMES Interesting observations on the extrusion of lysosomes and of their enzymes in leucocytes have been made by Woodin and Wienecke (1964a, b). They showed that after treatment of leucocytes with leucocidin or with vitamin A, the contents of the granules were extruded into the medium, and their membranes fused with the surface membranes of the cells. Calcium and adenosine triphosphate seemed to play a role in the process of extrusion which showed certain analogies to a secretion process. The extrusion of enlarged phago-lysosomes from the convoluted tubule cells of the kidney into the lumen was observed after high doses of peroxidase (Straus, 1964b). This process may be similar to "potocytosis" observed by Rouiller and Modjtabai (1958) after repeated injection of ovalbumin or 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 297 sucrose, resulting in extrusion of cell fragments together with hyaline drop- lets. Novikoff (1959) reported extrusion of enlarged lysosomes from kidney cells after ligation of the ureter. The extrusion of cell material from kidney cells occurring in vivo has to be distinguished from the extrusion that occurs post mortem (Hanssen, 1960; Parker, Swann and Sinclair, 1962; Longley and Burstone, 1963). D. POSSIBLE ROLE OF LYSOSOMES IN INFLAMMATION, NECROSIS, SHOCK AND VIRUS INFECTION Biochemical studies on lysosomal enzymes during liver injury by carbon tetrachloride and other poisons were reported by Slater and Greenbaum (1965) and Dianzani (1963). The relatively uncharacteristic changes in lysosomal enzymes were not considered by these authors to be primary causes of cell injury. Nagel and Willig (1964a) concluded that lysosomal enzymes did not play an essential part in ischaemic necrosis of rat kidney. These investigators observed a rapid inactivation of lysosomal cathepsins during the development of necrosis, whereas aminopeptidases and neutral proteases of the microsomal fraction retained most of their activity 48 hr after treatment. Trump, Gold- blatt and Stowell (1962), made careful observations by electron microscopy of early cytoplasmic alterations in parenchymal cells of mouse liver during necrosis in vitro, and concluded that the release of lysosomal enzymes was a secondary process, damage to mitochondria and endoplasmic reticulum occurring earlier. Van Lancker and Holtzer (1959a, 1963), by studying the release of lysosomal enzymes during autolysis in vitro, also concluded that the lysosomes were not the primary cause of necrosis. A role of lysosomes of leucocytes in inflammation is indicated by the work of Janoff and Zweifach (1964). They observed that cationic proteins isolated from lysosomes of leucocytes caused the adhesion of leucocytes along the endothelial surface of capillaries and venules and the subsequent emigration of the leucocytes into the perivascular tissue. Janoff, Schaefer, Scherer, and Bean (1965) reported that a lysosomal extract from leucocytes caused disruption of mast cells. The increase of vascular permeability by lysosomes of leucocytes may be mediated, according to these investigators, by the release of vasotropic agents from mast cells. According to Halpern (1964), tissue damage in haemorrhagic shock may be due to the release of cathepsin, since the effect was counteracted by a poly- peptide possessing anti-protease activity. Bitensky, Chayen, Cunningham and Fine (1963) proposed that in haemorrhagic shock the lysosomes of the reticulo-endothelial cells were disrupted and were no longer able to detoxify endotoxin. Janoff, Weissmann, Zweifach and Thomas (1962) earlier sug- gested that endotoxin caused release of lysosomal cathepsin. 298 W. STRAUS A possible injurious effect of released lysosomal enzymes during virus infection was discussed by Allison and Sandelin (1963), who observed the release of these enzymes in mouse liver cells and monkey kidney cells infected with hepatitis and vaccinia virus, respectively. Release of lysosomal enzymes was also reported in virus hepatitis by Pagliaro, Giglio, Moli, Catania and Citarrella (1964) by taking needle biopsies from human liver. IX. Appendix: Methods for the Study of Lysosomes and Phagosomes a. isolation of lysosomes The theoretical problems connected with the isolation of lysosomes and of other cytoplasmic particles by differential centrifuging or by density gradient centrifuging have been discussed by Schneider and Hogeboom (1951), Allfrey (1959), de Duve (1964), and by others. In the following paragraphs, only a few practical considerations on the isolation of lysosomes and some examples from the literature will be given. In view of their heterogeneity, it will be difficult or even impossible to isolate most of the lysosomes from a complex tissue as a homogeneous population of granules. Only a proportion of them which fall into a similar range of size and density, may be separated as a single, highly purified fraction. The lysosomes which fall outside this range have either to be dis- carded, or to be collected separately. This difficulty does not exist for mito- chondria or nuclei which can be isolated as a relatively homogeneous class of particles. The difficulties in preparing highly purified lysosomes may be illustrated by a few examples from the literature. The "light mitochondrial" fraction, prepared by de Duve and co-workers (1955) contained 30-40% of the lyso- somal enzymes of the total homogenate but was contaminated by an excess of mitochondria. The lysosomes of this fraction were probably derived mainly from the parenchymal cells, whereas the larger lysosomes from the Kupffer cells sedimented with the heavy mitochondrial or nuclear fraction. S^want et al. (1964c), by using a combination of differential centrifuging and of discontinuous gradient density centrifuging, succeeded in obtaining relatively pure lysosomes from liver (with 6 to 10% microsomal contamina- tion) in relatively good yields. Kidney lysosomes were separated from "nu- clear" and "mitochondrial" fractions by differential centrifuging in good purity but poor yield (Straus, 1954, 1956). Fractions containing "large" lysosomes (1-5 )u. diameter), "intermediate-sized" lysosomes (0-5-1 -5 /i diameter), and "small" lysosomes (0- 1-0-5 /li diameter) were collected separately from the same homogenate. Figure 1 shows one of the fractions. The purification was facilitated by the property of the granules to form com- I 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 299 pact, dark brown layers at the bottom of the centrifuge tubes. Smaller lysosomes, present in the microsomal fraction of kidney homogenates could not be purified sufficiently. Very large lysosomes (phago-lysosomes) formed after injection of egg white were purified by differential centrifuging combined with filtration through a column of non-absorbent cotton. The contaminating nuclei were retained by the cotton, whereas the "droplets" passed into the filtrate (Straus, 1954). Kidney lysosomes of high purity, but probably low yield, were also isolated by Maunsbach (1964a) using differential centrifuging. Shibko and Tappel (1965) have recently obtained highly purified preparations of large kidney lysosomes. These investigators also made use of the fact that the granules form dark brown sediments at the bottom of the centrifuge tube during differential centrifuging. The puri- fied fractions contained 1-2% of all the lysosomes of the homogenate. Since the lysosomes are larger and more numerous in the kidney than in the liver, the purification of kidney lysosomes presents much less difficulty than that of liver lysosomes. Lysosomes isolated from blood cells, pancreas (van Lancker and Holtzer, 1959b), thymus, brain, and intestine (see Section III.) probably were contaminated by other types of granules. The degree of purification of isolated lysosomes can be estimated by com- paring the specific activities of lysosomal enzymes in the isolated fractions . with those of the original homogenates. The increase in specific activity \ depends not only on the degree of purification but also on the number of * lysosomes in the tissue. Of course, only those enzyme activities which are predominantly localized in the lysosomes can be used as a measure of purity. \ For example, the specific activities of hydrolytic enzymes in "pure" liver lysosomes were found to be 60 times higher than in the original homogenate ' (Sawant et al., 1964c) and in "pure" kidney cortex lysosomes 10-15 times higher than in the original homogenate (Straus, 1956). From these figures, it may be estimated that there are five times more lysosomes in the kidney cortex than in the liver, if the average concentration of hydrolytic enzymes in the lysosomes of both tissues is approximately the same. The contamination of isolated lysosomes by other cytoplasmic granules can be estimated by using characteristic markers for the contaminating particles: DNA^for nuclei and cytochrome oxidase (1.9.3.1.) for mitochondria, for example. ! The purification of "large" and "intermediate-sized" kidney lysosomes may be followed by microscopic examination. The large kidney lysosomes can be distinguished in sucrose suspension from mitochondria by their greater refractility and greater resistance towards swelling (Straus, 1954, 1956). Isolated fractions of small lysosomes (0-5 /x or smaller) require electron microscopy for morphological evaluation of their purity. Electron micro- graphs of liver lysosomes, reproduced from the paper by de Duve (1963a) and of kidney lysosomes, reproduced from the paper by Shibko, Pangborn and Tappel (1965), are shown in Figs. 17 and 18. V 300 W. STRAUS 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 301 B. CYTOCHEMICAL REACTIONS FOR LYSOSOMAL ENZYMES Cytochemical staining reactions for acid phosphatase and, to a lesser extent, for esterase, have greatly facilitated the study of the lysosomes (see Novikoff, 1963). Many applications of these cytochemical methods may be found in the articles cited in Section III. Since the technical details for the staining procedures for acid phosphatase are discussed in books on histo- chemical methods (Pearse, 1960; Burstone, 1962; Barka and Anderson, 1963), only a few comments on the principles of the methods will be made here. The factors should be pointed out which have made these cytochemical methods so valuable for the study of the lysosomes. There are adequate histochemical methods for relatively few enzymes, among them for phospha- tases and esterases. Until a few years ago, the resolution of staining reactions for most of these enzymes was only sufficient for localization at the level of the tissue but not in individual cells. The methods for phosphatases and esterases have now been improved to such an extent that the cellular sites of these enzymes may be visualized not only under the light microscope but also at the dimension of the electron microscope. The second favorable factor for enzyme cytochemistry of lysosomes is the fact that a considerable portion of lysosomal enzymes survives the conventional fixation of the tissue with formaldehyde (approximately 50% in the case of acid phosphatase. Holt and Hicks, 1961a). Thus the cytochemical reactions for lysosomal enzymes may be performed with well-fixed tissues. It may be mentioned, for comparison, that significant amounts of mitochondrial enzymes usually do not survive formaldehyde fixation and that cytochemistry for mitochondrial enzymes, therefore, is only possible with poorly fixed, disintegrating mito- chondria. The procedure for acid phosphatase described by Gomori (1952) is based on the principle that inorganic phosphate, split by acid phosphatase from jS-glycerophosphate, or from another phosphate ester, is trapped as lead phosphate at the site of acid phosphatase activity, and lead phosphate is transformed into lead sulphide by ammonium sulphide. Lysosomes are stained dark brown by this reaction. Since the reaction product (lead sulphide) is electron dense, the reaction can be applied to studies at the electron micro- scope level (Holt and Hicks, 1961b; Miller and Palade, 1964). In the azo-dye procedures (Burstone, 1958; Barka and Anderson, 1962), phosphate esters of naphthol-AS compounds are used as substrates. Enzymic hydrolysis of the phosphate group of the ester by acid phosphatase releases the naphthol- AS compound which couples with a diazotized aromatic amine resulting in a deeply-stained reaction product at the site of acid phosphatase activity. Staining for esterase also permits precise localization of lysosomes in M Fig. 17. Electron micrograph of isolated liver lysosome, x 21,000. (Reproduced from the paper of de Duve, 1963a, with the permission of the Ciba Foundation.) ,v-rt-^^ G 302 W. STRAUS Fig. 18. Electron micrograph of isolated kidney lysosomes. x 22,000. (Reproduced from the paper by Shibko, Pangborn and Tappel (1965), with the permission of the J. Cell Biol.) 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 303 many tissues although the exact nature of the enzyme and its lysosomal localization is still controversial (see Section II. a. 1., and Holt, 1963). Methods for esterase using indoxyl acetate as substrate were developed by Holt (1956). These and the azo-dye procedures for esterase were used by Shnitka and Seligman (1961) who identified esterase-positive granules (lysosomes) in kidney cells. Great progress was made recently in the cytochemical procedure for /S-glucuronidase using naphthol-AS-BI-glucuronide as substrate and hexa- zonium pararosanilin as coupler (Hayashi, Nakajima and Fishman, 1964; Hayashi, 1964). Hayashi (1964) reported that the granules which were stained for ^-glucuronidase in different tissues had a similar intracellular location as those stained for acid phosphatase. This confirmed the results of bio- chemical experiments showing that both enzymes were present in lysosomes. Pugh and Walker (1961) had previously used naphthol-AS derivatives for the demonstration of )3-glucuronidase in granules of chondriocytes of hyaline cartilage. Aronson, Hempelman and Okada (1958) and Vorbrodt (1961) reported a cytochemical method for acid deoxyribonuclease, another lysosomal en- zyme. In this procedure, highly polymerized DNA and acid phosphatase were added to an incubation mixture similar to the one used for acid phos- phatase by Gomori. According to the principle envisaged by Aronson et al. (1958), nucleotides split from DNA by deoxyribonuclease are further degraded by acid phosphatase, and the inorganic phosphate is transformed into lead sulfide at the site of DNase activity. The same principle was applied by Atwal, Enright and Frye (1964) to the cytochemical detection of ribonuclease, and ribonuclease-positive granules were demonstrated by these authors in leucocytes. Naphthol-AS-BI-sulfate-K was introduced recently by Woohsmann and Hartrodt ( 1 964) as substrate for the cytochemical detection of aryl-sulphatases. Goldfischer (1965) demonstrated aryl sulphatase activity in lysosomes of various tissues by using /7-nitrocatechol sulphate as substrate in conjunction with lead nitrate in a Gomori-type reaction. Hopsu, Arstila and Glenner (1965) detected aryl sulfatase activity in lysosomes at the electron microscope level by using sulphate esters of 8-hydroxy-quinoline or /7-nitrophenol as substrates and barium salts as precipitating agents. Hayashi (1965) was able ta demonstrate iV-acetyl-^-glucosaniimdase (3.2.1.29) in lysosomes of kidney and liver employing naphthol AS-BI iV- acetyl-)8-glucosaminide as substrate. C. MARKERS FOR PINOCYTIC AND PHAGOCYTIC VACUOLES Since foreign materials (proteins, colloidal particles of heavy metals, carbon, or dyes) ingested by pinocytosis or phagocytosis are concentrated in phagosomes, such materials may be used as markers. Many examples may be 304 W. STRAUS found in the articles reviewed in Sections V. b., V. d., V. e., and V. f. The most useful markers are those ingested materials which can be detected by light or by electron microscopy in very low concentrations. By labelling proteins or other materials with fluorescent or radioactive groups, it is often possible to determine the intracellular location of these substances by fluorescence microscopy or autoradiography. Phagosomes, marked by fluorescent-labelled proteins may be seen with great clarity (see for example, illustrations in Holtzer and Holtzer, 1960; Cormack and Ambrose, 1962; and Payne and Marsh, 1962). Antigens may be detected in isolated phago- somes by reaction with antisera (Straus and Oliver, 1955). or, in situ, by the fluorescent antibody procedure (Coons, 1956). Injected ferritin is a useful marker for phagosomes since it can be identified in electron micrographs by the characteristic shape of the iron micelles (see, for example, Farquhar and Palade, 1962). Ingested haemoglobin, segregated in phagosomes, may be recognized in electron micrographs by its slightly increased electron den- sity (Miller, 1960; Miller and Palade, 1964; Ericsson, 1964). Colloidal particles of HgS, Th02, Au, or carbon, segregated in phagocytic vesicles or vacuoles, may also be readily identified in electron micrographs. Iron com- pounds such as iron-dextran and iron saccharide may be stained in phago- somes by the Prussian blue reaction (Kent et al., 1963a). Wattiaux, Wibo and Baudhuin (1963) observed that rat liver lysosomes accumulated injected Triton WR-1339. The altered granules showed increase in size and decrease in density which facilitated their separation from mitochondria and micro- somes by density gradient centrifuging. The use of an enzyme (ribonuclease) as a marker protein for the study of pinocytosis was introduced by Brachet (1956). An injected enzyme is a good marker for phagosomes if no similar enzyme is present in the cells before treatment, if it can be visuaUzed by a sensitive cytochemical staining reaction, and if it is not toxic to animals. Plant peroxidase was found to be favorable in this respect, and cytochemical procedures for the use of this marker were reported (Straus, 1964c, d). Staining for injected peroxidase with benzidine can be combined with staining for acid phosphatase, or another lysosomal enzyme, by an azo-dye procedure in the same tissue section. Since the staining for the two enzymes can be made to result in contrasting colors, the reaction products for the exogenous protein (peroxidase) and for the endogenous lysomal enzyme can be recognized in the same granules, after fusion of phagosomes with lysosomes. In the experiments illustrated in Figs. 3, 11, 12 and 15 staining for injected plant peroxidase with benzidine alone was applied and Figs. 5, 6, 7 and 13 show successive staining for acid phos- phatase and for peroxidase in the same tissue sections. Graham and Karnov- sky (1966) observed that 3,3'-diaminobenzidine resulted in a very electron dense reaction product with peroxidase after post-fixation with OSO4. They used this substrate for the detection of injected horse-radish peroxidase by 5. LYSOSOMES, PHAGOSOMES AND RELATED PARTICLES 305 electron microscopy. A further advantage of the use of peroxidase as a protein marker is the possibility of correlating cytochemical observations with colorimetric assays for the injected protein in isolated fractions (Straus, 1957b, 1962b). As mentioned in Section V. a. 3., an endogenous enzyme, nucleoside phosphatase, may also be used in some cases as a marker for studying phagosomes under the electron microscope. X. Concluding Comments In looking back on lysosome research over the last ten years, it must be recognized that great progress has been made. With the aid of cytochemistry and electron microscopy, lysosomes have been identified in many types of epithelial and reticulo-endothelial cells. The heterogeneity of the granules is better understood, and the definition of lysosomes has been broadened to include sub-types of different age and function. It has been observed that pre- existing lysosomes fuse with newly-formed phagosomes, and that this might be a general process by which phagocytizing or absorbing cells incorporate such diverse materials as proteins, vital dyes, various colloidal particles, or bacteria. These results have established the relationship of the lysosomes to the well-known function of the reticulo-endothelial system in protecting the organism against foreign material. The discovery of the fusion of newly- formed phagosomes with pre-existing lysosomes has settled the old contro- versy as to whether ingested material is segregated in newly-formed or in pre-formed cytoplasmic granules. It has also been found that lysosomes, in addition to breaking down ingested foreign material, take part in the autolytic degradation of components of the cells themselves. This may reflect a role in the renewal of cell constiluents or in the alteration of meta- bolic pathways. Finally, lysosomes have been shown to take part in physio- logical regression, for example, in the resorption of embyronic organs. Other properties of the lysosomes are less well understood. Our ignorance j^ apphes especially to the origin of lysosomes and of their enzymes. The relationship of the lysosomes to cell injury requires further study, especially the question as to whether cell injury is initiated by the release of lysosomal enzymes in vivo. The possible role of lysosomes in the early stages of antibody formation has not yet been studied systematically. It also is not known whether lysosomes take part in the degradation of serum proteins. The function and properties of the phagosomes also raises new questions. IThe relationship between small and large phagosomes, pre-existing lysosomes, ftnd the fusion of phagosomes and lysosomes in the same cells, (i.e., the relationship between uptake, intracellular digestion, and transport into and put of the cells) is not fully understood. The process of fusion between lyso- somes and phagosomes has not been suflficiently studied. Although it has been observed that uptake of injected proteins occurs mainly in the cells of the t 306 W. STRAUS reticulo-endothelial system, in the epithelial cells of the kidney, and in the intestine of new-born animals, electron micrographs show micropinocytic vesicles and vacuoles in many other cells. This raises the question as to whether endogenous macromolecules are absorbed by this pathway, and if so, what is the significance of this process. Thus there is no lack of problems waiting for those interested in the study of lysosomes and phagosomes. 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Z. allg. Path. Bakt. 13, 146. Zucker-Franklin, D. and Hirsch, J. G. (1964). J. exp. Med. 120, 569. Note Added in Proof Maunsbach [/. Ultrastr. Res. 15, 197-241 (1966)]. presented the first definite proof for this hypothesis by microperfusing single proximal tubules of the kidney with 1125-iabelled homologous serum albumin and by demonstrating the uptake of the protein in apical phagosomes of the proximal convoluted tubules by electron microscopic autoradiography. il I I Chapter 6 MEMBRANE SYSTEMS E. REID Biochemistry Department, University of Surrey, London, England I. Introduction .......... 321 A. Nature of Membrane Systems . . . . .321 B. Cytomembranes ........ 325 II. Cytochemical Observations ....... 330 A. Methodology ........ 330 B. Enzymes Associated with Membranes . . . .331 III. Biochemical Observations ........ 337 A. Isolation and Characteristics of Hepatic Microsomal Fractions 337 B. Isolation and Characteristics of Hepatic Cytomembrane Fractions with Few Ribosomes ..... 346 C. Membrane-containing Fractions (Vertebrate), other than Hepatic Cytomembrane Fractions .... 371 D. Membrane-containing Fractions, (Invertebrate) . . 382 Correlations and Functional Aspects ...... 384 A. Normal Tissues ........ 384 B. Pathological Tissues 395 Summary . 397 Acknowledgements .......... 399 References ........... 399 I. Introduction A. NATURE OF MEMBRANE SYSTEMS Scope of Present Survey The term "membrane" in the present context connotes the classical triple- layer structure made up of lipid sandwiched between two protein layers, which is manifest in electron micrographs as two electron-opaque lines in close apposition and which limits the passage of solutes. On morphological grounds, the survey excludes cell walls (for which there are examples of penetrability even by substances of high molecular weight) ; reviews that deal with cell walls include those of Wolfe (1964) for bacteria and of Whaley, MoUenhauer and Leech (1960) for plant cells, It should be noted that some I 321 322 E. REID authors regrettably use the term "cell membrane" as a synonym not for the plasma membrane but for cell wall ; the term is best avoided altogether. The term "systems" has now been interpreted rather arbitrarily as including the plasma membrane (cell membrane, plasmalemma), the cytomembranes (here treated as distinct from the plasma membrane; see Section I. b.), and the nuclear membrane, but as excluding the membrane of mitochondria, lysosomes and other organelles (see Fig. 3). The fact that the lysosomal membrane appears to arise from the plasma membrane illustrates the arbitrary nature of the distinction now made. There is, of course, no nuclear membrane in bacteria. Cytomembranes, in contrast with free ribosomes, are lacking or deficient in most bacteria, but are present to at least some extent in some algae, fungi and other protista, as well as in the cells of higher organisms. However, they are lacking in mam- malian red blood cells, and are scanty in leucocytes, in some plantc ells (Whaley et ah, 1960) and in the proximal tubule cells of kidney. The plasma membrane may well be the basic structure from which, with the evolution of large and metabolically complex cells, cj^omembranes and the nuclear mem- brane have developed. The plasma membrane itself may become differenti- ated ; for example, the myelin of nerve tissue represents regularly layers of Schwann-cell plasma membranes. To lay a foundation for the rest of the chapter, the nature of membranes and of membrane systems will be touched on. Consideration will then be given to cytochemical work and (in more detail) to biochemical work on enzymes, with emphasis on liver — a tissue which lends itself to biochemical experi- mentation (partly because it is fairly homogeneous in cell type) and which has furnished a core of knowledge to which observations on other tissues can be related. Questions of terminology will be considered in later sections. Locke (1964) may be consulted for fuller treatments, particularly of morpho- logical aspects. Recently, Ryter and Jacob (1966) have discussed bacterial membranes. • 2. General Morphological Background (a) Membranes. The "unit membrane" as described by Robertson (1959) represents a universal structure which, however, has been said to vary in dimensions from 4-5 to 11 m/x (Stoeckenius, 1964). Typical thicknesses for the plasma membrane are 7-5 — 9 m/x overall and 2 m/z for each of the two protein layers. Tashiro (1957) quotes values of the order of 20 m/x (for overall thickness), but Palade and Siekevitz (1956) and other authors reckon the thick- ness to be about 5 m/x; cytomembranes and especially the nuclear and mito- chondrial membranes are reckoned to be thinner than the plasma membrane. From work with model systems it is conceivable that membranes vary in respect to the nature of the lipid and of the outer (protein) layers ; the latter may differ from each other, and there may even be partial replacement of protein 6. MEMBRANE SYSTEMS 323 by other macromolecules such as polysaccharides (Stoeckenius, 1964). From measurements of sterol incorporation with a sterol-requiring insect, Clayton (1965) concluded that there may well be "a repeating unit of membrane structure which is common to all of the subcellular membrane systems of a particular tissue or cell type, but is different in different tissues." The idea that there is homogeneity in a given membrane with respect to appearance is now back in the melting-pot. There may be a "mosaic" structure with local specializations. There are thought to be actual "pores", which in excitable membranes are reckoned to have a diameter of the order of 1 m/z (Mullins, 1960). Moreover, Sjostrand (1963) has performed high resolution electron microscopy on mitochondrial membranes and smooth-surfaced cytomembranes in kidney cells (Fig. 1), and has interpreted the pictures, particularly those with permanganate staining, as showing globular com- ponents, possibly lipid micelles, separated by stained septa, possibly protein. From observations such as the latter, and from biochemical findings (e.g. that the protein and lipid are intimately linked). Green and Perdue (1966) have argued — with force rather than lucidity — that a membrane is "a fused continuum of repeating particles" each made up of an invariant base-piece and of a "detachable sector" ; the former, on exposure to a bile salt or acetone, can form a "vesicle". The term "vesicle" may here have a different connota- tion to that conveyed by the use of the term elsewhere in this chapter. (b) Membrane systems. The following structures will be considered (cf. Figs. 2 and 3) : Plasma membrane. This structure is distinguished here from cytomembranes (cf. NovikofT, 1964), although a sharp distinction is difficult to make (Emme- lot. Bos, Benedetti and Rumke, 1964b). The erythrocyte membrane, as dis- cussed by Wolfe (1964) inter alia, will not be considered. Cytomembranes. This term is used here in preference to e.r., an abbreviation for either the "endoplasmic reticulum" (the equivalent in skeletal muscle being the "sarcoplasmic reticulum") or the "ergastoplasm" [although the latter term refers to basophilic material and hardly includes "smooth-sur- faced" cytomembranes; see Haguenau (1958)]. Cytomembranes are sub- classified in Section I.B.I. Nuclear envelope. This term will not be restricted here merely to the outer of the two membranes surrounding the nucleus. The outer surface of the plasma-membrane is usually closely apposed to that of adjoining cells, and actual structural links (desmosomes) are reckoned to exist. In many cell types there are convolutions of the membrane, forming tubular ramifications (micro-villi) which comprise, in the case of hepatic cells, the bile canaliculi, and in the case of kidney tubular-cells, the "brush- border" structures. These "convolutions" are thicker than the non-convoluted portion of the plasma membrane, and may have hair-like protrusions; the lumen may contain hollow rod-like elements. » 6. MEMBRANE SYSTEMS 325 The cytomembranes, which will be further considered in Section Lb. [for historical aspects see Haguenau (1958)], are paired structures, and in sections of tissues such as liver they appear as parallel lines (often grouped in stacks) or as flattened rings. The central space between each pair of membranes is termed a cisterna. Each element (sac) can be visualized as resembling a rubber hot- water bottle; the appearance of a particular unit thus depends on the plane in which it is cut. In qualifications of this simile, it should be pointed out that the sacs are typically interconnected with one another to form sponge- like reticulum, ramifications of which connect with the nuclear envelope and probably also with pores in the plasma membrane. Evidently, then, the perinuclear space enclosed by the double membranes is in direct communica- tion with the exterior of the cell, by way of the cisternae of the cytomembranes, so that there is a line of transport which has no direct link with the cell sap (Watson, 1960). Cytomembranes are often seen to surround the mitochondrial membrane (Fig. 2), but no actual connections appear to exist. In the nuclear envelope there appear to be pores which directly connect the nuclear sap to the cell sap (Fig. 2), and which might well serve to allow passage of macromolecules [Watson (1960); see also Haguenau (1958) and Whaley et al. (I960)]. Biochemical evidence largely supports the view that there is little restriction on interchange of molecules between nucleus and cytoplasm; observations such as the high concentration of Na^ ions in nuclei are explicable in terms of binding to nuclear constituents (Siebert and Iumphrey, 1965). B. CYTOMEMBRANES Classification On the basis of their appearance (Figs. 2 and 3), depending particularly on hether or not the membrane surface is studded with ribosomes, cytomem- branes may be classified as follows (see, inter alia, Haguenau, 1958): — ^^H Rough-surfaced (granular, a-) cytomembranes. These are commonly found ^^■1 regular stacks, and in profile each pair typically has a thickness of 50 mix l^where the cisternal separation is narrowest. Smooth-surfaced (agranular, j8- andy-) cytomembranes. These form irregular ramifying agglomerations located largely in the outer areas of the cytoplasm and in profile, have a narrowest thickness ranging from 40 to 100 m/x; the prefix ^- has been used to denote cytomembranes which seem to represent ^- Fig. 1. (left). Smooth-surfaced cytomembranes in proximal convoluted tubule of mouse kidney, showing globular pattern in both oblique and tangential sections. From Sjostrand (1963), courtesy of author, x 400,000. Fig. 2 (right). Portion of a mouse liver cell. BC = bile canaliculus, G = Golgi region, L = lyosome, NE = nuclear envelope, PM = plasma membrane, R = rough-surfaced cytomembranes, S = smooth-surfaced cytomembranes, U = uricosome (micro-body). • >. Courtesy of M. %. C. Birbeck. x 32,000. I 326 E. REID 6. MEMBRANE SYSTEMS 327 invaginations of the plasma membrane. Golgi cytomembranes comprise a cluster ("complex") of membranes designated y-cytomembranes lying close to (but not linked with) the plasma membrane, and are particularly evident in some plant cells (Whaley et al., 1960). The above sub-division is somewhat arbitrary, particularly since tissue sections sometimes show rough- and smooth-surfaced strips in contiguity on the same membrane; the different types of cytomembrane may in fact be local differentiations within a common system, and be interconvertible in the day-to-day functioning of the cell. There may be tissue differences, to which scant regard can be paid here; thus in certain secretory organs such as pancreas, zymogen granules may be present in the cisternal spaces within smooth-surfaced cytomembranes (Palade and Siekevitz, 1958a), and in liver these cytomembranes are concerned in glycogen storage. In at least some plant cells there appear to be few rough-surfaced cytomembranes (Whaley et al., 1960). Steroid-producing and certain other vertebrate organs likewise contain cells in which there is a predominance of smooth-surfaced cyto- membranes— an "organelle" the functions of which have been reviewed by Jones and Fawcett (1966). In the living cell, as distinct from the dead cells seen in tissue sections, structures such as mitochondria and the plasma membrane may change from one moment to another in shape and position. It is difficult to obtain evidence for a dynamic state of the cytomembranes [see however, III, A.3 (a) and IV. B. 1]. From work in vitro there are indications that they may have osmotic properties (Palade and Siekevitz, 1956; Wallach and Kamat. 1964), but some of this work (e.g. Packer and Rahman, 1962; Tedeschi, James and Anthony, 1963) is rather inconclusive, and it has been claimed that there is no hindrance to access of sucrose (Share and Hansrote, 1960). Certainly it would be unwise to assume a static, structural role which would justify the term "cytoskele- ton", which is sometimes used; probably "the membranes . . . are in constant flux and motion" (Siekevitz, 1965). 2. Experimental Approaches (a) Cytochemistry. Ideally a cytochemical assay should be available for each enzyme investigated which, with whole tissue, furnishes quantitative unambiguous information about its intracellular location. This ideal is a long way from fulfilment; nevertheless for some enzymes there are qualitative but valuable cytochemical observations which are probably unblemished by artifacts such as can arise from diverse causes — e.g. partial destruction during fixation, or diffusion of the reaction product. Such observations, as briefly surveyed in Section II, have come mainly from the recent application of elec- tron microscopy to this field. Little attention will be paid to histochemical observations, since their value lies largely in ascertaining which type of cell in a heterogeneous tissue contains the enzyme question; nevertheless the light 328 E. REID microscope can give cytochemical help unless the enzyme in question is associated with cytomembranes. Some workers (see Haguenau, 1958) have subjected suspensions of intact cells to ultracentrifugation and obtained distinct layers, one of which repre- sents cytomembranes as judged by staining properties and electron-micros- copic appearance. This early approach, semi-biochemical in nature, may warrant rescue from its recent neglect. (b) Biochemistry. The only strictly biochemical approach at present available for the study of intracellular membranes entails deliberate break- down of the plasma membrane. The method of choice is gentle homogeniza- tion in an isotonic or hypertonic medium, so that there is minimal damage to organelles such as mitochondria. Techniques for homogenization and for differential centrifugation will be surveyed in Section III. Here it may be pointed out that, as discussed by J. Chauveau and colleagues in 1955 [see Haguenau (1958)], Palade and Siekevitz (1956) and Tashiro (1957), there appears to be fragmentation of the cytomembranes, the double membranes becoming transected and "pinched together" at intervals so as to furnish "vesicles". These vesicles are the main constituent (on a weight basis) of the "microsomal fraction" isolated from the homogenate by high-speed centri- fugation (Figs. 7a and 8a). A tissue made up of diverse types of cells is hardly likely to give clear-cut biochemical results. Even with liver, which consists substantially of cells, of one type (parenchymal, hepatic), non-parenchymal cells could conceivably be the source of certain enzymes — although there is no well authenticated example. Moreover, there may be diversity of enzyme pattern within each lobule (cf. Fig. 4). With other tissues, cytological interpretation of biochemical results needs great caution, although for tissues such as kidney and pancreas the difficulties are not insurmountable. The pioneer work, such as that by Claude (1946) suffered from the handicap that the morphology of microsomal fractions — i.e. of the pellet obtained after removal of mitochondria — could be studied only by light microscopy, which is a poor tool for this purpose. Even in 1952, the possibility that "micro- somes" are really fragmented mitochondria was serious mooted (Green, 1952). Only with the advent of electron microscopy did it become clear that Fig. 4. Enzyme localizations in liver lobule as shown by light microscopy. From Rcid et al. (1964), courtesy of Walter de Gruyter & Co. Top left (a) 5'-Nucleotidase with UMP as substrate: there is some staining in nuclei (an artifact probably due to an insufficiently high concentration of Vb^+ ions) as well as in plasma membranes and sinusoids; the cytoplasm is unstained, x 540. Bottom (b). 5'-Nucleotidase: note that the staining is predominantly in centrolobular plasma membranes and sinusoids, and in blood-vessel walls (central vein at top right, portal vein at bottom left), x 270. Top right (c). Glucose- 6-phosphatase: note that there is staining throughout the cytoplasm but not in nuclei, x 270. 6. MEMBRANE SYSTEMS 329 330 E. REID microsomal fractions from liver and certain other tissues consist mainly of fragmented cytomembranes. For some tissues this conclusion may not hold, and indeed the lack of electron-microscopic data in some biochemical studies (even with liver) seriously detracts from their value. There has even been neglect to verify in each case the classical observation that material designated "microsomal" (by the criterion of centrifugal behaviour) is characteristically rich in RNA and phospholipid. (c) Nomenclature for sub-cellular fractions. In pleading for the term "micro- somal fraction" rather than "microsomes", de Duve (1964) has pointed out: "Possibly the most troublesome word in our cytological vocabulary is represented by the term 'microsomes'. Originally proposed as synonym of 'small granules', it had a purely operational significance and served simply as an inclusive denomination for all subcellular entities requiring a relatively high centrifugal force for complete sedimentation. The term .... was used by many biochemists to designate what was believed to be a distinct group of intracellular components . . ." But in view of the heterogeneity disclosed by the electron microscope, ". . . the term 'microsomes' has become extremely hard to define since it has lost part of its purely operational significance, while gaining nothing in cytological precision." Occasional use of the term is perhaps permissible if it is clearly intended as an abbreviation for "micro- somal fraction". The term "ribosome", which has superseded the term "Palade particle", can justifiably be used both in cytological and in operational contexts. However, use of the classical term "RN P particle" is sometimes still warranted, for example when the material has been exposed to detergents that might have damaged the ribosomes (Campbell, Colper and Hicks, 1964). As discussed in Section III., there is no uniformity in the centrifugal condi- tions used by different workers to sediment the microsomal fraction. Where different microsomal "cuts" were taken, the later cuts have been variously termed "light-microsomal", "ultra-microsomal" and "post-microsomal". The policy in the present chapter (cf. Table III.) is to avoid the term "ultra- microsomal", to use the term "light-microsomal" where "heavy" and "light" fractions each rich in phospholipid have been prepared, and to apply the term "post-microsomal" to a final cut known or inferred to be poor in phospho- lipid. The terms as used in this article are in some instances different from those used by the authors concerned. II. Cytochemical Observations *w A. METHODOLOGY The general procedure in cytochemical work is to incubate a tissue sample, already lightly fixed, in the presence of the substrate and of a "product- 6. MEMBRANE SYSTEMS 331 capturing" agent (usually Pb2+ ions, if phosphate liberation is being studied); finally, after further fixation, sections are examined. (No consideration will be given here to autoradiography, of which increasing use is being made by electron microscopists.) One point which warrants mention is that tissue sections (cut frozen) are now preferred (Goldfischer, Essner and NovikolT, 1964) to the tissue blocks used for incubation in early cytochemical work with the electron microscope (Persijn, Daems, de Man and Meijer, 1961). Another approach, which is complementary to this rather than an alternative, is to incubate suspensions of subcellular fractions for the enzyme assay, re- centrifuge them and then section the pellet (see Section IYa. 1). There has been much debate about the merits of different fixatives (Saba- tani, Miller and Barrnett, 1964), and it is doubtful whether a perfect fixative of universal applicability will ever be discovered. Many authors have neg- lected to check biochemically the degree of enzyme destruction caused by the fixative, or indeed, by the incubation medium itself. For example, there can be significant impairment of nucleotide dephosphorylation through the use of Os04 (Persijn et al, 1961) or glutaraldehyde (Goldfischer et al., 1964). Destruction may be less with formaldehyde-Ca2+ or hydroxyadipaldehyde, and indeed the latter fixative is thought to be advantageous if glucose-6- phosphatase (3.1.3.9) or mitochondrial ATPase (3.6.1.3.) is to be demon- strated (Goldfischer et al., 1964; Sabatani et al., 1964; cf. Tice and Barrnett 1962). However, results of fair reliability can often be obtained by judicious use of glutaraldehyde, as in Figs. 5 and 6 below; for glucose-6-phosphatase the pre-fixation should be very brief (Ericsson, 1966). One serious source of unreliability is possible diffusion of the reaction product. Such diff"usion could explain an observation shown in Fig. 4 — that nuclear staining for 5'-nucleotidase (3.1.3.5.) is sometimes demonstrable (and also for glucose-6-phosphatase), for example if the concentration of Pb2+ ions in the incubation medium is rather low (Reid, El-Aaser, Turner and Siebert, 1964) or if the incubation time is prolonged (Goldfischer et al., 1964). Much patient work is necessary to eliminate the diverse sources of arte- facts. Even the choice of buffer may be important; /8, ^-dimethylglutarate is a particularly versatile buffer for which, unlike tris, no inhibitory actions have so far been reported. With over-incubation there may be cellular damage which, with intestine, could be a cause of failure to detect invertase (3.2.1.26) in the brush border (Hubscher, West and Brindley, 1965). B. ENZYMES ASSOCIATED WITH MEMBRANES As is evident from Table I and Figs, 5 and 6, only dephosphorylating enzymes have so far been shown to have a membranous location, at least with electron as distinct from light microscopy. [For hepatic cytochemistry with 332 E. REID c (U ttJ — -^ -C _ ^ V5 c ^ ^ en -'^ ,.- o ^ 'a .^ r"i ON *- ■3 "sj ^H ^ ON 1> ^ PQ S5 ■OS OS )S ^ ^ i8 fe c fe fe ^ S c« ^ o1 ° 2^ -i •:: ^ -i G O wW w "3 UJ O s On ^.^ . ^ _o "^ = > O u n.= "o -«i — C/5 1) ri-i v^ rf M "d t/s 3 3 C JLo ^ 0) •- > 2 «*j o o o I ^ ^ Id SQ < £ N C UJ £.2 ■>r| u, ■§3 c x> p. 3 a c (U •-- > ^ a s: a o a- 2^ 1 o c 2i La u b u T3 1 u u c > .^ f« o J t-l cu o 0 ^^ • Q •^ cu 3" cu NO oTQ NO Q <^ Q^ < 0 _^ ^ a> s: c ;S 9- o 'S C/J o £ 60 o C/3 C "1 1 '-o i3 ^ 6. MEMBRANE SYSTEMS 333 0\ c z- X, I a- -- — ^ < .a > }_ ^ x: cc cc •S'-r -O TJ s 2 <: o o ^ Oj *J 0^ -< 3 "5 OS •-< ^ !S jSf U o so 3 o 1/5 O .^ D. O ^ O ? c . .^ (U D •Q (/5 _> o c t3 o (/) C/5 o c c C/5 C 3 u .b o PQ u £= > I ^ t/5 2 Bii fe i> >- o .. o JJ o D, O -JS •- >» ."S 4) •S.S 8 ■«5 '5 ^ ^ a- iSH i^ Q. i; c« o ■§& ah- ■n < 5§ 'K 60 ^ o c u • - VO *" y r<^ 3 ca •>-• O 73 'u 3 c2 c ^ ii O rn o c • 3 O '^. c u: 60 C > c 1 O - 13 c s: o o >> c/5 — (U d 3 3 c/5 4> o c/5 c/5 > c 3 'Z! >> ^— 'w U ei «J 3 >> c3 g s >, >, -c "S x: JS u 5 o o C X) c c (U 3 u p w -i-> u b> c^ rt ca O. D. a >> C/3 V) SJ Ui 4> h< u Ln .-•§ -d 1 3 i-i ■n 1 3 c/5 ^"5 ^ a> •-" c c/5 1) c _t/5 d) c >^ rt O ■-S > «j o '■3 > 03 O .£ — ' c u E plasmic ein and dfischer 0 ■'-' -^ ■« 0 20 "S t/i a — J c« b> 3 c/3 A 60 3 O .2 .JD c ^ ul « -O 2 u E 0 J= 0 ^ u 1) X E 2 si e3 -C c/5 <« « 60-" cQ 0, 3 t« "E a o'C ^^ <« E "^ S E •^^s^-? u ■s ^ - a 0 "^ d « o •« oj c^ c •0 ~^ « .. ^ >r 0 « Q u « < ■" -ft =« — 's «5 2 uj s 3 0 jg -c C s: S" "> CO ^^ 2 •2 s £ i CJ 1, c + "60 '&b<'S "3 c 0 .2 « -S •S^ E = g-g oS 0 § *" 2 l«c| a Cm 0 « C 0 3 u T3--3 c u <*. « C3 .- !5 8 c 3 S^ -3 .- "S 2 -- • f •s X X < ■s ^ 0 s E< sifli £3^ 2 ' es A 2 t2^ £3 6. MEMBRANE SYSTEMS 335 the latter, see Wachstein (1963). Note that the references in Table I are not intended to be comprehensive]. So far, there has been no demonstration of a membrane-linked esterase. It will be noted that the difference shown by hght microscopy between glucose-6-phosphatase and 5'-nucleotidase — the former having a diffuse cytoplasmic distribution and the latter being ap- parently in the plasma membrane (Fig. 4) — is supported and clarified by electron-microscope findings. These are shown in Figs. 5 and 6 together with findings for nucleoside di- and tri-phosphatases. (It may be pointed out here that the term "nucleoside phosphatase", as used by same authors, is ambigu- ous and deplorable.) The illustrations support the conclusions of Goldfischer et al. (1964) and other authors, that glucose-6-phosphatase is intimately associated with cytomembranes, whereas "UTPase" (or 'ATPase') and "ADPase" (not illustrated) are mainly in the plasma membrane especially where it folds to form micro-villi, and that the enzyme which splits GDP, IDP or UDP (but not ADP or CDP) is located in both cytomembranes and the plasma membrane, Enzymic activities demonstrable in cytomembranes are likewise evident in the nuclear envelope. Staining in the non-convoluted regions of the plasma membrane, with substrates such as UTP or UMP, may not be obvious if a short incubation time is employed. From an examination of some 60 cell types in Novikoff 's laboratory, the generalization has been made that in no cells can the cytomembranes dephos- phorylate nucleoside mono- or tri-phosphates or ADP or CDP (Goldfischer et al., 1964). Novikoff (1964) also believes that plasma membrane contains a 5'-nucleotidase which is activated by Co2+ as distinct from Mg2+ ions. Thiamine pyrophosphatase is unusual in being located in the Golgi cytomem- branes; staining sometimes observed in other structures (such as the nucleus) may be an artefact (Goldfischer et al., 1964). The electron microscopic results for cytomembrane-located enzymes point to a location on the side of the membrane facing into the cisterna (see Figs. 5a and 6a), but do not conclusively establish that the enzymes indeed have a membranous as distinct from a cisternal location (Tice and Barrnett, 1962; Goldfischer et al., 1964; Ericsson, 1966). This cursory survey, in which scant justice has been done to individual papers as distinct from reviews, serves a background to the biochemical observations which will now be presented (Section III), and which later (Section IV.A.) will be dove-tailed with the cytochemical observations. Fig. 5. Enzyme localizations in hepatic cells as shown by electron microscopy (El-Aaser, 1965), courtesy of A. A. El-Aaser and M. S. C. Birbeck. x 73,000. Top (a). Glucose-6-phosphatase: note staining in cytomembranes and nuclear envelope. Bottom (b). 5'-NucIeotidase with UMP as substrate: note staining in bile canaliculus, in plasma membrane (faint), and also (due to the non-specific acid phosphatase) in lysosomes. 6. MEMBRANE SYSTEMS 337 III. Biochemical Observations A. ISOLATION AND CHARACTERISTICS OF HEPATIC MICROSOMAL FRACTIONS 1. Methods of Isolation {a) Preparation of homogenates. Mild conditions such as are necessary to / preserve the integrity of nuclei, mitochondria and lysosomes are desirable^ and effective for cytomembranes also. The formation of vesicles from cyto- membranes by a pinching-off process is in artifact that must be accepted, and at least this process appears to be reproducible if use is made of a Potter- Elvejhem homogenizer and of a^ucrose medium. Surprisingly, however, this homogenizer was found by Leadbeater and Davies (1964) to be inferior _to a Waring blendor with respect to the yield and the stability in vitro of licrosomal demethylases [see also III.c.l(c) below]. For subtle studies such as are considered in III. b. below, 0-88 m sucrose lay be preferable to 0-25 m, although its effectiveness in giving flattened bther than swollen vesicles (cf. Palade and Siekevitz, 1956) may be poor |see Fig. 7a compared with Fig. 8a). However, for routine preparation of licrosomal fractions, 0-25 m sucrose solution is more convenient, a trace of iDTA being permissible (de Duve, Pressman, Gianetto, Wattiaux and Lppelmans, 1955). Rupture of a high proportion of the cells is not too hard \o achieve, particularly if the first "nuclear" pellet is rehomogenized (de Duve )t al., 1955) and the g values adopted for the centrifugations need be only 'about half of those for isolation in 0-88 m sucrose. Some workers compromise _by using 0-4 m sucrose for example. Unfortunately the "nuclear fraction" will, whichever strength of sucrose is used, be contaminated with cytomembrane fragments, as shown by assays for glucose-6-phosphatase; only by the use of unorthodox media such as organic solvents (the tissue being first freeze- dried) or 2-2 m sucrose can virtually uncontaminated nuclei be obtained, at (^ the expense of the integrity of the nuclear envelope [Reid et al. (1964), inter alia]. With a raffinose-dextran-heparin medium which Birbeck and Reid (1956) found to be advantageous for isolation of mitochondria, contamination of the mitochondria with cytomembrane fragments is low, but the latter are themselves not readily sedimentable and, moreover, are rounded in shape just as with 0-25 m sucrose medium. Media lacking sucrose but containing salt(s), as used by A. Claude and other pioneers of differential centrifugation (cited by Moule, Rouiller and Chauveau, 1960), may resujt in less sharp separations i Fig. 6. See legend to Fig. 5. Top (a). Nucleoside diphosphatase with UDP as substrate: note staining in cytomembranes and also in bile canaliculus. Bottom (b). Nucleoside triphosphatase with UTP as substrate: note staining in bile canaliculus and adjoining plasma membrane, with little cytomembrane staining. 338 E. REID than sucrose media although for microsomal fractions the difference may be small (Takanami, 1959). Nevertheless, trial of such media may be helpful where it is suspected that the presence of an enzyme in the microsomal frac- tion is an artefact due to redistribution (de Duve, Wattiaux and Baudhuin, 1962; Schneider, 1963; see also Section III. b. 4). Recently, particularly in studies of amino-acid incorporation, increasing use has been made of media containing both sucrose and a trace of neutral buffer, often supplemented with KCl and MgCl2 (inter alia, Campbell et al., 1964; cf. Tashiro and Ogura, 1951; and Dallner, 1963); with such a medium in place of a medium con- taining sucrose only there is a difference in the yield of the 'post-microsomal'" sediment, if not of the microsomal sediment (Hird, McLean and Munro, 1964). The MgCl2 serves to minimize damage to ribosomes and polysomes. With use of phosphate instead of bicarbonate or tris buffer, there is some risk of inadvertent elution of cytomembrane constituents (Reid, 1961a). (b) Centrifugation conditions. Some comments are warranted about the choice and description of conditions of centrifugation. Firstly, the choice of ^'-min values [namely the product of the relative centrifugal force {g) value and the time of centrifugation, ideally with allowance for the acceleration and deceleration periods] for differential centrifugation inevitably entails some compromise. This depends on whether the aim is an "analytical frac- tionation" (so that a balance-sheet can be drawn up for the distribution of the constituents analysed) or a "preparative fractionation" with interest centred on a particular fraction (de Duve, 1964). Secondly, runs at nominally similar ^-min values may differ in effectiveness according to the geometry of the rotor (de Duve, Berthet and Beaufay, 1959). Fortunately, however, angle (rather than swing-out) rotors of comparable geometry have been used in much of the published work entailing high-speed centrifugation without a density gradient. Thirdly, some authors regard the value g as referring to the bottom of the tube and others as referring to the centre (sometimes with disregard of any incompleteness of filling of the centrifuge tube); such ambiguity is lessened if the actual rotors, speeds and times employed are specified. Nevertheless, values for ^-min are valuable as a rough yardstick for comparing results obtained in different laboratories at least for the non-gradient procedures now to be considered. [Gradient centrifugation re- presents a powerful tool (see de Duve et al., 1959, and de Duve, 1964), but only certain applications will be considered here.] Light microscopy, as used in the pioneer work, is almost valueless in connection with cytomembrane studies. The classical paper of de Duve et al. (1955) illustrates how careful application of purely biochemical tech- niques, particularly enzymic, may be rewarding in choosing conditions for differential centrifugation (see also de Duve, 1964). In connection with the desirability of obtaining a cytomembrane fraction little contaminated with lysosomes, enzymic assays (particularly for acid phosphatase (3.1.3.2.)) are in 6. MEMBRANE SYSTEMS 339 fact the only reliable guide, since lysosomes are too few to be enumerated by electron microscopy. For examination of actual cytomembrane elements electron-microscopy is, of course, a valuable tool, unfortunately neglected in some studies where its use would have been of notable benefit. When use is made of the conventional 0-25 m sucrose medium, with no gradient, removal of mitochondria together with the bulk of the lysosomes can be effected by centrifugation at a value in the range of 100,000-170,000 ^-min, as used by most authors [de Duve et al. (1955) and Ernster, Siekevitz and Palade (1962), inter alia]. With a higher value — e.g. 600,000 ^-min, as deliberately used by Campbell et al. (1964) with 0-3 m sucrose medium — there is likely to be complete sedimentation of all lysosomes, but some loss of microsomal material must be accepted. When 0-88 m sucrose medium is used, a value of the order of 500,000 ^-min is suitable. The nature of the "fluffy layer" which overlies the mitochondrial pellet, at least with 0-25 m sucrose medium, was a matter of controversy until it became evident that it consists largely of damaged mitochondria. In the method of Palade and Siekevitz (1956) it is left behind with the mitochondria, but some authors transfer it to the next centrifugation stage or give no indication of the procedure followed. For sedimentation of the microsomal fraction with 0-25 m sucrose, a value of the order of 6,000,000 ^-min has been used by many authors (e.g. Ernster et al., 1962); a 60-min run at 40,000 revs/min in a No. 40 angle rotor (Spinco) is customary, the nominal g being 105,000 (centre of tube) or 145,000 (bottom of tube). However, a value of only 3,000,000 g-min — as used in the study by de Duve et al. (1955) — is ample as judged by assays for glucose- 6-phosphatase, and in at least some contexts use of a much lower ^r-min value (as employed by the pioneer workers in the field) may be sufficient or even preferable [see Reid (1961a), and Section III. b. \{b)]. Moule et al. (1960) shrewdly remark: — "The centrifugal forces used to carry out the sedimentation of microsom.es have increased as the centrifuges have become more powerful so that, with 0-88 m sucrose as suspending medium, the earlier centrifugation of 2 hours at 40,000^ . . . has been changed to 130,000^^ • . • and 145,000^ for varying times . , . [Usually] no rational explanation has ever been given to justify the experimental conditions." One practice common when only low-speed centrifuges were available — the sedimentation of micro- somal material by acidification and low-speed centrifugation — ^is now used only occasionally (e.g. Gorlich and Heise, 1962, 1963) and is to be deprecated. Thus with citrate at pH 5-4 there can be enzyme solubilization (Gosselin, Podber- Wagner and Waltregny, 1962); on the other hand, a study of deCDP- choline synthesis indicated that there was enzjmie adsorption on to micro- somal material at pH 5-2 (Schneider, 1963). The microsomal fraction is normally obtained as a gelatinous reddish pellet, underlying which there may be a pellet of particulate glycogen if the Q 340 E. REID animals were not fasted. There is no excuse for the vagueness in some papers as to whether the animals were fasted, or for apparent surprise that the "microsomal" pellet showed a lower layer which consisted of glycogen (cf. Trivus and Spirtes, 1964). Most authors do take the sensible precaution of fasting the animals overnight, although this failed to depress the level of total glycogen in one strain of rat (E. Reid and G. Siebert, unpublished experiments). The microsomal material is best resuspended mechanically with a loosely fitting pestle, inserted directly into the centrifuge tube after addition of the suspending medium. 2. Characteristics of the microsomal fraction {liver) (a) Morphology. Electron-microscope examination of sections of micro- somal material, after fixation (usually with osmium tetroxide) and resedi- mentation, discloses mainly "vesicles" representing cytomembrane frag- ments— these are mostly "rough-surfaced" (i.e. ribosome-studded) — together with free ribosomes (Fig. 4a). From cytochemical examination (see Fig. 10), glucose-6-phosphatase appears to be localized in the membrane fragments, in accordance with the cytochemical observations on intact cells. Besides the vesicles — which are rounded, at least if a medium containing 0-25 m sucrose is employed, there is a varying admixture of miscellaneous elements, including so-called amorphous material. (b) Constituents found by chemical analysis. In view of the morphological heterogeneity of microsomal fractions and of differences between labora- tories in the conditions of isolation, it would be pointless to tabulate values for protein or RNA. Typically, about ^ of the liver protein and up to f of the RNA is recovered in the microsomal fraction, the "microsomal" RNA being mainly in the ribosomes. The ratios of RNA and of phospholipid to protein (or to lipid-free solids) are relatively high for the microsomal fraction (for examples of actual values, see Table III). The microsomal fraction is likewise rich in free and esterified cholesterol (for values see Reid, 1961b). Microsomal fractions are low in cytochrome c, but contain cytochrome 65 (cytochrome m) (Palade and Siekevitz, 1956; Ernster et al., 1962) and a CO-binding pigment (citations in Orrenius, Ericsson and Ernster, 1965). They contain coenzymes Qg and Qio (Leonhaiiser et al., 1962), and NADP (see Reid, 1961b) and inosine (Siekevitz, cited by Ernster and Jones, 1962). Immunological studies on microsomal material (Westrop and Green, 1960; see also Reid, 1961b and Jungblut, 1963) have not so far been very illuminat- ing, except to demonstrate the existence of proteins characteristic of this material. Recently discovered constituents include cytochrome P-450 ("reticulochrome" ; Omura, Sato, Cooper, Rosenthal and Estabrook, 1965) and o-tyrosine (Feuer, Golberg and Gibson, 1965). (c) Enzymes. Table II lists enzymic activities reported to be present in microsomal fractions, not necessarily in a high amount relative to the 6. MEMBRANE SYS.TEMS 341 o 1/3 -4-> 3 o3 O O cd C »-i fl3 X) ^ s c»-( u 2 S «50 c c •- 11 «_ 11 1 » (U 1 [> 03 8 o 3 a "eojc -^ ^ o^ \0 a o^ vo «S c3 g C O ^ o ^ o 6 >. N C 0< (U si O D T3 C D.T3 Jo " « o > u I c o T3 C o '> o o H (/J C _ 3 O "" i \o C l-l ON <^ c x> ^ " C T3 I— .JC -r - - Oh 2? C —I l-c ^^ !LL c ^ 35 Vi ^s V ■St 2 5 .-2 ^ —I Tf to 'O o o 5 !>- ^ XJ rf 4> ' X) >> VO S< CO ^5 cj O :s a 3 »5 O ES I::; ^^ c to 2 £ >^ ^ .S •53 .2 -S > 3 ^ <3 D ^ -d W CLi \0 •— 'ON i" 1^ ■o^^fe CO CO m 3 ON ON rf "^ 1^ ^ 13 ^o^Q Q|0N ^ 1^ c3 -^ 3 «• 2 s o o o c 6 60 2 2 -2 JS >. >> 0) o o ^ ffi DC J2 Q Q i2< < 342 E. REID O cfl O c '•3 ^ o •-I _ S.2 B U Ok \o \5>0. .5 o 6 a o^ .a -J <3 o c Ij M On «2 o •J 5 ••5 2 43 o rr, (U m Uh (/I D OS 3 "-^ r^ s^ -H 0\ d) I vo PQ 2 «J a > J2 Oh <3 -d <4) § B •*-» w i- c4 2 a > .S o rf OS ^ S ^ sJ O It e o I o < a: Ok Q < Z ^^ ^ 73 to s ■3 "^ rn C t=P X 2 Ss « S^ 3-'S >r TO •« r; 9 ^ 2|. Z ■<-•'<-> -J CO V3 C/3 c « o .2 II ON oT ^^ !^ S «*-! ^ 2 ;=^ J^ ^ ^ J2 >*^ c C 1 o >« s etf O C " wo ^ rt T wON C c o x; D. t/5 O Xi a o H 8 ^ M l?" >^ S O H< I ^^ 8 3) ; Stei 1955] \0 /-~s v^^ •2 • ^ ^-H 60 ♦J ►7 NO 03 A ov ^. u, <^ 0 . -^ Si S x: 0 i-» ^-' ?.au --0 c dPr and oma Han Aric -u c "^ U _4 C rt e 9 ^ ^ Wilgram a Komberg Brawermai ^.a S elude 1 R. H. M Nordlie j 0\ o ^ ^ o CO o 4S o. s ^ < ^^ _c >^ ^ 0 43 s C^ ••3 U 00'.; «N >8 ^ 60 >. « ■qo£ c ^-^ CO (U O to X g D. ^ So — O 2^ c w :2 a '4-> CO a o j:: 43 D. a JO o 43 >. CU Oh 6. MEMBRANE SYSTEMS 343 1-^ ^> c S^ 6 o a o c o c T3 c 3 -o o o o ^ "^ OS V So « o o U ^ c u l-l C OS O 4) S so 60 in O Os o ON bO CO :i ^ X) •a "O O 03 ^ (Z! :? J s 60 ;: i-i h -5 55 ii ■= « c « o K r: 00 6 o\ o o '^ 60 o\o\ > Tf \0 ^ ^ r^ oo t^ w-i Tt VO lO VO 00 a — I 1 c c o u 3 -a Q D 1 = I 344 E. REID s n < 6. MEMBRANE SYSTEMS 345 ^ a X) •a D. O \0 O ON =« a IT- OS / — , 3 •a o a Ov ON ii: t« ON Q z: C 1> 1— 4> U > C C C^ S £ Q li: o ""^ PQ U (U » U "? ^ 6 ^ < 0 1 u XI >> u 0 e J3 0) yo •- « s 53 o S ^ W O O 3 « rt t« n T-. ^ X O u •£ -c I- .y o .^-§ I ° S " c «^ «> ^-o «J ^ 4^ c S ^ O C >< £ - 00c ^ <" o b ^ > > " X O nj u U h> U T3 3 I- U ^ ■£ "3 « = ON " 3 X ^ T3 «» ^ « o «> u 60 3.S "60 tl = 1 ^- .5 85 o o o 4) "^ Ex ^ JO 5o^ > £?. S gx- "5 ** >^ t; 3 o -p 2 M X 0-£ _>> o c; '^ X -^ « '5 "> w c f^ c X 5 u o *^ o b o ^ '^ "O >o "^ oi 2 >^ O O « -2^ C >< ^x o O I. =1 > 3 N U 4) 7L C 3 •a ON y §5 U U PLh 4> c t; ■£ fe § °<^ fc- > c o ^^O E ^ on -J ° « O § « t« « h ^ u j: .a O 2x 2 8 R X ^ •= g o E b (X^ea ,S 1) c 01 > O « I- 1/5 £30 ir c« u- = Si E O ° 3 - I- O 8 « fe ,P X - >>2 " -a « a> ^ gc/3 4> . « C X.2 £ X — U nJ vo « UON (i> ■£ ^^- :2 - c > .£ >. w L^ "^ ^ o * « o u .2 3 60 •0 iZ C3 0 C Z 8 ■^^ X ? U5 g ^ - t^ S E. REID —1 -a "« c JD m a S ^ C o /-"V o CJ (U -o u X) J3 s T3 C c ^^ (J 'S 3 Q ^3 C S ^ C^ O. rt c/j d 3 ta iS 3 T CO bi) cA g «s 2 ^ su oo 30? O O 3 CO o <^ ^ 'C ■ < CO (U CO 3 C ^ S ?i ^ .•2 'C •■= u W-) oj ^ > C/3 -I ^ o I I ! l! 0^ I- CO O \o C/3 o\ •:= ^ T3 ~ CO S 73 5 - 6. MEMBRANE SYSTEMS 349 o Q t3 OS 12 -O OO ^ w o o so ON fe -. c ^ OS o O 00 o X « (U N .2 &. C ^ o\ ^ (N "5 '>o ^ 23 f*^ SOI30 o l-l 9 ^ c^ <« X3 OS D ■<-» °P 6 6 o „ „ O n T^ »^ + + n ^ Tt f^ OS 6 -' o ^ -^ o + + OO 6 g U O 3 8 a c o — + + -HS + + + + -h + 60 OOJO "eio "eo S, < fs < rs &<; Oh I •d J3 Oh £oi ^5 2§ bo2 C e3 1 ^" c a o it bo2 eg c; o O 1 5^ Q 350 E. REID I •S i a s So^ A 23 O X O C? S^ 2 .S •- « OO «0 4) r< + + + §1 s ex 6. MEMBRANE SYSTEMS 351 + + + + + + + + -H, + + + + + + + + + + + + a p: 352 E. REID centrifugal cuts may be increasingly rich in RNA (at least relative to phospho- lipid), but that there is no good morphological evidence for a predominance of smooth-surfaced fragments in the lighter fractions. It may also be pointed out when light elements rich in RNA have been sedimented, prolonged centrifugation of the supernatant gives a small pellet which is poor in cyto- membrane fragments as shown by electron microscopy (Fig. 8f); it may be low in RNA and phospholipid (Table III). This material truly warrants the term "post-microsomal". This fraction may have abundant ribosomes in the case of pancreas, which is rich in free ribosomes (Palade and Siekevitz, 1958b). A judicious choice of centrifugal "cuts" in the classical, non-gradient, procedure may, then, be helpful in ascertaining whether different groups of membrane fragments (perhaps differing in morphology) differ in their enzyme content. There may indeed be instances of such enzymic heterogeneity (Table III), but unfortunately in few studies has assessment been made of the mor- phology of the fractions, or even of biochemical parameters such as phospholipid and glucose-6-phosphatase which would facilitate the interpretation of the results and their comparison with results obtained in other laboratories. In one particularly careful study (Moule et al., 1960), close parallelism was actually found in the distribution of three different enzymes (Table III); but on applying the procedure of Moule and co-workers, Carruthers and Baumler (1962) observed that an intermediate "cut" — presumably represent- ing a mixture of rough- and smooth-surfaced fragments — was particularly rich in esterase. (c) Flotation with no gradient. Chauveau, Moule, Rouiller and Schneebeli (1962) ingeniously exploited the relatively low density of phospholipid- containing structures and succeeded in isolating membrane fragments almost free of ribosomes. The starting material was a "light" microsomal fraction already with a low content of ribosomes and rough-surfaced membrane fragments (see above). Centrifugation of this fraction for 20-40 hr in a concentrated sucrose medium gave, besides a pellet, a surface pellicle which was particularly firm and manageable if the sucrose density were 1 -25 rather than 1-21. More than half of the nitrogen of the original fraction was recovered in this pellicle, which appeared in the electron microscope to consist of smooth-surfaced cytomembranes (Fig. 7b). The chemical and enzymic composition of such material is given in Table IV. Fig. 7. Microsomal material isolated from liver in 0-88 m sucrose medium (Moule, 1964; see also Moule et al., 1960, and Chauveau et al., 1962). Courtesy of Y. Moule. Top (a). Whole microsomal fraction. R= rough-surfaced vesicle, S= smooth-surfaced vesicle, x 75,000 approx. Bottom left (b). Fraction obtained at 145,000 g (3 hr) after removal of a first microsomal "cut" at 40,000^ (2 hr): note predominance of smooth-surfaced vesicles, x 75,000 approx. Bottom right (c). Pellicle (floating) sub-fraction obtained by re-centrifugation, in sucrose medium of high density, of the second microsomal "cut" depicted in b): note that only smooth-surfaced elements are present, x 150,000 approx. I 6. MEMBRANE SYSTEMS 353 -'%' *-. #Kh^' 4 V" •#; l> -^ 354 '^ s: .a 5 Q - c =5 u z; c (U Cj o. o 2i o e *^ o Z62 a, " O rv C i- ^ "3 k- u> 60 D. oj 3 0 3 .0 U C _C 8 "^ a 3 0 ;^ (D VO _> On ^— ^^ 15 cfl X3 3 rt 0 k> Mh ^ -t-> '-^ t/3 — >> (U C OS CT3 > C i. "^ .JL •! I -^ g ^ ^ X o c o =: „ •^ Q ^ "« U N U 7; =^ ^ "SO Clfl £ c ^ ^ .S c ■^ -^ ^ :§ -^ '5 >1 >~- ON E. REID c "q 0 x; .23 "q E 0 E 0 c 0 (U > '5j E =5 ■> E ^ T3 c 0 °^ U so to 0^ fti Soj 2 w oi 0 — U^ W '^ A — c/) E 1^ E o E O tt o «^ . S x: 3 r— 'f -- o O O I ^•^^ 00 ig «j 00 ^ — ' -r -= -a t: o i:: 73 •i o .2 3 cj vo (u i^ "^ O x: .y o E oj 1/3 -^ *J T3 r- O 03 E KJ O O a> a 3 6 .2 ^-o -^ (M oQ _ x< g ^ 3 cd 3 (U ■^ a o ■t: «j r^ E E >. 3 N 73 E S O ^ c 2 « H U tn "3 2 0 '• E 2 'e O t^ c vS ■<-' ■S <= 53 x; 3 T 60^ I '■p ^ ^ o 6 o U 0) £13 o >. t/5 -^ ^ ^ •c a I o ^ C (u a> "3 Ji > C/5 c o D-:2 s:x 0. X3 O 3 . ^E 00 E o O X3 1/3 "" E 0 c f s Cm 8 c2 £ £ Q 0 356 E. REID •a 60 O T3 C « .a S3 Is c IS a ^ a S 1-2 0,43 ss ."2 ^ft -5 *? 60 «« « O G .22 So u W (1> 43 •♦S X) 60 o o X) X3 S A Q 2 Q- o " a> C j^ ON ^ 8 O CXi o. 43 b O 3 a Sx 43 ^^ u O 3 f*5 o |1 cu 1 8 3 C O .S 1 rf in ei3 jC o OS N a> 35 ^ 43 i i3 t3 .2 7< 3 S .ti ^ ►^ "O (u •^ ^ -^ 2 o Z! ' « 3 Ki So g o r' T3 V o 6 Ti p "55 3 Is 2 <=>, O 43 G O o 2 ^-BS !_, (/O .^ V3 -^ ^ S.43 t- «tJ S «« o "^ c Q 4> 2 K o W 3 2^ ^ (zi .5 ^ <=! G ^ O .3 •S C 1^1 Oh )-> cU _ (U 5 o «^ o -o o |3 X5 ON =3 G ^ ? o-o ^ "S o, ^ ^ & ^ S =;* ^ . H ^ = u cj 5 t. t) -2 C IM O O 60 ^^ ?i s c k! T-r -5 ^ o =5 H + ^ c: (90 ?g o !>) ■4-» I •^ o !3 <;j T 5 ^ VO C IN TO o^ OS 60 ^ ^ •- (U .-J ^O 73 t; s ''S "0 o t/3 •a 3 01 — «5 o V. c -2 J- ."§ "3 S^ « g , o B ^ o ,« «^ ^ « ■^ o S "*- o <-> '^ a s 52 ^ 3 i> Co TO ^ 53 H .5 ^^ « c c lT t^ — 1^ O,0m«oparo Q so 4 c 60 > . IT c ^ •- c u «5 >r> "O t3 i go CO O 0) O li u =5 >»J. ^ J- ^ ^ _ . . 60 Ui D.^ "' e ^ B ta^ ■<-»" "U •< ^■•00 ^ ^ c o 3= > « O (U O 00 -i- (U CX\0 2 S 60 c3 .2 § *-' *-• 60 o 3 D, u ex U o c j3 ^ a,^ P 2 o It; C S - 2 c o '^ oc C C • - fi 'i O Si o S 2 » 1-4 o a O -O o c o 60 .S e 2 60 (U 3 ;s 1=; 60 M >. a S-2 1.t£ Infer dens oft 13 s T3 -- a> cs c .a '5 2P ■w o oil III .SS& O 73 ps c t^«* c« 4> V^ — " P CTJ ^ u tc ,o t- 1: 60 D. cj 3 «*- "5 h5 o H B a "^ c ;2 13 8 3 «=^ O \0 I o\ O .^^ I 3 .S o a 60 O. 6 ^ 8 = J3 3 O) _u i 1 o CO •^/f .2 o o Ih 3 p. 60 g.S ■-» 1-1 .ts =^ ^ C .-3 -O .2 S g 60 U O O .-'.2'S^ =2 603 o — 'C h <^ "-^ .ape q T3 rt ."^ *j .— J3 c: > DO ^ -^ c § c-2 TD .5 60 c „ -d _ 3 :S o " "H 1 3^ S §2 3 C > « s O ^ ^ en 1 CO •a (U (U 73 vo c 3 03 _c C o> S .^ o o c 3 J^ JD 4> *S ^ ■^ *^ o PLh D 2 1 •^1 I T3 « -a i 1 C c 3 O rt 1 ft ,c S 13 ^^ c^ • #* a 1 e a a, o til > ft O Ma 'l-c c ft 60 u ON •n V 3 ;3 u o V— ^ CO o cn S ^ o (U li "O *j T3 3 ■ft" ^'^ - 60 > c c o "* ;:s X) rt 3.^ D. O 5 -c tj ^ i> V3 "5 ■ ■; (u u "o ) -2 *f -a i3 -2 . U 2 O « g ^ O ^ J= J= -^ ^ u .5 c ^ •=; lu i) Sri (D 4> < O Z T3 '^ =; o 6 V V I V := o5 S g o (u <4j M (« c 2 1 to > c ^ o sen »1h jS 60 B O + +^ o 73 G 50 S> 5 c (50 v>. D. g O^ 2 Ea~s, CO ro C G c C ro 56 T3 C *- S G J2 Ji G T3 « (U e >, .4.4 a 1/9 "u G D, 4^ 9 « 3 ^ o O §32 « 13 (U «u 3 f«^ "2 G I- j3 :" '^ J ^ 60'-' )$ + c o OU -^ !/3 T3 • Su G a> O — •G ^ ~ - 13 360 E. REID o o a ■»-» o o 6. MEMBRANE SYSTEMS 361 (d) Centrifugation with a gradient. As has been discussed by de Duve (1964) (see also Wallach and Kamat, 1964), density-gradient centrifugation is a powerful tool if the conditions are judiciously chosen. Even a simple layering procedure may be effective. Examples are given in Table IV. Thus, a technique previously tried by Dallner, Orrenius and Bergstrand (1963) was applied by Campbell et al. (1964) to a homogenate in 0-3 m sucrose which had been freed from mitochondria and heavier particles and was then layered over 1-5 m sucrose solution. Centrifugation in an angle rotor for 11 X 10^ ^-min in the presence of 10 mM Mg2+ ions gave, in the final upper layer, "smooth vesicles" together with some free ribosomes (see Table IV). As will be considered below, a few workers have already used such procedures while studying the enzymology of microsomal fractions. Increasing application is likely to be made of the ingenious "zonal" rotors designed by Anderson (see Anderson, Price, Fisher, Canning and Burger, 1964) which, with comparatively short centrifugation times, enable particles to be banded isopycnically or on the basis of differences in sedi- mentation rate. In experiments designed to separate plasma membrane fragments from cytomembrane fragments, this aim was partly realized (Table IV), but the most notable result was that discrete fractions were obtained corresponding to smooth-surfaced cytomembranes, rough-surfaced cytomembranes and ribosomes (El-Aaser, Reid, Klucics, Alexander, Lett and Smith, 1966b). 2. Re-centrifugation of Microsomal Material after Special Treatment (a) Deoxycholate treatment. Dallner (1963) and Ernster et al. (1962), inter alia, have tried deoxycholate concentrations lower than those cus- tomarily used to solubilize membrane fragments [see III. b. 4.(a)]. [Not only the concentration but also the ratio of deoxycholate to protein is important, as emphasized by Higashi and Peters (1963a) and earlier authors]. When a microsomal suspension in 0-25 m sucrose containing 0-024% deoxycholate was centrifuged (13 x 10^ g-vam), Ernster et al. observed a loose reddish sediment ("m fraction") overlying the ribosome-containing pellet. Table IV gives data for this fraction. Its yield was low, and it was not entirely free of ribosomes; possibly the smooth-surfaced cytomembranes had dissolved pre- ferentially on exposure to deoxycholate, leaving behind some of the rough- surfaced membranes. Supernatants thus obtained after deoxycholate treat- ment may themselves yield a pellet when exhaustively re-centrifuged after dilution with sucrose medium free of deoxycholate (Ernster et al., 1962), or after addition of extra deoxycholate (Moule, Bouvet and Chauveau, 1963). The latter workers (see also Moule et al, 1960) were able to obtain such a deoxycholate-treated pellet from a smooth-membrane fraction as described in Section III. b. \.{b) and (c), and considered it to represent particles derived from the smooth membranes and distinct from ribosomes. These particles 362 E. REID were apparently smaller and (or just lighter) than ribosomes, and were lower in RNA content and richer in phospholipid than ribosomes; they could be obtained even in the presence of Mg2+ ions [see also III. b. 3. (a)]. Particles rich in phospholipid have likewise been reported by Pollak and Shorey (1964) who obtained them by treating rough-surfaced microsomal material (Dallner, 1963) isolated from embryonic chick liver with deoxycholate and also with ribonuclease. Their assumption that the particles, given the unfortunate term "reticulosomes", have physiological significance is not well founded. (b) Iso-octane treatment. Use of iso-octane as described by Hawtrey and Schirren (1962) was found by Campbell et al. (1964) to be less effective than gradient centrifugation [see III. b. l.(fi?)] in giving membranes free from ribo- somes. In brief, a microsomal fraction was treated with iso-octane, and the emulsion centrifuged to give an interface layer containing membrane frag- ments. However, with a "light" microsomal fraction prepared by the method of Moule et al. (1960) as the starting material, Hallinan and Munro (1964, 1965) obtained a centripetally migrating fraction which was thought to represent smooth-surfaced membrane fragments of good morphological purity. (c) Other treatments. Dallner (1963) tried re-centrifugation in the presence of Cs+ and Mg2+ ions, which were believed to show selectivity in their binding to vesicles. The smooth-membrane fractions thus obtained — one binding Mg2+ but not Cs+, and the other binding neither cation — showed some difference in antigen and esterase patterns (Lundkvist and Perlman, 1965). When microsomal fractions are treated with certain buffers at an alkaline pH, there may be differential effects on the membranes, although such effects are not seen with tris (Palade and Siekevitz, 1956). Membrane rather than ribosomal constituents are perhaps preferentially extracted by bicarbonate buffer, as judged from assays for glucose-6-phosphatase (Hultin, 1957), or by Mg2+-containing phosphate or glycine buffer [Reid, 1961a; see III. b. 4.(c) and Fig. 8]. The converse may occur on extraction with pyrophosphate buffer (Sachs, 1958; Goswami, Barr and Munro, 1962). The latter treatment, Fig. 8. Microsomal material isolated from liver in 0-25 m sucrose medium at 20,000 g (90 min) (Reid, 1961a). x 50,000 approx. Top left (a). Freshly isolated microsomal fraction. Top right (b). Microsomal fraction after freezing and thawing. Centre (c, d). Residue from microsomal fraction after extraction with Mg2+-containing alkaline buffers (pH 7-4 phosphate, then pH 9-0 glycine — (c) and (d) respectively): note j>ersistence of the ribosomes. Bottom left (e). Residue from further extraction of the residue depicted in (d) with EDTA- containing pH 9-0 glycine buffer: note disappearance of the ribosomes. Bottom right (f). Material sedimented at 145,000 g (1 hr) from 20,000 g su]pematant i.e. after removal of material shown in a): note paucity of ribosomes. I 6. MEMBRANE SYSTEMS 363 364 E. REID as for treatment with EDTA (Palade and Siekevitz, 1958; Siperstein and Pagan, 1964) leaves mainly membrane fragments with a few attenuated, ribosomes; such fragments, like those in preparations mentioned under (a) and (b) above, would presumably correspond to rough-surfaced as well as smooth-surfaced cytomembranes. The lipoprotein "ghost" fractions ob- tained by Westrop and Green (1960) have not been adequately characterized, particularly with respect to morphology. 3. Constituents of Cytomembranes (a) RNA. The view that the RNA of microsomal fractions resides almost solely in ribosomes {inter alia, Palade and Siekevitz, 1956) was soon chal- lenged [see Reid and Stevens (1958) and Reid (1961a), and citations therein], and is now less tenable — although Campbell et al. (1964) consider that contamination with ribosomes could account for the RNA found in a mem- brane fraction by Hawtrey and Schirren (1962). Moule et al. (1960) found that a "light" microsomal fraction low in ribosomes contained as much RNA as a "heavy" fraction (Table III). Goswami, Barr and Munro (1962), Ernster et al. (1962), Dallner (1963) and Petrovic, Becarevic and Petrovic (1965) have likewise found RNA in fractions containing few ribosomes, although in some such fractions, such as that prepared by Schneider (1963), the RNA content was low (Table IV). The "light" microsomal fraction (0-88 m sucrose) of Moule et al. resembled the "heavy" fraction in that half, and only half, of the RNA could be recovered in the "ribosomal" pellet obtained with deoxycholate — the implication being that smooth-surfaced membranes may contain RNA which is sedimentable in the presence of deoxycholate. (They mention that the pellet from either fraction consisted of dense granular elements.) A "light" microsomal fraction prepared in 0-25 m sucrose and with a low content of ribosomes, as shown in Fig. 8f, was subjected to an extraction procedure which, when applied to a heavy fraction, removed one-third of the RNA and left ribosomes apparently intact (Reid, 1961a; Fig. 8). With the "light" fraction, two-thirds of the RNA was recovered in the extract (E. Reid, unpublished results). Moreover, the light fraction differs from the heavy fraction, and resembles the supernatant fraction, in its changes in the level of RNA and in the labelling of RNA after injection of [^'^Q-orotate in response to a change in hormonal status (Reid, 1956, 1961a). When fractions that are rich in RNA supposedly derived from membranes are prepared from fasting rats given [^^^cj.orotate, or ppj-orthophosphate the RNA labelling differs from that found for ribosome-rich fractions. For example, the labelling tends to be relatively high soon after orotate administra- tion (Reid, 1961a, and Petrovic et al., 1965; see also Fig. 2 in Bouvet and Moule, 1964), an observation compatible with the possibility that the RNA is of "messenger" type, as suggested by Pitot (1964). A similar result was 6. MEMBRANE SYSTEMS 365 obtained with fed rats given [i^CJ-adenine, but the converse was found with fasted rats (Hallinan and Munro, 1964), In the latter connection it is of interest that in the experiments of Petrovic et al. (1965) with labelled orotate, the turnover of membrane RNA was particularly high if the rats were fed rather than fasted. The RNA of membrane-rich fractions was found by Chauveau et al. (1962). to differ from the RNA of ribosomal or supernatant (cell sap) fractions in having a higher content of guanine and a lower content of uracil; but no such difference was encounted by Reid (1961a). Cytomembranes do, then, appear to contain RNA, values for which are given in Table IV; but little morphological change other than aggregation is evident after treatment in vitro with ribonuclease (Palade and Siekvitz, 1956; Tashiro and Ogura, 1957). However, little attack on this RNA by nucleases was found by Pitot (1964), who suggested that the RNA was a stabilized form of "messenger RNA". It may be of interest that in many ways the properties of RNA in the cytomembranes resemble those of membrane- bound mitochondrial RNA (see Chapter 3). {b) Various chemical constituents. Table IV gives phospholipid/protein ratios for membrane fractions isolated in apparently "intact" condition so that their morphology could be examined. It is generally agreed that micro- somal phospholipid is located in membranes rather than ribosomes, but the published values differ widely; perhaps there may have been inadvertent elution of protein from membrane fractions in some studies. The phos- pholipid/protein ratio was 0-08 for the "reticulosomes" of Pollak and Shorey (1964) [not tabulated; see III. b. .2a]. From experiments in which the non-ribosomal material of microsomal fractions was solubilized by use of agents such as deoxycholate [see III. B. A.b], it can be inferred that certain specific constituents are present in the membranes. These constituents include cytochrome b (cytochrome m) (Palade and Siekevitz, 1956; Ernster et al., 1962) — which, however, is only loosely linked to the membranes — coenzyme Q (Leonhauser et al., 1962), and a haem-protein termed Fcx (Nebert and Mason, 1964). There is evidence that one protein fraction of the membrane proteins is particularly rich in aspartic and glutamic acids (Cohn and Simson, 1963). {c) Enzymes. The biochemical evidence that microsomal enzymes are generally located in the membranes rather than in the ribosomes comes mainly from experiments in which the membranes were solubilized. There is the complication that some enzymes become unstable when, through use of an agent such as deoxycholate, the membranes are solubilized or are stripped of ribosomes, as in the "m fraction" of Ernster et al. (1962) (see also Siekevitz, 1962). Such instability is to be distinguished from that encountered for certain microsomal enzymes, e.g. demethylases (Leadbeater and Davies, 1964), when "intact" microsomal fractions are stored or incubated. In the list of enzymes in liver microsomal fractions (Table II), attention is 366 E. REID drawn to those enzymes for which there is evidence for location in the membranes. This evidence is based rather insecurely in some instances on results of analysis of different microsomal "cuts" (Table III). However the evidence is particularly good for glucose-6-phosphatase, NADHa-cyto- chrome c reductase and azo-dye reductase, which have been assayed in a membrane fraction obtained without use of deoxycholate by the procedure of Chauveau et al. (1962). The warning should be given that many experiments using deoxycholate without Mg2+ ions have been unaccompanied by electron-microscopy or even by RNA analyses, and do not show unequivocally that the constituent in question is membrane-linked. In comparison with treatment in the presence of Mg2+ ions, treatment in the presence of EDTA with a low concentration of deoxycholate was strikingly effective in solubilizing RNA, glucose-6- phosphatase, and 5 '-nucleotidase (El-Aaser, 1965). There is some evidence (e.g. Tashiro, 1958; Roth, 1960) that the relatively low "latent" ribonuclease activity of microsomal fractions [Morals and de Lamirande (1965), inter alia] is associated with ribosomes rather than with membranes, although Takanami (1959) found a light-microsomal fraction to be particularly active. Recent evidence points to a second ribonuclease and to a phosphodiesterase, apparently membrane-located (de Lamirande, Boileau and Morals, 1966). The energy-dependent incorporation of amino acids into microsomal protein [inter alia, Campbell et al. (1964) and Hallinan and Munro (1965)], and into serimi albumin (Decken and Campbell, 1961), is attributable to ribosomes rather than to membranes. Membrane-rich fractions obtained by a density-gradient procedure were able to incorporate phenylalanine into protein, particularly if polyuridylate were added ; but it was thought that contaminating ribosomes could be responsible for the activity (Campbell et al., 1964). Hendler (1965) argues that membrane-bound ribosomes are the sites of protein synthesis, both components being indis- pensable. At least membranes may have some auxiliary role (Campbell et al., 1964; Hendler, 1965); indeed Peters (1962) has shown that newly-formed albumin passes first to the rough-surfaced and thence to the smooth-surfaced membranes, and is secreted without ever being found in the supernatant fraction to a significant extent [also see Jungblut (1963) and, for pancreatic amylase synthesis, Redman, Siekevitz and Palade (1966)]. Pitot (1964) suggests that proteins destined to be secreted are actually synthesized on membranes, with participation of membrane-RNA as distinct from ribosomal RNA. As has already been indicated [see III. b, \.b], there may be differences between the enzyme patterns of different groups of membrane fragments, and in particular between smooth-surfaced and rough-surfaced elements. This may occur particularly in experimental situations (see IV.b.1 below). Pointers to such differences have come particularly from separations in high-density media as summarized in Table IV; but further study is needed before definite 6. MEMBRANE SYSTEMS 367 conclusions can be reached, even concerning glucose-6-phosphatase. From experiments with a zonal centrifuge (El-Aaser et al., 1966b and unpubUshed experiments) it appears that rough-surfaced and smooth-surfaced cyto- membrane elements are qualitatively, although not necessarily quantitatively, similar in enzymology, but that within the smooth-surfaced fraction there may be subtle differences in location, for example between glucose-6-phospha- tase and nucleoside diphosphatase assayed with UDP as substrate. One difficulty is that the actual membrane fragments arise not only from rough- and smooth-surfaced cytomembranes (including Golgi cytomembranes) but also from the plasma membrane. That certain "microsomal" enzymes such as 5 '-nucleotidase are largely attributable to plasma-membrane frag- ments seems certain from the cytochemical results surveyed in Section II, from the admittedly scanty biochemical evidence concerning plasma-mem- brane fractions (Table IV, and Section III. c. 1.), and from cytochemical studies of microsomal fractions in which 5'-nucleotidase has been shown to be in elements distinct from the vesicles containing glucose-6-phosphatase (see Section IV. a. 1.). For a different reason the results of Higashi and Peters (1963a) on the distribution of catalase (1.11.1.6) (Table IV) are in- conclusive ; here the observed activity may well have been in distinct organelles (microbodies, or uricosomes — see Fig. 2) that tend to sediment with cyto- membrane fragments (de Duve et al, 1962; See Appendix Chapter 3). 4. Solubilization and Activation of Microsomal Enzymes (a) Approach to the problem. Attempts to solubilize enzymes in the micro- somal-fraction have sometimes been prompted by a suspicion that the enzyme in question was present because of a re-distribution artifact, but more often by the hope that the enzyme could be purified — a hope so far seldom realized. Consideration may first be given to enzymes which are solubilized only with fairly drastic treatment, but which differ in their ease of solubilization. Such differences hardly establish that the enzymes are dif- ferently located among the diverse elements that are disclosed by morpho- logical studies. As Tashiro (1957) and other authors have recognized, en- zymes in membranes are presumably bound to lipid and resistant to solu- bilization, unless treatments are applied to remove the lipid or to break the bonds supposedly concerned in the linkage of lipid to protein (presumably hydrogen bonds and Van der Waals forces); the strength of the linkage is hardly likely to be the same for all enzymes. Siekevitz (1962) has pointed out some difficulties in the performance and interpretation of such experi- ments: "What are the criteria for solubility? When are enzymes soluble? Certainly not when they resist centrifugation in a specified gravitational field, for perhaps a higher field will cause them to sediment. I think that we can only say that a protein is soluble when it is completely surrounded by water molecules. And in too many cases when proteins have been termed 'soluble' there is really no good reason to think this might be so. . . . On the 368 E. REID Other hand, when we extract and really purify a protein, and measure certain parameters, like pH optima, reaction rates, equilibrium constants, substrate binding constants, we have no idea whether this same protein does have these characteristics when it is embedded within the cell." Apart from the question of solubility, Siekevitz (1962) and Ernster et al. (1962) have discussed the question of the activity of membrane-bound enzymes which may or may not be lipoproteins. The activity of such enzymes is profoundly influenced by their membranous location. Solubilization of a nucleoside diphosphatase was apparently preceded by activation (Ernster and Jones, 1962). It has been suggested that the bound form of glucose-6-phospha- tase differs from the free form in substrate affinity. For enzymes having non- polar substrates, concentration of the latter within membrane micelles may have an important effect on activity (Tashiro, 1957). (b) Deoxycholate. Biphasic effects on enzyme activity of increasing con- centrations of deoxycholate are shown in Table V. [No regard has been paid in Table V to differences between laboratories in the deoxycholate :protein ratio employed (cf. Moule et al., I960).] An irreversible fall in activity after treatment with deoxycholate in high concentration would of course be a stumbling-block to enzyme purification if this concentration were required to solubiUze the enzyme. The question of reversibility has seldom been checked, but certainly in one instance, NADH2-cytochrome c reductase, there may be restoration of activity on lowering the deoxycholate concentration (Ernster et al., 1962; Siekevitz, 1962). Beaufay and de Duve (1954) concluded that solubilization of glucose-6-phosphatase by deoxycholate renders the enzyme more susceptible to thermal denaturation, presumably because of instability of the lipid-free protein. The enzyme becomes more susceptible to destruction by deoxycholate itself when the microsomes are first exposed to bicarbonate buffer (Hultin, 1957). In view of the cytochemical evidence that ATPase and 5 '-nucleotidase are really located elsewhere than in cytomembranes, it is striking that the activity of the former enzyme is depressed even with the low concentration of deoxy- cholate that activates glucose-6-phosphatase. Also the activity of 5'-nucleo- tidase is only enhanced with a high concentration of deoxycholate which results in a reduction of glucose-6-phosphatase activity (Table V). [It is puzzling that Ernster and Jones (1962) found negligible 5 '-nucleotidase activity in the microsomal fraction for various authors have found high activity even without treatment with deoxycholate (Table II).] Although deoxycholate may cause instability, or at least inhibition (pos- sibly reversible), even at concentrations that do not solubilize enzymes [Ernster et al. (1962)], it is at present unsurpassed as a tool for solubilizing microsomal enzymes. The minimum concentration and amount required depend on the enzyme studied [see III. b. A.e]. Deoxycholate does not 6. MEMBRANE SYSTEMS 369 ^ ?\ "^ < T3 Pi 5 rj ta o A 60 c _o o ^ 6 fe o V o e N c u G ON w ^ ^ c i-! t^ 2 On a g pq 3 W -a ^ ^ ^cii^ 23 ^ 1 ( .« S ^ 1^ O O OON ^ O ^^ On c SJ 3 o) ~^ ;-C o cS hJ J 13 ^- "^ 4> X 1 ts; see e/ ai irnefelt ■!-> —> Ti c a 'c/5 c o similar e Muscat er (1963 ^ c ON > B gave alia) Dalln ■•*• c CO i^ < ^^ 00 C 60 C Pl^ W W U A _A. ^ r — 1 ^^~" ^"~' V— ' — 1 (U ^ /^^ cj U (D 2 « ^ o 0) V- "O ^ (U o H o 3 _c •^ 1) (u m !•= 1 u -a -i-i 3 T3 C^ a u ^ j2 (U •- 't 2m > c l^ o 60 •^ d o o o Z o c p 5o S QQ o -3 U 4> c c o o C/3 4> U (/5 "J 52 S2 c« o ^ ^ w iZ 3 SI Z o z O X! / / O 6 O^ ^^ ^N^ '^ 374 E. REID material, with the sucrose polymer Ficoll for the final gradient centrifugation. As they carefully explain, their approach entailed (1) disruption of the plasma membrane by a gas-cavitation procedure, into fragments sufficiently small to sediment with the microsomal rather than the nuclear material, (2) re- centrifugation of the microsomal pellet in a two-layer sucrose medium so that the smooth-surfaced vesicular elements floated, (3) "lysis" of the vesicular elements in a hypotonic medium, and (4) density-gradient centrifugation in Ficoll. The separation of the plasma-membrane elements from the other elements was based on differences in osmotic behaviour, although it is not clear from the paper why "lysed" vesicles should still, as is implied, act as semi-permeable sacs. No electron-microscope description of the final plasma-membrane fraction was given; the identification was based on biochemical criteria mentioned in (b) below. As is indicated in Table IV, Kamat and Wallach (1965) later modified their method: they claimed that with a suitable pH (8-6) and with divalent cations (Mg2+) present there was shrinkage and aggregation of cytomembrane fragments which would otherwise have sedimented with plasma membrane fragments. No such aggregation has been found with rat-liver fractions run in a zonal centrifuge (Table IV), although there were density changes. With microsomal rather than crude-nuclear starting material it is in fact difficult to separate plasma-membrane fragments from cytomembrane fragments in the case of liver ; nevertheless a vesicle-containing fraction low in glucose-6-phosphatase and rich in 5'-nucleotidase has now been obtained [unpublished experiments; cf. El-Aaser et al. (1966b) and Table IV]. {b) Biochemical characteristics. In the pioneer study of Neville (1960), no biochemical parameters were investigated. Herzenberg and Herzenberg (1961) found the expected high level of certain homograft antigens in their membrane fractions. With heterologous antibody preparations, Wallach and Kamat (1964) and Emmelot, Bendetti and Rumke (1964a) likewise found characteristic cell-surface antigens in their fractions, together with a Mg2+- activated ATPase dependent on Na+ and K+ (and sensitive to ouabain); indeed, these parameters were the criteria used by Wallach and Kamat in developing their isolation procedure. These authors, unlike Emmelot and co-workers, were unable to detect hexosamine in their fractions. It is un- fortunate that in the thorough survey made by Emmelot and co-workers of the biochemical characteristics of their fractions (Table IV) side-by-side assays on other cell fractions for sialic acid and for some of the enzymes studied were not reported. Emmelot and co-workers conclude, presumably taking into account past work in their laboratory, that plasma-membrane fractions are rich in certain enzymes such as 5'-nucleotidase, and contain small but significant amounts of other enzymes which are indicative of the presence of cytomembrane elements in their fractions, not necessarily as a "contaminant". Their results for ribonucleases, which showed definite 6. MEMBRANE SYSTEMS 375 activity particularly at an alkaline pH, are hard to assess, since the results of assays based on RNA breakdown are given in arbitrary values although they are vahd for comparative purposes. RNA itself seems to be virtually absent (Table IV). Observations by Peters (1962) preclude the possibility that the plasma membrane plays a role in the synthesis of protein, or at least of serum albumin. Plasma-membrane fractions typically have a high cholesterol/ phospholipid ratio (Coleman and Finean, 1965). In a comparison with whole liver, Skipski et al. (1965) found a relatively high content of free cholesterol (other lipids were also determined); their value for plasma-membrane phospholipid was 1 Img P/g protein. Emmelot et al. (1964b) subjected their plasma-membrane fractions to deoxycholate treatment, which enhanced the activities of certain enzymes such as phosphodiesterase (3.1.4.1) (measured at pH 8-9) and arylesterase, and to extraction with saline, whereby the "phospholipo-glycoprotein core" was stripped of antigenic proteins which are positively charged at a neutral pH but not of enzymes such as 5'-nucleotidase and ATPases. The activity of the latter, which — unlike that of 5'-nucleotidase — is altered after partial removal of the sialic acid by neuraminidase, may be governed by a protein- phospholipid orientation (Emmelot and Bos, 1965; cf. Green and Perdue, 1966). Emmelot and Bos (1966) also consider that glycolytic enzymes associ- ated with plasma-membrane fragments from normal liver are not true components of the plasma membrane, in disagreement with a view based largely on erythrocyte experiments (Green, Murer, Hultin, Richardson, Salmon, Brierly and Baum, 1965). (c) Indications for future work. The "rule-of-thumb" approach prevalent in past work on the isolation of plasma-membrane fractions is hardly ex- cusable in view of the ease with "marker" enzymes such as 5'-nucleotidase can be assayed. (The latter may, however, be partly extracellular in location.) Ideally it should not be necessary to use, at least in the initial stages of isola- tion, special conditions that preclude the simultaneous isolation of the con- ventional sub-cellular fractions, for example, exposure of the various organelles to an osmotically unfavourable environment. Exploitation of slight differences in density seems to be the best hope; the density values, of which estimates are given in Table IV, vary with different media. With conventional procedures for homogenization and differential centrifugation using 0-25 m sucrose as an isolation medium, followed by the "mitochondrial supernatant" being centrifuged in high-density media, the different types of vesicles in the microsomal material may prove to be resolvable without recourse to a hypotonic medium at any stage. This may also hold for the crude nuclear fraction, although there is the difficulty that the surviving plasma-membrane "sheets" seem to be rather similar in density to intact mitochondria and erythrocytes. Apparent disagreements between laboratories over density values for 376 E. REID plasma-membrane fragments (Table IV) could hinge not only on the medium, but also on the tissue and the conditions of homogenization. These conditions — although not (for a given Potter-type homogenizer) the number of strokes per se — may govern the distribution of plasma-membrane fragments between the nuclear and the microsomal fractions. Thus, with intestinal mucosa the use of a blendor tends to convert brush-border elements into vesicles which sediment partly with microsomal material (Hiibscher et al., 1965). However, since the vesicles which arise from the plasma membrane seem not to fall into a unique density class (the density being estimated from the position of 5'-nucleotidase activity after isopycnic centrifugation), some of the vesicles conceivably arise from particular regions (unconvoluted ?) of the plasma membrane, rather than indiscriminately from any region through over- drastic homogenization. Further cytochemical work, with due regard to isolated fractions, may elucidate the origin of vesicles in terms of biochemically specialized regions in the plasma membrane of a given cell. Hand-in-hand with such work, progress in the separation of different types of vesicles will come from increasing use of zonal rotors, which achieve large-scale yet sharp separations without the need for repeated centrifugations. 2. Golgi Cytomembrane Fractions The y-cytomembranes in the Golgi region are presumably recovered in the microsomal fraction, and in smooth-membrane fractions prepared therefrom. Epididymis is the only tissue from which a Golgi fraction has been successfully prepared (Schneider and Kuff, 1954). The fraction was rich in alkaUne phosphatase (3. 1 .3.1), as would be expected from cytochemical observations, and in RNA, which is unexpected. The concentration of phos- phoHpid was within the range found for other smooth-membrane fractions (Table IV). These results are best regarded as essentially a useful starting- point for future work. With liver, unfortunately, no unique enzjonic "marker" is available. 5. Non-Hepatic Microsomal Fractions (a) Approach to the problem. Differential centrifugation has been applied successfully to the isolation of mitochondrial and lysosomal fractions from tissues other than liver, but has so far given few definitive results for cytomembranes. The fact that the latter are scanty in some non-hepatic tissues has not been recognized by certain biochemists who have isolated a "microsomal" fraction but have failed to examine it under the electron- microscope. Indeed, a blind eye has often been turned to the serious hetero- geneity of cell type found histologically in most non-hepatic tissues. However, as will be evident from the following brief survey, a few biochemists have in fact performed thorough studies with due attention to histological and cytological aspects. In most tissues other than liver and kidney, glucose-6- 6. MEMBRANE SYSTEMS 377 phosphatase is unfortunately not present in adequate amount to serve as a cytomembrahe "marker" [guinea-pig, but not rat, intestine contains some (de Duve, 1964)] ; but phospholipid determinations can be a useful guide. With many non-hepatic tissues, notably muscle, there is the difficulty that use of a conventional homogenizer and of a sucrose medium may fail to give efficient cell breakage; one consequence is that a reliable "balance-sheet" of enzyme activities in the various sub-cellular fractions can hardly be drawn up. For skeletal muscle, the procedure of Kono and Colowick (1961) seems to be particularly reproducible; the product has awaited characterization by electron microscopy until recently (Table VI). Muscle preparations are liable to contain mitochondrial cristae (Siekevitz, 1965). (b) Isolation and characteristics. The data given in Table VI should be viewed in the light of the above cautionary remarks, and the original publica- tions should be consulted hopefully for details not given here. One par- ticularly common sin is vagueness about assay conditions, e.g. the presence or absence of Mg2+ ions, as in work on alkaline phosphatase. In summary, "microsomal" fractions have been empirically prepared from diverse tissues, and shown to be rich in enzymes such as alkaline phosphatase, "ATPase", acetylchohnesterase (3.1.1.7), and NADH2- or NADPH2-cytochrome c reductase; the differences from liver are quantitative rather than qualitative. In some experiments not in Table VI, Morton (1954a) isolated a microsomal fraction from buttermilk which, as judged from electron microscopy and enzyme assays, seemingly represented lipoprotein particles derived from the cytomembranes of the mammary-gland cells concerned in milk secretion. Few workers have verified that their "microsomal fraction" was rich in phospholipid. Some workers have verified that their fraction was rich in RNA (Table VI), but it would be pointless to cite values in view of the contamina- tion with ribosomes. Microsomal fractions from intestinal mucosa may show few ribosomes but may be rich in RNA, possibly membrane-located (Porteous and Clark, 1965). There is the complication that with tissues such as kidney (Reid, 1956) a fairly high proportion of the RNA is recovered in the super- natant rather than the microsomal fraction. The converse has been found for xanthine oxidase (1.2.3.2), which seems to be microsomal in mammary gland and milk (Morton, 1954a) and possibly in intestinal mucosa (Morton, 1954b), but is recovered in supernatants from liver (Reid, 1961b; de Duve et al, 1962). Increasing use is being made of electron microscopy, for example in con- nection with the fractionation of brain (Whittaker, 1963; Wolfe, 1964) and of ■■muscle (Muscatello, Anderson-Cedegren, Azzone and Decken, 1961), but Ktoo often the accompanying biochemical examination of the fractions has ^been inadequate in scope. It is a reasonable hope that membrane fractions well characterized both morphologically and biochemically, will soon become available even with intractable tissues such as muscle and that for the latter I 378 E. REID ^1 Is 5 s c Pi N C W uh o .2 2 Q^ 3 e %-( -o o 8 2 c c g _o (/] "3 o 13 C/9 c c« a bO Cj {/3 S ON -1 . _H ^3 "». Oca — rf *. So'" X C/3 X C^ a On 2 r- o\ Ov On Q « ^ ^ ^ N S 3 3 o o c (D Ui C h4 m O al Q O H ^2 a bo 4) "d 60 -r NO On > C ci u 60 C ^ ■^x NO On I «*- n.S o O "^ ^ 60 5 - «l 2 -eg .£ olS "^^ X! JIC U ... ■ o X Z3 V- t. 60 3 • a 3 ^ ^ o 2 ■M u ^ - ^ c"^ ^ t ^ «a "a On ^t(^ -a a I < 6. MEMBRANE SYSTEMS 379 as 6 "5 4> *■' U5 CS s §S c U c .a o J— ^-v ^ ^ ^ vo -S; H o x: a (/I o a 2 «> .S c x: "" x; o W ■!-> "^ ,« '^ H ^^ g< O O "^ ti X a c ^ 2 55 «* "O 4^ 2 - «> S-S + -js nI^ i — ^ _i 3 1 4) x> «2 ^ + ^ 5 "7 [-1 ftA. 60 + •^^ .£ 12 t« .9" o x: D. « u 1^ c ^ ON 0\ 0\ ^ (U J^ (J '^ 2j T3 O e bO o X! ^ <3 :s < ;SOh o c (U C/3 B5 t- >-i. x; ^ J << kd CQ *-* y o ^ X! 2 O CA CU U i o o ft «2 ll 1.S § a S c • E E .2 o Si o o o e E E 11 •§ '^ 380 E. REID as N a § o c ^ x; -^ a G -S O rt O — o g o J2 5^ «J 1 3 OS Xi s 1 ^ IH o (U On fe -d ^ "5 '^' a o^ ;3 Q '3 13 6 tt O O t-i Q> o ti 3 O T3 tZ3 ii S ^ 6. NfEMBRANE SYSTEMS 381 I Id c a o c o o ^ X o CO U Sic + «c t: ^ o ^ g I ^ o E 6 is I S u X) no O o o E o C/3 3 u) o oo 1 o 'C •d E 8 g u X) J: _ o -o ^ c bo c — a CL, 3 '£ (y2 H •5- I '5 382 E. REID tissue it will not be necessary to have recourse to the hypotonic conditions used by some workers, which yield "ghosts" that are possibly diverse in cytological origin. (c) Linkage of enzymes to membranes. In efforts to establish the localiza- tion and fastness of attachment of enzymes, techniques analogous to those employed for liver (Section III. b. and III. c.) have been applied to brain and certain other tissues. Thus, Schwartz, Bachelard and Mcllwain (1962) observed parallel behaviour for ATPase, acetylcholinesterase and cholesterol when various centrifugal procedures were applied to brain, and found apparent solubilization of the ATPase when the microsomal fraction was treated with deoxycholate, Lubrol W or digitonin. Further work, particularly on solubilization (Swanson, Bradford and Mcllwain, 1964; Bradford, Swanson and Gammack, 1964), led to the conclusion that "struc- tural factors are important in determining the behaviour of the Na+ ion- activated adenosine triphosphatase" ; with agents other than digitonin, "the relative proportions of different lipids solubilized were similar to those present in the original microsomes". For solubilization of the Mg2+-dependent alkaline phosphatase of mam- mary gland or intestinal mucosa, recourse must be had to agents such as proteolytic enzymes or «-butanol (Morton, 1954b). The jS-glucosidase (3.2.1.21) of intestinal microsomal fractions could be solubilized by use of trypsin or deoxycholate, but not by freezing and thawing or by exposure to water (Dahlqvist, 1961). With pancreatic microsomal fractions, in contrast to zymogen-granule fractions, solubilization of amylase, with release of "latent" activity, could not be achieved by use of Triton-X-100, but was effected by exposure to pH 6-7 buffer (Holtzer and van Lancker, 1963). D. MEMBRANE-CONTAINING FRACTIONS (INVERTEBRATE) 1. Micro-organisms For the study of amino-acid incorporation. Godson, Hunter and Butler (1961) [see also Hendler (1965)] exposed lysed protoplasts o^ Bacillus mega- therium to hypotonic buffers and obtained a centrifugal pellet, designated "membrane complex", which was apparently derived from the plasma membrane. Ultrasonic disintegration and re-centrifugation gave a ribosome- containing supernatant, together with a pellet the upper layer of which showed twisted, irregular filaments by electron microscopy and consisted of muco-protein; the lower layer consisted of phospholipid with virtually no RNA, and showed very small membranous vesicles. Enzyme studies on this "membrane complex" are needed, in view of the evidence given by Neu and Heppel (1964) that various degradative enzymes such as alkaline phosphatase and a 5'-nucleotidase are indeed present on 6. MEMBRANE SYSTEMS 383 bacterial plasma membranes but are readily detachable. For example, exposure of bacterial cells to tris buffer at pH 8 and then to water caused such detachment, leaving viable spheroblasts behind. It is arguable whether such enzymes, as are also found in yeast, can be validly regarded as membrane- located. However, the possibility remains that protoplast membranes are indeed the site in vivo of certain enzymes characteristic of membrane fractions [as documented by Wolfe (1964)]. These enzymes include an ATPase and various oxidases together with cytochromes. One reason for caution is that membrane fractions obtained in the early studies did contain measurable amounts of RNA, unlike the "membrane complex" prepared by Godson et al. (1961). Green et al. (1965) centrifuged a French-press homogenate of fermenting yeast cells, in a hypotonic medium, and obtained finally a pellet of "mem- branous nature" with a phospholipid :protein ratio of 0-2. Besides glycolytic enzymes, it contained nucleotide phosphorus in "substantial amounts". Their assumption that the material arose from the plasma membrane is a dubious one, and their conclusion (based also on erythrocyte experiments) that the plasma mambrane truly contains a complete "glycolytic complex" has been questioned (Emmelot and Bos, 1966). 2. Plant Tissues The scanty literature on membrane-containing (as distinct from ribosomal) fractions from plants was reviewed by Whaley et al. (1960) with particular reference to meristematic tissues. Beet-petiole and wheat-root microsomal fractions were studied by Martin and Morton (1956, 1957), who briefly discuss "evidence for the origin of the plant microsomes from a primitive type endoplasmic reticulum." The fractions were rich in certain cytochromes (particularly ^3), in NADH2-cytochrome reductases (assayed with cytochrome c, although possibly linked with bs in vivo), and in xanthine oxidase. In a careful study of pea-seedling root tips, with media containing 0-4 m or 0-5 M sucrose, Loenig (1961) recovered half of the RNA of the homogenate in a microsomal pellet which could be sharply sub-divided into "heavy" and "light" components. The former consisted mainly of ribosomes, whereas the latter contained vesicles together with some ribosomes and "dense bodies" and had a lower ratio (<0-8) of RNA to protein. The proteins of the "light" fractions had more complex electrophoretic patterns after ribonuclease treatment than those of the "heavy" fractions. A microsomal fraction isolated from a fungus {Neurospora crassa) by Shulman and Bonner (1962), showed membranous structures covered with ribosome-like particles in the electron microscope and was found to contain DNA. This was present as a "hybrid" with RNA and was perhaps nuclear in origin and fortuitously associated with the microsomal material. Neither this fraction nor those isolated by Loenig (1961) were examined for enzymes. 384 E. REID 3. Invertebrate Animal Tissues Except in a few studies centred on ribosomes and protein synthesis, such tissues have received Httle attention with respect to the biochemistry of microsomal fractions, Clayton's study of fractions from various tissues of a roach (Eurycotis floridan), prompted by an interest in sterol incorporation into membranes, did include analyses for constituents such as RNA, and points the way to future work (Clayton, 1965). IV. Correlations and Functional Aspects A. NORMAL TISSUES /. Biochemical Findings in Relation to Cytochemical Findings The membranous locations established cytochemically for phosphatases (Table I) broadly match those established biochemically (Tables II and VI), given that there is now much evidence to support the often expressed warning that fragments of the plasma membrane as well as of cytomembranes may be present in the microsomal fraction. As is evident from cytochemical examina- tion of hepatic microsomal fractions (Fig. 10), 5'-nucleotidase activity is associated with structures distinct from the vesicles showing glucose-6- phosphatase activity, that might have arisen from micro-villi. Similar obser- vations have been made with crude nuclear fractions, except that 5'-nucleo- tidase activity is found in sheet-like structures (Fig. 10c), the source of which awaits firm identification — there being possible artefacts in cytochemical work on isolated fractions (A. A. El-Aaser and M. S. C. Birbeck, unpublished work). For nucleoside di- and tri-phosphatases it cannot yet be asserted that biochemical findings fully agree with cytochemical findings : in zonal centri- fuge runs, ATPase and UTPase do not consistently run in the same positions as AMPase and ADPase, nor does UDPase consistently coincide with glucose-6-phosphatase — whereas the cytochemical literature suggests that ATPase is largely in the plasma membrane and that UDPase is largely in cytomembranes (El-Aaser et ai, 1966b). For most microsomal-fraction enzymes there is no independent informa- tion from cytochemical study of the whole tissue, mainly because of technical Fig. 10. Enzyme localization in sub-cellular fractions incubated with substrate after isolation. Courtesy of A. A. El-Aaser. Top left (a). Glucose-6-phosphatase, microsomal fraction: note staining of the vesicles, x 73,000. Top right (b). 5 '-Nucleotidase with UMP as substrate, microsomal fraction: note that staining is not in vesicles as in (a), but in elements conceivably derived from bile canaliculi x 73,000. Bottom (c). 5'-Nucleotidase, crude nuclear fraction: note that staining is in sheets which resemble plasma-membrane fragments as shown in Fig. 9 (although a derivation from fibrous tissue cannot be ruled out). X 23,000. ~'~^^^RP^ I^I^HHHj ^^ Cv^ >" 4fe ^ V • ■ ^ -1* • '^'*, *' -*l ^ «- % b 4» . ^*^ * . ^^^ -*v • w'^te*^ V> 386 E. REID difficulties. Conversely, as mentioned by Novikoff (1964), there may be cyto- chemical information but "difficulties in isolating the relevant cell structures", although in the case of liver such difficulties are being overcome. For aryl- esterase there is an unresolved conflict between the biochemical finding of a microsomal location and the cytochemical finding with the light-microscope of activity in organelles, possibly lysosomes, clustered near bile canaliculi (Underhay, Holt, Beaufay and de Duve, 1956). Conceivably there may be leakage from lysosomes in homogenates and subsequent adsorption on to microsomal material (P. C. Barrow and S. J. Holt, unpublished work). There may, however, be several distinct arylesterases (Lundkvist and Perl- mann, 1965), certain of which might be destroyed or inhibited by cyto- chemical procedures. This appears to be the case for mitochondrial ATPase. Plurality of enzymes is a problem often encountered in eff'orts to localize the site of a particular activity. For some of the enzymes listed as present in the microsomal fractions (Tables II and VI), the proportion of the total activity in the homogenate attributable to microsomal material is rather low; yet only in a few instances, for example catalase, is there reason to suspect that the microsomal activity may be due to elements other than membrane fragments. As suggested for j8-glucoronidase (3.2.1.31) (de Duve et al, 1955), it is likely that there is a subtle difference between the nature of the enzyme in the microsomal fraction and that elsewhere. Microsomal fractions as prepared in some laboratories contain a high proportion of the lysosome population; but for the sake of clarity, reports of the presence in microsomal fractions of enzymes now known to be lysosomal (e.g. acid phosphatase) have been disregarded in the compilation of Tables II and VI. These tables have been drawn up emphasizing the enzyme rather than the cell structure. Novikoff (1964) has given a useful summary from the viewpoint of locations (Table VII). It should be noted that this list represents an amalgamation of cytochemical with some biochemical observations, but excludes most of the observations derived purely from biochemical studies. One such omission is NADH2- cytochrome c reductase; cytochemical observations by light microscopy on diaphorase activity are compatible with, but hardly reinforce, the biochemical evidence for a cytomembrane location of this enzyme (Chang, 1960; Novikoff, 1960). Apart from any metabolic function that it may have, the Golgi com- plex may serve "to stock products elaborated elsewhere in the cell" (Rouiller, 1964) or, in connection with this, "to pump in or out the ingredients neces- sary to segregate or concentrate the final substance" (Haguenau, 1958). The above list does not touch on the question of possible enzymic differences between smooth-surfaced cytomembranes (other than Golgi) and rough- surfaced cytomembranes. For glucose-6-phosphatase, cytochemistry has disclosed no difference (Tice and Barrnett, 1962) and the biochemical evidence is contradictory [Tables III and IV; see Section III. a. 1.c\. Smooth-surfaced 6. MEMBRANE SYSTEMS 387 Table VII. Plasma Membrane Nucleoside phosphatases. Variable in different tissues: nucleoside tri- phosphatases (Mg++-stimulated; Mg++, Na+, K+-stimulated, ouabain- sensitive); Co++-stimulated nucleoside monophosphatase ; Mg++-stimu- lated nucleoside monophosphatase; etc. Alkaline phosphatase. Present in only a few cell types. Endoplasmic Reticulum Nucleoside diphosphatase. Hydrolyses disphosphates of guanosine, uridine and inosine. Present in liver, kidney, steroid-producing cells, and other cells in which glucuronyl transferase activity has been demonstrated. In these cells the endoplasmic reticulum also shows low levels of thiamine pyrophosphatase. Glucose-6-phosphatase, esterase, and acid phosphatase are present in the endoplasmic reticulum of some tissues. Golgi Apparatus Golgi saccules : Nucleoside diphosphatase (diphosphates of guanosine, uridine, and inosine are hydrolysed more rapidly than those of adenine and cytosine) ; thiamine pyrophosphatase ; acid phosphatase (in some saccules) is being demonstrated in increasing number of cells; alkaline phosphatase is found in intestinal mucosa. Golgi vesicles : Acid phosphatase is being demonstrated in an increasing number of cells. cytomembranes appear to be concerned particularly "in hydroxylation and elimination of lipid-soluble drugs, and in cholesterol and steroid hormone metabolism" (Jones and Fawcett, 1966). 2. Functional Aspects of Membrane-bound Enzymes (a) Energy supply. Shull (1959) stressed the need to examine microsomal fractions for dehydrogenases lending to the reduction of NAD ; but present- day evidence (Table II) indicates that NADPH2 is more important than NADH2 in cytomembrane enzymology. Various authors have commented on the apparent paradox that cytochrome c is lacking in microsomal fractions although NADPH2-cytochrome c reductase and particularly NADH2- cytochrome c reductase are present. Phillips and Langdon (1962) suggest that the latter enzyme acts in vivo merely as a mediator of electron transport, without participation by cytochrome c, in certain hydroxylations and reduc- tions that require NADPH2. There is the complication that microsomal fractions contain a diaphorase effective with either NADH2 or NADPH2 but ineffective if cytochrome c is used (Ernster et ai, 1962; Orrenius. Ericsson and Ernster, 1965). 388 E. REID There is still no reason to believe that cytomembranes make a large con- tribution to the energy supply of the cell, even by electron transfer through cytochrome bs instead of cytochrome c. The 65 pathway may lead to "ion flux" rather than to ATP formation [Siekevitz, 1965; for this and other pathways see also Strittmatter (1965) and Omura et al. (1965)]. The re- oxidation of NADH2 is considered to be mediated by substrate shuttles, in particular the "glycerophosphate shuttle" with mitochondrial participation, and that of NADPH2 by mechanisms such as the pentose-phosphate cycle, involving soluble enzymes. Nevertheless, the tendency for mitochondria to be closely surrounded by cytomembranes (Fig. 2) points to some kind of co-operation in energy metabolism; perhaps mitochondria supply cytomembranes with cytochrome c, and this is lost by leakage when microsomal fractions are isolated, a situation which would explain the above paradox. There is no evidence that cytomembranes are concerned in the synthesis as distinct from the storage of glycogen, or in glycogenolysis ; morphological findings (Porter and Bruni, 1959) are inconclusive (Jones and Fawcett, 1966). The fact that glucose-6-phosphatase — the determinant of supply of glucose to the blood stream — is located in cytomembranes is of uncertain significance. As yet there is no evidence for the possibility that glucose formed by the enzyme may leave the cell by way of cisternae which lead to the exterior, rather than by way of the extra-membranous cytoplasm or cell sap. The membranous location of the enzyme — a lipoprotein — may offer the possibility of control of enzyme activity through subtle changes in the configuration of the enzyme protein itself or of contiguous macromolecules ; such changes, perhaps analogous to those effected by agents such as deoxycholate, might well be induced or suppressed by specific hormones as a means of regulating glucose output (cf. Siekevitz, 1962). This line of thought is essentially a facet of the concept that one role of cellular membranes is to limit and regulate the interaction between enzyme and substrate; for the cytomembranes under discussion, it may be a matter of the membrane serving to accommodate within itself a lipoprotein enzyme, rather than to bar access to a soluble enzyme within the cisternal space. Moreover, within or adjoining the mem- brane there might be a pH very different from that in the cell sap but particu- larly appropriate for the action of certain membrane-located enzymes. (b) Transport. The preceding speculation that the membranous location of glucose-6-phosphatase may enable glucose to be channelled out of the cell, comes into the realm of transport. A further possibility, likewise unsub- stantiated although often mooted, is that the entry of glucose into the cell is mediated by a hexokinase situated in the plasma membrane — a possibility which is by no means disproved by the biochemical evidence that most of the hexokinase activity of the liver cell is not membrane-linked. The prevailing view concerning active transport of monosaccharides across membranes is r 6. MEMBRANE SYSTEMS 389 that the process requires the mediation of a "carrier" which is possibly mobile and the nature of which is unknown (Quastel, 1964). More generally, "It is becoming increasingly evident that cell membranes are equipped with substances that play specific roles in conveying organic molecules from the cell exterior to the cell interior or from one compartment in the cell to another. These substances play roles as important as those of the enzymes in the control of cell growth, metabolism, and function" (Quastel, 1964). The transport of K+ and Na+ ions, a process particularly important in nerve function, has received especial attention (Conway, 1960). The picture is still unclear, but the "ATPase" which shows optimal activity in the presence of Mg2+, K+ and Na+ seems to be implicated and does not act hydrolytically (Jarnefelt, 1964). "Labile phosphate" is involved. "ATP is involved in the process of transport though the involvement is probably an indirect one, which is reflected in the rapid and parallel turnover of phosphate in certain types of phospholipid and of a phosphoprotein fraction. Whether these two phenomena involve a precursor which is common also to a transport carrier or whether either is the actual carrier, is presently a matter of speculation, experimentation, and a challenge to the imagination" (Schole- field, 1964; see also Wolfe, 1964). As Whittam (1964) points out, "Membranes are spatially asymmetrical in selecting Na+ for outward transport and K^ for inward transport, and one kind of ion movement does not generally occur without the other. . . . Perhaps the simplest working hypothesis for the mechanism of action of the ATPase is to regard Na+ and K+ as enzymatic co-factors which must be in the right location to exert their effects. . . ." Given that "ATPase" is concerned in active transport and that the cyto- plasm differs sharply from the extra-cellular space and the nucleus in ionic composition, it is curious that in parenchymal cells the activity is highest in the micro-villi of bile canaliculi. Usually cytochemists have found poor activity in the smooth portions of the plasma membrane and especially in cytomembranes including the nuclear envelope, although under some con- ditions, for example with formaldehyde fixation, ATPase activity is demon- strable in cytomembranes (Wachstein and Fernandez, 1964; A. A. El-Aaser disagrees, from unpublished work). Although there is a vast hterature on "ATPase", especially on its biochemical aspects, continued and more subtle study is needed. The observed ATP-splitting activity reflects a more complex process or processes, concerned not only with transport but also ■M^ith the contraction and relaxation of myofibrils, mitochondria, and perhaps " membrane systems in general. One "ATPase" seems to be phospholipid- f"^ "pendent (Tanaka and Strickland, 1965; see also Emmelot and Bos, 1965). The transport of Ca2+ seems to be mechanistically similar to that of Na+ artonosi and Feretos, 1964). With regard to water movement, at present ieems likely that the passage of water molecules across membranes is a 390 E. REID field, 1964), In the transport of phosphate the Golgi elements might play a role by virtue of their high alkaline phosphatase activity, but proof is so far lacking (Goldfischer et ah, 1964). It should be stressed that substances not carried by active transport are either excluded by membranes, a function no less important than active transport, or go through by "passive transport". There is a further but unproved possibility that substances may pass between the cell exterior and the nuclear envelope via cisternal channels (cf. Section II. b.) without ever passing across membranes. It should also be emphasized that "active trans- port", as briefly surveyed above, can hardly be considered as a self-contained topic. Electron-transport as mentioned under {b) may be concerned in active transport, possibly ATP-dependent (cf. Ernster et al., 1962; Ernster and Jones, 1962), and processes such as bile formation, now to be touched on, may entail active transport. (c) Conjugation processes; bile secretion; nucleoside mono- and di-phospha- tases. These items are considered together because there are certain correla- tions between them, although the reason for this is as yet unknown. For different tissues there is a parallelism between the occurrence of nucleoside diphosphatase in membranes and that of UDP glucuronyl transferase (2.4.1.17) in microsomal fractions (Goldfischer et al., 1964). (The authors fail to draw attention to the different techniques for study of the enzymes.) That microsomal enzymes are important in furnishing bile constituents is indicated not only by their ability to convert bilirubin into its glucuronide, but also by their capacity to build the steroid skeleton (Table III ; see also Siperstein and Fagan, 1964) and to convert cholic acid into its CoA derivative and thence into glyco- or tauro-cholic acid (Table II). Another intriguing observation is that the concentration of deoxycholate that activates 5'-nucleotidase (cf. Table IV) is of the same order as the con- centration of deoxycholate in rat bile (Ku and Wang, 1963). Since the enzyme is located in bile canaliculi, its activity in vivo may thus be under biliary control. This line of thought seems more profitable than the suggestion by de Verdier and Potter (see Novikoff", 1960) that, particularly in tumours, the the enzyme serves to furnish nucleic-acid precursors, in the form of nucleo- sides, to neighbouring cells, the plasma membrane being impermeable to nucleotides. Other lines of thought have been developed by Ernster and Jones (1962). "The nucleoside diphosphatase may serve a function in promoting reactions involving the corresponding triphosphates as reactants by shifting the equilibrium toward the formation of the diphosphate. It is conceivable that the nucleoside diphosphatase present in the endoplasmic canals may have such a function in removing GDP formed in the GTP-dependent incorpora- tion of amino acids and/or in removing UDP formed in the varios uridine nucleotide-linked conjugation reactions taking place in the membranes. The 6. MEMBRANE SYSTEMS 391 GMP and UMP so formed may then leave the membranes and be trans- formed into the corresponding triphosphatase [sic] by way of the nucleoside monophosphate kinase system." They further speculate, because of the microsomal hydrolysis of ID? and an early observation by Siekevitz that bound inosine is present in microsomal fractions, that inosine nucleotides may have a function in microsomal activities. There is no experimental support for the assumption that the "endoplasmic canals" are the site of nucleoside diphosphatase activity. (d) Various biochemical processes. From observations mentioned earlier (e.g. in Table II), and also collated by Ernster et al. (1962), Rouiller (1964) and Moule (1964), it appears that microsomal, possibly cytomembrane, elements are concerned in ascorbic acid synthesis, in drug metabolism, for example, NADPH2-dependent hydroxylations, in the reductive stages of fatty acid and steroid synthesis, and in lipid peroxidation — a process that is perhaps "an important factor in the regulation of cytoplasmic structure and function" (Hochstein and Ernster, 1964). Mason, North and Vanneste (1965) have reviewed, with attention to the role of "Fcx", the complex topic of microsomal "mixed-function oxidases", concerned in the metabolism of fatty acids, steroids and foreign compounds. The hydrophobic character of the membranes might serve to provide the requisite milieu for reactions concerned with lipids; Jones and Fawcett (1966) have emphasized the role of the smooth-surfaced elements. These are only some of the more important of the diverse processes catalysed by microsomal elements. The interpretation of the esterase activity, notably the acetylcholinesterase, of microsomal fractions is as yet uncertain. Relaxation phenomena in skeletal muscle appear to depend on a transphory- lating system as studied in microsomal fractions by Molnar and Lorand (1962). It should be pointed out that, in general, cytomembrane-1 inked pro- cesses do not require a supply of ATP. There is already some evidence that cytomembranes, and possibly phospho- lipids, play a role in protein synthesis distinct from and possibly secondary to that of ribosomes [Godson et al., 1961 (bacteria); Higashi and Peters, 1963a, 1963b (liver); Redman et al., 1966 (pancreas); for other citations see Moule, 1964, and Hendler, 1965]. The postulate of Barbieri and di Marco (1963) that there is actual synthesis de novo of protein in smooth membranes has been criticized by Hallinan and Munro (1965). Pitot (1964) postulates that membranous structures serve to stabilize and store the messenger-RNA templates required for the synthesis of proteins that are to be secreted. Physiological Changes in Morphology and Biochemical Pattern. (a) Development and renewal. Our knowledge of the primary origin of jllular membranes has advanced little since, in the context of rough-surfaced ^tomembranes, Haguenau concluded in 1958 that there was insufficient 392 E. REID evidence to decide whether the rough-surfaced cytomembranes were formed from the nucleus, mitochondria or the plasma membrane. Essner and Novikoff (1962) interpreted their cytochemical observations on diphosphatases and other enzymes as indicating that normally there is transformation of smooth-surfaced cytomembranes into Golgi cytomem- branes and thence into primary lysosomes or secretory vacuoles (see also Goldfischer et al., 1964); enzymes such as acid phosphatases, formed on ribosomes initially, were envisaged as accompanying the membranous ele- ments through these transformations. The essence of this concept is the reasonable supposition that the cytomembranes are normally in a state of flux. More speculatively, Essner and Novikoff (1962) suggest that cytomem- branes may originate from the nuclear membrane, In kidney there is evidence that the brush border is the source of the phagosomes that appear in response to foreign protein injections (Straus, 1962). However, our knowledge con- cerning interconversions of membranous structures has really advanced little since Tashiro (1957) put forward an elaborate hypothesis concerning the "reversible microsome system", The subject of the relationship between cell membranes and lyosomes is also considered in Chapter 5. There is evidence, summarized by Moule (1964), that during cell differen- tiation, for example in the hepatocyte, there is development of cytomem- branes and ensuing attachment of ribosomes to the latter. Experiments with chick liver led Pollak and Shorey (1964) to conclude that the protein as distinct from the phospholipid moiety of cytomembranes is already present in early development stages, supposedly in the form of granules which they called "reticulosomes". In common with various other enzymes, some membrane-linked enzymes are low in foetal liver and show a dramatic rise after birth. Such observations have been made for alkaline phosphatase and glucose-6-phosphatase. The rise is not dramatic in the case of 5'-nucleotidase, nucleoside triphospha- tase (El-Aaser, 1965), and enzymes concerned in the oxidative metabohsm of drugs (Pouts, 1963). The rise in glucose-6-phosphatase has been found with kidney (in the guinea-pig but not in the rat) and intestinal mucosa as well as with liver (Lea and Walker, 1964). For certain electron-transport enzymes there is "the peculiarity that the enzymes do not seem to be all synthesized at the same time" (Siekevitz, 1965). In regenerating liver, cytomembrane development follows the cell hyper- trophy (Moule, 1964), but the yield of the smooth-membrane fraction actually drops initially (Delhumeau de Ongay, Moule and Frayssinet, 1965). Rough- surfaced cytomembranes reappear as clumps surrounding the mitochondria (Haguenau, 1958). There are differences among enzymes in the rate and extent of the changes in concentration. There is often a rise in enzyme activity (Reid, 1962), but the few biochemical observations of membrane- linked enzymes show no rise. Indeed, both glucose-6-phosphatase and 6. MEMBRANE SYSTEMS 393 5'-nucleotidase were found by El-Aaser (1965) to decrease after partial hepatectomy (for earlier work see Weber, Singhal, Stamm, Fisher and Mentendiek, 1964); moreover, Fouts (1963) found a fall in drug-metabolizing enzymes. Cytochemically, "ATPase activity is increased in the tortuous canaliculi" (Wachstein, 1963). For 5 '-nucleotidase in lactation, see Wang (1962). In Loenig's (1961) study of root-tip microsomal fractions, the yield of the "light" fraction increased rapidly at the onset of differentiation, as a reflection of cytomembrane formation and perhaps, at a later stage, of plasma-mem- brane development. For further information on the wide topic of membranes in development, Locke (1964) may be consulted (see also Burch, 1965). (b) Fasting and dietary changes. Fawcett (1955) and other authors have observed that with prolonged fasting there is a loss of hepatic cytomembranes, which may be preceded by aggregation of the smooth-surfaced elements to give "hyaline inclusions", and which can be reversed by re-feeding. Accom- panying the active protein synthesis during re-feeding, the region adjoining the nuclear envelope becomes filled with rough-surfaced cytomembranes enmeshing mitochondria [Bernhard and Rouiller, cited by Haguenau (1958)]. Mention has already been made in Section III b. 3 (a) of reported changes in the incorporation of precursors into non-ribosomal species of RNA, possibly located in the membrane. There is as yet no evidence for a change in the amount of RNA as distinct from changes in the rate of labelling of this RNA, despite a remark by Hallinan and Munro (1964) that the actual amount is influenced by dietary protein. The pattern of deoxycholate-soluble proteins may change with overnight fasting, but apparently becomes normal again if the fast is prolonged (Hultin, 1957). Glucose-6-phosphatase is a classical example of an enzyme that is, under- standably, sensitive to dietary conditions in respect of the level of activity [Weber et al. (1964), inter alia]. Although prolonged fasting depresses the content of glucose-6-phosphatase in cytomembranes, as represented by deoxy- cholate-soluble material, there is a rise in the normally low content of enzyme in the ribosomes, as if transfer of new enzyme to cytomembranes had become arrested (Busch, Weill and Mandel, 1960). However, no change in distribution is detectable cytochemically (Tice and Barrnett, 1962). Prolonged fasting also depresses the oxidative (but not the reductive) metabohsm of drugs (Fouts, 1963), whereas 5'-nucleotidase is little affected (Terroine, 1961). In a biochemical and electron-microscope study of guinea-pig pancreas, Palade and Siekevitz (1958a) found that fasting caused changes such as disappearance of intraci sternal zymogen granules and a fall in microsomal- fraction ribonuclease. With a deficiency of essential fatty acids, the incorporation of phosphate ^P) into the lipids of brain microsomal fractions, with or without supple- I 394 E. REID mentation with acetylcholine, shows changes which, according to de Pury and CoUins (1962), point to a weakening of lipoprotein membranes. With cysteine feeding, hepatic microsomal fractions showed decreases in RNA and phospholipid and increases in cytochrome reductases; moreover, tissue sections showed disorganization of the rough-surfaced cytomembranes and an increase in smooth-surfaced cytomembranes (Emmelot, Mizrahi, Naccarato and Benedetti, 1962). Cholesterol feeding causes "feed-back inhibition" of the synthesis of mevalonic acid and hence of cholesterol (Siperstein and Pagan, 1964). (c) Hormonal influences. One of the few instances of morphological changes in cytomembranes with a change in hormonal status is an observation by E. G. Ball and collaborators (see Tepperman and Tepperman, 1960), that treatment of adipose-tissue cells with insulin in vitro results in the formation of a system of canaliculi and vesicles suggestive of a stimulation of pino- cytosis. Certain hormonal treatments may alter the level or the labelling of microsomal RNA, sometimes with a difference in this respect between "heavy" and "light" microsomal fractions (Reid, 1961a); but there is no reason to believe that membrane-located RNA as distinct from ribosomal RNA is affected. There are scattered reports [cited by Tepperman and Tepperman (1960), Reid (1962), and El-Aaser (1965), inter alia] concerning hormonal effects on membrane-linked enzyme activities. For example, hyperthyroid liver may show elevated activities of glucuronyl transferase and of glucose-6-phos- phatase but not of 5'-nucleotidase, whereas with adrenalectomy or hypo- physectomy there may be a decrease in glucose-6-phosphatase and also in the ATPase of microsomal fractions. After the rise in glucose-6-phosphatase activity caused by thyroxine, the stimulatory action of deoxycholate is still demonstrable (El-Aaser, 1965). Similarly, with the rise accompanying experimental diabetes the stimulatory effect of trypsin (see III b. 4) can still be seen (Heise and Gorlich, 1963). The rise with diabetes (or with corticoid treatment) truly seems attributable to synthesis of new enzyme protein (Weber et ai, 1964). For certain enzymes of drug metabolism a rise may be induced by anabolic steroids (Gilette, 1963), but with alloxan diabetes the oxidative drug-metabolizing enzymes are depressed (Fouts, 1963). In adrenal tissue, the proportion of the glucose-6-phosphatase activity recovered in the microsomal fraction falls after ACTH treatment, the nuclear and mitochondrial fractions becoming richer in this enzyme (Breuer, Petershof and Knoppen, 1963). The converse is found for 5'-nucleotidase (Hilf, Breuer and Borman, 1961). In the absence of morphological and cytochemical information however, these observations are difficult to interpret. As pointed out by Emmelot et al. (1964a) (see also Leaf, 1960), there is evidence for activation of Na+ transport by thyroid-stimulating hormones and neurohypophyseal hormones, this activation being perhaps due to 6. MEMBRANE SYSTEMS 395 hormone interaction with -SH groups in ATPase. Attempts have been made to explain the actions of insulin and of ACTH in terms of transport changes (Tepperman and Tepperman, 1960). Future work may well establish hor- monal effects not mediated by a direct hormone-enzyme interaction: ". . . do hormones act upon a higher order of the cell, upon the framework in which the enzymes are enmeshed?" (Siekevitz, 1962). B . PATHOLOGICAL TISSUES 1. Effects of Toxins, Carcinogens and Drugs To summarize a vast body of ultrastructural, cytochemical and biochemical observations (cf. Rouiller, 1964, and Reid, 1965 ; see also Novikoff and Essner, 1962, and Jones and Fawcett, 1966), there is no early action of carcinogens on hepatic membrane systems that cannot likewise be produced by non-carcino- genic toxins, and there is virtually no action of the latter that cannot Hkewise be produced by "physiological" treatment such as fasting. "One may infer that the dedifferentiation, frequently occurring in tumours, is not specific for the cancerous process" (Moule, 1964). It is, nevertheless, striking that for hepatocarcinogens such as azo dyes or dimethylnitrosamine (but perhaps not ethionine), and hepatotoxins such as carbon tetrachloride, the cytomembranes "seem to be the first site of detectable morphological lesions of the hepatic cell" [Rouiller 1964; see also Haguenau (1958)]. The morphological changes may include detachment of ribosomes from rough-surfaced cytomembranes, and swelling, aggregation or other alterations in the actual cytomembranes, with a randomization of their distribution in the cytoplasm. There may also be damage to the plasma membrane, at least if necrosis sets in ; this damage could cause leakage which might account for observations such as decreased activities of esterases (Rouiller, 1964). Such leakage of cytomembranous elements, from mammary-gland secretory cells, has been thought to occur even during normal lactation (Morton, 1954a). It is, however, unlikely that such leakage could be the sole cause of hepatoxin-induced enzyme decreases such as the fall in glucose-6-phosphatase mentioned by Feuer et al. (1965). In no hepatoma is there the normal hepatic complement of cytomembranes, yet certain hepatomas have an almost normal level of glucose-6-phosphatase (Pitot, 1962; Reid, 1965), and even of NADH2-cytochrome c reductase (Novikoff, 1960). There may be faster synthesis of cholesterol in slices and loss of "feedback inhibition" (Siperstein and Fagan, 1964); but drug- metaboUzing enzymes are commonly depressed (Fouts, 1963). Biochemical results of analyses for ATPase and 5 '-nucleotidase in precancerous or cancer- ous liver are difficult to interpret because these enzymes are not confined to the membranes of parenchymal cells ; but the cytochemical evidence, which distinguishes the activity in sites such as connective tissues and blood-vessel I 396 E. REID walls, indicates that in many hepatomas the actual "parenchymal" cells have rather low activity, at least for nucleoside di- and tri-phosphatase as distinct from 5'-nucleotidase (inter alia Chang, 1960; Novikoff, 1960; Essner and Novikoff, 1962; Goldfischer et al, 1964; El-Aaser, 1965). On the other hand, a notably high level of alkahne phosphatase has been found in plasma- membrane fractions of hepatomas by Emmelot, Benedetti and Riimke (1964a), who further observed a very low level of esterase in the hepatoma material, and an abnormal antigen pattern. Many, but not all, hepatomas are deficient in actual bile canaliculi (Essner and Novikoff, 1962; Novikoff and Essner 1962; Goldfischer et al, 1964). Even with only 3 weeks of azo-dye feeding, a depletion is cytochemically demonstrable in the ATPase and 5'- nucleotidase of bile canaliculi, whereas alkaline phosphatase is increased (Novikoff and Essner, 1962). Emmelot and Bos (1965, 1966) later concluded that a Na+- and K+-insensitive ATPase in hepatoma plasma-membrane fragments is more sensitive to neuraminidase than is the liver enzyme, and that in hepatomas, unlike liver, the plasma membrane truly contains hexo- kinase. At least it would appear that, with the more subtle techniques now becom- ing available, membrane systems warrant continued study as possible primary sites for the actions of carcinogens and toxins. Cytomembranes have long been thought of as major sites for the "binding" of carcinogens such as azo dyes {inter alia Hultin, 1957; Westrop and Green, 1960; Rouiller, 1964). There is likely to be a revival of interest in cytomembranes, although the current emphasis is on possible derangements in ribosomal function. Pitot (1964) puts forward the suggestion that "chemical carcinogens would be expected to randomly alter the membrane of the ER [endoplasmic reti- culum], leading to random changes in template stability, resulting in a wide variation of molecular and biologic expressions of neoplasia." Among observations which seen unconnected with the intriguing topic of template stability, one which warrants further investigation is that "Fcx", possibly a haem-protein, was deficient in a microsomal fraction of a hepatoma (Nebert and Mason, 1964; cf. Mason et ah, 1965). Little work has been done on effects of administered detergents. Injection of Triton WR-1339 caused dilation of Golgi saccules but otherwise had little effect on cj^omembrane morphology (Novikoff and Essner, 1962). The effect of injected phenobarbital on hepatic cytomembrane morphology and on microsomal drug-metabolizing enzymes has been studied in the laboratories of Remmer (cf. Remmer, 1962), of Ernster (Orrenius et al., 1965) and of Fouts (Pouts and Rogers, 1965; cf. Hart and Fonts, 1965), with grati- fying agreement on the nature of the derangement. It is the smooth-surfaced cytomembranes that preferentially show proliferation and a rise in enzyme content, the rise being attributable to faster synthesis of the enzyme proteins. This synthesis, and that of the actual membrane proteins, perhaps occurs in 6. MEMBRANE SYSTEMS 397 contiguous rough-surfaced cytomembranes (Jones and Fawcett, 1966). The effect was equally striking with the drug chlordane, but not with benz- pyrene or methylcholanthrene (Fonts and Rogers, 1965; Hart and Rogers, 1965). Since, however, the liver is not the target for the actions of these various agents, the results throw little light on their mode of action. Other microsomal changes observed with phenobarbital or chlordane include rises in total protein, in NADPH2 oxidase, and in CO-binding pigment (Hart and Fouts, 1965). For this pigment, and for NADPHg-cytochrome c reductase, the rise after phenobarbital again occurs more in the smooth- than in the rough-surfaced fractions, although the fractions have an equivalent content in untreated animals (Orrenius et al., 1965; see also Jones and Fawcett, 1966). Phenobarbital also causes a marked rise in cytochrome 65 and glucose-6- phosphatase, but not in NADP nucleosidase or in ATPase — even in the smooth-surfaced fraction; this fraction shows a sharp rise in phospholipid but not in RNA (Remmer and Merker, 1963). r 2. Effects of Bile- Duct Ligation There are striking changes with bile-duct ligation, although Novikoff and Essner (1962) emphasize that these may not be of primary importance in the pathogenesis of jaundice. One such change is a complete loss of 5'-nucIeo- tidase activity from many of the bile canaliculi, together with morphological derangement in the latter. There is also a marked fall in ATPase, the fall being reversed if the bilary obstruction is released (Wachstein, 1963). Cyto- membrane enzymes concerned in the oxidative metabolism of drugs are likewise depressed (Fouts, 1963), although there is hypertrophy of smooth- surfaced cytomembranes (see Jones and Fawcett, 1966). In toxin-induced liver damage, loss of canalicular 5'-nucleotidase activity is a late effect, and is less conspicuous than loss of ATPase (Wachstein, 1963)]. Certain other changes, such as an increase in the alkaline phosphatase activity of sinosoidal surfaces, are at sites remote from the bile canaliculi. V. Summary Consideration has been given above to the nature of membrane systems, to methods for their study in situ, for their isolation, and to problems of nomenclature. Exception was taken to the terms "cell membrane", "micro- somes", and "nucleoside phosphatase". The following systems have been surveyed, with emphasis on hepatic cells : the plasma membrane, cytomem- branes (of rough- and smooth-surfaced types, the latter including the Golgi complex), and the nuclear envelope, which on morphological and cyto- chemical grounds can be regarded as part of the cytomembrane system. _ Cytochemical work with the electron microscope, so far confined to 398 E. REID contain gIucose-6-phosphatase and nucleoside diphosphatase, assayed with GDP, IDP or UDP as substrate ; that the Golgi complex contains thiamine pyrophosphatase and sometimes alkaline phosphatase but no glucose-6- phosphatase, and that the plasma membrane, especially where it folds to form micro-villi, contains a nucleoside monophosphatase (5 '-nucleotidase) together with a nucleoside triphosphatase, although the latter activity may also be demonstrable in cytomembranes and mitochondria. The question of artifacts has been briefly considered. Biochemical work has given results compatible with the cytochemical findings, assuming that plasma-membrane fragments are recovered in the microsomal fraction and in the crude nuclear fraction and account for the 5'-nucleotidase activity of these fractions. Processes which, from biochemical study of microsomal fractions, are largely attributable to cytomembranes include steps in lipid synthesis, ascorbate formation, deacetylation of acetyl- choline, NADPH2-dependent hydroxylation of aromatic compounds, and glucuronide formation. In general, microsomal fractions have low levels of enzymes requiring NAD or ATP as a reactant; they can catalyse the re-oxidation of NADH2 and NADPH2, but cytomembranes can hardly be regarded as important in energy supply, unless a supposition is made that there is transfer of cytochrome c from mitochondria to adjoining cytomembranes in situ. Cytomembranes seem to be concerned in glycogen storage, in the late steps of protein synthesis and in transfer of the protein to other sites. Questions yet to be decided include the location of esterase(s), the metabolic role of nucleoside mono- and di-phosphatases, and the extent to which smooth-surfaced cytomembranes, other than Golgi membranes, and rough- surfaced cytomembranes differ in their enzymology. Progress has been made in the isolation of membrane fragments corresponding to individual types of cytomembrane and to the plasma membrane. There is considerable variability among laboratories in the reported values for phospholipid and RNA in membrane fragments with a low content of ribosomes. There are indications that cytomembranes truly contain some RNA, different from that in the ribosomes; but the plasma membrane is low in RNA. In work with microsomal fractions from tissues other than liver, there has seldom been conjoint biochemical and electron-microscopic examination; there may be differences from liver with respect to the sub-cellular distribu- tion of some constituents (e.g. RNA and xanthine oxidase), but the findings for most microsomal constituents (e.g. acetylcholinesterase) agree with those for liver. Some enzymes found in membrane fractions are so readily detached that their presence there may be a re-distribution artifact. Other enzymes, such as glucose-6-phosphatase, appear to be tightly-bound lipoproteins; progressive damage to the membrane by an agent such as deoxycholate may first cause 6. MEMBRANE SYSTEMS 399 activation, but may be ineffective in achieving solubilization without loss of activity. One function of membrane lipids may be to promote ingress and metabolism of lipid substrates. Aspects of the selective permeability of membranes have been considered. Nucleoside triphosphatase(s) in membranes seem to be important in the "active transport" of cations. Possibly, however, some substances may travel between the exterior of the cell and the interior of the nuclear envelope by way of the cisternal spaces in the cytomembranes, without ever traversing a membrane. Altogether it appears that membranes, and in particular cytomembranes, have diverse functions, and may themselves be in a state of flux; there is no evidence that would justify use of the term "cytoskeleton". Mention has been made of the appearance, or disappearance, and behaviour of cyto- membranes and cytomembrane enzymes in situations such as cell develop- ment, fasting and re-feeding, changes in hormonal status, exposure to toxins and carcinogens, and bile-duct ligation. Damage to cytomembranes might well be an important concomitant property of carcinogenesis. Acknowledgements Valuable comments on the manuscript were received from Mr. M. S. C. Birbeck and Dr. Y. 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Yamada, K., Ishikawa, S. and Spimazono, N. (1959). Biochim. biophys. Acta 32, 253. t I Chapter 7 III. IV. V. VI. VII. VIII. RIBOSOMAL ENZYMESt DAVID ELSON The Weizmann Institute of Science Rehovoth, Israel I. Introduction .... II. The Structure of the Ribosome . A. The Isolation of Ribosomes B. Properties of Ribosomes . C. Ribosomal RNA . D. Ribosomal Proteins E. The Structure of the Ribosome F. Non-bacterial Ribosomes The Function of the Ribosome . Ribosomal Enzymes — General Considerations Ribosomal Enzymes — Enzymes of Protein Synthesis Ribosomal Enzymes — Nascent Enzymes A. Pancreatic Enzymes and Zymogens . B. Other Studies of Nascent Enzymes . C. Summary ...... Ribosomal Enzymes — Ribonuclease (2. 7. 7. -) A. Pancreatic Ribosomal RNase . B. E. coli Ribosomal RNase C. Biological Function of RNase . Ribosomal Enzymes — Other Enzymes which Act on A. Deoxyribonuclease (3. 1. 4. -) . B. DNA Polymerase (2. 1.1.1) . C. Polyribonucleotide Polymerases (2. 7. 7. -) D. Aminoacyl-sRNA Synthetases (6. 1 . 1 . -) . E. RNA Methylases F. Polynucleotide Phosphorylase (2. 7. 7. 8) . G. Phosphodiesterases (3. 1. 4. 1) . H. The Binding to Ribosomes of Enzymes which Act on nucleotides . IX. Ribosomal Enzymes — Other Examples A. Phosphatases (3. 1.3.-) . B. Peptidases , . 408 , , . 410 . 410 . , 411 , 413 413 , • 415 416 , 417 , 421 426 , 429 , , 431 , , 432 , , 437 , , 438 439 . 440 , , 447 Polynucleotide; i 450 450 ^ ^ 453 • • • 454 454 • • ■ 454 455 • • • 455 .h Act on Poly • • • 455 456 • 456 t This review was written during the tenure of research grants (GM 12588) from the U.S. Public Health Service, and (GB-1163) from the U.S. National Science Foundation. 407 408 DAVID ELSON C. NAD Pyrophosphorylase (2. 7. 7. 1) . . . . . 457 D. Adenosine Triphosphatase (3. 6. 1. 3) .... 460 E. Other Enzymes Found in Ribosome Preparations . . 460 X. Summary 461 References 468 I. Introduction It has been hardly more than ten years since several broad lines of research, cytological, cytochemical, and biochemical, converged to give us our present concept of the ribosome and its place in the economy of the cell. The earlier cytological studies were culminated by the simultaneous and independent recognition by Brachet (1942, 1960) and Caspersson (1941) that the capacity of a cell to synthesize proteins was directly related to a substance with a high affinity for basic dyes, the basophilia, and that the basophilic material was RNA. This first pronouncement of the involvement of RNA in protein biosynthesis became a guiding principle of fundamental importance, setting the course of research for investigators in many disciplines. At about this time Claude's discovery of the RNA-rich microsome (one of the most valuable artifacts of modern biology) opened a series of investi- gations by himself. Porter, Palade, Siekevitz and many others (see, e.g., Palade, 1958; Hogeboom and Schneider, 1955; Dounce, 1955) which led, through the separate or combined use of the then new techniques of cell fractionation and electron microscopy, to the development of a new cytology at a higher level of resolution. One consequence was the visualization of the endoplasmic reticulum, a cytoplasmic network of tubules and vesicles formed of lipid-containing membranes and found in a great variety of differentiated cells. It was later seen that portions of the reticulum were often covered with dense particles and that these particles were also found free in the cytoplasm both of such cells and of cells which contained no endoplasmic reticulum. (Fig. 1). These particles were shown to be made up of RNA and protein and were eventually given the name of "ribosome" (Roberts, 1958). Microsomes were recognized as heterodisperse fragments of the endoplasmic reticulum, formed when the cell was broken and consisting of two elements : pinched off segments of the reticular membrane, and ribosomes. When the membrane is dissolved (usually with sodium deoxycholate), pure ribosomes may be obtained. It is these particles which constitute the basophilic substance concerned with the synthesis of proteins, and they are located in both the nucleus and the cytoplasm. In the meantime, similar particles had been isolated from many forms of living matter, and rapid progress was made in elucidating their physical, chemical and biological properties (McQuillen, 1962; Petermann, 1964). 7. RIBOSOMAL ENZYMES 409 mm II I II I — b.ifii tjwi»g// ribosomes negatively stained with phosphotungstic acid and magnified x 133,000. The ribosomes are almost entirely in the form of 70 S and 100 S particles. By courtesy of Prof. D. Danon and Mr. Y. Marikovsky. Reproduced with permission from Elson (1964 c). 7. RIBOSOMAL ENZYMES 413 100 S particles would present such a uniform appearance if this were not so (Fig. 3). It is clear, of course, that the internal environment of the living cell is not identical to that of a test tube; and it is not yet clear to what extent the association-dissociation reactions observed in vitro mirror processes of physi- ological significance in vivo. The 70 S ribosome is the particle which partici- pates directly in the synthesis of proteins. However, all of the degrees of association mentioned above have also been observed in untreated whole cell homogenates, and a correlation has been noted between the physiological state of cells and the state of association of their ribosomes (McCarthy, 1960). It is possible, therefore, that the association-dissocation reactions of ribo- somes are of biological significance. It should be remembered that the particles discussed here associate by direct interaction with each other. They should not be confused with another type of ribosomal aggregate, the polysome, which is distinctly different in nature. In the polysome, which will be discussed below, a number of 70 S particles are held together not because they interact with each other but because they are apparently attached to the same strand of RNA, presumably a strand of mRNA. The specific interactions that the ribosomes undergo in these association reactions and in the precise and reversible binding of molecules during the process of protein synthesis (see section III) must be a function of the chemical fine structure of the ribosomal surface. As yet, we know nothing of this and, indeed, we do not know very much about the gross chemical structure of the ribosome. What is known is summarized briefly in the following two sections. (For general reviews see McQuillen, 1962; Roberts, Britten and McCarthy, 1963; Petermann, 1964). C. RIBOSOMAL RNA The 30 S ribosome contains a single RNA chain of molecular weight 0-5- 0-6 X 10^ (1500-1800 nucleotides). The 50 S particle contains about twice as much RNA, in some cases as a single continuous strand and in others as two strands of equal length, although it is not completely certain that these two strands are not produced by the breakage of a single strand during its isolation (Kurland, 1960; Petermann, 1964). The RNA moieties of the. 30 S and 50 S ribosomes are distinct chemical entities; neither is derived from the other. They have been reported to differ slightly in gross nucleotide compo- sition (Bolton, 1959a, cf. Roberts, 1964, p. 368) and definite differences have been discerned in their nucleotide sequences (Aronson, 1962), which appear to be prescribed by different genes (Yankofsky and Spiegelman, 1963). r D. RIBOSOMAL PROTEINS Considerably less is known about the proteins of the ribosome. To a large 414 DAVlD ELSON degree this is due to their insolubility in aqueous media under conditions where their properties can be easily investigated. When the ribosomal RNA is degraded at neutral pH and low ionic strength, much of the ribosomal protein precipitates and cannot be wholly redissolved except in such incon- venient solvents as acid, alkali or concentrated urea. Although this is annoy- ing to the chemist, it seems not unlikely that an understanding of this par- ticular behaviour may in itself contribute to an understanding of the structure of the native ribosome. Ribosomal proteins have been prepared in varying yield and purity (not always described) by extracting ribosomes with hot trichloroacetic acid (Crampton and Petermann, 1959) or cold 66% acetic acid (Waller and Harris, 1961; Spahr, 1962), dialysing them against urea solutions (Spahr, 1962) or concentrated neutral salt (Spitnik-Elson, 1962a) in order to allow an endo- genous RNase to degrade the ribosomal RNA, or extracting them with LiCl (Curry and Hersh, 1962; Mathias and Williamson, 1964) or LiCl and urea (Leboy, Cox and Flaks, 1964; Spitnik-Elson, 1965). The number of different amino end groups in such preparations is small, with methionine (and, in E. coli, also alanine) predominating (Waller and Harris, 1961 ; Waller, 1963, 1964; Mathias and Williamson, 1964; Petermann, 1964). The proteins appear to consist of relatively small molecules, and estimates of their molecular weight have ranged from 13,000 to 25,000, based on sedimentation data (Zillig, Krone and Albers, 1959; Curry and Hersh, 1962) and end group analyses (Waller and Harris, 1961; Mathias and Williamson, 1964). Amino acid analyses show the ribosomal proteins of different organisms to be similar in composition (e.g. Ts'o et al., 1958, summarized in Petermann, 1964). They contain large quantities of both basic and acidic amino acids. Since, however, a substantial proportion of the acidic amino acids are amidated, the proteins are predominantly basic. They appear to be somewhat less basic than the histones, which they resemble, but it is not yet clear to what extent these preparations contain proteins other than the true structural proteins of the ribosome. Consequently, a comparison of the basicities of the the histones and the ribosomal structural proteins cannot yet be made with certainty. Although the ribosomal proteins are mainly basic, acidic molecules are also present. This was predicted on the basis of their solubility properties and later demonstrated by fractionation studies (Spitnik-Elson, 1962a, b, 1963, 1964). Acidic components have also been shown by gel electrophoresis and other techniques (Waller, 1964; Cox and Flaks, 1964; Leboy et al., 1964; Duerre, 1964a). Comprising a small part of the total proteins (Spitnik-Elson, 1964), the acidic fraction can interact with the basic proteins to form in- soluble electrostatic complexes which are difficult to dissociate. It is, however, becoming increasingly apparent that the insolubility of the 7. RIBOSOMAL ENZYMES 415 ribosomal proteins cannot be explained on the sole basis of electrostatic interactions (Petermann, 1964). Anomalies in the behaviour of the proteins during ion exchange chromatography and starch gel electrophoresis show that other forces are also operating; the acidic fraction, in particular, appears to exhibit a high degree of non-electrostatic aggregation (Spitnik-Elson, 1964, 1965; Duerre, 1964a). The 30 S and 50 S ribosomes apparently contain different protein comple- ments (Spahr, 1962; Waller, 1964; Cox and Flaks, 1964). There is general agreement that the ribosomal proteins are heterogeneous (see the preceding references, and Waller and Harris, 1961; Spitnik-Elson, 1962b, 1963, 1964; Duerre, 1964a), but the degree of heterogeneity is in doubt. Several investi- gators have demonstrated a very large number of protein bands in gel electrophoresis — so large, in fact, that it approaches the number of protein molecules in a ribosome (about 40 in a 70 S particle). Thus it is conceivable that no two protein molecules in one ribosome are the same. However, it must still be shown that this multiplicity of bands is not an artifact. A small number of aggregating proteins could, for example, give rise to a larger number of separable aggregates. Further, ribosomal protein preparations have been found, when examined with this in mind, to contain small amounts of RNA and non-RNA phosphorus (e.g., Spitnik-Elson, 1965). These might contribute different charges to different molecules of the same protein species and might also act as electrostatic cross-linking agents. Thus we really do not yet know how many different species of structural ribosomal protein there are. In summary, the ribosomal proteins are small, and heterogeneous to an unknown degree. They are mainly basic but also contain acidic components. Once freed of the ribosomal RNA, they show a striking tendency to interact, both electrostatically and non-electrostatically. It seems not unreasonable to suspect that such interactions may play a decisive role in determining the structure of the intact ribosome. E. THE STRUCTURE OF THE RIBOSOME It was once felt that ribosomes might be similar to viruses in structure, but this is not the case. Both types of nucleoprotein have in common that they are made up of a long strand of nucleic acid and a number of relatively small protein molecules. In the virus, however, the nucleic acid forms an inner core which is insulated from the surrounding medium by an envelope of protein. In contrast, the ribosomal RNA is not covered in this way. Both the RNA and the protein of intact ribosomes are vulnerable to enzymic attack (Bolton, Hoyer and Ritter, 1958; Elson, 1959a, b; Petermann and Hamilton, 1961). Either both RNA and protein lie on the surface of the [particle, or else the structure of the ribosome is open enough to allow ^enzymes to penetrate. In either case, the structure of the ribosome is clearly [different from that of viruses. 416 DAVID ELSON We see, then, that the ribosome is a polynucleotide chain to which are attached a number of molecules of structural protein. That this attachment is independent of the three dimensional structure of the ribosome is suggested by recent experiments with ribosomes that had been exposed to a high concentration of salt. This treatment seems to loosen the structure of the particle, and when such ribosomes were suspended in distilled water, they showed the properties of a flexible linear polyelectrolyte. The ribosome had lost its structure and become an extended chain of RNA; however, the ribosomal proteins were still attached to it (Spirin, Kiselev, Shakulov and Bogdanov, 1963). The compact native particle would appear, then, to be formed by the coiling and folding of the chain, the exact structure being determined by specific protein-protein, RNA-RNA, and protein-RNA inter- actions. The marked tendency of the ribosomal proteins to aggregate in solution may be a reflection of such interactions, with the difference that in the intact ribosome the interactions are limited and controlled in such a way as to produce, together with the RNA, a specific structure. It will be seen in the following section that the ribosome probably functions by forming a surface which presents a unique pattern of precisely aligned chemical groups. It can reasonably be assumed that the entire chemical makeup of the ribosome is adapted to this end. In this view the length and nucleotide sequence of the ribosomal RNA, the arrangement of the ribosomal proteins along the RNA strand, and the composition, sequence and folding of each ribosomal protein would be such as to ensure that these structural elements will interact specifically with each other to form a particle of unique structure and surface. This is simply to say that the forces which determine the structure of the ribosome are those which are believed to operate in other biological macromolecules such as proteins and DNA. F. NON-BACTERIAL RIBOSOMES This discussion has been based mainly on the ribosomes of E. coli. The ribosomes of most other bacteria seem to be very similar in almost all respects (Petermann, 1964). However, virtually all non-bacterial ribosomes differ from the bacterial particles in containing a somewhat larger strand of RNA and in having a higher protein content (about 50%, compared with about 40% for E. coli). Since the non-bacterial particles are larger, they have higher sedimentation constants: about 40 S, 60 S, 80 S and 120 S instead of the corresponding bacterial values of 30 S, 50 S, 70 S and 100 S. The particle weight of the 80 S ribosome is about 4-4-5 x 10^ as compared with about 3 X 10^ for the equivalent bacterial 70 S particle. Except for this difference in size, non-bacterial ribosomes appear to be built in the same way as their bacterial counterparts. Under the same external influences they undergo the same reversible association-dissociation reactions. 7. RIBOSOMAL ENZYMES 417 but even interchangeable, for there are many reports of mixed systems which are perfectly capable of effecting the incorporation of amino acids into polypeptides in vitro. Thus, bacterial ribosomes are capable of functioning in conjunction with non-bacterial soluble components and vice versa. In what Ibllows I shall consider bacterial and non-bacterial ribosomes to be similar II structure and identical in function, and I shall not always distinguish ^tween them, id III. The Function of the Ribosome As noted above, the ribosome appears to have the sole function of pre- siding over the polymerization of amino acids into protein chains of precisely predetermined amino acid sequence. The involvement of the ribosome in this process was firmly established about a decade ago by the decisive experiments of Zamecnik and his colleagues (Littlefield, Keller, Gross and Zamecnik, 1955; Zamecnik, 1960), who followed the career of radioactive amino acids within the rat liver cell in vivo and showed by means of cell fractionation studies, that amino acids are first built into polypeptides while they are associated with ribosomes (Fig. 4). Many of the details of the process have since been worked out, particularly with the aid of cell-free systems which ac- complish the incorporation of amino acids into polypeptides in vitro. This work is still in full progress today, but enough is already known to allow the construction of a coherent scheme with some measure of confidence that it is not too different from what actually takes place in nature (see, e.g., Crick, 1964). In synthesizing protein molecules the cell employs an elaborate apparatus. The amino acids are first converted to an activated state; they are then fastened to carriers, brought to the site of polymerization (the ribosome), polymerized, and released into the cell medium. Each step is a chemical reaction catalysed by one or more specific enzymes. The early steps require different enzymes for the different amino acids, and since some twenty amino acids are involved, the number of enzymes participating is large. In addition, there must be some provision to ensure that the various amino acids enter the protein chain in the required sequence. In considering this aspect, it is convenient to divide the steps into two groups, selective and non-selective. I shall use these terms in the following sense: a selective reaction is one which plays a direct role in determining the unique amino acid sequence of a protein ; a non-selective reaction does not. In other words, both the selective and non-selective reactions are required for the polymeri- zation of amino acids, but only the selective reactions determine that only one particular amino acid will occupy a given position in the protein chain II 418 DAVID ELSON 40 Deoxycholafe soluble Deoxycholafe insoluble Soluble protein of cell 25 700 600 500 ^ 400 e 300 200 Deoxycholafe _, soluble Deoxycholafe insoluble Soluble protein of eel 5 10 15 Time (min) (b) Fig. 4. The incorporation in vivo of leucine-^''C into different cytoplasmic fractions of the rat liver cell: the ribosomes ("deoxycholate insoluble"), the non-ribosomal components of the microsomes ("deoxycholate soluble"), and the soluble proteins. The RNA content (as weight per cent) of the ribosomal samples is shown. A small dose of radioactive amino acid was administered in a and a large dose in b. In both cases the ribosome-bound proteins are labelled most rapidly, after which the label passes into the other cell fractions. Repro- duced with permission from Littlefield et al. (1955). 7. RIBOSOMAL ENZYMES 419 and nineteen others will not. In general, selectivity is a function of the nucleotide sequence of RNA, which is derived ultimately from the nucleotide sequence of the genetic material, DNA. Where an enzyme plays a selective role, it must recognize, whether directly or indirectly, a specific nucleotide sequence and relate this sequence to a specific amino acid. Before continuing with this matter, let us return to review the biochemical reactions of protein synthesis in more detail. Figure 5 is an outline of the process. The first step is the activation of the amino acid through the enzymic formation of an anhydride bond between its carboxyl group and the phos- phoryl group of the AMP moiety of ATP (Fig. 5, step 1). The same enzyme, ATP + HOOC— AAi— NH2 AMP— OOC— AAi— NH2 + PP + sRNAi sRNAi— OOC— AAi— NH2 + AMP mRNA + Ribosome Ribosome-sRNAi— OOC— AAi— NH2 mRNA + SRNA2— OOC— AA2— NHa Ribosome-sRNA^— OOC— AA2— NHCO— AAi— NH2 + sRNAi mRNA + SRNA3— OOC— AA3— NH2 Ribosome-sRNA3— OOC— AA3— NHCO— AA2— NHCO— AAi— NH2 + sRNA^ 6. mRNA + sRNA»— OOC— AA»— NH2 Ribosome-sRNA"— OOC— AA"— NH CO— AA^— NH2 + sRNA»-i HOOC— AA«— NH CO— AAi— NH2 + sRNA" + Ribosome (Protein) Fio. 5. An outline of the biochemical steps involved in protein biosynthesis. apparently, then transfers the amino acid residue to a small molecule of RNA, the "soluble" or "transfer" RNA (sRNA) (Fig. 5, step 2). The attach- ment is again through the carboxyl group, which is esterified to the terminal ribose moiety of the sRNA. There are many different species of sRNA — probably fifty or more (since at least some of the 20 amino acids involved 420 DAVID ELSON can be attached to more than one species of sRNA) — and each is reserved for one particular amino acid. The specificity of the responsible enzyme is such that it will act on only one species of amino acid and will attach it only to one of the sRNA molecules specific for that amino acid. This is one of the selective reactions: the pairing of specific amino acid and sRNA molecules. Out of the pool of amino acid-charged sRNA molecules, one is now fixed to a site on the surface of the ribosome (Fig. 5, step 3). In the rabbit reticu- locyte, at least, the binding appears to be catalysed by an enzyme (Arlinghaus, Shaeffer and Schweet, 1964). This too, is a selective reaction, for at any given time only one predetermined species of amino acid can be bound through its specific sRNA carrier. Logically, we must assume that the binding enzyme is responsible only for the mechanics of attachment, but not for the selection of the specific amino acid. The selection is believed to be governed by a molecule of mRNA which is attached to the ribosome. The mRNA is a complementary copy of the DNA of that gene which specifies the amino acid sequence of the protein in question. Its length is believed to consist of a series of codons (presumably adjacent trinucleotide sequences) which move across the face of the ribosome in linear succession. There are 64 different possible codons, and each sRNA molecule is assumed to have, in a specific part of the molecule, a trinucleotide sequence (the anticodon) which is complementary to one of the codons and which no other species of sRNA has in that position. It is postulated that only that species of sRNA can be bound whose anticodon is complementary to the mRNA codon which happens to be in register on the ribosome at the moment (Crick, 1958, 1964). The nature of the binding of sRNA to the ribosome is not known. How- ever, it involves more than codon-anticodon interaction since there is a binding site on the 50 S ribosome (Elson, 1961a, 1962a, b, 1964a; Gilbert, 1963b) and the mRNA appears to be attached to the 30 S ribosome (Okamoto and Takanami, 1963). Further, the binding is probably not covalent, for the sRNA can be detached merely by changing the ionic strength or Mg2+ con- centration of the medium (Gilbert, 1963b; Elson, 1964a). As each new molecule of sRNA becomes attached to the ribosome, the carboxyl group of the amino acid or polypeptide already present is trans- ferred from its ester linkage with the preceding sRNA molecule to the free amino group of the incoming amino acid, forming a peptide bond. By this enzymic reaction (e.g., Traut and Monro, 1964; Arlinghaus et al., 1964) the growing protein chain is handed from one molecule of sRNA to the next, being lengthened by one amino acid each time and always being bound to the ribosome by the sRNA molecule to which it is attached at the moment (Takanami, 1962; Gilbert, 1963b; Gierer, 1964). After a molecule of sRNA has given over the growing protein to its successor, it becomes detached from the ribosome. The process is shown in Fig. 5 for the first (step 3), second 7. RIBOSOMAL ENZYMES 421 (step 4), third (step 5) and last (step 6) residues of a protein composed of n amino acids. Finally, the completed protein must be released from both the ribosome and the last molecule of sRNA (step 7), a reaction about which nothing is yet known except that it may require an enzyme (Morris, 1964). I have given the above description in terms of a single ribosome interacting with a molecule of mRNA. It has, however, become apparent that proteins are usually synthesized by groups of ribosomes attached to the same molecule of mRNA and called, variously, polysomes, polyribosomes, ergosomes, clusters, etc. (Wettstein, Staehelin and Noll, 1963; Gierer, 1963; Gilbert, 1963a; Warner, Knopf and Rich, 1963; Marks, Burka and Schlessinger, 1962). It is believed that each ribosome becomes attached to the starting end of the mRNA and then moves along the successive codons. When it has gone far enough, a second ribosome is able to attach to the starting end, and so forth. All of the ribosomes in any one polysome would therefore turn out copies of the same protein, since they are being instructed by the same series of codons. The length of the mRNA would set an upper limit to the number of ribosomes in the polysome. There is no apparent reason to believe that proteins must be made on polysomes. A single ribosome should function regardless of whether other ribosomes are accompanying it (Munro, Jackson and Korner, 1964). It may, however, be important in the economy of the cell to make the most efficient use of the mRNA. Electron micrographs of polysomes are shown in Fig. 6. The scheme outlined here is the product both of much experimentation and much invention. It has gained general, though not universal, acceptance and it probably bears a substantial resemblance to what actually takes place in the living cell. In going through this scheme it will be seen that selectivity is not found in the ribosome. The high degree of selectivity required for the repeated manufacture of proteins of unique sequence resides in the activating enzymes which pair amino acids with molecules of sRNA in a unique way, and in the nucleotide sequences of the codons and anticodons of mRNA and sRNA. In this scheme the ribosome cannot exercise selectivity, for it must process all species of amino acid and sRNA in the same way. Apparently, its function is to provide a surface on which the reactions take place. This surface must be capable of binding and releasing mRNA, sRNA, and a number of enzymes and cofactors in a precise steric arrangement and temporal sequence. The elucidation of its structure remains as one of the more intriguing unsolved problems in biological chemistry. IV. RiBosoMAL Enzymes — General Considerations A ribosomal enzyme would presumably be one which has a functional connection >yith the ribosome. In this review, however, 1 shall include any 422 DAVID ELSON enzyme whose activity has been found in a preparation of ribosomes. This definition, broad to the point of absurdity, is nevertheless the most suitable for the present purpose at the present time. Since it says nothing about the purity of the preparation (except that it be a ribosomal and not, for example, a microsomal preparation), it will clearly include a number of enzymes which are simply impurities. On the other hand, our understanding of the various kinds of relationship that might exist between enzymes and ribosomes is still rudimentary. Thus, although a more restricted definition might be more Fig. 6. (a) Polysomes in situ. A thin section of a rabbit reticulocyte fixed with glutaraldehyde and osmium tetroxide and stained with uranyl acetate. The magnification is X 66,000. In addition to many single ribosomes, clusters of two, three, four and five ribosomes can be seen; these are cross sections of polysomes. Electron micrograph by courtesy of Prof. D. Danon. Reproduced with permission from Elson (1964 c). 7. RIBOSOMAL ENZYMES 423 •o t35 -SCO «3 .2 >>■(/) ui-l a 3 60 C 2 c^ o E 2 g S-i 13 D. c •£ SO O • T3 424 DAVID ELSON Fig. 6. (c) Polysomes in situ. A thin section of an epidermal cell of a seedling radish root fixed with glutaraldehyde and osmium tetroxide and stained with urany! acetate and lead citrate. The magnification is x 37,000. Reproduced with permission from Bonnett and Newcomb (1965). meaningful, it would also be unjustifiably arbitrary and would not cover a number of cases that, for one reason or another, should be covered. The problem stems to a large extent from the structure of the ribosome and its place in the organization of the cell. For example, it is far less difficult, in 7. RIBOSOMAL ENZYMES 425 principle if not always in practice, to define a mitochondrial enzyme, for this cell organelle is bounded by a membrane. Thus, an enzyme inside the mem- brane would be a mitochondrial enzyme, and one which is outside would not. Further, the location of an enzyme within the membrane is of itself a good reason for suspecting it to play a physiological role in the organelle. In practice it may sometimes be difficult to distinguish between an interior enzyme and one adsorbed on the surface of the membrane, and it may also prove necessary to stretch the definition in order to accommodate certain cases; but in any event, it is possible to employ a single broad definition as a starting point. The ribosome, however, is not bounded by a membrane and its principal metabolic activities appear to be carried out on its surface. This surface is in contact with the cytoplasm or nucleoplasm, and the soluble components of the surrounding medium have free access to it. The ribosome has been shown to adsorb many substances very effectively, including proteins (e.g., Petermann, 1964). In particular, since its RNA moiety has a high density of negative changes, it has a high affinity for basic proteins; and it has re- peatedly been demonstrated that when ribosomes and basic enzymes are mixed in vitro, ribosome-enzyme complexes are formed (e.g., Petermann, 1964; Petermann and Hamilton, 1961; Keller and Cohen, 1961; Keller, Cohen and Wade, 1963; Keck and Choules, 1962; Madison and Dickman, 1963a; Siekevitz, 1963; Neu and Heppel, 1964c). There is no question but that such artifacts may be formed when the cell is broken in order to isolate its components and such compartmentalization as may have existed is destroyed. Thus, its mere location on the ribosome is not proof that an enzyme is a true ribosomal enzyme. This is a serious and constantly present problem. On the other hand, since the ribosomal function in the cell probably in- volves the attachment and detachment of certain enzymes, the adsorption so often seen in vitro might reflect a phenomenon which is biologically important in vivo. Particularly striking are several examples in which the adsorption exhibits a high specificity of location, namely, that the enzyme in question is bound to either the 30 S ribosome or the 50 S ribosome, but not to both (see ATPase (3.6.1.3.), RNase (2.7.7.-) and cysteinyl-glycine dipeptidase (3.4.3.5.) below). In short, the problem is not only to decide whether adsorption may have taken place, but also to ascertain whether this adsorption points to a mere accident of chemical affinity or to an interaction of genuine physiological significance. We may consider the same problem, but in the reverse sense. The physical absence of an enzyme from the ribosome is no proof that the enzyme has no functional connection with the ribosome. It is quite conceivable that an enzyme whose site of action is the ribosomal surface might still perform this u 426 DAVID ELSON action during only a fleeting contact with the ribosome. The binding between particle and enzyme might be so tenuous as not to survive the isolation pro- cedure, and even in the intact cell only a small proportion of the total number of enzyme molecules might be situated on the ribosome at any given time. Functionally, however, such an enzyme could be considered to be a ribosomal enzyme. It is clear, then, that this problem in its several aspects is a subtle one; and it is probable that individual solutions will perhaps have to be sought for quite a few enzymes. In the sections that follow, a large number of ribosomal enzymes will be described, some in groups and others individually. There are two classes of enzyme which, according to our present knowledge of the function of the ribosome, we might expect to be associated with the ribosome. The first group includes those enzymes which participate in the ribosomal stages of protein biosynthesis and thus have a direct functional connection with the ribosome. The second group comprises the so-called nascent enzymes — enzyme molecules which, as proteins, are in the process of being synthesized on the ribosome and have not yet been released from the site of synthesis. The remaining enzymes do not appear to be nascent proteins and are of such a nature that there is no obvious reason to predict a priori that they would be attached to ribosomes. They have been divided arbitrarily into three addi- tional groups. The first is ribonuclease I probably the first ribosomal enzyme to be detected and the one on which the most work has been done. The next group includes other enzymes which act in conjunction with a polynucleotide that serves as either substrate, template or primer for the enzyme. There happens to be considerable information on such enzymes, and it is convenient to consider them together. Finally, in the last group are summarily grouped all examples which do not fit into the preceding groups. V. Ribosomal Enzymes — Enzymes of Protein Synthesis The enzymes considered here participate in those stages of protein synthesis which take place in association with the ribosome. They catalyse such reactions as the attachment to, movement on, and detachment from the ribosomal surface of mRNA and sRNA ; the transfer of nascent polypeptides from one molecule of sRNA to another and the concomitant formation of the peptide bond; perhaps the hydrolysis of GTP; and so on. There is as yet little evidence on the ribosomal location of such enzymes. Indeed, some of them have not yet been conclusively shown to exist, and none has been characterized more than perfunctorily. This section is then little more than a statement to the effect that the 7, RIBOSOMAL ENZYMES 427 general scheme of protein biosynthesis described earlier in this chapter implies that a number of enzymes must function on or near the ribosomal surface, since their substrates are located there. It is conceivable that in the interests of economy and efficiency such an enzyme might even be built into the permanent structure of the ribosome, but it is equally conceivable that it might not be attached to the ribosome with any degree of permanency. The matter must be examined experimentally in each case, with the attendant problems of non-specific adsorption, etc., that have already been mentioned. A case in point is a study of the amino acid activating enzymes (6.1.1.-) of rat liver (Decken, 1961). These enzymes operate at an early stage of protein synthesis where the ribosome is not yet involved. Liver ribosomes, however, showed considerable amino acid activating activity, presumably owing to contamination. When, however, the ribosomes were washed with 0-6 M KCl-O-Ol M MgCl2, several other enzymic activities were completely removed, but a small amount of amino acid activating activity remained bound to the ribosomes and could not be taken off entirely even when the KCl concentration was raised as high as 3-2 m. Such situations are not un- common, and the investigator may be pardoned if he abandons the matter at this inconclusive stage with the feeling that, for all that, the ribosome-bound activity probably represents contamination. If, however, the case should be one in which it is important to ascertain whether the activity is really ribo- somal, then the problem becomes intricate and non-routine. A somewhat similar situation has been reported for GTPase (3.6.1.-). It seems fairly certain in this case that such an enzyme is implicated in one or more reactions which directly involve the ribosome. GTP is invariably re- quired for the incorporation of amino acids on the ribosome. In the reticulo- cyte the breakdown of GTP has been implicated in the binding of aminoacyl sRNA to the ribosome (Arlinghaus et al., 1964) and also in the release of completed polypeptides from the ribosome (Morris, 1964). In this case there is considerable interest in the relation between the enzyme and the ribosome and the question is now being tenaciously pursued. Thus, a non-ribosomal protein factor of E. coli, active in stimulating ribosomal polypeptide synthesis (Allende, Monro and Lipmann, 1964), was shown to contain a ribosome- dependent GTPase activity (Conway and Lipmann, 1964). However, the factor could be further separated into two fractions: one stimulated poly- peptide synthesis but lacked GTPase, and the other showed GTPase activity (still ribosome-dependent) but did not stimulate polypeptide synthesis (Nishizuka and Lipmann, 1965). The washed ribosomes employed in this experiment themselves exhibited a slight GTPase activity. Thus, the nature of the relationship among GTPase, ribosomes and protein synthesis is still obscure. A number of investigators have shown that the incorporation of amino acids from sRNA into polypeptides, a process which takes place on the 428 DAVID ELSON ribosome, is stimulated by two separable non-ribosomal protein factors. These factors, often called the transfer factors or transfer enzymes, produce maximal stimulation only when both are present together; apparently, each serves a different function. They have been observed and studied in E. coli (AUende et al., 1964; Keller and Ferger, 1965), rat liver (Nathans and Lip- mann, 1960; Fessenden and Moldave, 1962; Moldave and Gasior, 1965), rabbit reticulocytes (Arlinghaus, Favelukes and Schweet, 1963; ArUnghaus et al., 1964; Lamfrom and Squires, 1962), and other tissues. A partial characterization of the reticulocyte factors has been achieved: one factor appears to catalyse the binding of sRNA charged with amino acids to the ribosome and the other may be the enzyme which forms the peptide bond (Arlinghaus et al., 1964). As noted, these factors appear to be enzymes which act on substrates attached to the ribosomal surface, yet they are present in the soluble fraction of the cell. However, after reacting in the isolated reticulo- cyte ribosomal system in vitro, both factors became attached to the ribo- somes to the extent that they sedimented with them in the centrifuge. There is additional evidence as to the location of the enzyme which synthe- sizes the peptide bond. The antibiotic puromycin interferes with protein synthesis by causing the release of unJ&nished polypeptides from ribosomes. It is believed that it does this by substituting for a normal amino acid and being itself attached in peptide linkage to the growing end of the nascent polypep- tide. Since, however, the puromycin is not attached to a molecule of sRNA, there is nothing to bind the polypeptide to the ribosome, and it comes off (Zamecnik, 1962). Thus, the release of polypeptides from ribosomes by puromycin appears to involve the enzymic formation of a peptide bond. The 50 S ribosomal sub-particle of E. coli can be isolated under conditions where it is devoid of mRNA and of the 30 S ribosome, but still has attached to it sRNA and nascent protein (Gilbert, 1963b; Elson, 1964a). Puromycin has been shown to cause extensive release of the nascent polypeptide from such 50 S particles (Traut and Monro, 1964). Since this would presumably require the formation of a peptide bond, it is possible that the enzyme which does this may be located on the 50 S ribosome. Finally, an investigation of haemoglobin synthesis in vitro with a system derived from rabbit reticulocytes has yielded results suggesting that a specific enzyme may be responsible for the release of completed polypeptide chains from the ribosome, and that this enzyme may be located on the ribosome (Morris, 1964). In sum, work on this group of enzymes is still at its beginning. Consequently, there is very little solid information on how, where and, above all, whether these enzymes are attached to the ribosome. For several of them, it is true, there are indications of a ribosomal attachment, either permanent or transient, but no systematic study of this matter has yet been brought to a decisive stage. 7, RIBOSOMAL ENZYMES 429 VI. RiBosoMAL Enzymes— Nascent Enzymes The concept of nascent protein — unfinished protein molecules attached to their ribosomal site of synthesis — has been firmly established since the classic experiments of Zamecnik and his colleagues, which showed that amino acids are first assembled into polypeptides attached to ribosomes in the liver (Littlefield et al., 1955; Zamecnik, 1960). Similar results were soon reported for the pancreas (Siekevitz and Palade, 1958b; 1960a), and were amplified by the work of McQuillen, Roberts and Britten (1959) on E. coll These workers showed radioactive protein precursors to be rapidly incorporated into the ribosomes, which, if the exposure to precursor was brief enough, assumed the highest specific radioactivity of all cell fractions. When the incorporation of labelled precursor was stopped under conditions which allowed the synthesis of proteins to continue (by the addition of an excess of non-radioactive precursor) the labelled protein was seen to pass rapidly from the ribosomes to the soluble fraction (Fig. 7.) They concluded that "this nascent protein is a polypeptide strand which is formed on the ribosome and r\ . - 1 ! 1 1 J L- jI ■ I\l — r-r^i 1 1 1 r 1 . 1 • 1 8 12 16 20 24 Fig. 7, Demonstration of nascent protein on ribosomes in sucrose density gradient centri- fuge patterns of the cell juice of E. coli. The unbroken line shows the distribution of material absorbing at 260 m/i ; the peak at the centre of the tube is the ribosomes. The broken line shows the distribution of radioactivity, (a). Cells incubated fifteen seconds with radio- active sulphate, (b). Cells incubated as in (a), followed by fifteen seconds with excess non- radioactive sulphate. Most of the radioactivity has left the ribosomes, indicating that the label represents protein molecules transiently bound to the ribosomes and removed from them by normal metabolic processes. Reproduced with permission from McQuillen et al. (1959). 430 DAVID ELSON is subsequently released as soluble protein" (see also Tissieres, Schlessinger and Gros, 1960; Duerre, 1964b). Later work showed that the nascent protein is attached to the ribosome by a molecule of sRNA (see section 3). This attachment is not very firm, and the sRNA and protein are released from the ribosome when the Mg2+ concentration is reduced or the ionic strength is raised (Gilbert, 1963b; Elson, 1964a). They appear to become re-attached when the change in the medium is reversed (Schlessinger and Gros, 1963; Tal and Elson, 1963a), but it is not known whether they become re-attached to the same site and in the same way. Obviously the lability of the attachment of the nascent protein to the ribosome can make it difficult to distinguish between a nascent and an adsorbed enzyme. Since every protein is presumably synthesized on a ribosome, the nascent proteins should, by definition, include precursors of every enzyme then being synthesized in the cell. It is not evident that a nascent enzyme will show enzymic activity at this stage in its career. It is conceivable, however, that some might acquire the ability to act as catalysts before being released from the ribosome. This would probably depend on the structural properties of each individual enzyme : one might be able to fold into an active tertiary configu- ration before being released, while another might not. If the rate of synthesis of an enzyme is known, the amount which might reasonably be expected to occur in nascent form can be calculated. It has been estimated, for example, that of any enzyme in a steady state culture of E. coli growing exponentially with a generation time of 50 minutes, about 0-6% would be nascent and bound to the ribosomes (McQuillen et al., 1959; Cowie, Spiegelman, Roberts and Duerksen, 1961). Since this estimate includes both active and inactive forms of the enzyme, the amount of active nascent enzyme, if any, would be far less. The matter is further complicated by the fact that many enzymes are polymeric, being made up of more than one polypeptide chain, and the subunits may not necessarily be synthesized on the same ribosome. For such an enzyme to be both nascent and active, polymerization would have had to occur while one enzyme subunit was still attached to a ribosome. This has been observed to occur in vitro in the case of E. coli /S-galactosidase (3.2.1.23) by Zipser and Perrin, (1963). Whether it occurs in vivo is not known. In a study of the synthesis in vivo of the L-histidine ammonia-lyase (4.3.1.3) of B. subtilis, Hartwell and Magasanik (1964) have concluded that the polymerization step which is necessary for the enzyme to become active does not take place on the ribosome. If this should be generally true, then the finding of an active polymeric enzyme attached to ribosomes would be an artifact, albeit, perhaps, a useful and instructive artifact. It is apparent that every enzymic activity found in a preparation of ribosomes might be due, wholly or partly, to nascent enzymes. In this section, however, Il 7. RIBOSOMAL ENZYMES 431 only those ribosomal enzymes are discussed which may reasonably be suspec- ted of being nascent or which have been investigated with this possibility in mind. A. PANCREATIC ENZYMES AND ZYMOGENS One of the more widely studied mammalian secretory organs, the pancreas, is adapted for the large scale synthesis of a relatively small number of enzymes and zymogen precursors of enzymes. These proteins have, in the main, been well characterized and considerable work has been done on their intracellular distribution. We might expect to find in such a tissue a rather high nascent ribosomal level of those few enzymes and zymogens which are synthesized in large quantities. The earliest work on this subject showed that guinea pig pancreas ribosomes are rich in two types of protein synthesized for secretion: RNase and protease zymogens ("trypsin-activable protease") (Siekevitz and Palade, 1958a). Later work in the same laboratory concentrated on a-chymotrypsinogen and added amylase (3.2.1.-) to the list of such enzymes (Siekevitz and Palade, 1960a, b). A study of the time course of labelling of the cellular proteins showed that at short times after the injection of a radioactive amino acid the a-chymotrypsinogen of those ribosomes which had formerly been attached to the membranes of the endoplasmic reticulum was more highly labelled than in any other cell fraction. This is a clear indication that ribosomal a-chymo- trypsinogen is a precursor of the rest of the zymogen in the cell. It is difficult, however, to recognize true nascent enzymes in this system. This becomes evident when the quantities of ribosomal enzymes are con- sidered. The guinea pig pancreas ribosomes employed carried some three to 7% of their total protein as RNase and from three to ten per cent as chymotrypsinogen and trypsinogen, and these proteins seemed quite firmly attached to the isolated ribosomes (Siekevitz and Palade, 1960b, 1962). Clearly, only a minute fraction could actually be nascent protein, and its properties would be obscured. The observed preferential labelling of the ribosomal a-chymotrypsinogen can perhaps be explained by postulating that the most recently synthesized molecules remained in the closest proximity to the ribosomes, in the endoplasmic reticulum, and were preferentially adsorbed by them when the reticulum and later the microsomes were destroyed. The problem of adsorption has been directly pointed out in a number of investigations of the ribosomes of beef pancreas. The cell juice and zymogen granules of this tissue contain large quantities of the basic proteins RNase, trypsinogen and chymotrypsinogen A, and the acidic proteins DNase (3.1.4.-), chymotrypsinogen B and procarboxypeptidases A and B. Although the acidic proteins outnumber the basic ones by about two to one in the cell and therefore, presumably, in the nascent protein, isolated ribosomes 432 DAVID ELSON contained only the basic ones, which amounted to nearly 9 % of the ribosomal protein (Keller et ai, 1963). Further, when labelled chymotrypsinogen A was added to ribosomes, it was adsorbed and was not displaced by subse- quently added unlabelled chymotrypsinogen A (Keller and Cohen, 1961). Thus the promised advantages of the pancreas as a source of nascent ribosomal enzymes have been far outweighed by the great quantities of non- ribosomal enzymes in the tissue, at least some of which are adsorbed in large amounts by the ribosome. It will be necessary to find effective ways of distinguishing between adsorbed and nascent enzyme or of selectively removing adsorbed enzyme from the ribosome before a conclusive study of the pancreatic nascent enzymes can be carried out. There are, in fact, a number of accounts of the removal of enzymes and zymogens from pan- creatic ribosomes by various means: e.g., by washing the ribosomes with salt (Keller, et al, 1963; Madison and Dickman, 1963a; Siekevitz, 1963), precipitating them with Mg2+ ions (Gazzinelli and Dickman, 1962; Siekevitz, 1963), or treating them with spermine (Siekevitz and Palade, 1962) or anti- serum against the adsorbed enzyme (Madison and Dickman, 1963b; Gazzinelli and Dickman, 1962). The enzymic activities of the ribosomes have been substantially reduced by such treatments; however, a low level of activity usually remains. Whether this remaining ribosomal activity represents an enrichment in nascent enzyme has not yet been examined in detail. B. OTHER STUDIES OF NASCENT ENZYMES The literature contains a number of examples of ribosomal enzymes which have been studied specifically to probe the possibility of their being nascent enzymes. Some of these investigations have been perfunctory, others, more extensive and searching. Among the criteria employed to distinguish nascent enzymes have been : that the enzyme is not easily removed from the ribosome, that specific antiserum against the enzyme precipitates not only the enzyme but also a small fraction of the ribosomes, that the ribosome-bound activity is only a small fraction of the total cellular activity, and that the ribosomal enzyme shows the characteristics expected of a precursor of the other enzyme molecules of the same species. Since this is relatively new ground, it is not surprising that some of these criteria have been found inadequate and others have proved unexpectedly difficult to apply. The work summarized below represents, essentially, a beginning. / . Viral Neuraminidase (3.2.1.18) When influenza virus is grown in allantoic membranes or monkey kidney cells, the enzyme neuraminidase (sialidase), an enzyme which forms part of the protein complement of the virus, is found associated with host cell ribosomes. Possibly nascent protein, the enzyme is released from the ribosomes 7. RIBOSOMAL ENZYMES 433 as a molecule of molecular weight 2 x 10^, and is later incorporated into larger complexes and built into the viral envelope (Noll, 1962). 2. Triosephosphate Dehydrogenase (1.2.1.-) Well washed yeast ribosomes have been shown to contain a low but signifi- cant level of triosephosphate dehydrogenase activity (Warren and Goldthwait, 1962), although crystalline enzyme added to ribosomes was easily washed away. Four to eight % of the washed ribosomes were precipitated by rabbit antiserum against the crystalline enzyme, and the precipitate was somewhat enriched in enzyme activity. The impUcation is, of course, that the ribosomes were precipitated by the antiserum because they were attached to nascent enzyme molecules. 3. Tryptophan Synthetase (4.2.1.20) The tryptophan synthetase of E. coli is made up of two separate and dis- similar protein chains, designated A and B. Employing immunological techniques to distinguish between the two proteins, Horibata and Kern (1964) have presented evidence which indicates that some ribosomes carry only the A protein and others only the B protein. Other ribosomes, however, contained both proteins. This is a matter of some interest, since the A and B proteins are governed by different but adjacent structural genes and there is a possibility that both proteins are synthesized by the same ribosome from a single polycistronic mRNA (i.e., a single molecule of RNA in which is transcribed the information contained in more than one structural gene). The authors suggest that this is what occurs and that those ribosomes which carried only one of the proteins originally contained both but lost one. However, it is entirely possible that the ribosome-A-B complexes result from an interaction between ribosome-A and free B or ribosome-B and free A, as has been observed in the case of j3-galactosidase (Zipser and Perrin, 1963). The data presented to ehminate this possibility are not convincing, and it is not certain that those A and B chains found on the same ribosome were in fact both synthesized there. 4. Pencillinase (3.5.2.6) Duerksen and O'Connor (1963) have studied the inducible penicillinase of ribosomes isolated from lysozyme spheroplasts of -5. cereus. The ribosomes were not washed, unfortunately, but the enzymic activity sedimented with the particles in a sucrose density gradient. The authors have calculated that there is one ribosome-bound enzyme molecule per uninduced cell, ten per fully induced cell, and fifteen per cell in a constitutively positive strain. 5. Lactate Dehydrogenase (1.1.1 .27) Some of the difficulties of distinguishing a true nascent enzyme are pointed out in a thoughtful paper by Keck and Choules (1962), who have investigated 434 DAVID ELSON the lactate dehydrogenase isoenzymes of guinea pig hver and kidney in an attempt to identify nascent precursors still attached to ribosomes. Ribosomes from these organs did contain isoenzymes, but predominantly the basic ones, suggesting the adsorption of free enzyme on the acidic ribosome. The point was not conclusive, since non-basic isoenzymes were also present. However, the level of ribosomal activity was such that if it were all due to nascent protein, at least one of every thirty kidney ribosomes would have had to be engaged in synthesizing this enzyme, and this does not seem reasonable. Consequently, the authors concluded that they were dealing with adsorbed rather than nascent enzymes, even where non-basic isoenzymes were involved. 6. fi-Glucosidase (3.2.1.21) The work of Halvorson and his colleagues (Hauge, MacQuillan, Cline and Halvorson, 1961; Kihara, Halvorson and Bock, 1961; Kihara, Hu and Halvorson, 1961) on the jS-glucosidase of a diploid strain of yeast constitutes one of the earliest and yet one of the fullest studies of a ^ascent enzyme now available. These workers showed that when ribosomes of a constitutively positive strain were washed by repeated sedimentation in sucrose density gradients, their specific ^S-glucosidase activity fell to a level not further reduced by continued washing. Control ribosomes from a constitutively negative strain could be washed free of added soluble enzyme. The identity of the ribosomal enzyme was examined by degrading the ribosomes and so releasing the enzyme (this treatment also results in a several fold rise in activity). The released enzyme was found not to differ from non-ribosomal j8-glucosidase in a number of physico-chemical properties and its affinity for a number of jS-glucosides. /?-Fluorophenylalanine was next employed in an attempt to determine whether the ribosomal enzyme could be a precursor of the soluble enzyme. This amino acid analogue is incorporated into proteins in place of phenyl- alanine and the cell produces an enzymically inactive version of j3-glucosidase. When the analogue was administered to a culture of cells, the quantity of non-ribosomal enzyme ceased to rise, and remained constant; but the amount of ribosomal activity dropped by a factor of ten. Upon the addition of excess phenylalanine to the culture, enzyme synthesis was resumed and the ribosome- bound activity rose to its former level. These results are consistent with the proposition that the ribosomal enzyme is the nascent precursor of the soluble enzyme. 7. ^-Galactosidase One of the most extensively studied enzymes known, the inducible j3-galactosidase of E. coli, has also been widely employed in investigations of nascent enzymes. Early work by Kameyama and Novelli (1960, 1962) demonstrated the presence of a particle-bound jS-galactosidase in E. coli which differed from the soluble enzyme in several respects and appeared 7. RIBOSOMAL ENZYMES 435 likely to be a precursor of the soluble enzyme. The quantity of particle bound enzyme was, however, much too high to allow it to be identified with nascent protein, and the particles examined were not purified ribosomes but something much more complex. Perhaps the first detailed study with purified ribosomes was that of Cowie et al. (1961). When they washed the ribosomes of a constitutively positive strain by repeated sedimentation, the ribosomal activity was reduced to a low but constant and significant level, and this activity sedimented with the ribosomes in sucrose density gradients. In the control, a mixture of ribosomes from a strain genetically incapable of producing the enzyme and free enzyme from a different strain, essentially all of the adsorbed activity was washed off (Fig. 8). Inducible but non-induced cultures were estimated to contain one ribosome-bound active enzyme molecule per cell. Constitutively positive 10 1-0 i 001 ML-308(Loc*) ribosomes in ML-308 supernate 0 2 4 6 Number of woshes Fig. 8. The effect of successive washings by centrifuging on the specific jS-galactosidase activity of ribosomes from genetically positive (#) and negative ( x ) strains of E. coli. Both sets of ribosomes were initially suspended in extracts containing large amounts of active enzyme. Reproduced with permission from Cowie et al. (1961). I cells carried about ten times more and induced cells, about twenty five. Rabbit anti-^-galactosidase serum reacted with the ribosomes but did not precipitate them. When, however, this treatment was followed with chicken anti-rabbit serum, about 1 % of the ribosomes precipitated and carried with them over 90 % of the ribosomal /3-galactosidase activity. Certain of these results have been confirmed in other laboratories (Proctor, 436 DAVID ELSON 1961; Fogel and Eison, 1964; Kiho and Rich, 1964), and in addition, the ribosomal /3-galactosidase has been shown to be located mainly in the poly- some fraction, the ultimate site of protein synthesis (Kiho and Rich, 1964). The indications were, then, that the ribosomal j3-galactosidase activity might well represent nascent molecules in a stage of synthesis sufficiently advanced to allow enzymic activity. Almost from the beginning, however, it became apparent that the matter was more complex than the results summarized above might suggest. In following up their earlier investigation, Duerksen and Cowie (1961 ; cf. Roberts, 1964, p. 407) studied the kinetics of the appearance of ribosomal j3-galactosidase during induction. If all the ribosomal enzyme were a nascent precursor of soluble enzyme, then the amount of ribosomal activity should follow the rate of enzyme synthesis. It should rise rapidly at the beginning of induction, reach a constant maximum level when the rate of enzyme synthesis becomes constant (about two and a half minutes after addition of the inducer), maintain this level during induction, and decline when the inducer is removed. The experimental observations were, however, quite different. The quantity of ribosomal enzyme continued to rise throughout the entire period of enzyme synthesis and did not decline when synthesis was stopped by the removal of the inducer. Thus the quantity of ribosomal enzyme ran parallel not with the rate of enzyme synthesis, but with the total amount of enzyme in the cell. Furthermore, the quantity of ribosomal enzyme was different when the cells were broken by different techniques. These findings led to the conclusion that "the newly formed enzyme can only be a minor fraction of the total quantity found to sediment at the same rate as the ribosomes." ^'^''Tliese results have been amplified by Zipser (1963), who was able to / detect two kinds of ribosomal /S-galactosidase : one fraction which remains / bound to the ribosomes and increases the longer the cells have been synthe- l sizing the enzyme, and another fraction which leaves the ribosomes after 1 the removal of the inducer and could be the precursor of the soluble enzyme. \_^ There are other indications, too, that the ^-galactosidase activity of well washed ribosomes may not all be due to nascent molecules. For one, it has been reported that when such purified ribosomes are sedimented in a caesium chloride gradient of density 1-3-1 -6 (over 2 m), the amount of ribosome- bound enzyme is reduced forty times. (Lederberg, Rotman and Lederberg, 1964). This does not prove that only 2 or 3 % of the ribosomal enzyme is nascent, since high ionic strength will detach nascent protein from ribosomes (Elson, 1964a), but it does suggest that not all of the ribosomal enzyme is bound to the ribosome in the same way. Also when purified ribosomes were separated into 30 S and 50 S particles by sucrose density gradient centrifuging, both particles showed activity (Fogel and Elson, 1964) although nascent protein remains bound only to the 50 S ribosome (Gilbert, 1963b). 7. RIBOSOMAL ENZYMES 437 Finally, another factor must be fitted into the picture before it will be possible to understand the nature of ribosomal jS-galactosidase. Like many other enzymes ^-galactosidase is polymeric and is active only in the polymeric form. Although /3-galactosidase subunits can polymerize in vitro while one ['IFsUITattached to a ribosome, it is not known whether this occurs in vivo or whether the process takes place at a completely post-ribosomal stage of protein synthesis. If the latter, then any association of )3-galactosidase activity with ribosomes is an artifact. This is still an open question (see the opening discussion of this section). It seems likely that the true nascent jS-galactosidase has so far eluded our grasp. The considerable amount of work, sometimes both ingenious and profound, which has been done has only cleared the approaches to the problem. 8. Latency of Nascent Enzymes Several ribosomal enzymes are activated by one treatment or another, treatments which either release them from the ribosome or apparently cause some change in their attachment to the particle. Before such treatment the enzyme is presumably bound to the ribosome in such a way as to reduce or prevent its enzymic action. Of the enzymes discussed in this section, two have been reported to be latent in this sense. As mentioned above, the )3-glucosidase of yeast becomes more active when it is liberated through the destruction of the ribosome (Kihara, Hu and Halvorson, 1961). There are conflicting reports concerning the ribosomal )3-galactosidase, to the effect that the destruction of the ribosome does (Proctor, 1961) or does not (Cowie et al, 1961 ; Fogel andElson, 1964) lead to an increase in enzymic activity. A different treatment, however, does cause the activity to increase several times. This is the treatment of the ribosomes with rabbit anti-jS-galactosidase serum, which reacts with the ribosomes but neither precipitates them nor causes their destruction (Cowie et al, 1961). This effect has now been further studied by Lederberg et al. (1964). Using a technique sensitive enough to detect single molecules of j3-galactosidase (Rotman, 1961), they succeeded in showing that the increase in activity is caused not by an increase in the activity of already active ribosomes, but by the appearance of enzyme activity on ribosomes which were previously inactive. It is to be hoped that the nature of this interesting phenomenon will soon be elucidated. C SUMMARY These examples of work aimed, sometimes more and sometimes less persistently, at characterizing the nascent enzymes show that we are still far from understanding the relationship between the ribosome and the growing 438 DAVID ELSON polypeptide chain, which at some point in its growth acquires the ability to catalyse a specific biochemical reaction. We have, in fact, not quite achieved the first necessary objective, the ability to recognize such a nascent enzyme and to distinguish it from other forms of the same enzyme. The criterion of ribosomal location is a necessary one but is not satisfactory alone, since it is easy to recognize a high level of adsorbed enzyme but can be quite difficult to discern a small amount. Also, the enzyme must be identified and measured by its activity. This probably makes it impossible to deal with certain enzymes in their nascent stages and introduces numerous uncertainties. Where polymeric enzymes are involved, the question of the polymerization step and its place in the normal sequence of events is a complicating factor whose severity cannot yet be accurately assessed. It would seem desirable to stress two criteria in this sort of investigation. The first is that a true nascent protein must have the properties of a precursor to other forms of the enzyme (even here there are pitfalls: see the section on pancreatic enzymes). The second is a criterion that has only recently become available and has not yet been applied, namely, that a nascent protein molecule should be attached to a molecule of sRNA (see section III). VII. Ribosomal Enzymes — Ribonuclease (2.7.7.-) The ribonucleases are a ubiquitous family of enzymes which differ in several respects from other enzymes which degrade RNA. The RNases are endonucleases which produce mono- and oligonucleotide fragments whose terminal phosphate residue is attached to the 3' position of the nucleoside ribose. Most other enzymes which degrade RNA are exonucleases, and most of them form 5'-mononucleotides. What appears to distinguish the RNases absolutely from the others is their mechanism of action, which proceeds in two steps. The first step, a transfer reaction, involves the splitting of an internucleotide phosphoester bond between phosphate and the 5' position of the adjacent nucleoside, and the simultaneous formation of a phosphoester bond at the 2' position of the nucleoside to which the phosphoryl group is already attached. Thus, the first degradation product is a fragment whose terminal phosphate group is esterified to both the 2' and 3' positions of the same ribose moiety. The enzyme subsequently hydrolyses this cyclic diester to form the 3'-phosphomonoester. This mechanism is specific and it would seem proper to reserve the name "ribonuclease" for those enzymes which form the cyclic diester intermediate. The various RNases share this reaction mechanism; they differ in that some will split only those bonds which are adjacent to certain nucleosides but not to others. In practice, of course, the reaction mechanism is rarely defined, and it is common to identify RNases by another property which distinguishes them ' 7. RIBOSOMAL ENZYMES 439 from all other nucleases known at present, namely, that they do not require Mg2+ or any other divalent cation. Thus, a nuclease which degrades RNA in the presence of an excess of a complexing agent such as EDTA is generally considered to be an RNase. (For reviews see, e.g., Anfinsen and White, 1961 ; Roth, 1963). RNase is probably the first enzyme shown to be specifically associated with ribosomes. In 1950, Pirie (1950, 1957) reported ribonucleoproteins prepared from tobaco leaf to contain the enzyme in a tightly bound form. This unambiguous observation did not become generally known, and some seven or eight years later, after a number of intensive investigations of the ribosome had been undertaken, the phenomenon was inevitably rediscovered in several laboratories (Elson, 1958, 1959b; Bolton, 1959b, cf. Roberts, 1964, p. 362; Siekevitz and Palade, 1958a; Stenesh, 1958; Tashiro, 1958; Ts'o, Bonner and Vinograd, 1958; ZilUg et al., 1959; Roth, 1960a, b). These findings stemmed generally from the common observation that ribosomes, which are quite stable at low temperature and ionic strength, become unstable when either the temperature or the ionic strength is raised or the concentration of Mg2+ ions is reduced (e.g., Elson, 1959a). At such times the ribosomal RNA is degraded and an enzyme, soon identified as an RNase, was found to be responsible (Elson, 1958, 1959a; Bolton, 1959b, cf. Roberts, 1964, p. 362; Zillig et al., 1959; Tashiro, 1958). The fact that the ribosomes carry with them an enzyme capable of catalysing their own destruc- tion was startling enough to provoke several investigators into rather detailed examinations of the matter. By now, an association between RNase and ribosomes has been reported for a great many diffeient organisms (see Table I at the end of this chapter). These observations are so numerous and so widespread thoughout nature that at first it seemed that the phenomenon might be universal (Elson, 1961b). There are, however, exceptions, some better and some less well established, and these appear to be significant, as will be discussed below. A. PANCREATIC RIBOSOMAL RNaSE It is clear that the association between ribosome and enzyme is of at least two different types. One extreme is typified by the ribosomes of the pancreas (see section VI a.) RNase is one of several enzymes synthesized and secreted in large amounts by this organ. The pancreas contains a high concentration of the enzyme, and so do its ribosomes. Pancreatic ribosomes have, in fact, been reported to contain from 1-16% of their protein as RNase (Madison and Dickman, 1963a; Keller, Cohen and Wade, 1963, 1964; Siekevitz and Palade, 1960b; Siekevitz, 1963). This is probably due virtually entirely to non-specific adsorption. A basic protein, RNase would inevitably be bound by the acidic RNA of the ribosomal surface. It has been directly demonstrated 440 DAVID ELSON that extraneous RNase is adsorbed by pancreatic and other ribosomes (e.g., Madison and Dickman, 1963a; Siekevitz, 1963), and that most and often virtually all of the RNase of pancreatic ribosomes can be rather easily removed from the particles by salt (Madison and Dickman, 1963a; Siekevitz, 1963), magnesium ions (Keller, et al., 1964; Gazzinelli and Dickman, 1962; Siekevitz, 1963), spermine (Siekevitz and Palade, 1962), bentonite (Keller et al., 1964), anti-RNase serum (Gazzinelli and Dickman, 1962; Madison and Dickman, 1963b), and other agents. In short, the presence of large amounts of RNase in ribosomes isolated from the pancreas is probably of no direct physiological significance, reflecting only the normal affinity between cationic and anionic macromolecules. The same may well be true of at least a portion of the RNase activity of ribosomes from other organs. B. E. coli RIBOSOMAL RNaSE In certain other types of cell, however, a different relationship between ribosome and RNase can be discerned. This type is exemplified by E. coli, on which considerable work has been done. These cells contain comparatively little RNase ; however, most of it is attached to ribosomes isolated in the usual way, and it is attached so firmly that it can hardly be removed without destroying the ribosome (Elson, 1959b, 1961b; Bolton, 1959b, cf. Roberts, 1964, p. 362; Spahr and Hollingworth, 1961). This striking relationship, further documented below, has led to speculation that the function of the enzyme is connected with the function of the ribosome (e.g., Elson, 1961b; Zillig, et al., 1959; Tashiro, 1958; Roth, 1960b; Spahr and Hollingworth, 1961, Tal and Elson, 1963b), namely that it has to do with protein bio- synthesis. Recent developments, however, have cast doubt on both the ribosomal location of the enzyme and its proposed biological function (Neu and Heppel, 1964a,b,c; Gesteland, 1965). I shall treat this work in roughly chronological order, outlining first the findings which appeared to establish RNase as a true ribosomal enzyme and then those which have placed these conclusions and the attendant speculations in question. 1. Identity In the early work with the RNase ofE. coli, evidence was soon accumulated which indicated that the enzyme was a true RNase. Several other possibilities were eliminated by direct test; and the enzyme was shown to resemble pancreatic RNase in heat stability, in having no requirement for divalent cations, and in producing 3 '-nucleotides (Elson, 1958, 1959b; Bolton, 1959b, cf. Roberts, 1964, p. 362; Zillig et al., 1959). Later work with a purified enzyme confirmed these observations, and showed also that the enzyme degrades RNA, via cyclic phosphodiester intermediates, 7. RIBOSOMAL ENZYMES 441 completely to mononucleotides, thus differing from bovine pancreatic RNase and resembling certain plant RNases in specificity (Spahr and HoUingworth, 1961). 2. Latency A phenomenon eariy noted was the latency of this ribosomal enzyme (Elson, 1958, 1959b; Bolton, 1959b, cf. Roberts, 1964, p. 362). This concept has been widely misunderstood and should, perhaps, be clarified here. It was initially observed that the ribosomes were stable in the cold at low ionic strength. That is, the ribosomal RNase did not attack the ribosomal RNA under these conditions, nor did it attack added free RNA. Active enzyme was liberated by a variety of treatments : e.g. urea, increased ionic strength, tryptic digestion, EDTA (which removes the Mg2+ ions which stabilize the nucleoprotein structure), and increased temperature (to 37°). Following such treatments, which disrupt the structure of the ribosome, the enzyme became able to attack 0-2 0-1 ?<' 4m urea No urea U- o^P r 9 9 — -o 8 10 6 hr hr Fio. 9. The latency of ribonuclease in a highly purified ribonucleoprotein from E. coli, and the activation of the enzyme by 4m urea. "Acid-soluble A260" is a measure of the degradation of the ribosomal RNA by the enzyme. Conditions: a. 0°; 0-075 m tris buffer, pH 80. b. 37°; 0005 m MgS04. Reproduced with permission from Elson (1959 b). both the ribosomal RNA and added free RNA under conditions of tem- perature, pH, ionic strength and Mg2+ concentration where intact ribosomes showed no RNase activity (Elson, 1958, 1959b; Bolton, 1959b cf. Roberts, 1964, p. 362; Spahr and HoUingworth, 1961) (Fig. 9). Latency was thus defined in the following way: that there exist conditions under which the RNase can attack RNA but does not when the enzyme is associated with ribosomes (Elson, 1959b). This is not true of all conditions. For example, under the conditions usually employed for the assay of RNase (e.g., neutral 442 DAVID ELSON pH, ionic strength of about 0-1-0-2, 30° or 37°, Mg2+ concentration of 0-01 M or less) ribosomes will generally break down rapidly, their RNase will be liberated, and its latency will not be seen. Latency can be demonstrated only under conditions where the ribosomes are stable, and these are not the conditions of maximum enzyme activity. The conditions prevailing inside a living cell of E. coli are not those under which the ribosomal RNase would remain inactive outside the cell, and the demonstrable latency in vitro does not, of course, imply that the enzyme is inactive in vivo. It has been suggested, however, that there is an implication that the action of this enzyme in vivo may be controlled and limited by virtue of its association with the ribosome (Elsoh, 1961b). 3. Ribosomal Location The early work on the RNase of E. coli strongly suggested that virtually all of the enzyme was associated with the ribosomes (the cell debris was not examined in these investigations). This was shown in a variety of ways. When ribonucleoproteins were purified by a relatively drastic chemical procedure, all of the RNase activity of the crude cell extract remained in the nucleoprotein (Elson, 1959b). When the ribosomes were sedimented in the centrifuge, the supernatant fluid contained little or no RNase (Bolton, 1959b, cf. Roberts, 1964, p. 362; Spahr and Hollingworth, 1961); and when a crude extract was resolved in a sucrose density gradient, RNase activity was found almost exclusively in the fractions containing ribosomes (Tal and Elson, 1963b; Elson, 1964b). Later reports indicate that the soluble fraction may contain as much as 1 1 % of the RNase activity of the crude extract (Shortman and Lehman, 1964; Anraku and Mizuno, 1965) and, further, that the cell debris (presumbably fragments of cell wall and cell membrane) contains an appreciable quantity of an RNase which may be different from that of the other fractions (Anraku and Mizuno, 1965). In any event, all of these in- vestigations showed that the bulk, if not virtually all, of the RNase was associated with the ribosomes. The observed concentration of the RNase of E. coli in the ribosomes prompted a close examination of the localization of the enzyme, and it was then found to be localized exclusively in the 30 S ribosome (Elson and Tal, 1959). This has been confirmed in a number of ways. Purified 50 S ribo- somes contained only a low level of RNase activity and this could be accounted for by a small amount of contaminating 30 S particles (Elson and Tal, 1959; Tal and Elson, 1963b). Further, the RNase activity of mixtures of 30 S and 50 S ribosomes varied directly with the proportion of 30 S ribosomes in the mixture (Elson and Tal, 1959; Tal and Elson, 1963b; Spahr and Hollingworth, 1961) and extrapolated to zero activity for pure 50 S particles (Fig. 10). Again, when 30 S and 50 S particles were resolved by centrifuging in a sucrose density gradient, only the 30 S ribosomes 443 20 40 60 80 30 S ribosomes (%) Fig. 10. The intraribosomal location of ribonuclease in E. coli; a plot of the ribonuclease activities of 30 S and 50 S ribosomes mixed in diflferent proportions. The plot extrapolates to zero activity for pure 50 S particles. Reproduced with permission from Tal and Elson (1963 b). showed RNase activity (Tal and Elson, 1963b; Elson, 1964b) (Fig. 11). Finally, when the proteins derived from separate preparations of 30 S and 50 S ribosomes were subjected to electrophoresis in starch gel, only the 30 S particles showed a band with RNase activity (Waller, 1964). Opposed 6-90- 847 Tube number 'Fig. 11. The intraribosomal location of ribonuclease in E coli, as determined by sucrose density gradient centrifuging. The broken line shows the location of the ribosomes and the unbroken line shows that of the enzyme, which is only in the 30 S particle. Reproduced with permission from Tal and Elson (1963 b). I 444 DAVID ELSON to these results is the reported observation that the 30 S and 50 S ribosomes show equal levels of RNase activity (Bolton, 1959b, cf. Roberts 1964, p. 362). The reason for this discrepancy is not apparent; however, the bulk of the available evidence indicates strongly that the affinity of E. coli ribosomes for RNase is restricted to the 30 S particle. 4. Amount in the Ribosomes Since the RNase of E. coli has not been fully characterized, it has not been possible to determine unequivocally the quantity of enzyme in the ribosomes. What has been done is to compare the ribosomal RNase activity with that of known amounts of crystalline bovine pancreatic RNase. Three such estimates have been made. The results indicate that the ribosomes of E. coli contain about 0-08 molecules of enzyme per 70 S ribosome (Elson, 1959b), between 0-1 to 10 (Bolton, 1959b, cf. Roberts, 1964, p. 362) or 0-1 (Spahr and Hollingworth, 1961). The first two estimates were based on the assumption that the E. coli enzyme is identical with pancreatic RNase in both molecular weight and specific activity. The third estimate, however, was made with a purified enzyme whose specific activity had been approxi- mately determined, although here too the molecular weight could not be measured. The concensus is, then, that no more than one ribosome in about ten would carry a molecule of RNase. In the case of yeast, an estimate of one molecule per four ribosomes has been pubUshed (Ohtaka and Uchida, 1963). 5. Firmness of Binding The enzyme is bound to the ribosome very firmly, so that, until recently, its removal had not been accomplished without the destruction of the ribo- some. For example, in early chemical purifications in my laboratory, ribonucleoproteins of E. coli were exposed to 1 -25 % sodium deoxycholate, precipitated as the barium salt in 50% ethanol, redissolved with sodium sulphate, and repeatedly precipitated with ammonium sulphate in the saturation range of 25-60% (1 •2-2-6 m, ionic strength 3-6-7'8). This harsh and varied procedure was designed to remove extraneous proteins from the ribonucleoprotein ; yet the entire complement of RNase remained attached (Elson, 1959a, b). E. coli ribosomes have also been exposed to high con- centrations of caesium chloride (approximately 2 to 5 m) without losing RNase activity (Lederberg, et al, 1964). In view of these observations, a milder salt treatment might not be expected to remove the ribosomal RNase of £. coli. There are, however, reports that prolonged washing with 0-5 m ammonium chloride de-activates the ribosomal RNase (Jardetzky and Julian, 1964) or removes it (Spirin et al, 1963), although data on the effectiveness of the procedure were not pubhshed. In our hands the procedure described by Spirin et al. (1963) 7. RDBOSOMAL ENZYMES 445 has given variable results; some activity has been removed, but in all cases so far the ribosomes have retained a significant level of RNase (P. Spitnik- Elson, personal communication ; D. Elson, unpublished results). In contrast, when large quantities of bovine pancreatic RNase were adsorbed on E. coli ribosomes, a brief exposure to 0-5 m ammonium chloride reduced the riboso- mal activity to its low basal level. Apparently, the pancreatic enzyme was completely removed, while the endogenous E. coli RNase remained attached (D. Elson, unpublished results). However, what appears to be an effective technique for removing RNase from E. coli ribosomes has now been described by Salas, Smith, Stanley, Wahba and Ochoa (1965). After two prolonged washings with 0-5 m ammonium chloride, the ribosomes, still containing a high level of RNase activity, were chromatographed on DEAE-cellulose. This treatment reduced the RNase activity by about 99%. The ribosomes were active in promoting the incorporation of amino acids into peptides, and were successfully employed in an experiment in which the virtual absence of RNase activity was critical. Taken together, these observations show that RNase can be removed from the ribosomes of E. coli without causing their inactivation ; the enzyme is, however, bound very firmly to the ribosomes. ' 6. Location — Observations with Spheroplasts of E. coli The foregoing observations indicated that the bulk of the RNase of £. coli is bound firmly and specifically to the surface of the 30 S ribosome in such a way as to affect the action of the enzyme. This is certainly true after the breakage of cells and the isolation of ribosomes in the usual way. It has been assumed that the same is true of the intact cell, but this assumption has now been put in doubt by the findings of Neu and Heppel (1964a, b, c, d; 1965). It had previously been shown by Malamy and Horecker (1961) that E. coli cells release their entire content of alkaline phosphatase (3.1.3.1) into the external medium when converted to spheroplasts with lysozyme and EDTA in a sucrose-?rw medium. A number of other enzymes remain within the spheroplast. This observation was interpreted in terms of a compartment- alization of enzymes. It was postulated that the alkaline phosphatase is situated on the periphery of the cell, either associated with the cell wall or lying between it and the cell membrane, while the unreleased enzymes are in the interior of the cell. When Neu and Heppel (1964a, b, c; 1965) performed similar experiments, they found that several additional enzymes leaked out of the spheroplasts, I among them RNase. Spheroplasts made from cells in stationary phase released about 45-60 % of their RNase ; those from exponentially growing cells, up to 90%. The release of enzyme was rapid, reaching completion in a few minutes at room temperature. Ribosomes were not released from the 1 446 DAVID ELSON spheroplasts under these conditions and, compared with ribosomes isolated from cells broken in a different way, the spheroplast ribosomes were lower in RNase activity by an amount roughly equal to that released from the spheroplasts. It was not apparent at first whether the RNase might not, for all that, have been attached to the ribosomes within the spheroplast and have been released because of structural damage caused by the withdrawal of Mg2+ by the EDTA in the spheroplast medium (Neu and Heppel, 1964a). However, later control experiments showed that the spheroplast ribosomes have a normal ratio of RNA to protein and, by this criterion, are not grossly degraded (Neu and Heppel, 1964c). Furthermore, ribosomes isolated from broken whole cells did not release RNase when exposed to the reagents employed to form spheroplasts (EDTA, tris, sucrose and lysozyme), either singly or in combination (Neu and Heppel, 1964c; D. Elson, unpublished results). There is no indication, then, that the release of RNase from spheroplasts is caused by a direct effect on the ribosomes; at least, such an effect has not been observed in vitro. Accordingly, Neu and Heppel (1964b, c) have pro- posed that the RNase of E. coli may not, or may not all, be located on the ribosomes but may be segregated near the surface of the cell, while the ribosomes are situated in the interior. As an alternative they suggest that the enzyme may be concentrated on those ribosomes or polysomes which are attached to the cell membrane, and that this fraction of the ribosomal RNase is, somehow, easily detached. If the enzyme is not bound to the ribosome in vivo, it would appear some- what difficult to account for the several strikingly specific features of its ribosomal attachment in vitro. That they can nevertheless be accounted for is shown by the following observation (Neu and Heppel, 1964c). Purified E. coli RNase, added to E. coli ribosomes, became bound to the particles. The added enzyme was not removed during washing in sucrose; it was enzymically inactive unless the ribosomes were destroyed; and when 30 S and 50 S ribosomes were separated in a sucrose density gradient, the newly attached enzyme was found only in the 30 S particle (Fig. 12). Thus, the earlier noted characteristics of the binding between RNase and ribosome were reproduced merely by mixing the two components together. While this does not prove that the ribosomal location of the E. coli RNase is an artifact produced when the cell is broken, it clearly indicates that such could be the case. The release of enzyme from spheroplasts has several still puzzling features. For example, the six enzymes (including RNase) presently known to be released from EDTA-lysozyme spheroplasts (Neu and Heppel, 1964d, 1965) are not released from spheroplasts prepared by other methods (Neu and Heppel, 1964c). The other five enzymes are, however, released from cells which have been exposed to a shock treatment which does not convert them to spheroplasts, but in this case no RNase is released (Neu and Heppel, 7. RIBOSOMAL ENZYMES 447 0-2- E o 0-1- - 1 r 1 r/isOS ....,., ,, 1 -T- - / 30 S - / 1 H - - ^- 1 — ty 1 / f 1 1 / A\\ 1 1 1. "V. 1 500 -400 S -300 200 00 ■-0 8 20 24 28 32 Tube number Fig. 12. The specific adsorption of E. coli ribonuclease by the 30 S ribosome of E. coli. Sucrose density gradient centrifuge patterns show the distribution of ribosomes (O), of the ribonuclease activity of untreated ribosomes (A), and of the enzymic activity of ribosomes which had been mixed with soluble ribonuclease (•). Reproduced with per- mission from Neu and Heppel (1964 c). 1964d, 1965). Furthermore, although the balance studies indicate that it is the "ribosomal" enzyme which is released from EDTA-lysozyme sphero- plasts, this has been questioned by Anraku and Mizuno (1965), who suggest that it may be a different RNase, which they have discovered in the cell envelope of £. coli and which can be adsorbed by the ribosomes although it is not usually found in them. It has also been pointed out that the RNase released from spheroplasts is not chromatagraphically identical with RNase isolated from ribosomes (Neu and Heppel, 1964b; Anraku and Mizuno, 1965; Anderson and Carter, 1965), although this may mean that different treatments have wrought different changes in the molecules. From all of this, it is obvious that we still have much to learn about the intracellular location of RNase in E. coli, and the matter may not be fully clarified until new techniques have been applied to the problem. It seems clear that the enzyme has a high and specific affinity for the ribosome, but how much, if any, of the enzyme is attached to the ribosomes in the intact cell, is at present an open question. C. BIOLOGICAL FUNCTION OF RNaSE There has been much speculation on the function of RNase. A scavenger 448 DAVID ELSON function is an obvious and still eminently tenable possibility, and a number of others have also been proposed. Roth (1954) was probably the first to suggest that the enzyme might have the task of breaking down the template RNA in order to free the proteins which had been made on the template (see also Elson, 1959b), and I shall return later to a modernized version of this proposal. It has also been suggested that RNase acts in uncovering RNA templates (Leslie, 1961) or in modulating the interaction between ribosome and sRNA (Bosch, Huizinga and Bloemendal, 1962). A correlation between growth and RNase has also been noted (Roth, 1963). The apparent localization of the E. coli RNase in the ribosomes pointed to a ribosomal site of action for this enzyme. As noted above, the enzyme seemed to be bound exclusively to the 30 S ribosome with exceptional firmness and in such a way as to influence its activity. The properties of the bound RNase did not seem to be those of a nascent protein or of a non- specifically adsorbed protein. There were indications in the literature that a similar situation might hold for ribosomes and RNase in other organisms Bosch, et al, 1962; Datta, Bhattacharyya and Ghosh, 1964; Roth, 1960b; Burdon, 1963; Stavy, Feldman and Elson, 1964; Danner and Morgan, 1963; Ohtaka and Uchida, 1963). Taken as a whole, this body of data suggested a connection between the physiological role of RNase and that of the ribosome, that is, that the enzyme might have to do with the biosynthesis of proteins. The later observations of Neu and Heppel, mentioned above, which cast doubt on the ribosomal location of RNase do not eliminate this possibility, for it remains true that the enzyme exhibits a high and specific affinity for the ribosome. This may imply a functional relationship, whether or not the enzyme is permanently located on the ribosome. With the advent of messenger RNA and the elaboration of a detailed mechanism of protein synthesis, the number of acceptable ways in which RNase might participate in protein synthesis became rather strictly limited. Furthermore, it soon became evident that whatever its function might be, the enzyme could not be essential for the polymerization of amino acids. This arose from the observation that rabbit reticulocyte ribosomes, if washed, have very little RNase and quite possibly have none at all (Williamson and Mathias, 1963; Stavy et al, 1964; Mathias, WilHamson, Huxley and Page, 1964); nevertheless, they synthesize protein. This, together with another feature of the mammalian reticulocyte, suggested a role for the ribosomal RNase. In bacteria the mRNA is rapidly broken down and renewed (Levinthal, Keynan and Higa, 1962). This does not appear to take place in the mammalian reticulocyte (Marks, Willson, Kruh and Gros, 1962) and, indeed, it could hardly do so, since the cell lacks a nucleus and mRNA cannot be synthesized in the absence of DNA (Hurwitz and August, 1963). This suggested a cor- relation between RNase and the metabolic turnover of mRNA. It had been indicated that RNase is not the enzyme responsible for the 7. RIBOSOMAL ENZYMES 449 gross degradation of mRNA, since the end products of this degradation appear to be nucleoside-5'-phosphates (Sekiguchi and Cohen, 1963). We suggested, therefore, that the ribosomal RNase might inactivate mRNA, perhaps by cleaving no more than a single internucleotide bond, and so render the molecule unable to attach to ribosomes (Tal and Elson, 1963b, Elson, 1964b). The mRNA would then be exposed to the degradative action of other nucleases. The location of RNase on the 30 S ribosome of E. coli was in keeping with this suggestion, since the mRNA appears to be attached to the same particle (Okamoto and Takanami, 1963). It has also been pointed out that a molecule of mRNA survives, on the average, long enough to promote the synthesis of about ten to twenty protein molecules (in B. subtilis) and about one in ten ribosomes carries RNase (in E. coli). Thus, the lifetime of a molecule of mRNA might be governed by the random probabiUty of its becoming attached to a ribosome which carries RNase (Artman and Engelberg, 1964). Based on circumstantial evidence — affinity between RNase and the ribosome — this proposal requires that cells which possess a metabolically labile mRNA should also possess RNase. It can be argued, of course, that the function in question might be performed by RNase in certain organisms and by a different nuclease in others, but such an argument would seem less likely to be valid in the case of closely related organisms. With the discovery of bacterial strains which appear to lack RNase, a direct test of the matter became possible. There are several reports of bacteria which lack RNase. They include B. subtilis and St. albus (Neu and Heppel, 1964d) and Ps. fluorescens (Wade and Robinson, 1963). No data are available for the first two, but the case of Ps. fluorescens was investigated rather thoroughly, in that activity was sought over a wide pH range in the presence of EDTA, and was not found. On the other hand, ribosomal RNase has been reported in Ps. fluorescens (McCarthy, 1959, cf. Roberts, 1964, p. 370), and a cursory examination of B. subtilis ribosomes in my laboratory indicated the presence of an RNase active in excess EDTA (B. Benish and D. Elson, unpublished results). These would appear to be instances where RNase is present in one strain and absent in another of the same bacterial species. More recently three RNase-less strains of E. coli have been reported. Two, isolated in the laboratory of J. D. Watson, are related mutants of the same strain of E. coli (Gesteland, 1965; see also Haruna and Spiegelman, 1965). Both lack RNase and one also lacks polynucleotide phosphorylase. The third is an unrelated strain of E. coli shown, in the laboratory of H. E. Wade, to lack RNase (Cammack and Wade, 1965; Wade and Robinson, 1963). What remained was to examine the metabolic lability of mRNA in these RNaseless bacteria. This has been done with the three strains of E. coli mentioned above (T. Kivity-Vogel and D. Elson, 1966). The method of 450 DAVID ELSON Kepes (1963) was employed to measure the half life of the mRNA of induced ^-galactosidase. The cultures examined contained no more than a small fraction of the RNase activity of a control culture of E. coli B. Nevertheless their mRNA became inactivated at essentially the same rate as that of the control. Thus, the lack of RNase did not cause a derangement of mRNA catabolism, and the enzyme would not appear to be involved in the inactiva- tion of mRNA in E. coli. (one of the strains also appeared to lack polynucleotide phosphorylase, (2.7.7.8.). We have seen, then, that a number of biological functions have been pro- posed for RNase and have, with the accumulation of new data, been ruled out. Certain possibilities still remain. One — that RNase is concerned with the regulation of the concentration of ribosomes in the cell — is now being in- vestigated in my laboratory. But so far no ribosomal function has been demonstrated, and it remains possible that the striking aflEinity of RNase for the ribosome is merely a chemical accident of no physiological significance. VIII. Ribosomal Enzymes — Other Enzymes which Act on Polynucleotides The enzymes of this rather arbitrarily defined group have in common that they act in conjunction with a polynucleotide, which serves as either substrate, template or primer. Unlike RNase, these enzymes require magnesium or other divalent cations. These two features may not be unrelated to the presence of the enzymes in ribosomal preparations, a point that will be elaborated on later in this section. A. deoxyribonuclease (3. 1. 4. -) DNase activity was first observed in a ribonucleoprotein prepared from E. coli by a drastic chemical technique designed not to preserve the fine structure of the ribosome but rather to remove as much extraneous protein as possible (Elson, 1959a, b). The enzyme was latent, in that conditions could be found under which it was inactive until something was done to disrupt the structure of the nucleoprotein (Fig. 13). Ribosomes prepared conventionally by differential ultracentrifuging also contained DNase activity, but in varying amount. This was eventually found to depend on the degree of dissociation of the ribosomes, which is in turn controlled by the concentration of magnesium ions in the medium (Tal and Elson, 1961, 1963a). Ribosomes isolated in the form of 70 S and 100 S particles contained about 8 or 9% of the total DNase activity of the crude cell extract (from which cell debris had previously been removed), and their activity was not reduced by repeated washing in the same medium. When such purified ribosomes were dissociated to 30 S and 50 S particles 7. RIBOSOMAL ENZYMES 451 50 — r . .. J. . , , . 1 ^ 40 30 y{ - .• 4 m urea 20 '/ /• 1 10 No urea ^ ^jy' ^ A 0 9" 1 1 1 10 hr Fig. 13. The latency of deoxyribonuclease in a highly purified ribonucleoprotein from E. colt, and the activation of the enzyme by 4 m urea. The ribonucleoprotein was incubated with DNA at 37° in the presence of 0005 m MgS04. In the absence of urea the enzyme became active only after six hours of incubation. In 4 m urea there was maximum activity from the beginning. Reproduced with permission from Elson (1959 b). by lowering the Mg2+ concentration, about 90% of their DNase activity was released into the medium. When these dissociated ribosomes were re- converted to 70 S and 100 S particles in the presence of the previously released DNase, the enzyme became re-attached to the ribosomes, apparently as firmly as before (Fig. 14); and when the process was carried out with an Number of washes Fig. 14. The magnesium-dependent binding of deoxyribonuclease to E. coli ribosomes. The ribosomes were treated as follows: A. Maintained as 70 S and 100 S particles in 001 M magnesium acetate — 0001 m tris buffer, pH 7-4; D. Dissociated to 30 S and 50 S particles by dialysis against 0001 m tris; R. First dissociated as in D, then re-associated in the medium of A. After treatment the ribosomes were washed repeatedly by centrifuging. Reproduced with permission from Tal and Elson (1963 a). 452 DAVID ELSON excess of released enzyme, the ribosomes captured more than their original content of DNase. At intermediate concentrations of Mg2+, which caused only partial dissociation of the ribosomes, the release of DNase was also partial and was roughly equivalent to the degree of ribosomal dissociation. Further, when the concentration of Mg2+ was shifted either upward or downward, DNase activity moved into or out of the ribosomes accordingly. In short, like ribo- somal dissociation and association, the attachment of DNase to the ribosome is freely reversible in both directions, depending only on the final Mg2+ concentration. It is still not clear whether the primary factor governing the attachment is Mg2+ concentration or ribosomal association. It is, however, evident that the dissociation of ribosomes is not the only controlling factor, for enzyme release is much slower than ribosomal dissociation, and when complete dissociation is achieved in different media, the amounts of DNase released are also different (Tal and Elson, 1963a). Except for the quantity of DNase involved this phenomenon of reversible release is reminiscent of the behaviour of nascent protein. As mentioned above, nascent protein is attached to the ribosomes by sRNA, and both the ribosomal sRNA and the nascent protein have been shown to become detached from the ribosomes at low concentrations of Mg2+ and to return to them at high Mg2+ concentrations (Tal and Elson, 1963a; Schlessinger and Gros, 1963; Elson, 1964a). However, nearly 10% of the total DNase participates in this reversible release, and this is too much to be nascent protein, by several orders of magnitude. It seems more likely, then, that if the phenomenon is of biological significance (and it is not certain that it is), its significance may not be general but may be restricted to a small group of ribosomal enzymes, and perhaps only to DNase. It might, for example, have to do with ribosomal participation in the regulation of DNA metabolism — perhaps with its co-ordination with protein synthesis — but this is pure con- jecture, and more prosaic suggestions can also be advanced (see below). There are four known DNases in E. coli, one endonuclease and three exonucleases, each distinguishable from the other by virtue of their properties and specificity (Lehman, 1963). It would be instructive to know whether all or only certain of these enzymes undergo the reversible release. Unwashed ribosomes from E. coli have been found to carry all four enzymes, each in an amount ranging from 5-14% of the total activity of that enzyme in the crude cell extract (Shortman and Lehman, 1964). Since the endonuclease is by far the predominant DNase in the cell, it is almost certainly involved in the observed reversible binding. This supposition is strengthened by the work of Weissbach and Korn (1964) who extracted ribosomes from E. coli in the absence of magnesium (presumably as 30 S and 50 S particles) and found them to contain only a low level of DNase activity. When the extraction was carried out with 0-01 m Mg2+, the ribosomes (70 S and 100 S in this medium) 7. RIBOSOMAL ENZYMES 453 carried much more DNase; this DNase was latent and appeared to be the endonuclease. Whether the level of ribosomal exonucleases also changes with the Mg2+ concentration is not known. Upon the infection of E. coli with phage T4r, the quantity of ribosome- bound DNase rises markedly (Weissbach and Korn, 1964). The new ribo- somal enzyme, whose synthesis is induced by the infecting phage, resembles E. coli exonuclease II. This does not, of course, shed light on the situation in the normal uninfected cell. A comment may be inserted here concerning both the latency of the ribosomal DNase and the possible nature of its attachment to the ribosome. RNA was found long ago to inhibit DNase (Kozloff, 1953), and specifically the endonuclease (Lehman, 1963). Consequently, this enzyme is known to bind to RNA, and this might well account both for its binding to the RNA- rich ribosome and for its latency when so bound. Those conditions which have been employed to activate the ribosomal DNase are also conditions which activate the ribosomal RNase and lead to the degradation of the ribosomal RNA (Elson, 1959b; Weissbach and Korn, 1964). Magnesium is necessary for the action of DNase. Its function is unknown, but it might conceivably be required for the binding of the enzyme to its substrate, DNA. If this is so, then Mg2+ might function in a similar way to bind the enzyme to the RNA of the ribosome, and this might account for the reversible binding of DNase to the ribosome and for the fact that the extent of binding is a direct function of the magnesium ion concentration. This suggests that the binding of the DNase to the ribosome may be non-specific and that the association and dissociation of the ribosomes may not play a direct role in the binding. I Whether the phenomenon is of biological value is, of course, an open question. Bf It should be noted that the above suggestion has been framed essentially for the endonuclease. As for the exonucleases, there is no evidence as to whether or not their binding to the ribosome is reversible or magnesium-dependent. Further, since they are not inhibited by RNA, it is not certain that they bind to this substance. I r B. DNA POLYMERASE (2. 7. 7. 7) Unwashed 70 S and 100 S ribosomes of £. co/z (extracted with 0-01 m Mg2+) rry up to 40% of the DNA polymerase (DNA nucleotidyltransferase) activity of the crude cell extract (Weissbach and Korn, 1964). When the extract contains no magnesium ions, the ribosomal activity drops to less than 2%. Cells infected with T4r phage produce a new DNA polymerase. The phage-induced enzyme has a higher affinity for ribosomes, which now carry 30 % of the total activity in the absence of magnesium ions and over 50 % in 0-01 M Mg2+. The normal enzyme thus shows a magnesium-dependent affinity 454 DAVID ELSON for ribosomes reminiscent of that of DNase. The greater affinity between ribo- somes and the phage-induced enzyme appears less sensitive to the concentra- tion of magnesium ions. C. POLYRIBONUCLEOTIDE POLYMERASES (2.7 7. -) A magnesium-dependent enzyme which catalyses the synthesis of poly- ribo-adenylic acid from ATP in the presence of RNA has been observed to be concentrated in E. coli ribosomes. Some seventy to ninety per cent of the activity is ribosomal (August, Ortiz and Hurwitz, 1962; Ortiz, 1963). An enzyme which incorporates nucleotides from ATP or UTP into poly- ribonucleotides has been found on the ribosomes of the nuclei of Landschutz ascites tumour cells (Burdon, 1963). D. AMINOACYL-SRNA SYNTHETASES (6. 1. 1.-) The enzymes which activate amino acids and attach them to sRNA are soluble enzymes. As mentioned above, low levels of activity have been found in ribonucleoproteins prepared from rat liver (von der Decken, 1961). BeUeved to be a contamination, the activity nevertheless resisted strenuous efforts to remove it. E. RNA METHYLASES When the methionine-requiring mutant K 12 W-6 of E. coli is starved of methionine, it ceases to synthesize protein. Normally, the synthesis of RNA would also be blocked, but this strain carries a second mutation which causes an impaired control of RNA synthesis, and the production of RNA continues (Srinivasan and Borek, 1964). Under these conditions the cells accumulate RNA in the form of particles which resemble and may be identical with the particles produced in the presence of chloramphenicol (Nomura and Watson, 1959). These particles appear to be defective ribosomes which carry the full complement of ribosomal RNA, but less than the full complement of ribo- somal proteins. Ribosomal RNA contains a very small amount of methylated purines and pyrimidines ; these bases are methylated by specific enzymes which act on the fully polymerized RNA (Srinivasan and Borek, 1964; Gordon and Boman, 1964). Since, however, the source of methyl groups is a molecule derived from methionine (Srinivasan and Borek, 1964), the RNA of the particles lacks the methylated bases. When the purified particles are incubated with the methyl donor, they are able to accomplish the methylation of their own RNA in the absence of any soluble factors. This indicates that the required methylating enzymes are present on the defective ribosomes (Gordon and Boman, 1964). 7. RIBOSOMAL ENZYMES 455 F. POLYNUCLEOTIDE PHOSPHORYLASE (2.7.7.8.) Ribosomes (not always washed) of E. coli and Ps. aeruginosa have been found to contain as much as 40 % of the total polynucleotide phosphorylase activity of crude cell extracts (Wade, 1961; Wade and Lovett, 1961; Kimhi and Littauer, 1962; Strasdine, Hogg and Campbell, 1962; Grunberg-Manago, 1963). There are also indications of activity in ribosomes isolated from B. subtilis and St. aureus (B. Benish and D. Elson, unpublished results). On the other hand, the free ribosomes of lysozyme protoplasts of Strep, faecalis are devoid of activity and the enzyme appears to be concentrated on the cell membrane (Abrams and McNamara, 1962), The considerable variation in the amounts of polynucleotide phosphorylase found in different ribosome preparations indicates that the attachment is rather labile. Thus, Y. Kimhi and U. Z. Littauer (1962, and personal com- munication) have washed 70 S and 100 S E. coli ribosomes to constant activity and found them to contain from 5-15% of the total cellular activity. When such particles were centrifuged in a sucrose gradient of the same magnesium concentration, much of the residual enzyme was removed. When 70 S and 100 S ribosomes which had undergone either or both of the above treatments were transferred to a medium of lower magnesium ion concen- tration, most of the remaining ribosomal activity was released. Thus, the polynucleotide phosphorylase content of the ribosomes is greatly influenced by the way in which the particles are treated. Further, the binding of enzyme to ribosome appears to be magnesium-dependent, as previously noted for DNase and DNA polymerase. G. PHOSPHODIESTERASES (3. 1. 4. 1) Phosphodiesterase activity has been found in ribosomes of goat brain (Datta and Ghosh, 1963b) and E. coli (Wade and Lovett, 1961), but is probably absent from those of rabbit reticulocytes (Stavy et al., 1964). The recently discovered potassium-activated phosphodiesterase has been isolated from the soluble fraction of E. coli, although ribosomal activity was not ruled out (Spahr and Schlessinger, 1963). H. THE BINDING TO RIBOSOMES OF ENZYMES WHICH ACT ON POLYNUCLEOTIDES The enzymes listed in this section all interact with a polymeric nucleic acid substrate, template, or primer. It is not surprising, therefore, that they are often found attached to the RNA-rich ribosomes. This may simply follow from their necessary affinity for nucleic acid, and may or may not be signifi- cant biologically. Much remains to be learned of the firmness and extent of binding. In some cases this has been studied with some thoroughness, but 456 DAVID ELSON in others, in whicti this matter has not been the main burden of the work, there is Uttle more than an estimate of enzyme activity in unwashed preparations of ribosomes. Magnesium ions are required for the action of all of these enzymes. The role of this ion is not known. If it should promote the binding of enzyme to polynucleotide, then we might have a basis for understanding the effect observed with three of these enzymes — DNase, DNA polymerase and polynucleotide phosphorylase — namely, that the degree of their binding to the ribosome depends on the magnesium ion concentration of the medium. If this is true, we might expect the binding of other enzymes of this group to be magnesium-dependent, but this has not yet be«n tested. IX. RiBOsoMAL Enzymes — Other Examples A. PHOSPHATASES (3. 1. 3. -) Phosphatases, both acid and alkaline, have been found in ribosomes isolated from swine and rat kidney (Binkley, 1961), goat brain (Datta and Ghosh, 1963a), and E. coli (Spahr and HoUingworth, 1961 ; Warren and Goldthwait, 1961). Except for the kidney particles, the ribosomes had been purified by repeated sedimentation at low ionic strength. This treatment brought the alkaline phosphatase activity of E. coli ribosomes to a constant level under conditions where purified enzyme was not adsorbed (Warren and Goldthwait, 1961); this ribosomal activity was presumed to represent nascent enzyme molecules. The acid phosphatase activity of E. coli ribosomes was not removed by centrifuging through 50 % sucrose. It was present in both 30 S and 50 S ribosomes, with the 30 S particle showing a higher level of activity (Spahr and HoUingworth, 1961). B. PEPTIDASES 1. Leucine Aminopeptidase (3.4.1.1.) The first of several ribosomal peptidases found, the leucine aminopeptidase of E. coli, has also been the most extensively studied (Bolton and McCarthy, 1959, cf. Roberts, 1964, p. 365; Matheson, 1963). Unwashed ribosomes carry from 10-80% of the total cellular activity, and the quantity appears to depend on the physiological state of the cells. Thus, cells grown in glucose- rich media have a higher proportion of ribosomal enzyme than cells grown in media poorer in glucose (Bolton and McCarthy, 1959, cf. Roberts, 1964, p. 365). During the transition of a culture from exponential growth to stationary phase the enzymic activity of the free ribosomes falls and that of the membrane fraction rises ; it was suggested that this may come about through a transfer of enzyme to membrane-bound ribosomes or a binding of free ribosomes to the membrane (Matheson, 1963). 7. RIBOSOMAL ENZYMES 457 This ribosomal enzyme appears to be distributed equally between well washed 30 S and 50 S ribosomes. Its specificity may not be identical with that of the soluble enzyme. It has therefore been suggested that the ribosomal peptidase may play a role in protein synthesis (Bolton and McCarthy, 1959, cf. Roberts, 1964, p. 365). Leucine aminopeptidase has also been found to be attached to the ribosomes ofPs.fluorescens, B. megatherium and A. aerogenes, but not to those of yeast (Bolton and McCarthy, 1959, cf. Roberts, 1964, p. 365). 2. Cysteinyl-glycine Dipeptidase An enzyme which hydrolyses L-cysteinylglycine was first observed by Binkley (1961) to sediment with the ribosomes of swine and rat kidney. This enzyme was studied further in E. coli by McCorquodale (1963), who showed that ribosomes purified by repeated centrifuging through 10% sucrose at low ionic strength reached a constant activity at which they still carried some 30 % of the total cellular activity. When the ribosomes were dissociated, the enzyme appeared exclusively in the 50 S ribosome. When, however, the magneisum concentration was raised and the ribosomes under- went re-association to form a mixture of 70 S, 85 S, 100 S and larger aggregates, an unanticipated result was obtained. The 70 S ribosomes were apparently devoid of the peptidase; the enzyme was found only in 85 S and larger particles (Fig. 15). This was interpreted as indicating that those 50 S particles which carry the enzyme are incorporated into 70 S ribosomes which have a higher tendency to form larger aggregates. The nature of the 85 S particle is obscure (Roberts et al., 1963), but the 100 S particle is a dimer formed by the joining of two 70 S ribosomes through their 30 S sub-particles (Hall and Slayter, 1959; see also Figs. 1 and 2). It might be, then, that those 50 S ribosomes which carry the enzyme belong to a class of particles which selectively pair with those 30 S ribosomes possessing a high tendency to interact with each other. There is a hint here of a multiple relationship which might be interesting to clarify. 3. Iminodipeptidase (3.4.3.6) An enzyme which splits L-prolylglycine but not glycylglycine or poly-L- proline has been found in chemically purified ribonucleoproteins as well as in conventionally prepared ribosomes from E. coli (Elson, 1961b, 1962b, and unpublished results). C. NAD PYROPHOSPHORYLASE (2. 7. 7. 1) The NAD pyrophosphorylase of rat liver and Krebs-2 tumour is a nuclear enzyme. It has been shown to be associated with the nuclear ribo- somes, while cytoplasmic ribosomes do not carry the enzyme (Traub, 458 DAVID ELSON Xt]A!PD 9S093 U3 ^ 7/UJ092 - / rj o 1 / , ^ 1 / o ^ 1 / 30S o c / /%^ • 1 < o • / 1 &« y w o Si < E 45 / 0-2 1 / i. V -•^ o / b • -...-.-^^•^^^^-o-o , ° , 0 15 20 Fraction number Fig. 16. The intraribosomal location of ATPase in E. coli, as shown by sucrose density gradient centrifuging. Reproduced with permission from Schlessinger (1964). E. OTHER ENZYMES FOUND IN RIBOSOME PREPARATIONS There are a number of references to ribosomal enzymic activities which have not been mentioned. Some of them are : proteolytic activity in E. coli (Chaloupka, 1961); amylase (3.2.1.-) in well washed goat brain ribosomes (Datta and Ghosh, 1963a); glutamate dehydrogenase (1.4.1.-) and j8- glucuronidase (3.2.1.31) in calf thymus nuclei (Wang and Wang, 1962); traces of aldolase activity (4.1.2.7) in E. coli (Elson, 1961b; 1962b); peroxidase (1.11.1.7) in pea seedlings and rabbit liver (Matsushita and Ibuki, 1960b) and in wheat embryo (Lanzam* and Galante, 1964); and ribonucleoside triphosphatases (3.6.1.-) in pea seedlings (Raacke, 1963) and E. coli (Raacke and Fiala, 1964). In the last mentioned case the enzymes appeared to be bound only to polysomes. They were not found in 70 S ribosomes produced by breaking down the polysomes. I 7. RIBOSOMAL ENZYMES 461 X. Summary In this chapter I have tried to outhne what is known and what may reason- ably be inferred about the function and structure of the ribosome, and to describe the current, somewhat chaotic, status of ribosomal enzymology. The enzymes considered here fall into two broad classes : those which, from our present understanding of the role of the ribosome in protein synthesis, we might expect to be associated with ribosomes; and other enzymes which have been found associated with ribosomes although there seems no reason a priori to expect them to be there. The first class includes both the enzymes which catalyse those reactions of protein synthesis which take place on the ribosome and the nascent enzymes, the umeleased products of ribosomal protein synthesis. Many investigators have touched on the question of nascent enzymes, yet in no case has a nascent enzyme been unequivocally identified and its properties fully elucidated. What has been done so far has served, essentially, to define the problem,| to clarify the nature of certain difficulties, and to indicate experimenta approaches which may possibly be effective. As for the enzymes which mediate the ribosomal stages of protein syn- thesis, they are largely a matter of conjecture. Insofar as we know the reactions of protein biosynthesis, we assume that certain enzymes are involved. Since our knowledge is incomplete, we do not yet know exactly how many such enzymes there are. There is a still meagre body of indirect experimental evidence which testifies to the existence of some of these enzymes, but none of them has yet been isolated or characterized. Furthermore, there is no firm evidence as to whether these enzymes are actually bound to the ribosome. Our guiding principle here states merely that there is a functional association between the ribosome and these enzymes, but it says nothing about the physical or chemical nature of the association. Thus, even if we were to have a complete list of these enzymes, we would still be ignorant of exactly how they work. This would require a detailed understanding of the ribosomal structure' particularly its surface, and the complex interactions among ribosome, enzyme, substrate, etc. , and these are matters which will remain unclear for some time to come. The second broad class of ribosomal enzymes includes those which have been reported to be associated with ribosomes but which do not seem to fit into the first class; that is, they are enzymes for whose ribosomal association we have experimental evidence but no a priori guiding principle. Quite a few such enzymes are listed. It is certain that the list would be shorter if all the ribosome preparations involved had been washed thoroughly. It would be, however, unjustifiably glib to dismiss this entire class of ribosomal enzymes as adsorption artifacts. Some of them — such as, for example, ATPase, RNase and peptidases — may eventually find their way into the list of enzymes which participate in ribosomal protein synthesis. Others may 462 DAVID ELSON conceivably teach us about other presently unsuspected aspects of ribosome function, for it is not impossible that the ribosome, by virtue of its abun- dance, makeup and ability to interact with many kinds of molecule, may play a role in the regulation and co-ordination of metabolic processes other than protein synthesis. If this is true, this role may emerge in the course of time as a generalization from a mass of individual observations of which each, in itself, may not seem to be of general significance. Table I. A List of References to Enzymic Activity in Preparations of Ribosomes Enzyme Enzyme Source Commission of Number Ribosomes References Adenosine triphosphatase 3.6.1.3 E. coli Pea • Raacke and Fiala (1964), Schlessinger (1964) Raacke (1963) Alcohol dehydrogenase 1.1.1.1 Yeast Warren and Goldthwait (1962) Aldolase 4.1.2.7 E. coli Elson (1961b, 1962b) Aminoacyl-sRNA synthetase 6.1.1.- Rat liver von der Decken (1961) Amylase 3.2.1.- Goat brain Guinea pig pancreas Not specified Datta and Ghosh (1963a) Siekevitz and Palade (1960b, 1962) Siekevitz (1965) Cytidine triphosphatase 3.6.1.- E. coli Pea Raacke and Fiala (1964) Raacke (1963) Deoxyribonuclease 3.1.4.- E. coli Elson (1959b, 1961b, 1962b, DNA polymerase ^-Galactosidase 2.7.7.7 E. coli 3.2.1.23 E. coli Not specified 1964b), Lehman (1963), Short- man and Lehman (1964), Tal and Elson (1961, 1963a), Weissbach and Korn (1964) Weissbach and Korn (1964) Chaloupka ( 1 96 1 ), Duerksen and Cowie (1961), cf. Roberts 0964), p. 407 Elson (1964b), Fogel and Elson (1964), Kiho and Rich (1964), Lederberg,, Rotman and Lederberg (1964), Madison and Dickman (1963b), Zipser (1963), Zipser and Perrin (1963), Proctor (1961) 7. RIBOSOMAL ENZYMES Table I — continued. 463 Enzyme Source Enzyme Commission of Number Ribosomes References j3-Glucosidase 3.2.1.21 Yeast j3-GIucuronidase Glutamate dehydrogenase Guanosine triphosphatase 3.2.1.31 Calf thymus nuclei 1.4.1.- Calf thymus nuclei 3.6.1.- E. coli Pea Hauge, MacQuillan, Cline and Halvorson (1961), Kihara, Halvorson and Bock (1961), Kihara, Hu and Halvorson (1961) Wang and Wang (1962) Wang and Wang (1962) Conway and Lipmann (1964), Nishizuka and Lipmann (1965), Raacke and Fiala (1964) Raacke (1963) Lactate 1.1.1.27 Guinea pig Keck and Choules (1962) dehydrogenase kidney Guinea pig Keck and Choules (1962) liver NAD 2.7.7.1 Krebs-2 Ginzburg-Tietz, Kaufmann and pyrophosphorylase tumour Traub (1964), Kaufmann, nucleoli Traub and Ginzburg-Tietz (1964), Traub, Kaufmann and Ginzburg-Tietz (1964), Rat liver Ginzburg-Tietz, Kaufman and nucleoli Traub (1964), Kaufman, Traub and Ginzburg-Tietz (1964), Traub, Kaufmann and Ginzburg-Tietz (1964), Neuraminidase Penicillinase Peptidases Cysteinyl-glycine dipeptidase 3.2.1.18 Influenza virus 3.5.2.6 B. cereus Noll (1962) Duerksen and O'Connor (1963) 3.4.3.5 E. coli McCorquodale (1963) Rat kidney Binkley (1961) Swine kidney Binkley (1961) Iminodipeptidase 3.4.3.6 E. coli Elson (1961b, 1962b) 464 DAVID ELSON Table I — continued. Enzyme Source Enzyme Commission of Number Ribosomes References Leucine 3.4.1.1 aminopeptidase Peroxidase Phosphatases 1.11.1.7 3.1.3. Phosphodiesterases 3.1.4.1 Polynucleotide 2.7.7.8 phosphorylase Polyribonucleotide- synthesizing enzymes A. aerogenes McCarthy (1959), and cf. also Roberts, 1964 p. 370 B. megatherium McCarthy (1959) and cf. also Roberts, 1964 p. 370 E. coli Bohon and McCarthy (1 959) ; also cf. Roberts (1964, p. 365); Matheson (1963) Ps. fluorescens McCarthy (1959) and cf. also Roberts, 1964 p. 370 Pea Matsushita and Ibuki (1960b) Rabbit liver Matsushita and Ibuki (1960b) Wheat embryo Lanzani and Galante (1964) E. coli Spahr and Hollingworth (1961), Warren and Goldthwait (1961) Goat brain Datta and Ghosh (1963a) Rat kidney Binkley (1961) Swine kidney Binkley (1961) E. coli Goat brain B. subtilis E. coli Wade (1961), Wade and Lovett (1961) Datta and Ghosh (1963b) B. Benish and D. Bison, un- published Grunberg-Manago (1963), Kimhi and Littauer (1962), Wade (1961), Wade and Lovett (1961) Ps. aeruginosa Grunberg-Manago (1963), Strasdine, Hogg and Camp- bell (1962) St. aureus B. Benish and D. Elson, un- published E. coli August, Ortiz and Hurwitz (1962), Ortiz (1963) Landschutz ascites tumour nuclei Burdon (1963) Protease E. coli Chaloupka (1961) 7. RIBOSOMAL ENZYMES Table I — continued. 465 Enzyme Enzyme Source Commissioi 1 of References Number Ribosomes Beef pancreas Dickman, Holtzer and Gazzinelli (1962); Keller and Cohen (1961), Keller, Cohen and Wade (1963), Madison and Dickman (1963a) Guinea pig Siekevitz and Palade (1960a, pancreas 1962) Protease zymogens Chymotrypsinogen "Trypsin-activable protease" Trypsinogen Protein synthesis, enzymes of Ribonuclease Guinea pig pancreas Beef pancreas E. coli Rabbit reticulocyte 2.7.7.- A. aerogenes Apple leaves Beef pancreas B. mega- therium B. subtilis Calf liver Calf thymus nuclei E. coli Siekevitz and Palade (1958a, (1960b) Keller and Cohen (1961), Keller, Cohen and Wade (1963) Traut and Monro (1964) Arlinghaus, Shaeffer and Schweet (1964), Morris (1964) McCarthy (1959), cf. Roberts, 1964, p. 370 Kessler and Engelberg (1962) Dickman, Holtzer and Gazzinelli (1962), Gazzinelli and Dickman (1962), Keller and Cohen (1961), Keller, Cohen and Wade (1963, 1964), Madison and Dickman (1963a, 1963b), Siekevitz (1963) McCarthy (1959), cf. Roberts, 1964, p.370 B. Benish and D. Elson, un- published Siekevitz (1963) Wang and Wang (1962) Anderson and Carter (1965), Anraku and Mizuno (1965), Artman and Engelberg (1964, 1965), Beer, Highton and Mc- Carthy (1 960), Bolton (1 959b) (also, cf. Roberts, 1964, p. 362), 466 DAVID ELSON Table I — continued. Enzyme Source Enzyme Commission of Number Ribosomes References Ribonuclease (continued) Elson (1958, 1959b, 1961b, 1962b, 1964b), Elson and Tal (1959), Gesteland (1965), Jardetzky and Julian (1964), Lederberg, Rotman and Lederberg (1964), Lehman (1963), McQuillan (1961), Neu and Heppel (1964a,b,c,d), Shortman and Lehman (1964), Spahr and Hollingworth (1961), Spirin, Kiselev, Shakulov and Bogdanov (1963), Spitnik- Elsori (1962a), Tal and Elson (1963b), Thomas and Herbst (1963), Wade (1961), Waller (1964), Zillig, Krone and Albers (1959) Goat brain Datta, Bhattacharyya and Ghosh (1964), Datta and Ghosh (1963a, c; 1964) Guinea pig Martin, England, Turkington liver and Leslie (1963), Siekevitz (1963) Guinea pig Siekevitz (1963), Siekevitz and pancreas Palade (1958a, 1960b, 1962) Landschutz Burdon (1963) ascites tumour nuclei Pea Lett and Takahashi (1962), Matsushita and Ibuki (1960a), Ts'o, Bonner and Vinograd (1958b) Ps. fluorescens McCarthy (1959) and cf. Roberts, 1964, p. 370, Wade and Robinson (1963) Rabbit liver Stavy, Feldman and Elson (1964) Rabbit lymph node D. Elson, unpublished 7. RIBOSOMAL ENZYMES Table I — continued. 467 Enzyme Source Enzyme Commission of Number Ribosomes References Ribonuclease (continued) Rabbit spleen Rat Jensen sarcoma Rat liver Rabbit Elson (1964b), Farkas, reticulocyte Singer and Marks (1964), Mathias, Williamson, Huxley and Page (1964), Stavy, Feldman and Elson (1964), Williamson and Mathias (1963) D. Elson, unpublished Petermann and Pavlovec (1963a) Bosch, Huizinga and Bloe- mendal (1962), Madison (1962), Madison and Dickman (1963a), Petermann and Pavlovec (1963b), Roth (1960a, b), Stavy, Feldman and Elson (1964), Tashiro (1958) Rat spleen Wust and Novell! (1964) Rat uterus Greenman and Kenney (1963) Tobacco leaf Pirie (1950, 1957), Whitfeld and Williams (1963) Yeast Banner and Morgan (1963), Kihara, Halvorson and Bock (1961), McCarthy (1959) and of. 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Zipser, D. (1963). /. mol. Biol. 7, 739. Zipser, D. and Perrin, D. (1963). Cold Spr. Harb. Symp. Quant Biol. 28, 533. Chapter 8 THE SOLUBLE PHASE OF THE CELL^ NORMAN G. ANDERSON AND JOHN G. GREEN Molecular Anatomy Section, Biology Division^, Oak Ridge National Laboratory, and Technical Division^, Oak Ridge Gaseous Diffusion Plant, Oak Ridge, Tennessee U.S.A. I. Introduction .... II. Definition of the Soluble Phase . A. Classification of Components B. Interpretation of Results . III. The Composition of the Soluble Phase A. Micromolecules B. Mesomolecules C. Macromolecules IV. Enzymes of the Soluble Phase A. Enzyme Groups of the Soluble Phase B. Problems in Interpretation of Enzyme Data C. Control of Enzymic Activity in the Cell V. Physiology of the Soluble Phase .... A. The Effect of Heat B. The Effect of pH C. The Role in Membrane and Particle Formation VI. Conclusions References 475 476 477 481 483 483 484 491 496 496 497 499 500 500 501 503 503 504 I. Introduction The soluble or diffusing phase (the cell sap) is the main stream for the metabolic commerce of the cell and is the milieu in which cellular formed elements persist and function. Its composition reflects so much of cell biochemistry that a complete review of its role is no longer feasible. The 1 Work supported by the U.S. Atomic Energy Commission and by the Joint National Institutes of Health-Atomic Energy Commission Molecular Anatomy Program supported by the National Cancer Institute, the National Institute for Allergy and Infectious Diseases, and the U.S. Atomic Energy Commission. 2 Operated for the U.S. Atomic Energy Commission by the Nuclear Division of Union Carbide Corporation. 475 476 NORMAN G. ANDERSON and JOHN G. GREEN present chapter will therefore be concerned with a discussion of only a few salient aspects of the chemistry, physics, and physiology of this cell fraction. The soluble phase (SP) has been studied from several viewpoints which are difficult to interrelate. These may be grouped under physical and chemical analysis, immunoanalysis, enzymology, and physiology. Thus, the SP proteins may be grouped into ultracentrifugal or electrophoretic classes, may be studied as specific antigens, assayed for enzymic activity, or related to structural changes in cells. Unfortunately, rather few cytoplasmic proteins have been studied from each of these viewpoints. In examining a cell fraction which is related to such a wide spectrum of interests we shall adopt the following negative viewpoints and then examine them in the light of the available experimental results. 1. The SP is an intractable, irresolvable mixture including hundreds and perhaps thousands of substances. 2. Any given physical or chemical property is randomly distributed among these constituents. When examined by any physical or chemical separation method, essentially Gaussian results will be obtained. 3. (As a corollary to 1 and 2) Any substance isolated from the SP will, on more refined analysis, be found to be a mixture. Results which run contrary to these postulates must therefore indicate that the constituents of the SP may ultimately be reduced to some degree of order. II. Definition of the Soluble Phase The soluble phase is defined operationally as the supernatant remaining after the sedimentation of all particulate material. The definition applies in vivo to centrifugally stratified cells (Zalokar, 1960) and in vitro to centri- fuged cell homogenates. The central question is: What relation does the isolated soluble phase bear to the interparticulate fluid of the intact cell ? Any consideration of this question must deal first with the problem of whether particulate and non-particulate (soluble) cell constituents can be sharply defined (i.e., are two mutually exclusive sets of entities) and whether any interchange between these two sets occurs before, during, or after cell disruption. If, in a cell brei, there exists a sedimentation continuum (i.e., particles of all sedimentation coefficients are present) then the soluble phase can only be defined arbitrarily. However, if a discontinuity is observed in a plot of sedimentation coefficients vs mass in a brei, then a rational basis for both the definition and the separation of the soluble phase exists. For the most part the constituents of the soluble phase may be considered to be single "molecular" entities. However, numerous soluble proteins are known to be composed of covalently linked chains held together, in many instances, by secondary valencies. 8. THE SOLUBLE PHASE OF THE CELL 477 A few large complexes have been described in cells including, for example, the 19S thyroglobulin (Salvatore, Vecchio, Salvatore, Cahnmann, and Robbins, 1965) the 36 S a-ketoglutarate dehydrogenase CMukherjee, Matthews, Homey, and Reed, 1965) and 58 S pyruvate dehydrogenation complex (Koike, Reed, and Carroll, 1963) from E. coli. There is no reason to believe that other complex micro-organelles may not be found. Indeed, many of the "soluble" enzyme systems may exist in the form of labile aggregates in the cell. Part of the responsiveness of cells to alterations in the external environ- ment may be due to the reversible formation of such aggregates and may give rise to changes in the consistency or viscosity of the cytoplasm (Gross, Philpott, and Nass, 1960), a view supported by the experimental demon- stration of particle formation in soluble phase preparations (Anderson, 1959). I A. CLASSinCATION OF COMPONENTS The weights in daltons of a number of cell constituents are given in Fig. 1 These are taken from convenient sources. The plot suggests that the soluble Human cells Mitochondria I0'° 10" I0'2 I0'2 10"^ 10'^ 10'® 10' Weights of cells and cell constituents in daltons Fig. 1 . Plot of molecular weights of classes of particles found in cells and of cells them- selves. Note that few substances in the region of 5 x 10^ Daltons have been described in cells. Microsomes are omitted since analytical data on individual particles are not available. phase may consist of at least three general subfractions which are : 1. Micromolecular subfraction, including water, inorganic ions, and dis- solved gases. . 478 NORMAN G. ANDERSON and JOHN G. GREEN 2. Mesomolecular subfraction, including all metabolic intermediates, lipids, sugars, nucleotides, nucleotide derivatives, and other low-information- content molecules. 3. Free macromolecules. Chiefly proteins and RNA (including sRNA). The smallest well-defined particle which is generally described in electron micrographs is the 70-80 S ribosome. The ribosomal subunits (30-40 and 50-60 S) do not appear to exist in appreciable quantities in most cells. In vaccinia infected HeLa cells about 10% of the ribosomal mass appears as free subunits (Joklik and Becker, 1965). Since most studies with the soluble phase are done with the Spinco No. 40 rotor (or its equivalent) operating at 40,000 rev/min for one hour, it is of interest to consider what would be sedimented under these conditions. Neglecting the accelerated removal of particles by collision with the wall of an angle head rotor tube, the smallest particle completely sedimented according to the simple equation: — , 1 In jRmax — In Rmln where Ts = time in seconds S = sedimentation coefficient to = angular velocity in radians per second R = radius would be 120 S. Smaller particles are sedimented to a lesser degree, and the pellet is enriched with all sedimenting molecules, however small, to some extent. Under these conditions in an angle-head rotor, the majority of 80 S ribosomes would be sedimented. However, if the distribution of an enzyme known to be associated with ribosomes is being studied, the separations should be made in a zonal ultracentrifuge (Anderson, 1966) where the ribo- somes and ribosome subunits can be quantitatively removed from the soluble phase. As shown in Fig. 2, however, with rat liver a surprisingly flat plateau is reached after about 20 minutes at 105,000^. This suggests that if a continuum of sedimentation coefficients exists, it must reach a very deep minimum at some point to give the plateau observed. It might be thought that the amount of material having sedimentation coefficients in the 100-1000 S region could be determined using the analytical ultracentrifuge. This instrument is not well adapted to the study of hetero- disperse preparations, however. For example, if 5-10 % of the cell mass was distributed uniformly over the range 20-1000 S, it would be difficult to detect, i.e., only a small shift in base line would be observed. The zonal ultracentri- fuge (Anderson, 1966) is better suited to this problem since both preparative and analytical studies may be done in the same rotor on one experiment. It is emphasized here that the relation of the isolated fraction to the diff'usible 8. THE SOLUBLE PHASE OF THE CELL 479 I0< 9- 8 7- B c 5 o ■^ 4- z 3- 2 1 \ 15 30 60 Time ot 105,000 ^(min) 120 Fig. 2. Sedimentation of rat liver brei nitrogen as a function of time in a Spinco No, 40 angle head rotor (Anderson, 1956a). sap of the intact cell remains to be elucidated. However, as will be diseussed in a subsequent section, rather simple techniques are available for studying interactions between the formed elements of the cell and the cell sap. It is concluded that experimentally the soluble phase is a fairly well defined entity which may be easily and reproducibly isolated. We may ask whether the SP is resolvable experimentally into the subgroups suggested by Fig. 1, The results obtained with rat liver SP on Sephadex columns suggest that it is. Very little ultraviolet absorbing material is observed in the range between the macromolecular and mesomolecular peaks. Salts and micromolecules or ions are also a fairly discrete group. The soluble phase is therefore resolvable into rather well defined subgroups on the basis of molecular size. Unfortunately, preoccupation with studies on the distribution of substances and activities between centrifugally isolated fractions has obscured the fact that the first question to be settled is simply this : is the substance of interest bound to particles or not? Since complex isolation procedures obviously may give rise to artifacts, it would appear wise to determine the distribution between the sediment and the supernatant in the shortest possible period of time. Subcellular particles are more likely to be in a physiologically native state in the original homogenate than in any subsequent isolation medium. We therefore require very simple and extremely rapid methods for homo- genizing tissues, sedimenting particulate material, and (when required) freezing the supernatant. This is especially true when labile metabolic inter- mediates are being studied. In addition, simple particle-supernatant distribu- 480 NORMAN G. ANDERSON and JOHN G. GREEN tion studies done with breis containing different amounts of tissue can give clues to adsorption or (more likely) desorption. Thus if, over a wide range of homogenate tissue concentrations approximately the same percentage of an enzyme is found in the SP, then the curve may be extrapolated to 100 % tissue (i.e., to the intracellular state) with some confidence as shown in curve A in the illustrative diagram in Fig. 3. However, if the percentage in the super- natant increases on dilution (curve B) then the activity is loosely adsorbed on surfaces in the cell. lOOn 80- o 60 40 20- 100 80 60 — r— 40 — T" 20 % Tissue in homogenote Fig. 3. Diagramatic representation of changes in enzymic activity found in the soluble phase as a function of tissue concentration in the homogenate. Interpretation of curves described in text. If the percentage decreases on dilution (curve C) altered surfaces of sub- cellular components produced as artifacts of homogenization are removing previously soluble materials from solution. An example of a study of this type is shown in Fig. 4 where acid soluble nucleotides of the SP (expressed in terms of /^moles of adenylic acid per gram of liver) are plotted against the percentage of liver in a brei. It is evident that appreciable changes in the nucleotide composition of the SP occur when a homogenate is diluted. Unfortunately, studies of this type have rarely been done, and the chief contribution of this chapter is to indicate in some detail the present un- satisfactory state of research in this area. An additional problem arises from the rapid turnover rate and lability of many of the compounds found in the soluble phase and their effect on the stability of some of the proteins found in it. In studies on brain metabolites it has been found necessary to freeze the tissue very rapidly, and to make acid extracts of frozen powdered tissue as thawing occurs (Lowry, Passoneau, Hasselberger, and Schulz, 1964). Marked changes were observed, especially 8. THE SOLUBLE PHASE OF THE CELL 481 in the ATP level, if circulation was interrupted for a few seconds. It is obvious that very large changes in the metabolite composition of the soluble phase may occur during the isolation procedures presently in use. I-6- 1-5 1-4 10 20 10 Percentoge of liver in brei — I 2-5 iFiG. 4. Ultraviolet absorbing material from supernatant after centrifugation at 105,000^ for one hour of liver breis of different concentration. Results expressed as equivalent [amount of adenylic acid per gram of liver. B. INTERPRETATION OF RESULTS For many comparative purposes the method used to tabulate analytical results is of little consequence. However, if we desire to develop concepts concerning the composition of the cell as a functional unit then the results might be presented in terms of the number of molecules found in a cell. Unfortunately rat liver, which is the most widely studied tissue, contains many binucleate cells. For this reason, it is suggested that results be presented in terms of a 1 N cell mass equivalent. For a diploid cell this number would be doubled; for a tetraploid cell it would be quadrupled to give the quantity present per cell. Table I lists the number of molecules of several substances per 1 N [liver cell mass. For B12 only 25,000 molecules are present. The concentration i^hich a substance present in a tissue would have when only one molecule is [present per cell (the limiting molarity) is given by the equation 1015 1-66 X 10-» Mmm = ^.Q23 X 1023 xVc" Vc 482 NORMAN G. ANDERSON and JOHN G. GREEN Table I Number of molecules of various compounds in 1 N liver cell mass equivalent^ Inorganic phosphate 4-8 x 10* Glycerophosphate 1-2 x 10' AMP 4-5 X 108 ADP 2-5 X 10» ATP 11 X 109 NAD+ 4-2 X 108 NADH 2-3 X 108 NADP+ 60 X 106 NADPH 21 X 108 Glucose- 1 -phosphate 4-5 x 10''' Glucose- 1 :6-diphosphate 9-8 x 10^ Uridine diphosphoglucose 1-1 x 108 Vitamin A 2 x 108 Vitamin C 9 x 108 Vitamin E 3-8 x 10^ Thiamine M x 10' Riboflavin 60 x 10' Nicotinic acid 1-2x109 Biotin 3 0 x lO^ Choline 7-9 x lO^ Inositol 2-7 x 10« Bi2 2-5 X 104 ^Data from Biochemists Handbook, C. Long, ed., D. Van Nostrand Co., Inc., Princeton, New Jersey (1961). Calculations based on the assumption that 1 N DNA = 3 picograms, and that 100 mg of fresh tissue contains 208 ng DNA. where Mmin is the limiting molarity and Vc is the cell volume in cubic microns. For an 800 fj.^ cell the limiting molarity would be 2 x 10~i2 when one mole- cule is present per cell. For an amino acid with a molecular weight of 120, 2-4 X 10~i^ grams would be present in a kilogram of cells. This quantity is well below the limits of present detection methods. The interesting question arises, is a single molecule of any substance except DNA or viral RNA functionally important in a cell? If so, then present lists of the total number of compounds present in cells may be very far from complete. A second point regarding presentation of data concerns the possibility of constructing a balance sheet of cell constituents. Does the sum of the known acid soluble nitrogen-containing compounds equal and account for the total acid-soluble nitrogen found ? What percentage of soluble cytoplasmic proteins are accounted for by known enzymes? Until such balance sheets are con- 8. THE SOLUBLE PHASE OF THE CELL 483 structed it is difficult to determine what work remains to be done, and whether, as is the case in blood plasma, a large fraction of proteins are present which have functions other than cnzymic ones. III. The Composition of the Soluble Phase The soluble phase contains between 23-67 % of the total nitrogen, depend- ing on the tissue and the procedure used (Reid, 1961). The percentage in rat liver is 43% of which 87-8% is non-dialysable (Anderson, 1956a). While the concentration of a large number of substances in liver and other tissues is known (Long, 1961), with the exception of enzyme activities the percentage of each found in the soluble phase is known in only a few instances. Since many reviews listing cell fractionation results have appeared (Roodyn, 1965; de Duve, Wattiaux, and Baudhuin, 1962; Claude, 1948; de Duve and Berthet, 1954; Hogeboom, Kuff, and Schneider, 1957; and Roodyn, 1959) the distri- bution of enzymic activities between cell particles and the soluble phase will not be tabulated in detail here. A. MICROMOLECULES The inorganic ion content of cells'reflects the balance between rate of gain and rate of loss of individual ions (Ussing, 1960; Rothstein, 1961). Analytical data on inorganic substances has been reviewed (Long, 1961 ; Ussing, 1960), and the distribution of ions between plasma and tissue cells examined. Monovalent ions are thought to be largely free in the cell, while divalent cations such as magnesium and calcium appear to be chelated by nucleic acids and acid polysaccharides. Since the intracellular ion composition reflects that of the external medium, the effect of the contents of the extra- cellular spaces in tissues used in cell fractionation must be considered. If perfusion is used to remove blood, then the ionic composition, or the lack of ions when sucrose is used for perfusion, may cause changes in intracellular /^ ionic composition. In addition, the degree of binding of inorganic ions to subcellular particles is concentration dependent, i.e., there appears to be an equilibrium between bound and unbound calcium, for example. Dilution during homogenization may be expected to disturb such equilibria. In addition, selective accumulation of ions in subcellular particles may occur ; for example, K+, Na+, Ca2+, and Mg2+ levels are maintained or increased in isolated mitochondria under conditions where oxidative phosphorylation is taking place. Since this is dependent on the physical state of the mitochondria and the supply of ATP (Lehninger, 1964) alterations during or after homo- genization may produce either leakage or additional uptake. Studies on the inorganic ion composition of the soluble phase should therefore be plotted 484 NORMAN G. ANDERSON and JOHN G. GREEN both as a function of tissue dilution and of time after cell breakage, and should take into consideration the amount of extracellular fluid included in the SP. Since subcellular particles exist in the SP originally, we would Uke to devise an isolation medium which approximates to the SP in its composition and effects on subcellular components. Solutions containing the same salts found in ashed cells are notoriously poor isolation media, whereas the most successful ones are ion free. In fact, the SP of the intact cell may contain only traces of diffusible inorganic anions, chiefly the bicarbonate ion, while the remainder of the inorganic anionic sites appear to be fixed. Both chloride and free orthophosphate are present in extremely low concentrations in liver cells, the diffusible anions being chiefly low molecular weight organic acids. The problem of approximating to the SP with a solution in which the sub- cellular particles remain freely suspended and in which these particles experi- ence the same ionic environment found in the cytoplasm remains to be solved. The hydrogen ion concentration of the cell (and therefore of the SP) has been extensively reviewed (Wiercinski, 1955; Caldwell, 1956; Bittar, 1964). The pH of soluble phase in the cell in mo:t mammahan tissues appears to be very close to or slightly less than 7-0. Since at pH 7-0, one cubic micron con- tains approximately 60 hydrogen ions, local variations in pH in cells highly compartmentalized by cytomembranes is probable. The soluble phase as usually isolated in sucrose from rat liver has a pH of 6-45 (Anderson, Makinodan, and Norris, 1961). The pH of the SP of mammalian tissues needs to be re-examined in very rapidly prepared samples, (e.g. after 20-30 sec.) B. MESOMOLECULES Cellular mesomolecules are generally grouped by the analytical techniques used for their analysis. Most group analyses depend on sensitive separation techniques coupled to sensing systems which measure the concentration of a chemical group, element, or may measure total mass. Thus, automated analytical systems exist for amino acids and ninhydrin-positive compounds (Spackman, Stein, and Moore, 1958; Piez and Morris, 1960; Hamilton, 1963), nucleotides and ultraviolet absorbing derivatives (Anderson, Green, Barber, and Ladd, 1963; Green, Nunley, and Anderson, 1966) and sugars (Green and Anderson, 1965; Green, 1966). The detectors used in gas chroma- tography may be sensitive to mass, or to specific groups or elements (Juvet and Nogare, 1964; Giuffrida, 1964). Pre-fractionation or selective volatiliza- tion often allow known classes of substances to be studied. In the case of glycolytic intermediates specific fluorometric techniques may be preferable because of their extreme sensitivity (see Lowry and Passoneau, 1964), although chromatographic methods are available for their analysis (Schmitz and Walpurger, 1959). * 8. THE SOLUBLE PHASE OF THE CELL 485 /. Nucleotides and Related Compounds A large number of nucleotides and nucleotide derivates have been reported in acid extracts of mammalian tissues (Tunn and Dumazert, 1962; Gregoire, 1958; Henderson and LePage, 1958; Potter, 1958; Strominger, 1960; Brady and Tower, 1960; Mandel, 1964). The distribution of nucleotides among cell fractions, however, has been less frequently studied. The problem again is the rapid breakdown of the triphosphates during the centrifugal separations. The rate at which this may occur is illustrated by the studies of Lowry et al. (1964) who followed the change in ATP and glycolytic intermediates with time in ischemic brain. It is evident from their studies that marked technical advances will be required before the SP can be isolated without appreciable change in the nucleotide triphosphate content. The nucleotides found in tissues are not catalogued here; however, a summary of their functions is given in Fig. 5. Automated high-resolution chromatographic systems for nucleotides, nucleosides, and purine and pyrimidine bases (Anderson, et al. Control AMP Various Metabolic Cycles ■«- ATP Intracellular Membranes UDP Polysaccharides -«—— Coenzymes - ^ Phosphatides -<- Polysaccharides *- - CD? - Coenzymes - GDP Coenzymes -UTP — CTP RNA GTP Protein Synthesis-*- FiG. 5. Summary of the roles of the nucleotide di- and triphosphates in different metabolic cycles (From P. Mandel, 1964) 1963; Green, et al. 1966) will allow a larger number of these substances to be determined in tissue fractions than has been the case heretofore. The key role of adenylates as intracellular regulatory effectors has recently been reviewed by Atkinson (1965). The chromatography of extractable nucleotides has recently been reviewed by Saukkonen (1965). 2. Metabolic Intermediates No complete study of metabolic intermediates in rapidly prepared SP has appeared. It is evident that the levels of metabolic intermediates of the glycolytic pathway found in the soluble phase reflect not only the condition of the cells at the moment of rupture, but the metabolic changes which occur 486 NORMAN G. ANDERSON and JOHN G. GREEN during preparation of the fraction. It is probable, however, that tissue ana- lyses for glycolytic intermediates such as the fluorometric procedures used by Lowry and Passoneau (1964) reflect the concentration of these substances in the SP. The glucose level of the SP of the intact cell has a profound effect on the glycolytic pathway. In Ehrlich ascites tumor cell homogenates the glycolytic activity is controlled by the glucose supply at concentrations below 1-2 mM, although anaerobic glycolysis is already maximal at 0-2 mM glucose (Wu, 1965a). Under aerobic conditions inhibition of phosphofructo- kinase (2.7.1.11) occur after a short interval. Orthophosphate stimulates both aerobic and anaerobic glycolysis and phosphofructokinase. The effects of the intracellular inorganic phosphate level appear to be mediated through hexokinase, (2.7.1.1) phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase (1.2.1.9) in a co-ordinated manner. The effect of Pi on phosphofructokinase may be mainly responsible for the Pasteur effect (Wu, 1965b). Automated chromatographic systems for the glycolytic and Krebs' cycle intermediates which extend and make quantitative presently available techniques (Passera, Pedrotti, and Ferrari, 1964; Stern, Gramet, Trabal, Mennis, and Zinsser, 1965; Schmitz and Walpurger, 1959) are urgently needed. 3. Sugars While the glucose level of tissues has been repeatedly studied (Long, 1961) sensitive quantitative methods for all the sugars which might be found in mammalian tissues have not previously been available. With the development of automated carbohydrate analysers (Green and Anderson, 1965; Green, 1966), this problem can be examined in detail using media other than sucrose for the isolation of the soluble phase. 4. Amino Acids (and Related Compounds) Amino acids are probably the most studied mesomolecules of the cell. This volume of experimental effort has resulted largely from the availability of simple analytical techniques such as paper and column chromatography and the use of simple extraction procedures which yield reproducible results. A complete review and discussion of the vast body of literature available is not within the mission of this article, and when one applies rigidly the defini- tion of the soluble phase much of the Uterature is not applicable. Enough information is available, however, to permit discussion of the problems involved in the study of amino acids in the soluble phase (SP). For additional information the reader is referred to extensive reviews on the various aspects of amino acid research including analytical methodology (Light and Smith, 1963), occurrence in tissues (Christensen, 1964; Roberts and Simonsen, 1960; Mesister, 1965) and the pool concept (Holden, 1962). (a) Current problems — Although numerous technical problems have been met and solved in the field of amino acid biochemistry, only the problems of i 8. THE SOLUBLE PHASE OF THE CELL 487 terminology, sample preparation, and data interpretation will be considered here, since these problems relate directly to the consideration of amino acids in the soluble phase of the cell. Generally, amino acids from natural sources are placed in two classes with respect to the manner of isolation. "Free" amino acids occur in extracellular fluids such as sera, urine, or culture media, or can be removed from cells and tissues by mild extraction procedures with such materials as hot or cold water, alcohol solutions or dilute acids, Amino acids which are not extracted easily are classified as "bound" amino acids. We propose to revise the ter- minology used as follows : Free amino acids; This classification would be restricted to amino acids derived from clearly extracellular sources. Derived terms include free pep- tides, free amino sugars, and free amino acids (FAA); however, the abbrevi- ated form is the same one designating a commonly used fixative. Extractable amino acids; This classification would include amino acids obtained by extraction of cells and tissues and includes much sample material now called free amino acids. Derived terms include modifiers depicting the extractant, e.g., acid-extractable ; application of the term to other amino compounds, e.g., extractable peptides; and the abbreviated form, extractable amino acids (EAA). The term "easily extractable" amino acids or an equiva- lent form has appeared with increasing frequency in recent literature to define accurately the fraction within the designation of free amino acids. Soluble phase amino acids; This arbitrary classification would be used to designate amino acids obtained by sampling techniques directed specifically toward obtaining materials identical to their state in vivo. Derived terms include such items as soluble phase peptides, as well as the abbreviated form soluble phase amino acids (SPAA). Bound amino acids; This category includes amino acids associated with other materials such as protein in a manner preventing their appearance in other fractions. Derivatives include such terms as protein-bound, or lipid- bound amino acids or peptides, and it may be desirable to designate whether the compounds are intracellular or extracellular. This or similar classifications present some inadequacy. For example, it is conceivable that amino acids associated with subcellular particles may be "bound" during isolation of the particle yet be extractable in subsequent procedures. Nevertheless, the system permits more exact description of experimental work and facilitates evaluation of the vast literature on the basis of title alone. Comparable terminology may be used for other low molecular weight substances such as nucleotides, sugars, and metabolic intermediates. Experimentation has not established the true nature of intracellular ex- tractable amino acids. Recent studies, for example, have shown that (a) very rapid post-mortem, pre-extracting changes occur in tissues which may be 488 NORMAN G. ANDERSON and JOHN G. GREEN modified by techniques involving freeze quenching, and (b) the proportions of free and bound amino acids in homogenates of brain and other tissues vary with pH, ionic composition and temperature of the extraction medium (De Ropp and Snedeker, 1960; Stephen and Steinbauer, 1963; Mitchell and Simmons, 1962; Kolousek, Jiracek and Cizinsky, 1964; Elliott, Khan, Bilodeau and Lovell, 1965). Evidence has also been presented indicating that in crustacean neural tissue glutamic and aspartic acids appear to exist in a physiologically inactive form and that in the case of aspartic acid the physi- ologically active form is very unstable (Florey, 1964). The problems involved have been ably discussed by Holden (1962). Some attempts have been made to apply methods involving cryological techniques to the study of cell constituents to preserve their true state during sample preparation. They include freezing, sectioning, and extraction of the tissue (Tee, Wang and Watkins, 1964), freezing, pulverizing, and centrifugal extraction (Kolousek, et al., 1964), centrifugation and freezing of homo- genates in hematocrit tubes (Wittgenstein and Rowe, 1965), and freeze quenching, sectioning, and extraction at subfreezing temperatures (Bucher, Krejei, Russman, Schnitger and Wesemann, 1964). The results of applying such techniques to the study of the amino acids in cells have not yet appeared in the literature. Another approach to the problem which has not yet been applied involves the sampling of material from living cells by microsurgical techmques (Chambers and Chambers, 1961). In one such study, human tumor cells were stratified by centrifugation but only particulate materials were withdrawn by micropuncture for further study (Mateyko and Kopac, 1964). In similar studies Neurospora hyphae (Zalokar, 1960) or Paracentrotus eggs (Pasteels, Castiaux and Vandermeerssche, 1958) were centrifuged and subjected to cytochemical testing, but here, too, no effort was made to assay mesomolecular cellular components. In the case of amino acids, the correla- tive problem of minute samples has been reduced by the development of techniques for quantitative analysis of concentrations as small as 10 -^i molar (Hamilton, 1962). SampUng techniques are only part of the problem of determining amino acids in the soluble phase of the cell. The thoroughness of extraction tech- niques has been rarely checked. In potato tissue, amino acids are removed selectively, repeated extraction is required for quantitative removal, and the degree of difficulty and order of selectivity of extraction varies with different tissues (Talley, Carter and Porter, 1958). Although major findings have been obtained through the use of paper chromatography, the procedure lacks precision. In one example, paper chromatography showed the existence of 5 or 6 peptides in homogenates of larval and rabbit brain tissue but column chromatography yielded over 600 peptides and amino derivatives in the same tissues (Mitchell and Simmons, 1962). In another example, findings from paper chromatography agreed poorly with data obtained from column 8. THE SOLUBLE PHASE OF THE CELL 489 chromatography and microbiological assay, although the results with the latter two techniques were in close agreement (Dunn, Sakamoto, Sutaria and Murphy, 1963). In spite of the promise of new and improved techniques, the state of intracellular amino acids has not been adequately described. The question is, what, of the vast amount of data found in the literature, is applicable to this problem? It is logical to conclude that although much data may be subject to reinterpretation in the light of subsequent findings and discoveries yet to come, certain concepts and facts have been derived which will condition future study and interpretation of the soluble phase. Therefore, it is appro- priate to consider some of these concepts including analytical data, the pool concept and the discovery of complex forms. (b) Important findings — The vast literature giving extractable amino acid con- tents of tissues will not be reviewed here. Reviews listed previously contain many such tabulations and additional sources are widely distributed (see e.g.. Biochemist's Handbook). However, certain general observations merit cita- tion. It has been reported that extractable amino acids exist in a concentration of 15-30 mM in tissues and that 60-120 times that quantity is bound as |^b>rotein (Christensen, 1964). It has been found that the number of easily ^^^xtracted amino acids in tissues considerably exceeds the number found in proteins (Greenstein and Winitz, 1961 ; Steward and Pollard, 1962). In fact, it has been noted that new amino acids are being reported at the rate of ten a year; whereas the spectrum of amino acids found in proteins has not changed in 25 years (Meister, 1965). Some stable complexes of amino acids have been isolated by extraction procedures. In Escherichia coli covalently bonded RNA-amino acid complexes constitute about 5 % of the extractable amino acid pool (Lacks and Gros, 1959), and acid soluble cytidine nucleotide linked amino acids have been reported from rabbit liver extracts (Agren, 1960). Amino acid-phospholipid complexes have been isolated from ascites tumour cells (Bailey and Woodson, 1963), and lipid-bound amino acids have been recovered from extracts of broad bean leaves (Brady, 1964), and from hen oviduct (Hendler, 1961 ; Hendler, 1962). A very high turnover rate has been observed in the latter fraction (Hendler, 1962). Perhaps the most important principle evolved in the interpretation of extractable amino acids is the concept of intracellular amino acid pools. Although the subject has been extensively reviewed (Holden (ed.), 1962) it is appropriate to consider several aspects of this concept here. The amino acid pool is operationally defined to include the quantity of low molecular weight I material which is extracted from the cell under conditions which exclude the Contribution of material degraded from macromolecular constituents (Britten knd McClure, 1962). The principal features of the amino acid pool in E. coli, where it has been most extensively studied, are given by Britten and McClure 11962) as follows: 490 NORMAN G. ANDERSON and JOHN G. GREEN 1. Pool passage is an obligatory step for incorporation of extracellular amino acids into proteins. 2. Pool amino acids are incorporated at random into protein regardless of the length of time they have been in the pool. 3. An energy source is required for formation of a pool at normal rates but not for maintenance of the pool. 4. Specific pool formation mechanisms exist for each amino acid or structurally similar group. 5. For each amino acid, there exists a maximum pool size for saturated external concentrations. 6. Exchange between pool and external amino acids occurs at high rates when steady flow is occurring through the pool and when pool forma- tion is being suppressed. The dynamic nature of the pool is attested by the observation that in £. coli the pool is easily lost under conditions such as water washing and osmotic shock which do not cause cell death and that the pool in yeast actually increases under conditions such as exposure to ultraviolet radiation which induce leakage of nucleotides and amino acids from the cells (Siegel and Swenson, 1964). It has been suggested that maintenance of the pool including amino acid transport, binding phenomena and uptake is closely associated with alkahne phosphatase activity (3.1.3.1.) in rat brain (de Waart, 1964). The limitations of the pool concept are evident and some of them are given by Christensen (1964) as follows: 1. Cellular amino acids do not represent a functionally homogenous pool due to compartmentalization, e.g., mitochondrial pools. 2. The pool is not always diluted by amino acid precursors during in- corporation into bound forms. 3. The pool is defined on the basis of kinetic relationships rather than on the basis of physical barriers. 4. Undetected chemical differences such as labile complexes may exist between forms of an amino acid in the pool. Although many questions remain unanswered about the function, nature, and constituency of the amino acid pool, it is now the major vehicle for inter- preting the findings about easily extractable amino acids and future results will undoubtedly be interpreted in light of the pool concept. This section would not be complete without some consideration of the role of amino acids in the soluble phase. A comprehensive discussion of the biochemistry of amino acids has appeared (Meister, 1965) but some functions merit citation here. The role of amino acids in protein synthesis is well known. In addition, they may serve as substrates for the synthesis of peptides (e.g., glutathione) and related substances and, as respiratory substrates (via transamination), resulting in yields of ATP (Krebs, 1964). Considerable effort I I 8. THE SOLUBLE PHASE OF THE CELL 491 has been expended to associate extractable amino acid contents of tissue with osmoregulatory activity, especially in aquatic animals (Shaw, 1958; Jeuniaux, Duchateau-Besson and Florkin, 1961; Allen, 1961; Lange, 1963 and in bacteria (Kuczynski-Halmann, Avi-Dor and Mager, 1958). It has been suggested that lysine may replace lost intracellular potassium in maintaining cation-anion balance in potassium defficient tissue in Fundulus (Hanlon, 1960) and that glutamic and asparic acids may function as transmitter substances in crustacean nerve tissue (Florey, 1964). In plants, extractable amino acid and amide content has been correlated with cold hardening in winter wheat (Zech and Pauli, 1962). The great spectrum of amino compounds found in cells and tissues suggests that many more roles for them remain to be elucidated. Determination of the state of amino acids in the soluble phase of the cell in vivo presents a most pressing challenge. New approaches and new and refined techniques even now promise to solve this problem. C. MACROMOLECULES In this section macromolecules of the soluble phase will be considered from a physical, chromatographic, and immunochemical viewpoint. i. Proteins {a) Ultracentrifug alanalysis — Ultracentrifug alanalysis of mammalian tissue extracts reveals evidence for the existence of several classes of proteins. In rat liver three major and at least two minor electrophoretic peaks are distin- guishable (Sorof and Cohen, 1951a, b; Anderson and Canning, 1959). Schlieren patterns for rat liver, kidney, brain, and testis are shown in Fig. 6. The most important finding is that half or more of all the soluble proteins of brain, kidney, and liver have sedimentation coefficients between 3-4 and 4*2 S. In testis the amount of 4 S protein is slightly less, accounting for an average of 39-1 % of the total. The most characteristic differences between the various organs were in the amount and in the sedimentation coefficients of the more rapidly sedimenting material. The macroglobulin-like constituents of brain and testis deserve special attention since their concentration is much higher than in rat plasma. In rat liver, protein-bound amino azo dye derivatives of several hepato-carcinogens are found in the 3-6 S fraction (Sorof, Colder, and Ott, 1954). The lack of preparative centrifugal systems having a resolution compar- able to that of the analytical systems has made the task of determiningthe position of SP enzymes among the ultracentrifuge peaks difficult, although some progress has been made using swinging bucket rotors (Martin and Ames, 1961), most of which, however, use smaller samples than the analytical ultracentrifuge. With the development of higher speed zonal centrifuges I 492 NORMAN G. ANDERSON and JOHN G. GREEN 60S 9% Ultracentrifuge patterns of rat tissue soluble proteins Fig. 6. Ultracentrifugal patterns of soluble phase proteins from rat tissues (Anderson and Canning, 1959). the problem of identifying the members of the various sedimentation groups should be much easier. The findings of ultracentrifugal classes among tissue proteins indicates that size is not a randomly distributed property. This suggests that (a) tissue proteins fall into a number of general classes, (b) the peaks observed are caused by the presence of a disproportionately large amount of a few specific proteins that obscure a broad distribution of the majority of protein types, or (c) the observed patterns are a combination of the two. (b) Electrophoretic analysis — Electrophoretic studies on soluble phase pro- teins done before 1960 have been reviewed by Anderson and Swanson (1961). If a large number of proteins are present in low concentration, and if electro- phoretic mobilities are randomly distributed, then the electrophoretic pattern should also be a Gaussian curve. Examination of rat liver, brain, testis, and kidney soluble proteins in the Tiselius apparatus at pH 7-5 reveals the presence of distinct peaks, some of which are common to several tissues, and also characteristic differences between the preparations (Fig. 7). The presence of distinct peaks therefore suggests either (a) extensive complexing occurs in 8. THE SOLUBLE PHASE OF THE CELL 493 Serum Brain Testis n Testis I Kidney Liver +2 +1 -7 -£ 10 I -1 -2 -3 -4 -5 -6 Mobility (T units) Fig. 7. Electrophoretic analysis of soluble phase proteins of rat liver, kidney, brain, testis (I with, and II without tubular fluid), pH 7-5, 01 ionic strength. Rat serum proteins in- cluded for comparison (Anderson and Canning, 1961). solution giving rise to aggregates with distinctive average mobilities; (b) a few proteins are present in fairly high concentration and mask the random distribution of mobilities in the remainder of the proteins present, or (c) families of proteins with similar mobilities are present. Ultracentrifugal analyses tend to rule out (a). The choice between (b) and (c) cannot as yet be made. Inter-organ differences are indicated both by the shape of the Schlieren pattern and by the differences in median mobihties (liver, -2-0; kidney, -3-0; testis, -4-1; and brain, -5-2). A peak of low positive mobility is present in liver and kidney and almost totally absent in testis and brain. The location o catalase (1.11.1.6), rhodanese (2.8.1.1), acid phosphatase (3.1.3.2), nucleoside phosphorylase, esterase, adenosine deaminase (3.5.4.4), glutathione reductase (1.6.4.2), and /3-glucuronidase (3.2.1.31) in the electrophoretic pattern have been determined (Anderson, Fisher, and Anderson, 1961). The hypothesis that nuclear volume is related to the level of slightly basic cytoplasmic proteins present has been proposed (Anderson and Swanson, 1961 ; Swanson, 1960a). If this is correct, cells with large nuclei should have Y* 494 NORMAN G. ANDERSON and JOHN G. GREEN proteins which are more acid (more rapidly migrating electrophoretically) than cells of the same organism with small nuclei. The comparative experi- ments in rat tissue, and electrophoretic analysis of oocyte SP and its com- parison with embryonic extracts supports this view. In addition, evidence has been presented that the nuclear globulins are the more basic proteins of the soluble phase (Barton, 1960). The view has been expressed in some detail (Anderson, 1956b) that DNA is functional in an extended state, and non- functional in a condensed condition and has given rise to recent studies linking histone to repression of genetic expression. If nuclear volume of a somatic cell reflects the sum of the volumes of the genes, then factors con- trolling nuclear volume may be important in differentiation and gene control (Swanson, 1960b). Radioautographic evidence has been presented by Gold- stein (1963) and Prescott and Bender (1963) for the existence of a protein which is predominantly locaUzed in the nucleus during interphase, is uniformly distributed throughout the cell during division and returns to the nucleus after telophase. In addition, nuclear transplantation studies have shown that a nucleus transplanted into an enucleate host assumes some of the characteris- tics of the host (Lorch and Danielli, 1950) further supporting the view that the cytoplasm may produce physical changes in the nucleus, presumably through the mediation of slightly basic soluble cytoplasmic proteins. It is of interest in this regard that the hormonally-controlled "male" protein is among the sHghtly basic proteins of the soluble phase (Bond, 1962). Electrophoresis in starch (Smithies, 1962) or acrylamide gels (Raymond and Weintraub, 1959; Ornstein, 1964) depends on the electrophoretic mobility of the proteins being studied and their interaction with the stationary medium (Tombs, 1965). The result is a very high resolution separation which has been widely used to demonstrate isoenzymes and to evaluate purity of isolated protein preparations. As the resolution of this method is improved and preparative counterparts developed, the problem of identifying each of the bands observed may be approached. (c) Chromatographic analysis — In addition, chromatographic studies of sol- uble proteins on ion exchange media have been widely done as part of enzyme purification procedures. However, the initial extracts are often made in ways which do not minimize damage to subcellular components. In addition, interest in isolation leads to the development of isolation methods giving maximum purification of the enzyme in question and not to techniques for achieving the maximum resolution of the entire mixture. The latter problem has been studied in detail by Bond (1965) and an elution program for high resolution separation of rat liver SP on DEAE columns developed. This work led to the discovery of a protein in male rat liver which is absent or present in only very small amount in female liver (Bond, 1960, 1962; Barzilai and Pincus, 1965). It is induced in both sexes by androgens and repressed by estrogens. Its function is unknown. 8. THE SOLUBLE PHASE OF THE CELL 495 The detailed analysis of the distribution of enzymes of the SP by high resolution DEAE chromatography and by gel filtration remains to be done. With the introduction of low cross-linked beads, filtration through columns of particulate gels (Flodin, 1962) has been extended to the separation of large molecules. (d) Immunological analysis — Immunochemical and immuno-electrophoretic analyses of soluble phase antigens of normal and tumour tissues have been reported by many workers, (e.g. Perlmann and Hultin, 1958; Perlmann and D'Amelio, 1958; Perlmann, Hultin, D'Amelio, and Morgan, 1959; D'Amelio and Perlmann, 1960; Gazzaniga, Di Macco and Sonnino, 1963; Rossowski, Weinrander, and Hierowski, 1963; Baldwin, 1964). The results support the view that in liver a relatively small number of proteins are present in rather high concentration against a background of many which account for the biochemical versatihty of the organ. A minimum of 5 immunologically distinct non-plasma proteins can always be recognized in rat liver soluble phase (D'Amelio and Perlmann, 1960). The "h" protein described by Sorof (Sorof, Young, and Ott, 1958), is one of these, and is also found in liver microsomal fraction. A small number of common antigens occur in the deoxycholate extract of microsomal fraction and the cell sap. Several soluble phase antigens are deleted from liver SP during aminoazo dye carcinogenesis, while one antigen was shown to increase fourfold in the tumour (Baldwin, 1964). An increase in the number of soluble antigens in liver from the eighteenth day of intrauterine life to three months after birth has been demonstrated (Rossowski, et al., 1963). Using immuno-electrophoresis 7 to 10 lines are observed with rat liver soluble phase. In regenerating liver three of the Unes were frequently absent, one was always absent, three showed no change, and two increased in density (Gazzaniga, et al., 1963). The correla- ion of immunologically identified proteins with fractions isolated by centri- ugation, electrophoresis, and chromatography and with enzymic activities known to occur in the cell is urgently needed. 2. Lipids and Lipoproteins Studies on cell lipids have generally started with extracts of either whole tissues or isolated cell fractions excluding the fatty layer seen when whole breis are ultracentrifuged, or have utilized the surface lipids obtained after high speed centrifugation of whole homogenates. Evidence for a spectrum of particles having flotation rates sufficient to cause them to move centri- petally has been repeatedly obtained in the zonal ultracentrifuge. The centrifugal techniques for the isolation of plasma-lipoproteins (Lindgren, Elliott, and Gofman, 1951; De Lalla, Elliott and Gofman, 1954; Gustafson, Alanpovic, and Furman, 1965; Bobbitt and Levy, 1965) are also applicable to the soluble phase but thus far do not appear to have been used. 496 NORMAN G. ANDERSON and JOHN G. GREEN 3. Ribonucleic Acids (a) Separation ofRNAs — While amino acyl transfer RNAs (sRNA or tRNA) are largely located in the soluble phase, not all SP RNA appears to be active sRNA, Isolation of the soluble phase or at least removal of the major fraction of particulate material is generally the first step in sRNA isolation. The per- cent of total cell RNA found in the soluble phase varies widely depending on the tissue used, its physiological state, and the isolation methods em- ployed. Values ranging from as little as 5% to as much as 71 % of the total tissue RNA have been recorded (Reid, 1961). With the development of methods using the zonal centrifuge (Hastings, Parish, Kirby, and Klucis, 1965), and gel electrophoresis (Richards, Coll, and Gratzer, 1965) for separa- ting RNA according to size, the question of the nature of soluble phase RNA may now be re-examined. The number of species of sRNA present in a tissue is larger than the number of amino acids found in proteins (Morton and Rogers, 1965) making the problem of resolving all the species present more difficult than initially thought. The complete nucleotide sequence of alanine sRNA has been published (HoUey, et al, 1965). Operationally, ribosomal subunits, which may account for 10% of the total cytoplasmic ribosomal material in HeLa cells (Joklik and Becker, 1965), would be found in the soluble phase as ordinarily prepared, and their characteristic RNAs would therefore be found in total RNA extracts of this fraction. IV. Enzymes of the Soluble Phase Excellent reviews and tabulations of the enzymes found in centrifugally isolated fractions have appeared (Roodyn, 1965; de Duve, Wattiaux and Baudhuin, 1962). A listing of all enzymes which have been reported to occur in the soluble phase will therefore not be presented here. A. enzyme groups of the soluble phase 1. Protein Synthesis The amino acid activating enzymes are almost exclusively located in the SP (Gasior and Moldave, 1965) and in addition, are precipitable iso-electri- cally at pH 5 (see later section on pH stability of the soluble phase). 2. Nucleic Acid Synthesis All of the enzymes necessary to incorporate thymidine into DNA are present in the soluble phase of Walker 256 carcinoma, Flexner-Jobling carcinoma, rat thymus, spleen, small intestine, kidney, normal liver, brain, lung, testis, heart muscle, pancreas, skeletal muscle, regenerating liver (Bollum and Potter, 1958), and in adenovirus infected cells (Green, Pina, and Chagoya, 1964). Thymidine kinase (2.7.1.21), thymidylate kinase (2.7.4.9), thymidine I 8. THE SOLUBLE PHASE OF THE CELL 497 diphosphate kinase, thymidylate synthetase and deoxycytidylate deaminase have all been found to increase during cell proliferation (Myers, Hemphill and Townsend, 1961), and decrease during stationary phase (Kit, Dubbs and Frearson, 1965). In addition, part of the soluble DNA-dependent RNA polymerase (2.7.7.6) of rat prostate and liver nuclei may be recovered from isolated nuclei in a soluble form. The enzyme appears to be normally bound to its substrate, however (Doly, Ramuz, Mandel, and Chambon, 1965). Both replicative deoxynucleotidyl transferase (deoxyribonucleic acid polymerase) (2.7.7.7) and terminal deoxynucleotidyl transferase (end addition enzyme) are recoverable in a soluble fraction from calf thymus (Yoneda and Bollum, 1965). 3. Fatty Acid Synthesis The enzymes responsible for fatty acid synthesis appear to be largely confined to the soluble phase (Langdon, 1957). 4. Glycolysis In all animal cells thus far examined the majority of the glycolytic activity is present in the soluble phase. Recoveries range from 75-100% of the total homogenate activity in rat liver (Bucher and McGarrahan, 1956; Kennedy and Lehninger, 1949), rat brain (Abood, Gerard, Banks, and Tschirgi, 1952; Balazs and Lagnado, 1959), rat anterior pituitary (McShan, Rozich, and Meyer, 1953), and Flexner-Jobhng sarcoma (Le Page and Schneider, 1948). 5. Pentose-Phosphate Oxidative Cycle Studies on the enzymes of this cycle suggest that the major enzymes are found in the soluble phase of rabbit liver and kidney prepared with saline diluents in place of sucrose (Newburgh and Cheldelin, 1956). 6. Glycogen Metabolism The two most important enzymes in the synthesis and breakdown of glycogen (UDP-glucose-a glucan glucosyltransferase, 2.4.1.11; and a glucan phosphorylase, 2.4.1.1) are bound to the polymer itself in fed animals, and released into the soluble phase during starvation (Leloir and Goldemberg, 1960; Tata, 1964). Phosphoglucomutase (2.7.5.1) is characteristically an enzyme of the soluble phase (Hers, Berthet, Berthet, and de Duve, 1951), while glucose-6-phosphatase (3.1.3.9) is bound to cytomembranes (de Duve, Pressman, Gianetto, Wattiaux, and Appelmans, 1955) (see Chapter 6). B. PROBLEMS IN INTERPRETATION OF ENZYME DATA From a physiological or functional viewpoint the objective of enzyme studies on cell fractions is to find out where a given reaction occurs in the cell. Provided the natural substrates and cofactors and an approximation of intracellular pH and ionic environment obtain, it is of little interest whether 498 NORMAN G. ANDERSON and JOHN G. GREEN more than one enzyme is being measured. The question asked is where the cell carries out a particular process. However, if an enzyme assay is used to define one species of protein molecule, then it is of interest to show that only one is being measured. In addition, where similar enzymic activities are observed in several cell fractions, the question of leakage, adsorption, or cross contamination is easily solved if the enzyme molecules in different fractions differ in some physical property. The discovery of isoenzymes (Hunter and Markert, 1957; Shaw, 1965) adds both complexity and new experimental possibilities to cell fractionation studies. Quantitative methods for measuring the activities of many of the possible isoenzymes are not yet available, however. The question of whether enzymes tend to be characteristic of one cell fraction may be approached by plotting the number of enzymes against increments in the percentage found in the soluble phase (Fig. 8). There 35i 30- 25 0) E 20 15- 10- 20 30 40 50 60 70 80 90 100 %Total activity in soluble phase Fig. 8. Plot of % total activity of 102 enzymes found in the soluble phase of animal tissues (chiefly rat liver). 0-10% group includes instances where no activity was observed, 90- 100% group includes studies where more activity was found in the isolated soluble phase than was observed in the total homogenate. (Data from Roodyn, 1965). Biphasic distri- bution will probably be accentuated when specific isoenzymes are measured rather than total enzymic activity. is a marked tendency for an activity to be present (90 % or more) or absent (less than 10%) from the soluble phase. 1. Presence of Low Activity Levels Enzymes which are largely present in one cell fraction are usually stressed. A very small amount of an enzyme in the soluble phase however may be adequate to deal with the substrate levels present. The basic concept of the lysosome — that a series of otherwise deleterious enzymes are and must 8. THE SOLUBLE PHASE OF THE CELL 499 be compartmented within special bodies — hinges on this point. For this and other reasons low percentage recovery of enzyme activity in the soluble phase is often disregarded and attributed to isolation artifacts. 2. Localization of Activity Non-isoparticulate distributions may be attributed to one of several causes which include : (a) transport (the enzyme is being moved from site o f synthesis to site of action), (b) equilibrium (the structure containing an enzyme is in equilibrium with its constituents in solution), or (c) isolation artifacts. Rather few enzymes have been reported to be totally absent from this fraction. These include such enzymes as aryl-4-hydroxylase (1.14.1.1) (Brodie, et al., 1955), nucleoside diphosphatase (3.6.1.6) (Novikoff and Heus, 1963) using ADP and CDP as substrates, thyroxine degrading activity of rat liver (Wynn, Gibbs, and Royster, 1962), 4-monomethyl aminoazobenzene demethylase activity (Hultin, 1957), and the synthesis of ascorbic acid from glucuronolactone (Chatterjee, Chatterjee, Ghosh, Ghosh, and Guha, 1960). It would be of interest to re-check these very carefully for evidence of in- hibitors, or very low level activity to settle the point of whether any enzymic activity is ever totally excluded from a cell fraction. The question of whether the same enzymic forms or isoenzymes are being studied in the soluble and a particulate phase is directly relevant to the problem of whether an activity observed in the SP is particle derived Rat liver ^-galactosidase (3.2.1.23) from lysosomes and from the soluble pnase appear to be identical (Furth and Robinson, 1965). However, differences in soluble and mitochondrial aspartate aminotransferase (2.6.1.1) (Sheid, Morris, and Roth, 1965), phosphoenolpyruvate carboxykinase (2.7.1.40) (Holten and Nordlie, (1965), and alanine amino transferase (2.6.1.2) (Swick, Barnstein, and Stange, 1965) have been described suggesting but not proving that the SP enzymes were not bound to mitochondria in the cell (see Chapter 3). C. CONTROL OF ENZYMIC ACTIVITY IN THE CELL A few enzymes appear to be especially responsive to changes in the concentration of one or more metabolites related in some way to their own activity. Such enzymes appear to be composed of subunits (Gerhart and Schachman, 1965), undergo conformational changes when exposed to "effectors" (Changeux, 1964), and have a sigmoid-shaped substrate saturation curve under certain conditions (Atkinson, 1965). The "effectors" produce a conformational change or allosteric transition (Monod, Changeux, and Jacob, 1 963) by binding at a site distinct from the enzymic site. The result may be either activation or inhibition. Generally, the first enzyme of a sequence is inhibited by the end product of that pathway. Where several 500 NORMAN G. ANDERSON and JOHN G. GREEN isoenzymes are found, one may be a regulatory enzyme while another may not. Thus, in rabbit muscle lactate dehydrogenase (1,1,1.27) isoenzyme 5 is an allosteric protein and a regulatory enzyme while lactate dehydrogenase iso- enzyme 1 from rabbit heart is neither (Fritz, 1965), As higher resolution separations are attained, surprisingly large numbers of electrophoretically separable forms of a single enzyme may be found (Fritz and Jacobson, 1965) adding to the possibilities of selective regulation. In addition to feedback inhibition by single metabolites, a "concerted" feedback inhibition dependent on simultaneous presence of threonine and lysine has been described (Datta and Gest, 1964) for a bacterial aspartate kinase (2.7.2.4) while a contrasting form of multiple response has been observed in E. coli glutamine synthetase (6.3.1.2) where eight metabolites were found to have small additive effects, termed "cumulative" feedback inhibition (Woolfolk and Stadtman, 1964). An interplay between these forms of control, enzyme repression, and enzyme induction to achieve a fairly steady concentration of a series of metabolites while varying rate at which they flow through a metabolic pathway has been discussed by Atkinson (1965). The control of the level of specific isoenzymes by hormones is outside the scope of this review. Enzymes have long been known to be exquisitely sensitive to their chemical environment, and variation in pH, ionic strength, and cofactor concentration are routinely examined in the development of enzyme assays. Since it now appears that many alterations in enzymic activity are mediated through sites on the protein molecule other than the enzymic site, an enzyme molecule may, in fact, be considered as a sensor of the intracellular environment trans- lating one or many variations in milieu into a change in catalytic activity. The advantages of such direct and compact control mechanisms are at once apparent. It is not unlikely that all enzymes are in a wider sense regulatory enzymes, although a few have this function developed to a much higher degree. V. Physiology of the Soluble Phase A. THE EFFECT OF HEAT Proteins of rat liver soluble phase are surprisingly unstable (Anderson, Makinodan, and Norris, 1961). At 40 °C approximately 10% of the total nitrogen is precipitated in two hours, while at 100°C 86 % of the total nitrogen is flocculated. Precipitation rate at 40 °C is rapidly diminished as the pH of the SP is raised from 6-4 to 8-2. However, the flocculation observed, especially around 40 °C, is not attributable to a decrease in pH during incubation. When turbidity formation is followed an initial lag in the formation of a precipitate is observed suggesting that a protective substance initially present may be destroyed. Addition of ATP inhibited precipitate formation, but the effect disappears during incubation, possibly because of hydrolysis of ATP. Addition of polyanions (RNA, heparin, polymanuronic acid sulphate) markedly 8. THE SOLUBLE PHASE OF THE CELL 501 decreases the rate of precipitate formation, while RNase, CaCl2, and MgCl2 promote precipitation. The observation that a large fraction of the soluble proteins of rat liver precipitate in vitro during incubation at temperatures not lethal for an intact animal (Anderson, Makinodan, and Norris, 1961) raises several interesting questions. Is the precipitation due to enzymic digestion, thermal denaturation, or the removal of protective groups or substances ? What is the relation, if any, of this effect to physical changes in the ground substance of cells during cell division, after fertilizati©n, in response to chemical, physical and electrical stimuli, during amoeboid movement, and in incipient cytolysis? Work in this area has suffered from a lack of valid isolated model systems, and the effects observed in the SP deserve attention since they contain at least some of the reactants. The effect of incubating rat liver soluble phase prepared in 0-25 M sucrose at various temperatures is shown in Fig. 9. (Note that heating at times and temperatures empirically determined has long been used in the lOOn Time (hr) Fig. 9. Percentage of total nitrogen precipitated from soluble rat liver protein solutions as a function of time and temperature. Arrow along ordinate indicates percentage of total nitrogen not lost during dialysis (Anderson, et al., 1961). 100% = 3-76 of N/ml Av. isolation of enzymes from soluble tissue extracts). Sulfhydryl compounds and inhibitors of proteolytic enzymes and colchicine were without protective effect. B. THE EFFECT OF pH In phosphate buffers the SP of rat liver is stable for short periods at pH values between 1-26 and 3-5 and between 6-3 and 11. Between pH 3-5 and 6-3 502 NORMAN G. ANDERSON and JOHN G. GREEN marked precipitation occurs with a maximum of 24 % of rat liver SP nitrogen precipitated near pH 5 (Fig. 10). At acid pH values in the presence of HCl voluminous precipitates are obtained which are not seen with phosphoric lOO' so- so - 5 pH 7 8 10 II Fig. 10. Percentage of total nitrogen of rat liver soluble phase nitrogen precipitated as a function of pH. acid. The precipitation of part of the soluble phase at pH 5-0 has long been used in the purification of enzymes from tissues. Among those which precipi- tate are enzymes which incorporate amino acids into sRNA (Hoagland, Keller, and Zamecnik, 1956) and dihydrofolate reductase (Bertino, Perkins, and Johns, 1965). The percentage of soluble proteins iso-electrically precipi- tated at pH 5-0 in various tissues has not been examined in detail; however, voluminous precipitates have been observed in supernatants from Ehrlich ascites tumour (E. C. Horn, personal communication). The effect of pH on the SP is of interest because parallel effects are seen in cells where a slight decrease in pH produces a marked increase in viscosity. After injury, cell pH often drops and the cytoplasm is observed to coagulate (Heilbrunn, 1956). The precipitation observed in vitro parallels the intra- cellular change in that precipitation is initially easily reversed, but becomes irreversible with time. It is evident that if cell fractionation of a homogenate is attempted in the neighborhood of pH 5-0 that gross cross contamination with precipitated protein will occur. The nuclear isolation methods developed by Dounce (1955) involved the use of solutions on either side of the precipi- tation region shown in Fig. 10. The proteins of the mitotic apparatus are thought to be iso-electric at a pH below neutrality on the basis of studies on proteins solubilized from the isolated mitotic apparatus (Went and Mazia, 1959) and because of a sharpening of the mitotic apparatus observed in the light microscope in dividing cells exposed to slightly acid solutions. If the 8. THE SOLUBLE PHASE OF THE CELL 503 proteins of the mitotic apparatus persist in a soluble form in the interphase cell (Went and Mazia, 1959), they may well be found in the SP fraction isoelectrically precipitated at pH 5-0. C. THE ROLE IN MEMBRANE AND PARTICLE FORMATION The proposal that some cell structures may exist in equilibrium with their constituent macromolecules present in the SP (Anderson, 1956a, b, 1959) is based on the concept of coacervation (Bungenberg de Jong, 1949). This mechanism would allow changes in structure to occur in response to altera- tions in the composition of the SP during differentiation, growth, starvation, cell division, or in response to external agents or stimuli. If specific structures are to form in place of simple coacervates, then molecular surface specifi- cities must exist on structure forming elements which, in response to discrete micro-environments in the cell, result in the formation of characteristic structures in their proper places. The alternative to this view is that the molecular elements of subcellular structures are formed in situ. All presently available evidence suggests that this is not the case but that protein synthesis for example is confined to specific structures devoted to that purpose. The movement of structure-associated proteins to their structure-position must therefore occur by diffusion in the SP. Their concentration will depend on the binding equilibrium constant. If this is shifted far in the direction of a stable structure, the amount of structure-forming protein in the SP will be very small. Destruction of formed elements may then require special intracellular digestion mechanisms. Conceivably structure-forming proteins may, like fibrinogen, require activation to form insoluble configurations. Experimentally, the formation of structures not unlike those seen in homogenates has been observed in the soluble phase of rat liver (Anderson, 1959) and in clear bacterial extracts after the addition of magnesium (Bolton, et ai, 1957). Somewhat similar structures have also been reported from mixtures of haemoglobin and DNA (Nelson, 1958a and b). For these observa- tions to have any relevance to events in vivo it must be shown that the same molecular species are involved. The observation that the plasma membrane of the cell can rapidly reform after cell rupture (Heilbrunn, 1956) and that antisera against isolated cell particles cross react with the SP suggest that this is the case. From the point of view taken here the only interesting and un- expected finding in a cell fractionation study would be the total absence of a protein from the soluble phase. VI. Conclusions With the use of high resolution separation techniques including zonal ultracentrifugation, automated chromatographic systems, and gel electro- phoresis and Immunoelectrophoresis, the molecular anatomy of the soluble 504 NORMAN G. ANDERSON and JOHN G. GREEN phase may now be worked out in a manner analogous to blood plasma. A purely descriptive approach is essential initially since the functions of many of the constituents observed will not be known. On the basis of work already done it is concluded that the soluble phase is a tractable mixture which should now be resolved. If the results are to reflect the intracellular composition it is imperative that extremely rapid methods for the isolation of the soluble phase be worked out, if possible compressing homogenization, centrifugation, and freezing into a single process requiring only a few seconds. Centrifuge systems on which to base such studies have been described (Kolousek, Jiracek, and Cizinsky, 1964). 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The list that follows is not intended as an arbitrary "ruling" on such questions, since all language is to some extent subjective and individualistic. Also some of the terms at the moment escape precise definition and other terms, although inadequate or even incorrect (e.g. homogenate) have become firmly entrenched in our parlance. What I have tried to do is to present what seem to be reasonable definitions, derived partly from my own predudices, and also by abstraction from the various chapters in "Enzyme Cytology". Many of the terms are more fully explained by the various authors, and the reader is referred to the appropriate chapter for a full discussion. I am grateful to the various authors for their suggestions and comments, and I fully accept the responsibility for any violent reactions to this attempt to discipline our language. If this list does nothing more than provoke controversy, at least something will have been achieved. Throughout the list I have tried to distinguish between "cytological" and "opera- tional" terms (see Chapter 1). The superscript*^* after a term indicates that the component is being described in cytological terms, and the superscript* °> indicates an operational description (for example "the material sedimenting after 1 hr at 1(X),(X)0 g). The operational component may have a cytological counterpart (e.g. "mitochondrial fraction" and "mitochondria") and the two terms may then be used interchangeably with little confusion. However, it may have little or no cytological meaning (e.g. "fluffy layer" or "pH 5 fraction"). Conversely the cytolo- gical term may have no operational counterpart (e.g. "Golgi apparatus"). A com- plete separation of operational and cytological terms is of course impossible be- cause of the great overlap between the two fields of differential centrifuging and cytology, but in some cases the difference is clear and should be stressed. Standardi- zation in the use of the two terminologies at the present time may also prevent future confusion. D.B.R. TERMINOLOGY LIST Agranular cytomembranes^^^ See "smooth surface cytomembranes" Aleuroplast^'^^ Leucoplast in which protein granules predominate as a storage product. Amyloplasi^^^ Leucoplast in which starch predominates as a storage product. Area of focal degeneration^'^'' See "autophagic vacuole" Autolysosome^^^ See "autophagic vacuole". Autophagic vacuole^'^^ Enlarged lysosome containing mitochondria or other cell components. Essentially the same as "cytolysome", "area of focal degeneration", "cytosegresome", "composite bodies" or "autolysosomes". Cell sap^^^ Interparticulate fluid of the cell. Equivalent to hyaloplasm**^) and soluble phase"'**"* and not to be used synonymously with soluble fraction*"*. 511 512 APPENDIX Chloroplast^^^ Cell particle characterized by a limiting double membrane and an internal, chlorophyll-containing lamellar structure embedded in a protein-rich stroma. The chloroplast is capable of light-dependent photophosphorylation and of photosynthetic CO2 fixation. Chloroplast fraction^°^ Preparation of cell particles consisting predominantly of chloroplasts. Chromatin^^^ Material that includes spiralized (condensed chromatin) and de- spiralized (extended chromatin) parts of chromosomes. Chromatin threads <°* Chromosomal material obtained by differential centrifuging of disrupted nuclei. Chromatophores'^^^ Particles isolated from photosynthetic bacteria and containing photosynthetic pigments. Chromoplast^'^'^ Coloured plastid. Chromosome^^^ This term should only be used to refer to the discrete chromosome. In other cases the term "chromatin" should be used. Cisterna of cytomembranes^'^^ Space between paired cytomembranes. Composite body''^^ See "autophagic vacuole" Cristae of mitochondria'-'^'' Characteristic internal double membrane structure of mitochondria, generally thought to be continuous with the inner membrane. Cytolysome^''^ See "autophagic vacuole". Cytomembrane^''^'-°^ Membrane, or membrane system, in the cytoplasm. The term is probably more meaningful and less ambiguous than endoplasmic reticulum. May consist of paired structures separated by a space (cisterna). Divided into rough surface (a, granular) cytomembranes, and smooth surface (/3, y agranular) cyto- membranes. /3 cytomembranes are membranes which appear to represent invagina- tions of the plasma membrane, y cytomembranes are a cluster (or "complex") of membranes making up the Golgi cytomembranes. Cytoplasmic fraction'-'^'' It is a frequent practice to remove unbroken cells, cell debris and nuclei from tissue homogenates before fractionation. Since this is not strictly a total homogenate, the fraction may best be called the "cytoplasmic fraction". Cytosegresome^'^^ See "autophagic vacuole". Cytosome'''^^ See lysosome. Differential centrifuging This is a general term for the fractionation of tissue homo- genates by centrifugal methods. Dispersate''"^ The term "homogenate" is preferred. Z)/5/je/-5/o/7<°> Again, the term"homogenate" is preferred, although "tissue dispersion" is probably a more correct definition of the material than "tissue homogenate". See below, under "homogenate". DNP-fibril <''> (deoxyribonucleoprotein fibril) A fibrillar structure 100-250A in diameter consisting of deoxyribonucleoprotein. It may also be called an "elementary chromosomal fibril". Elaioplast i'^' Leucoplast in which oil predominates as the storage product. Electron transport particle *o> : ETP A particle derived from mitochondria, and capable of carrying out electron transport from suitable substrates to oxygen. In general the use of abbreviations to describe cell particles is to be avoided, in order to prevent difficulty in reading papers with too many such abbreviations. However, ETP has such general use that it should be retained. Endoplasmic reticulum ^''^ A system of cytoplasmic membranes, generally considered to consist of two components: 1. smooth surface endoplasmic reticulum, which are arrays of smooth membranes and 2. rough surface endoplasmic reticulum, arrays APPENDIX 513 of membrane studded with ribosomes. Endoplasmic reticulum is not synonymous with microsomes, or microsomal fraction («) — see "microsomes" below. Enzyme cytology The study of the intracellular localization of enzymes. E.R. or e.r. This abbreviation is sometimes used for endoplasmic reticulum, or ergastoplasm, but is best avoided or at least restricted to the labelling of electron micrographs. Ergastoplasm ^^^ System of cytoplasmic membranes. Cytomembranes or endo- plasmic reticulum preferred. Not synonymous with microsomes or microsomal fraction <°) — {see "microsomes" below.) Generally does not include smooth surface cytomembranes. Ergosomes <«') <°> See "polysomes". Fluffy layer <°> Material lying above the densely packed mitochondrial pellet after decanting the supernatant fluid. It is undoubtedly a heterogenous fraction and should not be described as a separate sub-cellular component. If possible, the term is best avoided, since it is very difficult to prepare the fraction in a reproducible way. Fraction Material separated from a homogenate by diff"erential centrifuging. Since some of the fractions have a clear content of well defined cell components (e.g. the mitochondrial fraction) there has been a very great tendency to describe the fraction in terms of the most abundant sub-cellular component present and even to use the terms synonymously (e.g. nuclei for nuclear fraction, ribosomes for microsomal fraction, lysosomes for lysosome-rich mitochondrial fractions etc.) Since there is usually cross-contamination between components during isolation of the fractions, and since they are frequently damaged more or less severely by the isolation pro- cedure, a clear distinction must be made between the isolated sub-cellular fraction and the original cell component. Although somewhat cumbersome, it is better to use the composite term "nuclear fraction", "mitochondrial fraction", "soluble fraction" etc. where any danger of such confusion exists. g Taken to mean the gav at the centre of the tube. A precise description of centri- fugal forces employed would include the rmax and rmin values, the type of rotor, and the forces developed during acceleration and deceleration. g min The product of the integrated centrifugal force in g and the time it was applied, in minutes — see "Kg min" below. Golgi apparatus <*=) A cytological term referring to a specialized system of cyto- plasmic membranes. Not to be used synonymously with smooth membrane frac- tion <°> unless the fraction is derived exclusively from the Golgi apparatus. Golgi cytomembranes are a cluster (or "complex") of so-called "y-cytomembranes". Gradient differential centrifuging Centrifugal fractionation based on differences in sedimentation rates of cell components in a density gradient medium. The gradient is generally employed to stabilize the various layers and the particles do not reach density equilibrium with the medium — see "isopycnic gradient centrifuging" below. Gradients Some standardization of the presentation of gradients would be desirable. For example, it might be suggested that the figures are always drawn with the lightest fractions to the right and the heaviest to the left i.e. the density-gradient increases from right to left. Also, the position of fractions along the gradient are best marked in some meaningful manner e.g. cm from meniscus or, better cm from the axis of rotation, rather than simply in fraction numbers. Granular cytomembranes See "rough surface cytomembranes". Heavy microsomal fraction See "microsomal fractions". Homogenate <°> Preparation of disrupted cells suspended in an appropriate medium. The term is strictly incorrect, since no homogenates are homogeneous. However, 514 APPENDIX the very wide use of the term must justify its retention. It need not strictly be applied to preparations of disrupted cells, since it is convenient, for example, to talk of a homogenate of disrupted mitochondria. The homogenate of cells or whole tissue is best called a "cell-homogenate" or "tissue homogenate". Hyaloplasm ('*> The inter-particulate fluid of the cell, equivalent to "cell sap" and "soluble phase". Not to be used synonymously with soluble fraction <°). Inner compartment of mitochondria (^> Space bounded by cristal and inner mem- brane. Equivalent to "matrix". Intracellular localization. This term should only be applied to the distribution of a given enzyme or chemical substance in sub-cellular fractions if these fractions have been examined cytologically. Preferably, its use should be restricted to methods in which the actual localization can be examined (e.g. microspectrophotometry) and not used at all for experiments based on differential centrifuging. "Subcellular distribution" or "Distribution in subcellular fractions" would be better terms to describe work based on fractions derived from homogenates. Isoenzymes, Preferred to "isozymes" since it retains the original term "enzymes". Isopycnic gradient centrifuging. Centrifugal fractionation in which cell components reach density equilibrium in the density gradient. This should be distinguished from gradient differential centrifuging {see above). Isotonic sucrose. The true tonicity of sub-cellular particles is generally not known, and it is best to avoid this term in cell fractionation studies, stating instead the exact concentration of solute. Isozymes, See isoenzymes. Kg Centrifugal force in g x lO"^. Kg min. The product of the integrated centrifugal force in Kg and the time it was applied, in minutes. {Kg hr for hours). These terms are useful in providing a simple way of describing the procedure used in the isolation of cell components, and may be used to distinguish sub-cellular fractions. If used in conjunction with suitable standard symbols for the common sub-cellular fractions, considerable information can be conveyed. For example, two mitochondrial fractions isolated after 10 and 20 min at 5000 g could conveniently be designated 50 M and 100 M fractions respectively, indicating that they were mitochondrial fractions isolated after 50 and 100 Kg min. Leucoplast <<=> Colourless plastid. Light microsomal fraction. See "microsomal fraction". Lysate, Homogenate obtained by lysis of cells or cell particles; except where there is danger of ambiguity, "homogenate" is preferred. Lysophagosome ^^'> See "phagolysosome". Lysosome ^^'^ A particle rich in lytic enzymes, and showing latency of these enzymes. A young lysosome may be called a "protolysosome". The term "cytosome" is probably equivalent to lysosome. Lysosomal fraction <°) Preparation of cell particles consisting predominantly of lysosomes. It should be clearly distinguished from mitochondrial preparations rich in lysosomes. Matrix of mitochondrion. See "inner compartment of mitochondrion". Microbodies ('^^ Catalase-rich cell particles, distinct from lysosomes and small mitochondria. They have a characteristic dense core and belong to the general class of particles known as "peroxisomes". Microbody fraction (°> Preparation of cell particles consisting predominantly of microbodies. Microsomal fraction (°'> Sub-cellular fraction obtained by centrifuging the super- APPENDIX 515 natant fraction obtained after sedimenting the mitochondria. It usually consists of a mixture of ribosomes and pinched off elements of the reticular membrane, but is an entirely operational component and should never be used synonymously with endoplasmic reticulum, ergastoplasm or cytomembranes. Microsomal subfractions <°> Phospholipid-rich sub-fractions may be called "heavy" or "light" microsomal fractions. The final microsomal fraction, poor in phospho- lipid, may be called the "post-microsomal fraction". Microsomes, There is no such structure as a microsome and the term should be avoided. If used, it should be synonymous with microsomal fraction, and not endoplasmic reticulum, ergastoplasm, or cytomembranes. Mitochondria (<=) Cell particles characterized by double outer and inner membranes (cristae) and capable of oxidizing suitable substrates to CO2 and water. Since most mitochondrial fractions consist predominantly of mitochondria, it is usually reasonably safe to use the two terms synonymously. If there is some doubt as to whether the respiratory activity observed in the isolated cell particles is due to mitochondria, the term "respiratory particles" is preferred {see below). Mitochondrial fraction <°) Preparation of cell particles consisting predominantly of mitochondria. Mitochondrial sap «=> Intramitochondrial fluid with dissolved substances. Mitochondrial soluble fraction <<*> Easily extractable soluble components of the mitochondrion. Mitochondrial supernatant <«> This term is sometimes used to describe the fraction remaining after sedimenting the mitochondria. However, it implies that the super- natant contains mitochondria and some alternative is preferred. Nuclear envelope <*=> Preferred to nuclear membrane in order to emphasize the complexity of the structure and the fact that it is not a simple lipoprotein mem- brane. Nuclear fraction <°) Isolated sub-cellular fraction consisting predominantly of nuclei. Because of difficulties experienced in the isolation of pure nuclei until fairly recently, many so-called nuclear fractions were in fact grossly contaminated with cell debris and unbroken cells, and are best called "whole cell-nuclear fractions" {see below). Nuclear sap «=' Intranuclear fluid in which nuclear structures are bathed. Nuclear soluble fraction ^°'> Soluble components of the nucleus not bound to the fibrillar structures of chromosomes and nucleoli. This roughly corresponds to material extracted from nuclei by dilute salt media. Nucleolar fraction <°) Fraction obtained by diflferential centrifuging of disrupted nuclei. Enriched in nucleoli, but generally containing significant non-nucleolar contamination. Nucleolar-chromosomal complex <°) The main structural material of the nucleus, containing chromosomal and nucleolar nucleoproteins. It corresponds to material (apart from membrane) remaining after removal of the nuclear soluble fraction. Nucleoli «=> Nuclear organelles containing ribonucleoproteins, usually organized in fibrillar structures. Active in RNA synthesis. Nucleolonema <<=) (pi. nucleolonemata) Fibrillar structures present in nucleoli, 500-1000 A in diameter, and possessing high electron density. They consist of fibrils and granules {see nucleonema). Nucleonema <'') Term suggested by Professor Georgiev and Dr. Chentsov for fibrillar structures containing 100 A fibrils (probably protein in nature) with attached ribosome-like granules. These elements are found in nucleoli (where the bundles of nucleonemata form nucleolonemata), and in chromosomes. 516 APPENDIX Nucleoplasm (^) The extra nucleolar zone of the interphase nucleus. It includes despiralized chromosomes and nuclear sap and is not identical to "nuclear sap". Nucleolus associated chromatin <*=> <°) Chromatin closely associated with the nucleo- lus, and usually isolated with the nucleolar fraction. It is not necessarily concerned with nucleolar function and should not be confused with the "nucleolar organizer". Outer compartment of mitochondria <«>) <°> Space between inner and outer mito- chondrial membranes. Pelleted, Sedimented is preferred. Peroxisome <<') (°) Particle rich in enzymes of peroxide metabolism, in particular catalase, urate oxidase and d amino acid oxidase. Microbodies are peroxisomes and have a characteristic internal core. This may or may not be present in all peroxisomes. pH 5 fraction (°> Sediment obtained after adjusting soluble fractions to pH 5. Phagolysosome ^''^ Fused lysosome and phagosome. Also called lysophagosome or telolysosome. Phagosome (^) Phagocytic or pinocytic vacuole. Polyribosome, See polysome. Polysome ^^'^ (°) Aggregate of ribosomes bound by a strand of RNA, presumably mRNA. A special example of a ribosomal aggregate {see below). Post-microsomal fraction <°) See microsomal fraction. Protolysosome, See lysosome. Relative concentration. The amount of an enzyme or chemical substance per unit weight of the sub-cellular fraction divided by the amount per unit weight in the homogenate. The term may also be used to describe the properties of fractions derived from sub-cellular components e.g. sub-mitochondrial fractions may be defined using the mitochondrial homogenate as standard. Relative concentration X 100= % specific activity {see below). Respiratory particle (°) An operational term to describe any sub-cellular particle isolated from cell-homogenates that can carry out the oxidation of suitable sub- strates. Respiratory particles may be intact or damaged mitochondria, sub-mito- chondrial particles (e.g. ETP) or non-mitochondrial material derived, for example, from bacteria. Ribosomal aggregate <°) Group of ribosomes, not necessarily bound by a strand of RNA. Ribosomal fraction (°) Preparation consisting predominantly of isolated ribosomes. Since most ribosomal fractions have a relatively high standard of purity, the term is usually synonymous with ribosomes. Ribosome (°' ^) Discrete particle consisting of RNA and protein. It should not be used to describe any RNA-rich sediment. RNP particle <<') <°> (Ribonucleoprotein particle) General term for all ribonucleo- protein particles. It should be used when the precise nature of the RNA rich particle is not known. Rough surface cytomembranes <^) System of cytomembranes lined with attached ribosomes. It should not be used to describe the microsomal fraction. Rough surface endoplasmic reticulum. See "rough surface cytomembranes". Sarcoplasmic reticulum («=> Endoplasmic reticulum system in muscle. Smooth-membrane fraction <°) Preparation consisting predominantly of cell mem- branes free of ribosomes. Smooth surface cytomembranes ^''1 Agranular cytomembranes forming irregular ramifications near the outer area of the cytoplasm. ^ cytomembranes represent APPENDIX 517 invaginations of the plasma membrane and y cytomembranes make up the Golgi cytomembranes. Soluble fraction <") The non-sedimentable material in a tissue homogenate may also be referred to as the "soluble phase". A usual definition of non-sedimentable is remaining in the supernatant after at least 1 hr at iOO Kg. Not to be used syno- nymously with cell sap or hyaloplasm {see above). Soluble phase (*=> (°' (S.P.) Cytologically, the soluble phase is the interparticulate fluid of the cell. Operationally it is defined as the supernatant remaining after the sedimentation of all particulate material. If it is intended to refer to the cytological meaning, the term "cell sap" is suggested. Conversely, "soluble fraction" is the unambigious operational term. Specific activity. The amount of an enzyme or chemical substance per unit weight of the sub-cellular fraction. % Specific activity The specific activity of the fraction divided by that of the homo- genate X 100. {see relative concentration above). Supernate "Supernatant fluid" is preferred. "Supernatant" should strictly only be used as an adjective, but its use as a noun is so common that it is probably accep- table. Tissue breis. Alternative term for homogenate. Total homogenate (°* Homogenate of the entire cell or tissue, to be distinguished from "cytoplasmic fraction" {see above) which is a homogenate from which the whole cells and nuclei have been removed. Whole cell-nuclear fraction <°) Fraction obtained by centrifuging the homogenate at low speed for short time. It should be clearly distinguished from "nuclear fraction" {see above) which is reserved for fractions consisting predominantly of nuclei. AUTHOR INDEX Numbers in italics refer to the page at the end of papers where complete references can be found Abood, L. G., 381, 399, 497, 504 Abraham, S., 380, 399 Abrams, A., 455, 468 Abrams, R., 59, 60, 61, 75, 95, 96, 99 Ackerman, R. F., 272, 319 Ada, G. L., 279, 291, 294, 306, 314 Adams, J. G., 272, 317 Adler, F. L., 289, 310 Adloff, E., 82, 88, 92, 96, 100 Afifi, F., 280, 316 Afzelius, B. A., 106, 146, 149, 178 Agranoff, B. W., 129, 176 Agren, Gunnar, 489, 504 Aird, G. L.. 51, 54, 98 Alanpovic, P., 495, 506 Albers, M., 414, 439, 440, 466, 473 Alberti, K. G. M. M., 137, 138, 175 Albertsson, P-A., 10, 24 Aldridge, W. N., 378, 399 Alexander, K. D., 269, 282, 313 Alexander, P., 344, 345, 357, 361, 367, 371, 372, 374, 384, 401 Alfin-Slater, R. B., 343, 404 Alivisatos, S. G. A., 79, 100 Alksne, J. F., 267, 306 AUard, C, 343, 344, 379, 399, 400 Allen, J. M., 249, 255, 306 Allen, Kenneth, 491, 504 Allen, M. B., 197, 205, 208, 228, 232 Allende, J. E., 427, 428, 468 Allfrey, V. G., 6, 9, 10, 16, 18, 24, 29, 30, 31, 35, 44, 45, 54, 57, 68, 69, 70, 71, 76, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 90, 91, 93, 95, 96, 97, 99, 101, 127, 178, 298, 306 Allison, A. C, 256, 276, 291, 298, 306 Allison, F., 266, 312 Ames, B. N., 491, 507 Altschul, A. M., 258, 310 Amano, M., 58, 95 Ambrose, E. J., 263, 271, 304, 308 Ananieva, L. N., 45, 64, 66, 80, 100 Anders, M., 60, 61, 97 Anderson, J. H., 447, 465, 468 Anderson, M. L., 493, 504 Anderson, N. G., 6, 9, 24, 32, 102, 249, 316, 361, 399, All, 478, 479, 483, 484, 485, 486, 491, 492, 493, 494, 500, 501, 503, 504, 506 Anderson, P., 301, 307 Anderson, P. J., 255, 295, 301, 306, 307 Anderson-Cedergren, E., 369, 377, 380, 403 Andres, A. G., 292, 306 Andresen, N., 276, 306 Andrews, R. D., 271, 278, 309 Anfinsen, C. B., 439, 468 Angeletti, P. U., 249, 313 Anraku, Y., 442, 447, 465, 468 Anstadt, G. L., 283, 310 Anthony, V., 327, 405 Apgar, J., 496, 506 App, A. A., 224, 232 Appelmans, F., 7, 23, 24, 137, 176, 240, 242, 244, 249, 253, 298, 309, 337, 338, 339, 341, 342, 344, 345, 346, 386, 400, 497, 505 Archer, G. T., 245, 256, 306 Archibald, F. M., 372, 375, 405 Arhelger, R. B., 264, 307 Arion, W. H., 342, 344, 345, 403 Arlinghaus, R., 420, 427, 428, 465, 468 Amon, D. L, 189, 197, 199, 200, 202, 203, 204, 205, 206, 207, 208, 209, 211, 212, 213, 214, 215, 216, 221, 223, 228, 229, 232, 232, 233, 235, 236, 237 Aronow, R., 263, 266, 306 Aronson, A. I., 413, 468 519 520 AUTHOR INDEX Aronson, H., 263, 266, 306 Aronson, J., 303, 306 Aronson, N. N., 245, 306 Arrhenius, E., 170, 180 Arstila, A., 303, 311 Artman, M., 449, 465, 468 Aschoff, L., 262, 306 Ashford, T. P., 262, 288, 295, 306 Ashwell, G., 341, 381, 402, 403 Ashworth, C. T., 273, 275, 306, 316, 399 Asrijan, I. S., 45, 71, 100 Astaurova, O. B., 77, 98 Aterman, K., 295, 306 Atkinson, D. E., 485, 499, 500, 504 Atwal, O. S., 303, 306 Aub, J. C, 263, 271, 315 Aubert, J. P., 211,252 Auerbach, V. H., 2, 25 August, J. T., 61, 62, 98, 448, 454, 464, 468, 470 Aurisicchio, A., 641, 101 Austin, C. M., 279, 291, 294, 306, 314 Avi-Dor, Y., 491, 507 Avignon, J., 341, 399 Avron, M., 204, 205, 208, 227, 232, 233 Axelrod, J., 343, 345, 399, 405, 499, 505 Azzone, G. F., 134, 175, 369, 377, 380, 403 B Bachelard, H. S., 378, 382, 405 Bachmann, E., 151, 159, 175 Baeckeland, E., 146, 176 Bahr, G. F., 17, 24 Bailey, J. M., 489, 504 Bainton, D. F., 259, 306 Bairati, A., 263, 306 Baird, G. D., 137, 175 Bak, I. J., 289, 309 Balazs, R., 497, 504 Baldwin, R. W., 495, 504 Balis, J. U., 272, 307 Ball, E. G., 278, 307, 378, 405 Ballard, P., 61, 62, 95 Baltscheffsky, M., 202, 236 Baltus, E., 47, 88, 95, 101 Balz, J. P., 258, 312 Bangham, A. D., 169, 175 Banks, J., 497, 504 Banon, P., 255, 287, 307 Barany, W. C, 16, 25 Barber, G. A., 221, 222, 223, 237 Barber, M. L., 484, 485, 504 Barbieri, G. P., 347, 391, 399 Barclay, M., 372, 375, 405 Bardawil, W. A., 278, 316 Bargoni, N., 343, 402 Barka, T., 241, 249, 254, 256, 259, 273, 295, 301, 306, 307 Barnett, R. J., 167, 178 Bamett, S. R., 29, 96 Barnhart, M. L , 284, 315 Barnstein, P L., 132, 779, 499, 508 Barnum, C. P., 343, 403 Barr, G. C, 362, 364, 401 Barrnett, R. J., 255, 267, 272, 278, 307, 312, 315, 317, 331, 332, 335, 386, 393, 404, 405 Barron, E. J.,. 230, 233 Barron, K. D., 255, 294, 307 Bartley, W., 137, 138, 175 Barton, A. D., 494, 504 Barzilai, D., 494, 504 Bassham, J. A., 209, 210, 212, 215, 218, 220, 224, 233 Bassler, K. H., 82, 92, 100 Battifora, H., 251, 307 Baudhuin, P., 3, 4, 7, 8, 9, 10, 19, 20, 22, 23, 24, 25, 26, 105, 117, 118, 133, 137, 172, 173, 174, 175, 175, 176, 180, 197, 233, 286, 304, 307, 318, 338, 341, 343, 345, 346, 367, 371, 377, 381, 400, 483, 496, 505 Baum, H.. 375, 383, 401 Baumler, A., 343, 346, 350, 352, 370, 400 Bautz, E., 64, 95 Bazil, G. W., 55, 95 Bean, M. A., 297, 311 Beam, A. G., 92, 101 Beaufay, H., 7, 8, 9, 10, 19, 20, 22, 23, 24, 24, 118, 133, 137, 172, 174, 175, 175, 242, 245, 251, 307, 314, 316, 317, 338, 343, 360, 368, 369, 370, 386, 400, 405 Becarevic, A., 364, 365, 404 Becker, M. J., 199, 233 Becker, N. H., 255, 307 Becker, Y., 71, 98, 478, 496, 506 Beer, M., 465, 468 AUTHOR INDEX 521 Behki, R. M., 51, 95 Behnke, O., 254, 259, 288, 307, 313 Behrens, M., 6, 24, 29, 95, 193, 194, 196, 236 Beinert, H., 85, 95 Bell, A. L., 267, 268, 269, 279, 313 Bell, D., 50, 101 Bellamy, D., 134, 177 Benacerraf, B., 256, 280, 317 Benckhuysen, C, 129, 777 Bendall, D. S., 23, 24, 118, 133, 134, 137, 175 Bender, M. A., 68, 99, 494, 508 Bendich, A., 273, 307. 308 Benditt, E. P., 279, 318 Benedetti, E., 323, 356, 358, 372, 375, 401 Benedetti, E. L., 356, 358, 374, 394, 396, 401 Benhamou, J. P., 280, 316 Bennacerraf, B., 292, 313 Bennett, H. S., 252, 263, 307 Bennum, A., 227, 232 Bensch, K., 273, 307 Bensch, K. G., 243, 244, 259, 260, 266, 273, 310 Bensch, V. G., 404 Bensley, R. R., 2, 24 Benson, A. A., 186, 209, 233 Benson, B., 257, 260, 261, 308 Berg, P., 60, 62, 65, 95, 96 Bergstrand, A., 57, 95, 355, 356, 361, 400 Berkaloff, A., 274, 309 Bernhard, W., 172, 179 Bernheimer, A. W., 251, 252, 296, 311, 318 Berthet, J., 6, 7, 9, 16, 17, 19, 22, 23, 24, 24, 25, 172, 175, 242, 259, 307, 338. 360, 379, 400, 402, 483, 497, 505, 506 Berthet, L., 7, 25, 379, 402, 497, 506 Bertini, F., 255, 257, 287, 307, 308 Bertino, J. R., 502, 504 Bessis, M. C, 270, 307 Bessman, M. J., 50, 98 Bessman, S. P., 78, 97 Beyer, G.T.,8 1,96 Beyer, R., 82, 92, 100 Bhattacharya, D., 448, 466, 469 Bianchi, G., 134, 175 Biggins, J., 184, 186,255 Billen, D., 51, 55, 95 Bilodeau, F. B., 488, 505 Binkley, F., 379, 399, 456, 457, 463, 464, 468 Birbeck, M. S. C, 337, 399 Bird, J. F., 217, 219, 220, 233 Birke, G., 283, 318 Birns, M., 257, 307 Bimstiel, M. L., 46, 47, 73, 74, 79, 80, 95,96 Bishop, N. I., 199, 233 Biswas, B. B., 60, 61, 95 Bitensky, L., 252, 293, 297, 307 Bittar, E. E., 484, 504 Bjorkerud, S., 286, 307 Bjur, R. A., 134, 777 Blair, P. V., 154, 166, 167, 775, 775 Black, C. C, 203, 235 Blackwell, J. B., 293, 307 Blaschko, H., 7, 24 Blech, W., 379, 402 Bloch, D. P., 79, 95 Bloemendal, H., 448, 467, 468 Bobbitt, J. L., 495, 504 Bock, R., 434, 463, 467, 470 Bock, R. M., 131, 144, 154, 158, 162, 164, 176 Boezi, J. A., 468 Bogdanov, A. A., 416, 444, 466, 472 Boileau, S., 343, 344, 366, 400 Boler, R. K., 264, 307 Bollum, F. J., 50, 51, 54, 95, 496, 497, 504, 509 Bolton, E. J., 64, 99, 413, 415, 439, 440, 441, 442, 444, 456, 457, 464, 465, 468, 503, 504 Bowman, H. G., 454, 467, 469 Bond, H. E., 494, 505 Bonner, D. B., 63, 100 Bonner, D. M., 383, 405 Bonner, J., 46, 57, 60, 61, 63, 66, 69, 70, 71, 79, 95, 98, 100, 211, 237, 414, 439, 466, 472, 473 Bonner, W. D. Junr., 207, 208, 234 Bonnett, H. R., Junr., 424, 468 Booth, J., 341, 399 Boothroyd, B., 288, 310 Bopp-Hassenkamp, G., 37, 95 Borek, E., 47, 74, 95, 96, 454, 472 Borenfreund, E., 273, 307, 308 Bornig, H., 343, 344, 399 522 AUTHOR INDEX Borman, A., 378, 394, 402 Borsook, H., 2, 24 Bortignon, C, 175 Bos, C. J., 323, 356, 358, 372, 375, 383, 389, 396, 401 Bosch, L., 448, 467, 468 Bottke, I., 286, 316 Bouvet, C, 364, 399, 403 Bove, C, 199, 233 Bove, J., 224, 233 Bove, J. M., 199, 225, 236 Bowers, W. E., 6, 7, 24, 245, 307 Boyden, S. V., 283, 293, 307, 317 Boyer, P. D., 123, 175 Boyland, E., 341, 399 Brachet, J., 58, 96, 99, 224, 234, 257, 276, 286, 304, 307, 315, 408, 468 Bradford, H. P., 382, 400, 405 Brady, C. J., 489, 505 Brady, R. O., 485, 505 Brambell, F. W. R., 270, 278, 280, 307 Brand, T., von, 151, 180 Brandes, D., 254, 255, 257, 258, 259, 287, 307, 308, 316 Brandt, P. W., 263, 308 Brandster, M. J., 87, 96 Braunstein, A. E., 133, 175 Brawerman, G., 224, 233, 342, 400 Bremer, H., 61, 64, 96 Bremer, J., 115, 175, 343, 345, 400 Breuer, C, 378, 394, 402 Breuer, H., 345, 378, 394, 400 Breuer, M. E., 68, 96 Brewer, D. G., 295, 308 Brierley, G., 159, 161, 162, 163, 175, 176 Brierley, G. P., 151, 175 Brierly, G. P., 375, 383, 401 Brindley, D. N., 331, 332, 359, 376, 379, 402 Brinton, C. C, 423, 472 Britten, R. J., 413, 429, 430, 457, 468, 471, 472, 489, 503, 504, 505 Brodie, B. B., 342, 401, 499, 505 Brodksy, V. B., 78, 96 Bronk, J. R., 127, 175 Brosemer, R. W., 134, 175, 343, 400 Brown, D. D., 73, 96 Brown, J. H. U., 381, 402 Brown, R., 96 Brown, R. R., 345, 400 Brummond, D. O., 211, 234 Bruni, A., 159, 175 Brunngraber, E., 381, 399 Bruns, R. R., 267, 314 Bucher, N. L. R., 116, 117, 175, 341, 400, 497, 505 Bucher, Th., 157, 158, 175, 178, 488, 505 Buetow, D. E., 257, 259, 307 Bungenberg de Jong, H. G., 503, 505 Buras, N. S., 293, 314 Burch, H. B., 393, 400 Burdon, R. H., 75, 96, 448, 454, 464, 466, 468 Burger, C. L., 361, 399 Burk, D., 10, 25 Burka, E. R., 421, 470 Burke, J. S., 292, 313 Burma, D. P., 217, 220, 233 Burns, J. J., 341, 402 Burstone, M. S., 256, 266, 284, 297, 301, 308, 309, 312, 319 Burton, A. D,, 92, 96 Busch, H., 33, 46, 49, 72, 99, 101 Busch, S., 393, 400 Butler, J. A. V., 60, 63, 96, 97, 382, 383, 391, 401 Butt, V. S., 223, 233 Byers, S. O., 272, 310 Byrne, J. M., 276, 308 Cabrini, R. L., 288, 316 Cagan, R. H., 245, 281, 308 Cahnmann, H. J., 477, 508 Cairns, J., 52, 53, 96 Caldwell, K. A., 247, 257, 316 Caldwell, P. C, 484, 505 Callan, H. G., 37, 68, 96, 97 Calo, N., 216, 233 Calvet, F., 57, 99 Calvin, M., 209, 210, 212, 215, 218, 220, 224, 233 Cameron, P., 242, 243, 309 Cammack, K. A., 449, 468 Campbell, D. H., 289, 291, 308, 310 Campbell, J. J. R., 455, 464, 472 Campbell, P. N., 330, 338, 339, 348, 356, 361, 362, 364, 366, 400 Canellakis, E. S., 50, 51, 98, 99 Canning, R. E., 361, 399, 491, 492, 493, 504 AUTHOR INDEX 523 I I Cantero, A., 343, 344, 379, 399, 400 Capindale, J. B., 208, 220, 221, 232, 235 Carafoli, E., 128, 129, 175 Carasso, N., 258, 308 Cardini, C. E., 220, 235 Cardini, C. S., 218, 235 Carroll, W. R., 156, 777, 477, 506 Camithers, C, 343, 346, 350, 352, 370, 400 Carter, C. E., 447, 465, 468 Carter, F. L., 488, 508 Carvalho, S., 90, 96 Caro, L. G., 3, 25, 60, 96, 259, 282, 308 Casley-Smith, J. R., 267, 272, 308 Caspersson, T., 56, 76, 96, 408, 468 Castiaux, P., 488, 507 Castro, A., 268, 312 Casu, A., 161, 175 Catania, A., 298, 314 Caulfield, J. B., 263, 271, 315 Chadwick, C. S., 295, 313 Chagoya, Victoria, 496, 506 Chaikoff, I. L., 31, 96, 380, 399 Chalkley, G. R., 79, 96 Chaloupka, J., 460, 462, 464, 468 Chamberlin, M., 60, 62, 65, 96 Chambers, E. L., 488, 505 Chambers, R., 488, 505 Chambon. P., 497, 505 Chance, B., 105, 123, 124, 125, 159, 160, 167, 175, 176, 179, 202, 233 Chang, J. P., 386, 396, 400 Changeux, J. P., 499, 505, 507 Chao,F-C., 410, 411,-^65 Chapeau, M. L., 292, 306 Chapman-Andresen, C, 243, 262, 263, 271. 276, 277, 278, 308 Chappell, J. B., 116, 134, 138, 140, 151, 152, 159, 176 Chargaff, E., 342, 400 Chatterjee, I., 349, 401 Chatterjee, G.C., 499, 505 Chatterjee, I. B., 349, 402, 499, 505 Chauveau, J., 32, 91, 96, 99, 165, 176, 337, 339, 341, 343, 344, 347, 348, 349, 350, 352, 354, 361, 362, 364, 365, 366, 368, 400, 403 Chayen, J., 146, 176, 297, 307 Cheldelin, V. H., 497, 507 Chentzov, J. S., 32, 37, 38, 41, 42, 44, 45, P7 Chemick, S. C, 273, 315 Chevremont, M., 146, 176 Chevremont-Comhaire, S., 146, 775 Chipchase, M., 46, 79, 95 Chipchase, M. I., 31, 100 Chipchase, M. I. H., 46, 47, 73, 74, 80, 95,96 Chlopin, N., 275, 276, 308 Choppin, P. W., 274, 309 Chorazy, M., 273, 308 Chouinard, L. A., 255, 308 Choules, E. A., 425, 433, 463, 470 Christensen, Halvor, N., 486, 489, 490, 505 Citarrella, P., 298, 314 Cizinsky, B., 488, 504, 506 Clark, B., 358, 372, 377, 379, 404 Clark, S. L., Junr., 263, 267, 270,278,505 Clarke, D. A., 256, 577 Clarke, E., 272, 575 aaude, A., 2, 24, 328, 400, 483, 505 Clayton, R. B., 323, 384, 400 Cline, A. L., 434, 463, 469 Cochrane, C. G., 284, 292, 308 Cohan, M. S., 222, 236 Cohen, E., 425, 432, 439, 440, 465, 470 Cohen, P. P., 491, 508 Cohen, S., 283, 295, 306, 308 Cohen, S. S., 449, 472 Cohen-Bazire, G., 186, 233 Cohn, P., 365, 370, 400 Cohn, Z. A., 245, 248, 250, 256, 257, 260, 261, 264, 284, 285, 308 Cole, L. J., 51, 99, 249, 577 Coleman, R., 358, 375, 380, 400 Coll, J. A., 496, 508 Collins, F. D., 394, 400 Collins, J. v., 261, 572 Colodzin, M., 379, 400 Colowick, S. P., 377, 380, 402 Colper, C, 330, 338, 339, 356, 361, 362, 364, 366, 400 Comb, D. G., 73, 74, 96 Conchie, J., 7, 24 Confer, D. B., 261, 308 Connelly, J. L., 124, 777 Conney, A. H., 345, 400 Conover, T. E., 86, 96 Contessa, A. R., 159, 775 524 AUTHOR INDEX Conway, E. J., 389, 400 Conway, T. W., 427, 463, 468 Cooksey, K. E., 343, 400 Coons, A. H., 268, 289, 304, 308, 318 Cooper, B., 202, 234 Cooper, D. Y., 340, 378, 388, 403 Cooper, J. R., 499, 505 Coote, J. L., 32, 99 Cordier, R., 262, 274, 310 Cormack, D. H., 263, 271, 304, 308 Cottone, M. A., 31, 96 Coutsogeorgopoulos, C, 51, 98 Cowie, D. B., 430, 435, 436, 437, 462, 468, 469, 503, 504 Cox, E. C, 414, 415, 468, 470 Crampton, C. F., 414, 468 Crane, F. L., 161, 167, 176, 179 Craston, A., 344, 404 Creasy, W. A., 86, 96, 345, 400 Crick, F. H. C, 52, 101, 417, 420, 468 Criddle, R. S., 131, 144, 154, 158, 162, 164, 176 Crocker, T. T., 58, 97 Crofts, A. R., 116, 151, 152, 159, 176 Cunningham, G. J., 297, 307 Curry, J. B., 414, 469 D Daems, W. T., 242, 253, 275, 288, 292, 295, 308, 331, 332, 333, 404 Dahlquist, A., 379, 382, 400 Dales, S., 274, 308, 309 Dallam, R. D., 141, 176 Dallner, G., 338, 341, 347, 355, 356, 357, 361, 362, 364, 369, 400 Dalq, A. M., 270, 309 Dalton, A. J., 24, 25, 348, 349, 402 Daly, M. M., 76, 79, 96 D'Amelio, V., 495, 505, 507 Daniel, M. R., 257, 313 Danielli, J. F., 494, 507 Dannenberg, A. M., Junr., 256, 309 Danner, J., 448, 467, 469 Danon, D., 263, 266, 306 Darnell, J. E., 72, 100 Darnell, J. E., 59, 72, 97, 100 Das, M. L., 161, 176 Das, V. S. R., 190, 234 Datta, A., 136, 178 Datta, P., 500, 505 Datta, R. K., 448, 455, 456, 460, 462, 464, 466, 469 Davenport, H. E., 201, 202, 233 Davenport, J. B., 10, 24 Davidson, A. G., 350, 401 Davidson, E. A., 245, 254, 306, 310 Davidson, J. N., 50, 51, 57, 76, 79, 101 Davidson, O. W., 268, 312 Davies, D. D., 221, 222, 223, 233 Davies, D. R., 337, 345, 365, 402 Davies, J., 250, 309 Dawson, I. M., 57, 101 Day, A. J., 272, 309 Deane, H. W., 287, 291, 309, 312 Deasey, C. L., 2, 24 De Bernard, B., 119, 176 Debov, S. S., 40, 44, 45, 702 Decken, A., von der, 348, 366, 369, 377, 380, 400, 403, All, 454, 462, 469 Decroly-Briers, M., 286, 307 de Duve, C, 3, 4, 5, 6, 7, 8, 9, 10, 16, 17, 19, 20, 21, 22, 23, 24, 24, 25, 105, 117, 118, 133, 134, 137, 172, 173, 174, 175, 175, 176, 197, 233, 240, 242, 243, 244, 245, 247, 249, 251, 252, 253, 261, 262, 288, 296, 298, 299, 301, 307, 309, 310, 311, 314, 316, 318, 330, 337, 338, 339, 341, 342, 343, 344, 345, 346, 360, 361, 367, 368, 369, 370, 371, 377, 379, 381, 386, 399, 400, 402, 405, 483, 496, 497, 505, 506 de Kouchovsky, Y., 201, 233 Delaccha, J. M., 268, 312 de la Huerga, J., 253, 275, 295, 304, 311 De Lalla, O. F., 495, 505 de Lamirande, G., 343, 344, 366, 379, 399, 400, 403 Del Campo, F. F., 227, 235 Delhumeau de Ongay, G., 392, 400 DeLuca, H. F., 129, 176 de Man, J. C. H., 331, 332, 333, 404 Denstedt, O. F., 85, 100 de Pury, G. G., 394, 400 de Reuck, A. V. S., 242, 243, 309 de Robertis, E., 7, 25 De Ropp, R. S., 488, 505 Desai, I. D., 251, 252, 261, 288, 298, 299, 316 De Salle, L., 148, 779 AUTHOR INDEX 525 Deshpande, P. D., 107, 176 de Terra, N., 282, 309 Devel, H. J., 343, 401 de Waart, C, 490, 505 Diamondstone, T. I., 133, 177 Dianzani, M. U., 297, 309 Dickman, S. R., 425, 432, 439, 440, 462, 465, 467, 469, 470 Dieckmann, M., 65, 95 Diengdoh, J. V., 256, 291, 309 Diezel, P. B., 286, 316 DiMacco, G., 495, 506 di Marco, A., 347, 391, 399 Din, G., 221, 235 Dingle, J., 251, 252, 318 Dingle, J. T., 251, 252, 257, 309, 312, 313 Dintzis, H. M., 414, 472 Ditzion, B., 257, 271, 315 Dixon, F. J., 284, 292, 308, 309 Dodd, M. C, 277, 312 Dodgson, K. S., 343, 370, 400 Doly, J., 497, 505 Doniacii, J., 295, 309 Domer, R. W., 211, 253 Doty, P., 37, 40, 70, 102 Dounce, A., 32, 81,96 Dounce, A. L., 29, 31, 32, 44, 81, 96, 408, 469, 502, 505 Dow, D. S., 129, 777 Doyen, A., 245, 316 Doyle, W. L., 262, 312 Druga, A., 257, 271, 313 Dubbs, D. R., 497, 506 Duchateau-Bosson, Gh., 491, 506 Duerksen, J. D., 430, 433, 435, 436, 437, 462, 463, 468, 469 Duerre, J. A., 414, 415, 430, 469 Dulit, E., 350, 401 Dumazert, C., 485, 508 Dumonde, D. C, 293, 309 Dunn, M. S., 489, 505 Durham, L. J., 208, 232 Durueux, J., 289, 312 Dutton, G. J., 343, 400 Duysens, L. N. M., 199, 200, 202, 229, 233 Eason, R., 51, 101 Easton, J. M., 263, 271, 293, 309 Easty, G. C., 270, 271, 278, 308, 309 Eck, H., 228, 236 Edmonds, M., 75, 96 Edstrom, J-E., 66, 96 Eekhout, Y., 287, 309 Eichel, H. J., 381, 400 Eisenstadt, J. M., 224, 233 Eisenstein, R., 251, 307 El-Aaser, A. A., 328, 331, 332, 333, 335, 337, 344, 345, 346, 351, 357, 360, 361, 366, 367, 369, 370, 371, 372, 374, 384, 392, 393, 394, 396, 401 El-Aaser, A. B. A., 62, 75, 87, 100 Eliasson, N. A., 57, 95 Elliot, A. M., 289, 309 Elliott, H. A., 495, 505, 507 Elliott, K. A. €., 488, 505 Elliott, W. B., 255, 311 Elliott, W. H., 345, 401 Ellis, R. J., 221, 222, 223, 233 Elsbach, P., 272, 285, 309 Elson, D., 410, 412, 420, 422, 428, 430, 436, 437, 439, 440, 441, 442, 443, 444, 448, 449, 450, 451, 541, 453, 455, 457, 460, 462, 463, 466, 467, 469, 472 Elvehjem, C. A., 31, 99 Emanoil-Ravicovitch, R., 341, 350, 354, 401 Emanuel, C. P., 31, 96 Emmelot, P., 323, 356, 358, 372, 374, 375, 383, 389, 394, 396, 401 Engelberg, H., 449, 465, 468 Engelberg, N., 465, 470 England, H., 466, 470 Englard, S., \U, 179 Ennis, R. S., 251, 311 Enright, J. B., 303, 306 Epstein, M. A., 274, 309 Ericsson, J. L. E., 242, 243, 254, 263, 264, 265, 266, 267, 268, 269, 274, 278, 304, 309, 331, 332, 335, 340, 355, 387, 396, 397, 401, 403 Ernster, L., 105, 170, 776, 180, 339, 340, 341, 342, 344, 354, 355, 361, 364, 365, 368, 369, 370, 371, 387, 390, 391, 396, 397, 401, 402, 403 Errera, M., 58, 72, 76, 99 Essner, E., 20, 25, 286, 288, 295, 309, 314, 331, 332, 333, 335, 390, 392, 395, 396, 397, 401, 403 I 526 AUTHOR INDEX Estable, C, 41, 96 Estabrook, R. W., 155, 176, 340, 378, 388, 403 Evans, H. M., 275, 309 Evans, W. H., 281, 309 Everett, G. A., 496, 506 Pagan, V. M., 341, 364, 390, 394, 395, 405 Fahay, J. L., 283, 317 Fancher, H., 65, 95 Farkas, W., 342, 403 Farkas, W. R., 467, 469 Famham, A. E., 281, 314 Farquhar, M. G., 246, 259, 263, 267, 269, 279, 304, 306, 309, 310 Faulkner, R., 71, 95 Fauvert, R., 280, 316 Favard, P., 258, 308 Favelukes, G., 428, 468 Fawcett, D. W., 257, 318, 327, 387, 388, 391, 393, 395, 397, 401, 402 Fedorko, M. E., 250, 310 Feingold, D. S., 218, 220, 233 Feldherr, A. B., 93, 96 Feldherr, C, 93, 97 Feldherr, C. M., 93, 96 Feldman, J. D., 269, 312 Feldman, M., 448, 455, 466, 467, 472 Fell, H. B., 251, 312 Fennell, R. A., 257, 312 Fennell, R. H., 292, 310 Feretos, R., 380, 389, 403 Ferger, M. F., 428, 470 Femandex, C, 333, 389, 405 Femandez-Moran, H., 154, 165, 166, 167, 175, 176 Fernando, N. V. P., 267, 310 Ferrari, G., 486, 507 Fessenden, J. M., 428, 469 Feuer, G., 340, 345, 395, 401 Fiala, J., 460, 462, 463, 467, 471 Fiala, S., 10, 25 Ficq, M., 76, 97 Fiedler, F., 214, 215, 216, 236 Fine, J., 297, 307 Finean, J. B., 358, 375, 400 Fischer, R., 285, 310 Fisher, E. A., 393, 394, 406 Fisher, E. R., 254, 268, 278, 310 Fisher, F., 88, 96 Fisher, H. W., 32, 75, 96, 98 Fisher, W. D., 361, 399, 493, 504 Fishman, M., 289, 310 Fishman, W. H., 303, 310 Fitzgerald, P. J., 57, 96 Fitzsimons, J. T. R., 357, 372, 401 Flaks, J. G., 414, 415, 468, 470 Flaxman, A., 257, 259, 310 Fleischer, S., 105, 155, 156, 161, 162, 163, 176, 179 Fleissner, E., 47, 74, 95, 96 Flodin, Per., 495, 505 Florey, E., 488, 491, 505 Florey, H., 275, 311 Florkin, Marcel, 491, 506 Fogel, Z., 436, 437, 462, 469 Foldes, F. F., 380, 405 Foldes, v., 380, 405 Folkers, K., 365, 378, 403 Fork, D. C., 201, 233 Forro, F., 60, 96 Fouts, J. R., 350, 354, 356, 392, 393, 394, 395, 396, 397, 401, 402 Francavilla, A., 134, 178 Frankland, M., 245, 310 Franklin, R. M., 59, 66, 100 Franks, J. J., 283, 310 Franz, I. D., 350, 401 Frayssinet, C, 392, 400 Frearson, P. M., 497, 506 Frederic, J., 264, 315 Freeman, K. B., 131, 158, 171, 176, 179 Freeman, T., 283, 308, 310 Freer, R. M., 29, 96 French, C. S., 200, 225, 233, 236 French, J. E., 272, 310 Frenster, J. H., 35, 44, 54, 68, 69, 71, 78, 97,99 Fresco, R., 57, 97 Frey-Wyssling, A., 186, 233 Fridovitch, I., 342, 403 Friedman, M., 272, 310 Friend, J., 235 Fritz, LB., 115, 775 Fritz, P. J., 500, 505 Frost, J. K., 255, 287, 307 Fry, K. T., 202, 233 Frye, F. L., 303, 306 Fuchs, E., 64, 97 AUTHOR E^JDEX 527 Fuller, R.C., 211, 213, 2i5 Fure-Fremiet, E., 54, 55, 97 Furman, R. H., 495, 506 Furth, A. J., 499, 506 Furth, J. J., 60, 61, 62, 63, 97 Fuxe, K., 255, 285, 310 Gabara, B., 258, 314 Gahan, P. B., 146, 176 Gaines, D., 245, 254, 312 Gaizhoki, V. S., 85, 97 Galante, E., 460, 464, 470 Gale, P. H., 365, 378, 403 Gall, J. C, 68, 97 Gall, J. G., 66, 96 Gammack, D. B., 382, 400 Ganchery, M., 54, 55, 97 Gansler, H., 268, 310 Garbus, J., 129, 176 Garvey, J. S., 289, 291, 308, 310 Gasior, E., 428, 471, 496, 506 Gaudette, L., 499, 505 Gauguly, J., 343, 401 Gauze, G. G., 80, 102 Gazzaniga, P. P., 495, 506 Gazzinelli, G., 432, 440, 465, 469 Gedigk, P., 285, 310 Geiduschek, E. P., 61, 64, 65, 97, 101 Georgiev, G. P., 30, 32, 35, 37, 38, 41, 42, 44, 45, 47, 52, 57, 58, 59, 63, 64, 66, 71, 72, 73, 74, 75, 78, 80, 97, 98, 100 Gerowin, L. D., 129, 180 Gerard, P., 262, 274, 310 Gerhard, J. C, 499, 506 Gerard, R. W., 497, 504 Gest, H., 187, 233, 500, 505 Gesteland, R. F., 440, 449, 466, 469 Geyer, R. P., 272, 318 Ghosh, J. J., 448, 455, 456, 460, 462, 464, 466, 469, 499, 505 Ghosh, N. C, 349, 401, 402, 499, 505 Gianetto, R., 23, 24, 137, 176, 240, 242, 244, 245, 249, 251, 253, 298, 390, 310, 318, 337, 338, 339, 341, 342, 344, 345, 346, 386, 400, 497, 505 Gibbs, M., 213, 214, 215, 216, 220, 233, 234 Gibbs, R., 499, 509 Gibor, A., 224, 234 Gibson, K. I., 340, 345, 395, 401 Gibson, Q. H., 370, 371, 402 Gierer, A., 420, 421, 469 Giglio, F., 298, 314 Gilbert, W., 420, 421, 428, 430, 436, 469 Gilden, R. V., 66, 70, 95 Gilette, J. R., 342, 394, 401 Ginzburg-Tietz, Y., 459, 463, 496, 470, 472 Girard, M., 59, 97 Giri, K. V., 217, 235 Gitlin, D., 268, 310, 312 Gitzelmann, R., 254, 310 Giuditta, A., 378, 401 Giuffrida, L., 484, 506 Gladstone, L., 60, 102 Glenner, G. G,, 303, 311 Glick, D., 343, 403 Gockerman, J., 249, 306 Godman, G. C, 79, 95 Godson, G. N., 60, 97, 382, 383, 391, 401 Goffeau, A., 224, 225, 234, 236 Gofman, J. W., 495, 505, 507 Golberg, L., 340, 345, 395, 401 Goldberg, A. F., 256, 257, 261, 291, 311 Goldberg, B., 263, 271, 293, 309 Goldberg, I. H., 59, 60, 61, 97 Goldblatt, P. J., 297, 318 Goldemberg, S. H., 343, 403, 497, 507 Golder, R. H., 491, 508 Goldfeder, A., 87, 99 Goldfischer, S., 255, 258, 303, 307, 308, 310, 331, 332, 333, 335, 390, 392, 396, 401, 403 Goldstein, L., 57, 58, 97, 494, 506 Goldthwait, D. A., 433, 456, 462, 464, 467, 473 Gomez, C, 268, 312 Gomori, G., 242, 301, 310 Gonatas, N. K., 276, 315 Gonzales, F., 288, 310 Gonzales, I. E., 272, 310 Good, J. J., 372, 375, 405 Goodman, H. M., 59, 66, 97 Goodwin, T. W., 187, 226, 234 Gordon, A. H., 283, 310 Gordon, G., 273, 307 Gordon, G. B., 243, 244, 259, 260, 266, 273, 310 528 AUTHOR INDEX Gordon, J., 454, 467, 469 Gorlich, M., 339, 370, 394, 401, 402 Gorry, J. D., 343, 349, 371, 404 Gosselin, L., 339, 341, 401 Goswami, P., 345, 349, 351, 362, 364, 401 Gottlieb, B., 343, 349, 371, 404 Gotto, A. M., 223, 237 Goutier, R., 343, 401 Goutier-Pirotte, M., 343, 401 Graham, R. C, 304, 310 Grampp, W., 66, 96 Granick, S., 132, 176, 182, 187, 224, 234 Gratzer, W. B., 496, 508 Gray, E. D., 50, 101 Gray, R., 245, 254, 312 Graziosi, F., 64, 101 Greco, A. E., 138, 178 Green, C, 273, 310 Green, D. E., 2, 25, 105, 131, 144, 151, 154, 155, 156, 158, 159, 161, 162, 163, 164, 166, 167, 175, 176, 179, 323, 328, 371, 375, 383, 401 Green, H., 263, 271, 293, 309 Green, H. N., 340, 364, 396, 406 Green, J. G., 484, 485, 486, 504, 506 Green, M., 65, 97, 496, 506 Green, M. H., 257, 310 Greenawalt, J. W., 10, 25 Greenbaum, A. L., 7, 25, 297, 317 Greenberg, D. M., 343, 400 Greenberg, S. D., 272, 317 Greengard, P., 344, 404 Greenman, D. L., 467, 469 Greenspan, C. M., 65, 99 Greenstein, J. P., 489, 506 Greenwood, A. D., 190, 234 Gregoire, J., 485, 506 Greville, G. D., 113, 134, 138, 176 Griffiths, S. K., 245, 314 Gros, P., 430, 448, 452, 470, 472, 489, 507 Gross, J., 2, 25, 417, 418, 429, 470 Gross, P. R., 477, 506 Gross, J. A., 199, 233 Grossman, D., 89, 97 Groth, D. P., 255, 307 Grumet, G., 486, 508 Grunberger, E., 286, 316 Grunberg-Manago, M., 455, 464, 469 Guha, B. C, 349, 401, 499, 505 GuUberg, R., 283, 318 Gurdon, J. B., 73, 96 Gustafson, A., 495, 506 Gustafsson, R., 170, 176 Gvozdev, V. A., 32, 77, 79, 97, 98 Gyorkey, F., 255, 307 H Haagen-Smit, A. J., 2, 24 Haak, E. D., 161, 176 Hackett, D. P., 229, 234 Hagen, J. M., 7, 24 Hagen, P., 7, 24, 378, 401 Haguenau, F., 323, 325, 328, 386, 391, 392, 393, 395, 401 Hajra, A. K., 129, 176 Hale, D. M., 269, 282, 313 Hall, B. D., 63, 101 Hall, C.E., 411,457, ^69 Halliday, R., 280, 307 Hallinan, T. H., 362, 365, 366, 391, 393, 401, 402 Halpern, B. N., 297, 310 Halvorson, H. O., 434, 437, 463, 467, 469, 470 Hamel, C. L., 78, 97 Hamilton, M. G., 44, 78, 99, 347, 404, 415, 425, 471 Hamilton, P. B., 484, 488, 506 Hammarsten, E., 57, 95 Hampton, J. C., 275, 310 Hancox, N. M., 288, 310 Handelman, C. S., 288, 310 Handler, P., 87, 99, 342, 403 Hanlon, David, P., 491, 506 Hannig, K., 10, 25 Hannover, R., 82, 92, 100 Hanson, H., 379, 402 Hansrote, R. N., 327, 405 Hanssen, O. E., 297, 310 Harbers, E,, 59, 97 Harding, B. W., 381, 402 Harding, C. V., 92, 97 Harrington, J. F., 258, 310 Harris, A. Z., 209, 233 Harris, E. J., 128, 777 Harris, H., 32, 58, 59, 71, 75, 96, 97, 98 AUTHOR INDEX 529 Harris, J. I., 414, 415, 473 Hart, L. G., 396, 397, 402 Hartmann, J. F., 37, 98 Hartrodt, W., 303, 319 Hartwell, L. H., 430, 469 Haruna, I., 449, 469 Haselkom, R., 148, 179 Hasselberger, F. X., 480, 485, 507 Hassid, W. Z., 218, 220, 233 Hastings, J. R. B., 496, 506 Hauber, J., 114, 179 Hauge, J. G., 434, 463, 469 Haurowitz, F., 268, 310 Hawthorne, J. N., 342, 403 Hawtrey, A. O., 362, 364, 402 Hay, A. J., 7, 24 Hayashi, H., 291, 317 Hayashi, M., 28, 56, 64, 65, 98, 101, 303, 310 Hayes, E. R., 255, 311 Haxo, F. T., 182, 234 Heath, D., 295, 308 Heber, M., 195, 211, 212, 2i^ Heber, U., 195,211,212,254 Hedman, R., 170, 180 Heilbrunn, L. V., 502, 503, 506 Heise, E., 339, 370, 394, 401, 402 Heldt, H. W., 122, 139, 140, 177 Helge, H., 129, 130, 178 Hell, A., 58, 72, 99 Heller, L., 343, 402 Hempelman, L, H., 303, 306 Hemphill, C. A., 497, 507 Henderson, J. F., 485, 506 Hendler, P., 165, 172, 777 Hendler, R. W., 366, 382, 391, 402, 489, 506 Hendley, D. D., 286, 310 Henly, W. S., 272, 319 Henshaw, E. C, 71, 98 Heppel, C. A., 382, 403 Heppel, L. A., 75, 98, 425, 440, 445, 446, 447, 449, 466, 471 Herbst, E. J., 466, 472 Herisson-Cavet, C, 341, 350, 354, 401 Hermann, P., 379, 402 Hers, H. G., 7, 25, 245, 286, 307, 312, 379, 402, 497, 506 Hersh, R. T., 414, 469 Herzenberg, L. A., 356, 372, 374, 402 Herzenberg, Leonore, A., 356, 372, 374, 402 Hess, E. L., 268, 316, 381, 402 Hess, R., 245, 272, 310 Hesselbach, M. L., 10, 25 Heus, M., 344, 403, 499, 507 Hiatt, H., 72, 98 Hiatt, H. H., 71, 95 Hickmann, D. D., 107, 176 Hicks, M., 330, 338, 339, 356, 361, 362, 364, 366, 400 Hicks, R. M., 20, 25, lAl, 253, 301, 311 Hierowski, M., 495, 508 Higa, A., 448, 470 Higashi, T., 341, 355, 361. 367, 391, 402 Highton, P. J., 465, 468 Hilf, R., 378, 394, 402 Hill, R., 201, 202, 207, 208, 233, 234 Hilmoe R. J., 76, 95 Hinegardner, R. T., 51, 99 Hinton, R. H., 357, 372, 401 Hird, H. J., 165, 777, 338, 349, 402 Hirsch, J. G., 245, 245, 248, 250, 251, 256, 264, 266, 274, 296, 306, 308, 311, 319 Hirschhorn, K., 256, 257, 261, 291, i77 Hirschhom, R., 256, 257, 261, 291, i77 Hnilica, L. S., 55, 73, 95, 99 Hoagland, M. B., 77, 98, 502, 506 Hochstein, P., 341,391,-^02 Hoerr, N. L., 2, 24 Hofscheider, P. H., 64, 97 Hogeboom, C. H., 2, 4, 6, 7, 17, 24, 25, 26, 32, 85, 87, 98, 100, 111, 112, 117, 118, 128, 133, 134, 777, 779, 248, 298, 316, 408, 470, 483, 506 Hogg, L. A., 455, 464, 472 Holden, J. T., 486, 488, 489, 506 HoUey, R. W., 496, 506 HoUingworth, B. R., 410, 440, 441, 442, 444, 456, 464, 466, 472 Hollunger, G., 124, 125, 775 Holowinsky, A., 155, 176 Holt, S. J., 20, 25, 241, 242, 245, 253, 301, 303, 311, 318, 343, 386, 405 Holten, D. D., 138, 777, 499, 506 Holter, H., 262, 271, 278, 308, 577 Holtzer, H., 268, 269, 293, 304, 577 Holtzer, R. L., 288, 296, 297, 299, 318, 382, 402, 465, 469 530 AUTHOR INDEX Holtzer, S., 268, 269, 293, 304, 311 Honig, G. R., 80, 98 Hopkins, J. W., 73, 77, 78, 93, 95, 98, 99 Hopsu, V. K., 303, 311 Horecker, B. L., 210, 211, 215, 216, 234, 237, 445, 470 Horibata, K., 433, 467, 470 Horn, R. G., 266, 311 Home, R. W., 169, 175 Homer, M. W., 248, 313, 314 Horney, D. L., 477, 507 Horton, A. A., 206, 232, 237 Hosie, R. J. A., 378, 402 Hoskins, D. D., 134, 777 Hosoya, T., 255, 311 Houst, M. D., 272, 307 Hoyer, B. H., 68, 99, 415, 468 Hoyez, J., 286, 307 Hmban, Z., 243, 288, 294, 295, 311 Hsu, K. C, 292, 306 Hsu, L., 245, 254, 311 Hu, A. S. L., 434, 437, 463, 470 Huang. R. C, 60, 61. 63, 66, 69, 70, 71, 95,98 Hiibscher, G., 245, 316, 331, 332, 342, 359, 376, 379, 380, 400, 402 Hughes, W. L., 101 Huizinga, F., 448, 467, 468 Hulsmans, H. A. M., 380, 402 Hiilsmann, W. C, 129, 777 Hultin, H. O., 162, 163, 779,375,383,^07 Hultin, T., 2, 25, 362, 368, 370, 393, 396, 402, 495, 499, 506, 507 Hummeler, K., 274, 309 Humphrey, G. B., 29, 100, 325, 405 Humphrey, J. H., 283, 310 Hunter, A. R., 370, 402 Hunter, G. D., 382, 383, 391, 401 Hunter, R. L., 498, 506 Hurlbert, R. B., 73, 99 Hurwitz, J., 60, 61, 62, 97, 98, 210, 211, 216, 234, 237, 448, 454, 464, 468, 470 Hutchison, D. J., 273, 308 Huxley, H. E., 448, 467, 471 Hyde, B. B., 46, 47, 74, 79, 80, 95 Ibuki, F., 460, 464, 466, -^77 lio, M., 280, 318 Imai, Y., 275, 312 Ingraham, J. S., 268, 311 Irving, J. T,, 288, 310 Ishikawa, S., 345, 406 Isselbacher, K. J., 379, 405 Ittensohn, O. L., 273, 308 Ivanov, v., 54, 98 Jackson, R. J., 421, ^77 Jacob, F., 322, 404, 499, 507 Jacob, J., 66, 74, 707 Jacobi, H., 211, 212, 214, 234 Jacobson, K. B., 344, 378, 402, 500, 505 Jacques, P., 7, 9, 19, 22, 23, 24, 118, 172, 174, 775, 180, 245, 251, 256, 577, 316, 318 Jagendorf, A. T., 204, 205, 224, 232, 233, 234 Jahr, K., 286, 316 Jakoby, W. B., 211, 254 James, A. T., 225, 236, 342, 403 James, J. M., 327, 405 James, W. O., 190, 234 Janeway, C. A., 268, 572 Janigan, D. T., 295, 577, 318 JanoflF, A., 250, 257, 271, 297, 577, 57P Jansco, M., 263, 268, 283, 577 Jansco-Gabor, A., 268, 283, 577 Jardetzky, O., 444, 466, 470 Jarnefelt, J., 369, 389, 402 Jayaraman, J., 148, 180 Jenkin, C. R., 277, 577 Jenner, R., 57, 98 Jennings, M. A., 275, 577 Jeuniaux, Ch., 491, 506 Jibril, A., 251, 572 Jiracek, V., 488, 504, 506 Johns, D. G., 503, 504 Johnson, A. B., 134, 777 Johnson, D., 124, 135, 777 Johnson, R. M., 129, 777 Johnston, F. B., 46, 98 Johnston, M. K., 378, 399 Joklik, W. K., 71, 98, 478, 496, 506 Jones, A. L., 327, 387, 388, 391, 395, 397, 402 Jones, K. W., 74, 707 Jones, L. C, 340, 344, 368. 369, 370, 371, 390, -^07 AUTHOR INDEX 531 Jordan, P. H., 272, 319 Jordan, S. W., 273, 311 Journey, L. J., 264, 311 Judah, J. D., 128, 777 Julian, G. R., 444, 466, 470 Jung, G., 267, 311 Jungblut, P. W., 340, 355, 366, 402 Jurshuk, P., 161, 179 Juvet, R. S., 484, 506 Kagamiyama, H., 132, 178 Kahn, A., 190, 191, 195, 211, 233, 234 Kajioka, R., 274, 309 Kakefuda, T., 379, 406 Kalckar, H. M., 343, 405 Kalf, G. F., 130, 177 Kalinsky, H. J., 344, 404 Kamat, V. B., 327, 357, 361, 372, 374, 402, 405 Kamen, M. D., 187, 233 Kamin, H., 370, 371, 402 Kameyama, T., 434, 470 Kanczak, N. M., 255, 311 Kandler, O., 213, 234 Kanfer, J., 341, 402 Kaplan, J. M., 256, 257, 261, 291, 311 Kaplan, M. H., 289, 308 Kaplan, N. O., 344, 378, 402 Kar, N. C, 349, 401, 402 Karlin, L. J., 267, 272, 314 Karmon, E. A., 201, 234 Karnovsky, M. J., 288, 304, 310 Karnovsky, M. L., 245, 277, 281, 283, 308,309,311,314,316 Kato, K. I., 66, 74, 101 Katoh, S., 200, 201, 234 Katoh, T., 164, 777 Katz, J., 283, 284, 311, 316 Katz, S., 73, 74, 96 Kaufmann, E., 459, 463, 469, 470, 472 Kaufmann, B. P., 37, 98 Kawamura, H., 138, 178 Kay, L. D., 209, 233 Kaye, G. I., 267, 275, 311, 314 Kazakova, T. B., 136, 178 Keck, K., 425, 433, 463, 470 Kedrowski, B., 262, 268, 272, 274, 280, 311 Kedrowsky, B. v., 76, 95 Keighley, G., 2, 24 Keir, H. M., 30, 50, 51, 54, 98, 101 Keiser, H., 251, 252, 311, 318 Keister, D. L., 204, 234 Keller, E. B., 2, 25, 417, 418, 428, 429, 470, 502, 506 Keller, P. J., 425, 432, 439, 440, 465, 470 Kennedy, E. P., 2, 25, 245, 318, 342, 379, 400, 406, 497, 506 Kenney, F. T., 133, 177, 467, 469 Kent, G., 253, 275, 295, 304, 311 Kepes, A., 450, 470 Kern, M., 433, 467, 470 Kerur, L., 129, 777 Kessler, B., 465, 470 Kessler, E., 199, 227, 234 Keynan, A., 448, 470 Khan, R. T., 488, 505 Khessin, R. B., 77, 98 Kielley, R. K., 2, 25, 131, 777 Kielley, W. W., 2, 25 Kiessling, K. H., 344, 402 Kihara, H. K., 434, 437, 463, 467, 470 Kiho, Y., 436, 462, 470 Kimball, R. F., 54, 99 Kimhi, Y., 455, 464, 470 Kimura, K., 66, 100 King, W. D., 273, 307 Kingsley, J. W., 256, 309 Kinuwaki, Y., 291, 317 Kirby, K. S., 496, 506 Kirk, J. T. O., 224, 234 Kirkland, R. J. A., 378, 406 Kiselev, N. A., 416, 444, 466, 472 Kit, S., 497, 506 Klamer, B., 257, 312 Kleinfeld, R. G., 243, 288, 294, 295, 311 Klingenberg, M., 124, 139, 140, 144, 157, 159, 777, 178 Klouwen, H., 161, 162, 163, 176 Klucies, E., 344, 345, 357, 361, 367, 371, 372, 374, 384, 401 Klucis, E. S., 496, 506 Knopf, P. M., 421, 473 Knoppen, R., 345, 378, 394, 400 Koen, P. F., 263, 316 Koenig, H., 245, 250, 251, 254, 276, 312 Koike, M., 156, 777, 477, 506 Kojima, M., 275, 312 Kok, B., 201, 202, 234 Koketsu, K., 381, 399 532 AUTHOR INDEX Kolousek, J., 488, 504, 506 Konrad, M. W., 61, 63, 64, 96, 98 Kono, T., 377, 380, 402 Kopac, M. J., 8, 9, 25, 488, 507 Kom, D., 452, 453, 462, 473 Kom, E. D., 273, 313 Kornberg, A., 50, 87, 98, 100, 342, 402 Kornberg, R. D., 65, 95 Korner, A., 370, 402, 421, 471 Kozloff, L., 453, 470 Krakow, J., 51, 98 Krakow, J. S., 71, 98 Krall, J. L., 255, 311 Krebs, H. A., Ill, 112, 134, 177, 490, 507 Krejei, K., 488, 505 Krichevskaya, A. A., 71, 100 Krisch, K., 343, 365, 378, 402, 403 Krivit, W., 256, 318 Krogmann, D. W., 203, 206, 228, 234, 235 Krone, W., 414, 439, 440, 466, 473 Kroon, A. M., 127, 130, 149, 158, 165, 777 Krug, E., 286, 316 Kruh, J., 448, 470 Kruse, H., 268, 269, 312 Ku, K-Y., 344, 360, 390, 402 Kuczynski-Halmann, M., 491, 507 KuflF, E. L., 24, 52, 249, 255, 312, 316, 348, 349, 358, 376, 402, 404, 410, 471, 483, 506 Kumpta, U. S., 250, 253, 398, 299, 316 Kunisawa, R., 186, 233 Kurahasi, K., 381, 399 Kurland, C. G., 413, 470 Kurtz, S. M., 269, 312 Labbe, R. F., 132, 178 LaBella, F. S., 381, 402 Lacks, S., 489, 507 Lacy, D., 272, 312 Ladd, Sr. E. C, 484, 485, 504 Ladman, A. J., 273, 312 La Du, B. N., 342, 401, 499, 505 Lagg, S. E., 381, 402 I^agnado, J. R., 497, 504 Lagunoff, D., 257, 312 Laing, G. H., 251, 307 Lajtha, A., 285, 312 Lambert, P. P., 269, 279, 312 Lamfrom, H., 428, 470 Landing, B. H., 268, 310 Landon, E. J., 380, 402 Lands, W. E. M., 342, 402 Lang, K., 82, 89, 97, 98 Langdon, R. G., 342, 387, 404, 497, 507 Lange, R., 491, 507 Lanzani, G. A., 460, 464, 470 Lapresle, C., 289, 312 Lardy, H. A., 2, 25, 124, 135, 138, 177, 178 Latham, H., 72, 100 Latta, H., 268, 295, 312, 313 Lea, M. A., 392, 402 Leadbeater, L., 337, 345, 365, 402 Leaf, A., 394, 402 Leahy, J. J., 503, 504 Leake, E. S., 250, 312 Leblond, C. P., 58, 95, 263, 266, 313, 315 Leboy, P. S., 414, 470 Lebrun, J., 277, 314 Lederberg, S., 436, 437, 444, 462, 466, 470 Lederberg, V., 436, 437, 444, 462, 466, 470 Leduc, E. H., 249, 289, 308, 312 Lee, C. P., 105, 176 Lee, Y. C, 268, 269, 314 Leech, J. H., 321, 322, 325, 327, 383, 406 Leech, R. M., 190, 193, 234, 235 Lehman, I. R., 50, 98, 442, 452, 453, 462, 466, 470, 472 Lehmann, F. E., 263, 306 Lehninger, A. L., 2, 10, 25, 105, 118, 119, 123, 128, 136, 154, 159, 160, 775, 777, 779, 205, 235, 345, 406, 483, 497, 506, 507 Lejeune, N., 245, 312 Leloir, L. F., 218, 220, 235, 343, 403, 497, 507 Leonard, M. R., 293, 315 Leonhauser, S., 365, 378, 402 Le Page, G. A., 82, 98, 485, 497, 506, 507 Lerman, M. I., 45, 47, 59, 64, 66, 72, 73, 75, 80, 97, 98, 100 Leslie, L, 448, 466, 470 Lett, J. T., 344, 345, 357, 361, 367, 371, 374, 384, 401, 466, 470 AUTHOR INDEX 533 Levin, E., 162, 177 Levinthal, C, 448, 470 Lewy, G. A., 7, 24 Levy, R. S., 495, 504 Lewis, W. H., 262, 271, 312 Leybold, K., 365, 378, 403 Liau, M. C, 73, 99 Lieberman, L, 59, 99 Liesenk otter, J., 213, 234 Light, Albert, 486, 507 Lilga, K., 346, 400 Liljedahl, S. O., 283, 318 Lindberg, O., 105, 170, 176, 180 Lindgren, F. T., 495, 507 Lindner, E., 243, 312 Lipmann, F., 427, 428, 463, 468, 471 Littauer, U. Z., 455, 464, 470 Littau, V. C, 44, 54, 69, 70, 78, 95, 99 Littlefield, J. W., 2, 25, 417, 418, 429, 470 Litwack, G., 133, 777 Lobel, B. L., 287, 312 Locke, M., 261, 312, 322, 393, 403 Lockwood, W. R., 266, 312 Loeb, H., 286, 307 Loenig, U. E., 383, 393, 403 Logan, R., 57, 76, 99, 101 Loh, P., 61, 62, 63, 97 Long, C. N. H., 482, 483, 486, 507 Longley, J. B., 297, 312 Loomans, M. E., 129, 176 Loomis, W. F., 2, 25 Lorand, L., 381, 391, 403 Lorch, I. J., 494, 507 Lorini, M., 122, 179 Losada, M., 198, 200, 205, 208, 209, 211, 212, 213, 214, 215, 216, 221, 223, 227, 228, 229, 232, 235, 236. 237 Lota. M., 223, 235 Loud, A. v., 16, 25 Lovell, R. A., 488, 505 Lovett, J. S., 467, 470 Lovett, S., 455, 464, 473 Lowenstein, J. M., Ill, 112, 177 Lowry, O. H., 480, 484, 485, 486, 507 Lowy, P. H., 2, 24 Luciani, S., 159, 175 Luck, D. J., 130, 148, 149, 777, 343, 403 Luck, D. J. L., 3, 10, 25 Lucy, J. A., 251, 312 Luibel, F. J., 399 Lukes, R. J., 256, 314 Lundegardh, H., 208, 235 Lundvist, U,, 362, 386, 403 Luria, S. E., 65, 99 Lutzner, M. A., 247, 256, 312 Lynen, F., 10, 25, 114, 116, 117, 775, 777, 341, 400 Lyttleton, J. W., 185, 211, 235 M Mabry, D. S., 277, 312 McBride, O. W., 273, 313 Mc Candless, E. L., 272, 319 McCann, W. P., 343, 403 McCarthy, B. J., 64, 68, 99, 410, 413, 449, 456, 457, 464, 465, 466, 467, 468, 471, 472 McClure, F. T., 489, 503, 504, 505 McCluskey, R. T., 292, 313 McConkey, E. H., 73, 99 McCorquodale, D. J., 457, 459, 463, 471 McCreary, P., 251, 507 McDonald, M. K., 37, 98 McDonald, T., 245, 254, 312 McDowell, M. €., 268, 269, 314 McEntegart, M. G., 295, 313 McEwen, B. S., 82, 83, 84, 85, 86, 91, 99 McFarlane, A. S., 278, 283, 308, 313, 317 McGarrahan, K., 497, 505 Machinist, J. M., 119, 161, 777 Mcllwain, H., 378, 382, 405 Mclndoe, W. M., 57, 707 Mclndoe, W. N., 57, 76, 79, 707 Mackaness, G, B., 266, 314 Mackenzie, C. G., 134, 777 McLean, E. J. T., 165, 777, 338, 349, 402 MacLennan, D. H., 165, 178 McLeod, R. M., 342, 403 McMaster, P. D., 268, 269, 312 MacMorine, D. L., 292, 313 McMurray, W. C, 135, 777 McNabb, J. D., 263, 316 McNamara, P., 455, 468 McQueen, J. D., 284, 313 MacQuillan, A. M., 434, 463, 469 McQuillen, K., 408, 413, 429, 430, 466, 468, 471 534 AUTHOR INDEX McShan, W. H., 497, 507 McSwain, B. D., 203, 232 Madden, S. C, 295, 313 Madison, J. T., 425, 432, 439, 440, 462, 465, 467, 470, 496, 506 Maeir, D. M., 271, 312 Magasanik, B., 430, 469 Mager, J., 491, 507 Maggio, R., 32, 35, 36, 37, 46, 49, 99 Maheshwari, N., 60, 63, 95, 98 Mahler, H. R., 148, 178, 180 Main, R. K., 51, 99 Majno, G., 267, 275, 279, 312 Makinodan, T., 484, 500, 501, 504 Malamy, M., 445, 470 Malec, J., 32, 99 MalkoflF, D. B., 257, 259, 307 Malucci, L., 256, 276, 291, 306 Mancini, R. E., 268, 312 Mandel, P., 393, 400, 485, 497, 505, 507 Mano, Y., 341, 405 Mantieva, V. L., 45, 47, 52, 57, 62, 63, 66, 72, 97, 98, 99 Mantsa vinos, R., 50, 51, 99 Marchesi, V. T., 267, 275, 311, 312 Marcus, A., 224, 225, 235 Marcus, A. J., 256, 312 Marcus, P. I., 276, 315 Marcus, S., 285, 317 Markert, C. L., 498, 506 Marks, N., 285, 312 Marks, P. A., 421, 448, 467, 469, 470 Marmur, J., 64, 99 Marquis, N. R., 115, 176 Marquisee, M., 496, 506 Marsh, C. L., 270, 304, 315 Marsh, J. B., 342, 403 Marshak, A., 57, 97, 99 Marshall, J. M., Junr., 263, 313 Martin, E. M., 32, 99, 383, 403 Martin, R. G., 491, 507 Martin, S. J., 466, 470 Martonosi, A., 380, 389, 403 Mason, H. S., 345, 391, 396, 403 Mason, W. S., 365, 396, 403 Massey, v., 113, 178 Mast, S. O., 262, 312 Master, R. W. P., 79, 100 Masters, B. S. S., 370, 371, 402 Mateyko, G. M., 8, 9, 25, 488, 507 Matheson, A. T., 456, 464, 470 Mathias, A. P., 414, 448, 467, 470, 471, 473 Matthews, J., 477, 507 Matile, P., 258, 312 Matushita, S., 460, 464, 466, 471 Matthes, K. J., 380, 399 Maunsbach, A. B., 250, 254, 295, 312, 313 Maurer, H. R., 79, 96 Maurer, P. H., 280, 317 Maver, M. E., 137, 178 Maxwell, E. X., 343, 381, 403, 405 Mayersbach, H., 269, 313 Mazelis, M., 227, 235 Mazia, D., 51, 99, 502, 503, 508 Mego, J. L., 284, 313 Meijer, A. E. F. H., 331, 332, 333, 404 Meisel, E., 249, 254, 318 Meister, A., 379, 403, 486, 489, 490, 507 Menefee, M. G., 267, 268, 269, 279, 313 Meng, H. €., 273, 313 Menke, W., 185, 235 Mennis, A., 486, 508 Menon, T., 88, 101 Mentendiek, M. A., 393, 394, 406 Merker, H. J., 130, 178, 354, 397, 404 Merkl, L, 342, 402 Merrill, S. H., 496, 506 Meselson, M., 52, 99 MetchnikoflF, E., 262, 313 Meudt, R., 93, 95 Meyer, R. K., 497, 507 Michaelson, I. A., 378, 406 Michell, R. H., 342, 403 Micou, J., 57, 58, 97 Midgley, J. E., 468 Milhaud,G.,21 1,252 Miller, A. T., Junr., 269, 271, 282, 313, 314 Miller, E. C, 345, 400 Miller, P., 241, 242, 246, 254, 259, 263, 264, 267, 268, 269, 284, 288, 290, 301, 304, 313, 331, 404 Miller, J. A., 345, 400 Miller, L., 273, 307 Miller, L. A., 87, 99 Miller, L. R., 243, 244, 259, 260, 266, 273, 310 Miller, T. B., 267, 268, 313 Millet, J., 211, 2i2 AUTHOR INDEX 535 Mills, G. T., 88, 99, 101 MUofsky, E., 281, 314 Minick, O. T., 253, 275, 295, 304, 311 Mirsky, A. E., 29, 31, 32, 35, 40, 44, 45, 53, 54, 57, 68, 69, 70, 71, 76, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 90, 91, 93, 95, 96, 97, 99, 100, 101, ni, 178 Mitchell, H. K., 488, 507 Mitchell, P., 120, 121, 171, 178 Mitchison, J. M., 276, 313 Mitoma, C, 499, 505 Miyawaki, H., 286, 313 Mizen, N. A., 347, 404 Mizrahi, I. J., 349, 394, 401, 403 Mizuno, D., 442, 447, 465, 468 Mizuno, N., 257, 314 Mobberly, M. L., 350, 401 Modena, B., 161, 175 Modjtabai, A., 296, 315 Moe, H., 254, 259, 288, 307, 313 Moldave, K., 428, 469, 471, 496, 506 Moli, S. L., 298, 314 MoUendorflF, W., 262, 275, 313 MoUenhauer, H. H., 321, 322, 325, 327, 383, 406 Molnar, J., 381, 391, 403 Monod, J., 499, 507 Monro, R., 427, 428, 468 Monro, R. E., 420, 428, 465, 472 Monty, K. J., 31, 96 Moohr, J. W., 65, 97 Moore, B. W., 249, 313 Moore, D. H., 267, 313 Moore, G., 263, 316 Moore, R. D., 291, 316 Moore, S., 484, 508 Moore, T., 251, 309 Morals, R., 343, 344, 366, 400, 403 More, R. H., 272, 307 Moret, v., 122, 179 Morgan, R. S., 448, 467, 469 Morgan, W. S., 495, 507 Morino, Y., 132, 178 Morris, A. J., 421, 427, 428, 465, 471 Morris, B., 272, 310 Morris, F. G., 280, 307 Morris, H. P., 132, 179, 499, 508 Morris, L., 484, 507 Morrison, A. B., 251, 254, 260, 312, 316 Morse, A., 288, 310 Morse, S. I., 250, 310 Mortimer, D. C, 217, 220, 233 Morton, M. J., 496, 507 Morton, R. K., 87, 88, 96, 99, 188, 235, 377, 379, 380, 382, 383, 395, 403 Mosser, E. L., 283, 310 Mottet, N. K., 275, 317 Moule, Y., 32, 91, 96, 99, 165, 176, 337, 339, 341, 343, 344, 347, 348, 349, 350, 352, 354, 361, 362, 364, 365, 366, 368, 391, 392, 395, 399, 400, 403 Movat, H. Z., 267, 292, 310, 313, 318 Mueller, B., 267, 268, 313 Mueller, C. B., 269, 279, 313 Mueller, G. C, 80, 101 Mugnaini, E., 149, 150, 178 Mukherjee, B. B., 477, 507 Muller, M., 257, 266, 271, 313 MuUer, W., 59, 97 MuUins, L. J., 323, 403 Mulnard, J., 257, 259, 310, 313 Mumaw, V. R., 291, 316 Munch-Peterson, A., 88, 101 Munro, A. J., 421, 471 Munro, H. N., 165, 777, 338, 345, 349, 351, 362, 364, 365, 366, 391, 393, 401, 402 Munro, T. R., 257, 313 Muramatsu, M., 33, 46, 49, 99 Murer, E., 159, 175, 375, 383, 401 Murphy, E. A., 489, 505 Murray, M. I., 277, 313 Muscatello, U., 369, 377, 380, 403 Mustard, J. P., 292, 313 Myers, D. K., 136, 178, 497, 507 Myers, J. K., 267, 268, 269, 279, 313 Myers, L. T., 341, 379, 403 Myrvik, Q. N., 250, 312 N Naccarato, R., 394, 401 Nachmias, V. T., 263, 313 Nadakavukaren, M. J., 167, 169, 178 Nadler, N. J., 266, 313 Nagel, W., 285, 297, 313 Nairn, C. 3., 295, 313 Nakajima, Y., 303, 310 Nakamoto, T., 61, 97 536 AUTHOR INDEX Nakamura, H., 200, 235 Naora, H., 58, 99 Naora, H., 58, 99 Napolitano, L., 288, 289, 313 Nass, M. M. K., 106, 146, 149, 178 Nass, S., 106, 146, 149, 178, 477, 506 Nathans, D., 428, 471 Nebert, D. W., 365, 396, 403 Neifakh, S. A., 136, 178 Neil, M. W., 248, 313, 314 Nelson, E. L., 293, 314, 503, 507 Nelson, R. A., 277, 314 Neu, H. C, 382, 403, 425, 440, 445, 446, 447, 449, 466, 471 Neubert, D., 129, 130, 178 Neufeld, E. P., 218, 220, 233 Neutra, M., 263, 315 Neville, D. N., 356, 358, 372, 374, 403 Newburgh, R. W., 497, 507 Newcomb, E. H., 424, 468 Newton, G. A., 186, 235 Newton, J. W., 186, 235 Nichols, G., 288, 319 Nichols, G., Junr., 288, 319 Nicol, T., 277, 314 Nieman, R. H., 200, 235 Niemi, M., 269, 314 Nilsson, O., 255, 285, 310, 314 Nishida, G., 132, 178 Nishizuka, Y., 427, 463, 471 Nodes, J. T., 248, 315 Noe, E., 343, 344, 403 Nogare, S. D., 484, 506 Noll, H., 421, 423, 433, 463, 471, 472, 473 Nomura, M., 454, 471 Norberg, B., 57, 95 Nordlie, R. C, 138, 777, 178, 342, 344, 345, 403, 499, 506 Norris, C. B., 484, 500, 501, 504 Norris, J. L., 380, 402 North, J. C, 345, 391, 396, 403 North, R. J., 266, 314 Norum, K. R., 115, 775 Nossal, G. J. v., 279, 291, 294, 306, 314 Novelli, G. D., 434, 467, 470, 473 Novikoff, A. B., 3, 4, 20, 25, 105, 77.5, 241, 242, 243, 250, 255, 259, 285, 288, 294, 295, 297, 301, 307, 309, 314, 323, 331, 332, 333, 335, 343, 344, 386, 390, 393, 395, 396, 397, 401, 403, 499, 507 Nunley, C. E., 484, 485, 506 Nusslein, G., 267, 577 O Oaks, B. A., 213, 234 Oberling, C, 152, 775 Ochoa, S., 211, 234, 445, 472 O'Connor, M. L., 433, 463, 469 Oda, T., 154, 155, 166, 167, 775, 176 Ogawa, K., 167, 178, 257, 314 Ogren, W. L., 203, 235 Ogston, A. G., 9, 25 Ogura, M., 338, 365, 405 6hEocha, C, 182, 234 Ohnishi, T., 137, 178 Ohnishi, T., 137, 775 Ohtaka, Y., 444, 448, 467, -^77 Okada, S., 303, 306 Okagaki, H., 66, 100 Okamoto, M., 257, 314 Okamoto, T., 420, 449, 471 Okamura, N., 72, 101 Okunuki, K., 283, 314 Old, L. J., 256, 577 Olhagen, B., 283, 575 Oliver, J., 240, 268, 269, 304, 314, 317 Olivero, E., 206, 235 Olszewska, M. J., 258, 314 Omachi, A., 343, 403 Omura, T., 340, 378, 388, 403 Ondarza, R., 88, 99 O'Neal, R. M., 272, 577 O'Neill, C. H., 263, 314 Ongun, A., 218, 220, 236 Oort, J.. 292, 505 Oren, R., 281, 314 Orfei, E., 253, 275, 295, 304, 577 Ornstein, L., 494, 507 Orrenius, S., 340, 355, 356, 361, 387, 396, 397, 400, 401, 403 Ortiz, J., 249, 254, 575 Ortiz, P. J., 454, 464, 468, 471 Osawa, S., 31, 44, 45, 70, 76, 79, 80, 81, 85, 93, 95, 99, 127, 775 Osborne, P. J., 271, 314 Osinshak, J., 254, 255, 259, 570, 314 on, Marilyn, 491, 505 Ott, M. G., 495, 505 » AUTHOR INDEX 537 Oudea, P. R., 295, 314 Ove, P., 59, 99 Overath, P., 116, 117, 175, 341, 400 Pack, B. A., 16, 25 Packer, L., 123, 124, 178, 327, 369, 404 Padykula, H. A., 273, 312 Page, A. C, 365, 378, 403 Page, S., 448, 467, 471 Pagliaro, L., 298, 314 Paigen, K., 245, 314 Palade, G. E., 2, 3, 6, 25, 26, 32, 35, 36, 37, 46, 49, 98, 99, 105, 178, 242, 246, 254, 259, 263, 264, 267, 269, 275, 279, 282, 284, 290, 301, 304, 308, 309, 310, 312, 313, 314, 111, 111, 328, 337, 339, 340, 341, 342, 351, 352, 354, 361, 362, 364, 365, 366, 368, 369, 370, 381, 387, 390, 391, 393, 401, 404, 408, 429, 431, 432, 439, 440, 462, 465, 466, 471, 472 Palay, S. L., 267, 272, 314 Palk, B. A., 188, 235 Palmieri, F., 134, 178 Paneque, A., 227, 235 Pangborn, J., 251, 299, 302, 316 Panner, B. J., 254, 260, 313 Papa, S., 105, 133, 134, 178, 179 Pappas, G. D., 263, 267, 275, 308, 311, 314 Parish, J. H., 496, 506 Park, R. B., 184, 185, 186, 190, 198, 235 Parker, F., 272, 314 Parker, J. W., 256, 314 Parker, M. V., 297, 314 Parsons, C. H., Junr., 410, 471 Parsons, D. F., 167, 175, 176, 179 Parsons, J. A., 130, 775 Passera, C, 486, 507 Passonneau, J. V., 480, 484, 485, 486, 507 Pasteels, J. J., 488, 507 Patterson, M. K., 165, 178 Patterson, R., 283, 291, 315 Pauli,A.W.,49 1,509 Pavan, C, 68, 96 Pavlovec, A., 467, 471 Payne, L. C, 270, 304, 315 Peachey, L. D., 150, 151, 178 Pcarse, A. G. E., 245, 257, 269, 301, 310, 314, 315, 318 Pearson, P. B., 342, 401 Pease, D. C, 288, 316 Pedersen, K. O., 9, 26 Pedersen, S., 170, 180 Pedrotti, A., 486, 507 Peel, M., 223, 233 Pelling, G., 57, 99 Penefsky, H. S., 136, 178 Penman, S., 59, 97 Penn, N. W., 137, 178, 285, 315 Penswick, J. R., 496, 506 Perdue, J. F., 323, 371, 375, 401 Pereira, A. da S., 148, 178 Perevoshchikova, K. A., 76, 79, 102 Perkins, E. H., 293, 315 Perkins, J. P., 502, 504 Perlman, P., 362, 386, 403, 495, 505, 507, Perner,E., 211,212, 214, 2i^ Perrin, D., 430, 433, 462, 473 Perry, R. P., 58, 66, 72, 99 Persijn, J-P., 331, 332, 333, 404 Peterkovsky, A., 214, 215, 216, 235 Petermann, M. L., 32, 100, 347, 404, 408, 410, 413, 414, 415, 416, 425, 467, 468, 471 Peters, T., 341, 349, 354, 355, 361, 366, 367, 370, 371, 375, 391, 402, 404 Petershof, I., 345, 378, 394, 400 Peterson, E. A., 410, 471 Petrack, B., 344, 404 Petrovic, J., 364, 365, 404 Petrovic, S., 364, 365, 404 Pette, D., 157, 177, 178 Pfaff, E., 139, 140, 144, 159, 777 Pharo, R. L., 119, 178 Phillips, A. H., 342, 387, 404 Philpot, J. S. L., 55, 95 Philpott, D. E., 477, 506 Pickels, E. G., 9, 25 Pierpoint, W. S., 222, 235 Piez, K. A., 484, 507 Pina, M., 496, 506 Pincus, G., 494, 504 Pinet, J., 271, 277, 315 Pinna, L. A., 122, 179 Pirie, N. W., 439, 467, 471 Pirson, A., 199, 235 Pistorius, E., 200, 236 538 AUTHOR INDEX Pitot, H. C, 364, 365, 366, 391, 395, 396, 404 Plantin, L. O., 283, 318 Plate, S., 31, 96 Platova, T. P., 78, 99 Plaut, W., 58, 97 Podber- Wagner, E., 339, 343, 344, 351, 401, 403 Pogo, A. O., 44, 78, 99 Pogo, B. G. T., 44, 78, 99 Polgar, M., 257, 271, 313 Pollak, J. K., 362, 365, 392, 404 Pollard, J. K., 489, 508 Pollister, A. W., 40, 44, 45, 99 Pon, N. G., 185, 190, 195, 198, 211, 212, 234, 235 Popper, H., 241, 256, 307 Porteous, J. W., 358, 372, 377, 379, 404 Porter, H. K., 217, 219, 220, 233 Porter, K. R., 261, 263, 268, 270, 278, 288, 295, 306, 315 Porter, W. L., 488, 508 Possingham, J. V., 199, 201, 236 Potter, V. R., 2, 25, 31, 58, 99, 100, 485, 496, 504, 507 Preiss, J., 87, 99 Prescott, D. M., 54, 58, 68, 99, 494, 508 Pressman, B. C, 23, 25, 137, 176, 240, 242, 244, 249, 253, 298, 309, 337, 338, 339, 341, 342, 344, 345, 346, 386, 400, 497, 505 Preuss, A., 64, 97 Price, C. A., 361, 399 Pricer, W. E., 342, 402 Proctor, M. H., 435, 437, 462, 471 Prose, P. H., 293, 309 Pruzansky, J. J., 283, 291, 315 Pugh, D., 303, 315 Pullman, E., 136, 178 Pullman, M. E., 136, 178 Quagliariello, E., 105, 133, 134, 178, 179 Quantock, D. C, 277, 314 Quastel, J. H., 282, 315, 389, 404 Quertier, J., 257, 276, 315 Raacke, I. D., 224, 233, 460, 462, 463, 467, 471 Rabinovitz, M., 59, 80, 97, 98 Rabinowitz, M., 148, 179 Racker, E., 123, 136, 167, 178, 179, 214, 215, 216, 235 Ragab, H., 246, 317 Rahman-Li, Y., 118,775 Rahman, M. M., 327, 369, 404 Rahman, Y. E., 252, 256, 315 Raimondi, A. J., 270, 315 Raison, J. K., 188, 235 Rakita, L., 129, 108 Rail, T. W., 88, 101 Ramasamna, T., 217, 235 Rambourg, A., 263, 315 Ramirex, J. M., 227, 235 Ramuz M,, 497, 505 Randerath, E., 269, 315 Rather, L. J., 269, 315 Raymond, S., 494, 508 Razzell, W. E., 344, 379, 404 Redfearn, E. R,, 235 Redman, C. M., 366, 391, 404 Reed, L. J., 156, 177, All, 506, 507 Reen, R., 342, 401 Rees, K. R., 86, 99, 100 Reich, E., 59, 66, 97, 100, 130, 143, 149. 777 Reichard, P., 57, 95 Reichman, E. S., 372, 375, 405 Reid, E., 62, 75, 87, 100, 248, 315, 328, 331, 337, 338, 339, 340, 341, 344, 345, 346, 348, 357, 351, 362, 364, 365, 367, 370, 371, 372, 374, 377, 379, 381, 384, 392, 394, 395, 399, 401, 404, 483, 496, 508 Reis, P. J., 110, 138, 779 Reller, H. H., 268, 310 Remmer, H., 343, 350, 354, 396, 397, 404 Revel, J. P., 272, 314 Revel, M., 71, 98 Rho, J. H., 31, 46, 57, 95, 100 Rhodin, J., 268, 288, 315 Rice, L. I., 343, 404 Rich, A., 59, 66, 97, 421, 436. 462, 470, 473 Richards. E. G., 496, 508 AUTHOR INDEX 539 Richards, J., 50, 101 Richardson, C. C, 50, 100 Richardson, S. H., 162, 163, 179, 375, 383, 401 Richter, G., 214, 235 Riddle, J. M., 284, 315 Rieske, J. G., 165, 178 Riley, V. T., 10, 25 Rimington, C, 132, 779 Ris, H., 35, 37, 38, 40, 45, 99, 100 Ritossa, F. M., 73, 100 Ritter, D. B., 415, 468 Rizak, M. A., 285, 309 Robbins, E., 276, 315 Robbins, J., 477, 508 Roberts, E., 379, 406, 486, 508 Roberts, J., 282, 315 Roberts, R. B., 408, 410, 413, 429, 430, 435, 436, 437, 439, 440, 441, 442, 444, 449, 456, 457, 462, 464, 465, 466, 467, 478, 471, 472, 503, 504 Robertson, J. D., 322, 404 Robineaux, R., 264, 271, 277, 315 Robinson, D., 499, 506 Robinson, D. S., 273, 315 Robinson, H. K., 449, 466, 473 Rodbell, M., 273, 315 Rodgers, A., 75, 98 Rogers, L. A., 396, 397, 401 Rogers, W. I., 496, 507 Rohlich, P., 257, 263, 266, 271, 315, 317 Rohringer, R., 215, 216, 228, 229, 236 Rolfe, R., 52, 100 Rolon, C. I., 249, 257, 266, 315 Rongine de Fekete, M. A., 218, 235 Roodyn, D. B., 5, 25, 29, 32, 81, 82, 85, 90, 91, 92, 100, 104, 105, 110, 113, 114, 118, 128, 130, 131, 137, 138, 141, 143, 146, 158, 161, 164, 165, 171, 176, 179, 293, 309, 345, 346, 404, 483, 496, 498, 508 Rose, F. A., 343, 370, 400 Rose, G. G., 264, 315 Rosenbaum, R. M., 249, 257, 266, 271, 315 Rosenberg, L. L., 213, 220, 221, 235 Rosenbluth, J., 263, 270, 315 Rosenfeld, S., 283, 284, 311, 316 Rosenthal, O., 340, 343, 349, 371, 378, 388, 403, 404 Ross, H. F., 86, 99 Rossi, C, 1 14, 179 Rossi, C. S., 10, 25, 128, 159, 175, 179 Rossi, F., 245, 281, 315 Rossowski, W., 495, 508 Rostgaard, J., 272, 315 Roth, J. S., 132, 179, 343, 366, 404, 439, 440, 448, 467, 472, 499, 508 Roth, L. E., 267, 315 Roth, T. F., 263. 268, 270, 278, 315 Rothenberg, M. S., 292, 306 Rothschild, J. A., 360, 404 Rothstein, A., 483, 508 Rotman, B., 436, 437, 444, 462, 466, 470, 472 Rouiller, Ch., 32, 54, 55, 96, 97, 165, 172, 176, 179, 268, 296, 310, 315, 337, 339, 341, 343, 344, 345, 347, 348, 349, 350, 352, 354, 361, 362, 364, 365, 366, 368, 386, 391, 395, 396, 400, 403, 404 Rowe, K. W., Junr., 488, 508 Rowland, G. F., 86, 99, 100 Rowley, D., 277, 311 Roy, A. B., 245, 315 Royster, B., 499, 509 Rozich, R., 497, 507 Rubinstein, D., 85, 100 Rudkin, G. T., 67, 68, 100 Rumke, Ph., 323, 356, 358, 372, 374, 375, 394, 401 Rurainski, H. J., 201, 234 Ruska, H., 267, 313 Russman, W„ 488, 505 Rustad, R. C, 276, 280, 282, 309, 315 Rusza, P., 263, 317 Rutgeerts, M-J., 10, 26, 295, 318 Rutter, W. J., 343, 400 Ruyter, J. H. C, 256, 315 Ryan, J., 343, 344, 403 Ryser,H., 263, 271,5/5 Ryter, A., 322, 404 Sabatani, D. D., 331, 404 Sabesin, S. M., 291, 315 Sabin, F. R., 268, 289, 315 Saccone, C, 134, 178 Sachs, H., 362, 404 Saetren, H., 29, 81, 87, 88, 90, 91, 95, 101 Safier, L. B., 256, 312 540 AUTHOR INDEX Sager, R., 202, 233 Saito, K., 281, 314 Sakamoto, K., 489, 505 Salas, M., 445, 472 Salmon, B., 375, 383, 401 Salvatore, G., 477, 508 Salvatore, M., 477, 508 Salzmann, R., 287, 316 Samarina, O. P., 32, 35, 44, 45, 57, 59, 64, 66, 71, 72, 74, 78, 79, 80, 97, 100, 102 Samuels, L. T., 381, 402 Sanadi, D. R., 105, 113, 119, 122, 154, 156, 178, 179 Sandborn, E., 263, 316 Sandelin, K., 298, 306 Sanders, E., 273, 306, 316 San Pietro, A., 202, 203, 204, 233, 234, 235 Santamaria, A., 292, 295, 310, 311 Sanukida, S., 164, 777 Sanwal, B. D., 221, 223, 235 Sarkar, S. K., 266, 313 Samat, M,, 64, 101 Samat, M. T., 65, 97 Sato, R., 340, 378, 388, 403 Saukkonen, J. J., 485, 508 Sawant, P. L., 245, 247, 250, 251, 252, 253, 257, 261, 287, 288, 298, 299, 316 317 Sbarra, A. J. 277, 278, 281, 316 Scarisbrick, R., 201, 254 Schachman, H. K., 9, 25, 410, 411, 468, 499, 506 Schaeffer, J., 420, 427, 428, 465, 468 Schaefer, S., 297, 311 Schaffner, P., 241, 256, 307 Schajowicz, P., 288, 316 Schaub, M. C, 245, 316 Schayer, R. W., 268, 316 Schechtman, A. M., 270, 316 Scheib, D., 286, 288, 316 Scherer, J., 297, 311 Scherrer, K., 72, 100 Schildkraut, C. L., 50, 100 Schiller, A. A., 268, 316 Schirren, V., 362, 364, 402 Schlessingcr, D., 410, 421, 430, 452, 455, 460, 462, 470, 472 Schlicht, I., 295, 316 Schmidt, W., 275, 276, 316 Schmitt, A., 286, 316 Schmitz, H., 484, 486, 508 Schneebeli, J., 165, 176, 341, 343, 344, 350, 352, 354, 365, 366, 400 Schneeberger, E. E., 251, 316 Schneider, J. H., 58, 100 Schneider, R. M., 32, 100 Schneider, W. C, 2, 4, 6, 7, 17, 25, 26, 32, 33, 51, 82, 85, 87, 95, 98, 100, 111, 112, 117, 118, 128, 131, 133, 134, 177, 179, 248, 249, 255, 298, 312, 316, 338, 339, 342, 351, 355, 358, 364, 371, 376, 404, 408, 470, 483, 497, 506, 507 Schnitger, H., 488, 505 Schoefl, G. I., 279, 312 Schoenberg, M. D., 291, 316 Scholefield, P. G., 389, 390, 404 Schollmeyer, P., 124, 177 Scholtissek, C, 58, 100 Schor, N., 66, 96 Schotz, M. C, 343, 404 Schuel, H., 249, 316 Schulman, H. M., 63, 100 Schulz, D. W., 480, 485, 507 Schulz, H., 243, 316 Schumaker, V. N., 282, 316 Schuster, P. L., 148, 179 Schwartz, A., 378, 382, 405 Schweet, R., 420, 427, 428, 465, 468 Schweiber, H. G., 79, 100 Scott, B. L., 288, 316 Scott, J., 245, 254, 312 Scott, K., 275, 309 Scow, R. O., 273, 315 Seaman, G. R., 257, 316 Sears, M. L., 133, 777 Sedgwick, B., 245, 316 Seegal, B. C, 292, 306 Sekiguchi, M., 449, 472 Sekuzu, L., 161, 779 Seligman, A. M., 245, 254, 303, 316 Sellers, A. L., 283, 284, 311, 316 Sellinger, O. Z., 7, 9, 19, 22, 23, 24, 118, 172, 775, 245, 316, 378, 404 Semadeni, E., 258, 312 Senior, J. R., 379, 405 Setlow, J. K., 52, 100 Sctlow, R. B., 52, 100 Scttcrfield, G., 46, 98 Shahar, A., 263, 266, 306 AUTHOR INDEX 541 Shakulov, R. S., 416, 444, 466, 472 Shapiro, B., 342, 405 Share, L., 327, 405 Sharman, I. M., 251, 309 Shatkin, A. J., 59, 66, 100 Shavit, M., 232, 233 Shaw, C. R., 498, 508 Shaw, J., 491, 508 Shefner, A. M., 199, 233 Sheid, B., 132, 179, 499, 508 Sheldon, H., 254, 316 Shibko, S., 245, 246, 247, 248, 250, 251, 253, 254, 257, 287, 298, 299, 302, 316, 317, 343, 344, 350, 405 Shimazono N, 341, 405 Shin, M., 203, 204, 235, 236 Shin, W. Y., 255, 288, 307, 314 Shiratori, J., 200, 234 Shirley, W., 278, 316 Shnitka, T. K., 245, 254, 303, 316 Shorey, C. D., 362, 365, 392, 404 Shortman, K., 442, 452, 462, 466, 472 Shull, K. H., 341, 387, 405 Shulman, H. M., 383, 405 Sibatani, A., 66, 100 Sicot, C, 280, 316 Siebert, G., 29, 30, 51, 62, 75, 82, 83, 84, 85, 86, 87, 88, 89, 92, 96, 98, 100, 101, 249, 286, 316, 325, 328, 331, 337, 404, 405 Sieffert, U. B., 129, 176 Siegel, L., 113,779 Siegel, S. J., 490, 508 Siekevitz, P., 2, 3, 25, 26, 32, 35, 36, 37, 46, 49, 99, 139, 141, 143, 154, 179, 180, 311, m, 328, 337, 339, 340, 341, 342, 346, 351, 352, 354, 361, 362, 364, 365, 366, 367, 368, 369, 370, 387, 381, 387, 388, 390, 391, 392, 393, 395, 401, 404, 405, 425, 429, 431, 439, 440, 462, 465, 466, 472 Siliprandi, N., 122, 179 Simmons, J. R., 488, 507 Simms, E. S., 50, 98 Simon, E. H., 52, 101 Simonis, W., 208, 213, 236 Simonsen, Daisy, G., 486, 508 Simpson, M. V., 285, 317 Simson, P., 365, 370, 400 Sinclair, J., 148, 179 Sinclair, J. G., 297, 314 Singer, M. S., 467, 469 Singer, T. P., 114, 119, 161, 777, 77P Singhal, R. L., 393, 394, 406 Siperstein, M. D., 341, 364, 390, 394, 395, 405 Sirlin, J. L., 66, 74, 707 Sissakian, N. M., 217, 218, 219, 226, 236 Sizer, I. W., 341, 405 Sjostrand, F. S., 323, 325, 405 Skipski, V. P., 372, 375, 405 Sklar, S., 255, 294, 307 Slater, E. C, 105, 123, 125, 126, 127, 136, 178, 179 Slater, J. J., 255, 306 Slater, T. F., 7, 25, 297, i77 Slautterback, D. B., 161, 162, 163, 775 Slayter, H. S., 411, 457, 469 Smellie, R., 85, 707 Smellie, R. M. S., 30, 50, 51, 57, 75, 76, 79, 98, 101 Smetana, H., 269, 317 Smetana, K., 33, 46, 49, 99 Smillie, R. M., 195, 196, 197, 211, 212, 213, 214, 215, 216, 218, 221, 228, 230, 236 Smirnov, M. N., 59, 66, 72, 97 Smith, A. D., 256, 317 Smith, E. E. B., 88, 99, 101 Smith, E. L., 486, 507 Smith, E. W., 287, 308 Smith, J., 344, 345, 357, 361, 367, 371, 374, 384, 401 Smith, J. C., 380, 405 Smith, L., 202, 236 Smith, J. H. C, 225, 236 Smith, M. A., 445, 472 Smith, R. E., 259, 577 Smith, S. H. J., 51, 707 Smith, S. M. J., 54, 98 Smithies, O., 494, 508 Smyrniotis, P. Z., 215, 216, 234, 237 Snedeker, E. H., 488, 505 Solomon, A., 283, i77 Song, S. K., 255, 306 Sonnino, F. R., 495, 506 Sorkin, E., 283, 293, 307, 317 Sorof, S., 491, 495, 508 Sotelo, J. R., 41, 96 Spackman, D. H., 484, 508 542 AUTHOR INDEX Spahr, P. F., 414, 415, 440, 441, 442, 444, 455, 456, 464, 466, 472 Spargo, B., 243, 288, 294, 295, 311 Spark, J. v., 293, 207 Speirs, R. S., 291, 317 Spencer, B., 343, 370, 400 Spencer, D., 199, 201, 224, 225, 236 Spencer, T., 75, 98 Sperti, S., 122, 179 Spicer, S. S., 256, 266, 267, 284, 311, 318, 319 Spiegelman, S., 28, 56, 59, 63, 64, 65, 66, 73, 98, 100, 101, 102, 413, 430, 435, 437, 449, 468, 469, 473 Spimazono, N., 345, 406 Spirin, A. S., 416, 444, 466, 472 Spiro, M. J., 378, 405 Spirtes, M. A., 340, 347, 405 Spitnik-Elson, P., 414, 415, 466, 472 Spitznagel, J. K., 250, 319 Squires, R. F., 428, 470 Srinivasan, P. R., 454, 472 Stadtman, E. R., 500, 509 Staehelin, T., 421, 423, 472, 473 Stamm, N. B., 393, 394, 406 Stange, J. L., 132, 179, 499, 508 Stanley, W. M., Junr., 445, 472 Stare, F. J., 272, 318 Stasny, J. T., 167, 179 Staubesand, J., 267, 317 Staubli, W., 272, 310 Staudinger, Hj., 365, 378, 403 Stavy, L., 448, 455, 466, 467, 472 Stedman, E., 69, 101 Stedman, E., 69, 101 Steels, W. J., 72, 101 Steffen, K., 187, 236 Stein, W. H., 484, 508 Stein, Y., 342, 405 Steinbauer, A. L., 488, 508 Steinberg, D., 285, 317, 341, 399 Stembridge, V. A., 273, 306 Stenesh, J. J., 439, 467. 472 Stenger, R. J., 261, 308 Stent, G. C, 63, 98 Stephen, W. P., 488, 508 Stern, F., 486, 508 Stem, H., 29, 32, 46, 53, 81, 82, 84, 85, 87, 88, 90, 91, 95, 98, 101 Stetten, M. R., 342, 344, 345, 405 Stevens, B. M., 364, 404 Steward, F. C, 489, 508 Stewart, S. G., 399 St. George, S., 272, 310 Stinson, H. T., 185, 237 Stocken, L. A., 86, 96 Stocking, C. R., 193, 217, 218, 219, 220, 233, 236 Stockinger, L., 275, 276, 317 Stodolsky, M., 64, 101 Stoeckenius, W., 167, 179, 111, 323, 405 Stolzenbach, F. E., 204, 234 Storck, R., 63, 101 Storey, I. D., 343, 400 Stowell, R. E., 297, 318 Strasdine, G. A., 455, 464, 472 Straus, W., 9, 26, 240, 242, 243, 245, 246, 247, 248, 249, 250, 252, 253, 254, 256, 258, 261, 262, 263, 264, 266, 267, 268, 269, 274, 276, 279, 280, 284, 286, 293, 294, 295, 296, 298, 299, 304, 305, 317, 392, 405 Strauss, E, W., 273, 312, 317 Strecker, H. J., 134, 777, 378, 401 Strehler, B. L., 286, 310 Strelzoff, E.. 63, 101 Strickland, K. P., 379, 389, 405 Striebich, M. J.. 7, 25, 32, 85, 98, 117, 128, 177 Strittmatter, P., 341, 371, 388, 405 Strominger, J. L., 343, 405, 485, 508 Strong, F. M., 129, 176 Struchkov, V. A., 38, 101 Struck, J., 341, 405 Stumpf P. K., 214, 225, 236 Sueoka, N., 52, 101 Sugar, J., 200, 234 Suszko, J. M., 283, 291, 315 Sutarua, P., 489, 505 Sutherland, E. W., 88, 101 Suttie, J. W., 113, 131, 158,779 Suzuki, K., 341, 405 Suzuki, M., 272, 317 Svedberg, T., 9, 26 Swann, H. G., 297, 314 Swanson, H. D., 491, 493, 494, 504, 508 Swanson, P. D., 382, 400, 405 Swenson, P. A., 490, 508 Swick, R. W., 132, 179, 499, 508 Swift, H., 35, 37, 41, 101, 148, 179, 243, 288, 294, 295, 311 AUTHOR INDEX 543 Swingle, K. F., 249, 317 Szafarz, D., 57, 98 Tagawa, K., 202, 203, 204, 207, 236, 237 Tager, J. M., 105, 125, 126, 127, 179 Tahaba, Y., 291, 317 Taichman, N. S., 292, 313 Takahashi, N., 275, 317 Takahashi, W. N., 466, 470 Takamiya, A., 200, 201, 234 Takanami, M., 338, 343, 350, 366, 405 420, 449, 471, 472 Takeo, K., 137, 178 Tal, M., 430, 440, 442, 443, 449, 450, 451, 452, 462, 466, 469, 472 Talley, E. A., 488, 508 Tamaoki, T., 80, 101 Tanaka, H., 243, 276, 317 Tanaka, R., 379, 389, 405 Tandler, C. J., 74, 101 Tappel, A. L., 245, 246, 247, 248, 250, 251, 252, 253, 254, 257, 261, 287, 288, 298, 299, 302, 311, 316, 317, 343, 344, 350, 405 Tashiro, Y., 322, 328, 338, 365, 366, 367, 368, 392, 405, 439, 440, 467, 472 Tata, J. R., 131, 158. 170, 171, 176, 179, 180, 497, 508 Tatum, E. L., 59, 66, 100 Taylor, A. B., 272, 312 Taylor, J. H., 101 Tedeschi, H., 327, 405 Tee, D. E. H., 488, 508 Telfer, W. H., 270, 278, 317 Tennyson, V. M., 275, 314 Tepperman, H. M., 394, 395, 405 Tepperman, J., 394, 395, 405 Terebus-Kekish, O., 372, 375, 405 Terroine, T., 393, 405 Terskich, V. V., 72, 73, 75, 98 Tessenow, W., 250, 317 Tewari, K. K., 148, 180 Tewfik, S., 214, 236 Thalacker, R., 193, 194, 196, 236 Thines-Sempoux, D, G., 245, 312 Thomas, G. H., 466, 472 Thomas, J., 343, 370, 400 Thomas, J. B., 185, 210, 212, 214, 215, 216, 218, 219, 223, 228, 229, 236 Thomas, L., 251, 252, 297, 311, 318 Thomas, L. E., 162, 777 Thomson, W. W., 184, 237 Thorbecke, G. J., 256, 280, 317 Thorpe, B. D., 285, 317 Tice, L. W., 331, 332, 335, 386, 393, 405 Tietz, A., 342, 405 Tilander, K., 344, 402 Timonen, S., 85, 101 Tisdale, H., 131, 144, 154, 158, 162, 164, 176 Tishkoff, G. H., 29, 96 Tissieres, A., 410, 430, 472 Tocchini-Valentini, G. P., 64, 65, 97, 101 Tolbert, N. E., 222, 223, 236 Tombs, M. P., 494, 508 Tomkins, G. M., 342, 405 Tomlin, S. G., 37, 96 Tooth, B. E., 132, 179 Torack, R. M., 255, 317 Toro, I., 257, 263, 266, 271, 313, 317 Toschi, G., 378, 405 Toth, J., 257, 266, 271, 313 Touster, O., 165, 178 Tower, D. B., 485, 505 Townsend, C. M., 497, 507 Trabal, F., 486, 508 Traub, A., 459, 463, 469, 470, 472 Traut, R. R., 420, 428, 465, 472 Treadwell, P. E., 293, 317 Trebst, A., 200, 207, 214, 215, 216, 228, 236 Trebst, A. V., 209, 211, 212, 213, 214. 215, 216, 228, 229, 235, 236 Trivus, R. H., 340, 347, 405 Trotter, N. L., 273, 317 Trowell, O. A., 295, 318 Trudinger, P. A., 211, 236 Truman, D. E. S., 158, 180 Trump, B. F., 243, 254, 275, 276, 279, 295, 297, 309, 318 Tschirgi, R. D., 497, 504 Ts'o, P. O. P., 211, 235, 414, 439, 466, 472, 473 Tsujimoto, H. Y., 203, 207, 232, 236 Tulkens, P., 10, 26, 295, 318 Tumanjan, V. D., 45, 64, 66, 80, 100 Tunn, v., 485, 508 544 AUTHOR INDEX Turk, J. L., 256, 291, 309 Turkington, V., 466, 470 Turner, J. F., 213, 214, 215, 220, 234 Turner, M. K., 62, 75, 87, 100, 328, 331, 337, 404 TzagolofF, A., 165, 178 U Ubich, H. von, 57, 95 Uchida, K., 444, 448, 467, 471 Udenfriend, S., 499, 505 Ullman, H. L., 256, 312 Ullrey, D., 357, 405 Underhay, E., 245, 318, 343, 386, 405 Urata, G., 132, 176 Urbach. W., 208, 236 Urinhara, T., 292, 313, 318 Ussing, H., 483, 508 Vaes, G., 174, 180, 256, 288, 318 Vandermeerssche, G., 488, 507 Vaneste, M., 345, 391, 396, 403 Van Lancker, J. L., 249, 261, 288, 296, 297, 299, 318, 382, 402 VanNoort, G., 211,25<5 Vars, H. M., 343, 349, 371, 404 Vaugham, M., 285, 317 Vaugham, R. B., 277, 318 Vecchio, G., 477, 508 Veerabhadrappa, P. S., 134, 175 Velick, S. F., 341, 405 Vennesland, B,, 200, 235 Verney, E. L., 257, 310 Vernier, R., 256, 318 Vernon-Roberts, D. C, 277, 314 Verster, F. de B., 378, 405 Vesell, E. S., 92, 101 Viala, R., 245, 318 Villar, O., 268, 312 Vincent, W. S., 35, 46, 47, 101 Vinijchaikul, K., 57, 96 Vinograd, J., 414, 439, 466, 473 Vladimirzeva, E. A., 72, 73, 75, 98 Volini, F. I., 253, 275, 295, 304, 311 Von Korff, R. W., 107, 176 Vorbrodt, A., 303, 318 W Wachstein, M., 249, 254, 318, 333, 335, 389, 393, 397, 405 Wachtler, K., 257, 318 Wada, H., 132, 178 Waddell, W. R., 272, 318 Wade, H. E., 449, 455, 464, 466, 468, 473 Wade, R. D., 425, 432, 439, 440, 465, 470 Wagner, H. N., 280, 318 Wagner, S., 229, 236 Wahba, A. J., 445, 472 Wakasa, H., 256, 314 Wakid, N. W., 380, 405 Walek-Czernecka, A., 258, 318 Waldman, T. A., 283, 577 Walker, D. G., 392, 402 Walker, P. G., 303, 315, 344, 405 Walkinshaw, C. H., 249, 318 Wallace, J. H., 277, 312 Wallach, D. F. H., 281, 311, 311, 357, 361, 372, 374, 402, 405 Waller, J-P., 414, 415, 443, 466, 473 Walpurger, G., 484, 486, 508 Walter, H., 268, 310 Walter, P. C, 256, 309 Waltregny, A., 339, 351, 401 Wang, C-T., 344, 360, 390, 402 Wang, D. Y., 7, 25, 393, 405 Wang, K. M., 460, 463, 465, 473 Wang, M., 488, 508 Wang, T-Y., 44, 78, 91, 101, 460, 463, 465, 473 Warburg, O., 199, 236 Ward, R. T., 152, 153, 180 Warburg, O., 2, 26 Warner, J. R., 421, 473 Warren, W., 456, 464, 467, 473 Warren, W. A., 433, 462, 473 Watanabe, S., 137, 178 Watkins, J., 488, 508 Watson, J. D., 52, 101, 410, 454, 471, 472 Watson, M., 154, 179 Watson, M. L., 37, 101, 139, 141, 143, 180, 325, 405 Wattiaux, A., 338, 341, 343, 345, 346, 371, 337, 381, 400 AUTHOR INDEX 545 Wattiaux, R., 3, 4, 7, 10, 23, 24, 25, 26, 105, 118, 133, 137, 172, 175, 176, 180, 197, 233, 240, 242, 244, 249, 251, 252, 253, 257, 295, 298, 304, 309, 318, 337, 338, 339, 341, 342, 344, 345, 346, 367, 386, 400, 483, 496, 497, 505 Wattiaux-De Koninck, S., 1 0, 25, 295, 318 Watts, J. W., 75, 98 Waygood, E. R., 215, 216, 228, 229, 236 Webb, J. A., 273, 310 Weber, G., 393, 394, 406 Weber, R., 287, 316, 318 Webster, G. C, 77, 101 Webster, L. T., 129, 180 Weichart, G., 213, 236 Weier, T. E., 184, 237 Weigle, O., 278, 318 Weigle, W. O., 284, 308 Weill, J. D., 393, 400 Weinbach, E. C, 151, 180 Weinbren, K., 295, 309 Weinrauder, H., 495, 508 Weinstein, H., 379, 406 Weintraub, L., 494, 508 Weiss, J. M., 276, 318 Weiss, L. P., 257, 318 Weiss, S., 60, 61, 101, 102 Weiss, S. B., 61, 64, 65, 98, 101 Weissbach, A., 210, 211, 215, 216, 234, 237, 452, 453, 462, 473 Weissberger, A. S., 291, 316 Weissmann, G., 242, 251, 252, 256, 257, 261, 291, 296, 297, 311, 318 Wellenskiek, H. J., 268, 289, 318 Wellman, H., 2, 25 Went, H. A., 502, 503, 508 Wernley, D. L., 346, 400 Wesemann, W., 488, 505 West, G. R., 331, 332, 359, 376, 379, 402 Westrop, J. W., 340, 364, 396, 406 Wetterfors, J., 283, 318 Wettstein, D. von, 190, 191, 195, 234 ^ Wettstein, F. O., 421, 423, 472, 475 tWetzel, B. K., 266, 267, 311, 318 IWhaley, W. G. 321, 322, 325, 327, 383, 406 ^hatley, F. R., 189, 197, 198, 199, 203, 205, 206, 208, 220, 221, 228, 232 233, 235, 237 Whipple A. 268, 310 White, F. H., Junr., 439, 468 White, J. G., 256, 318 Whitehead, E. P., 156, 157, 180 Whitfield, P. R., 467, 473 Whittaker, V. P., 7, 26, 105, 106, 108, 165, 167, 169, 180, 311, 378, 406 Whittam, R., 389, 406. Wibo, M., 10, 26, 112, 180, 304, 318 Wibo, R., 251, 252, 309 Wienecke, A. A., 282, 296, 319, 359, 406 Wiener, E., 245, 248, 256, 285, 308 Wildman, S. G., 21 1, 224, 225, 233, 236, Wilbur, K. M., 32, 102 Wildman, S. G., 21 1, 224, 225, 233, 236, 237 Wilgram G. F., 245, 318, 342, 406 Wilkie, D., 104, 113, 114, 146, 158, 164, 179 Wilkins, M. F. H., 90, 96 Williams- Ashman, W. G., 61, 62, 95 Williams, C. H., 370, 371, 402 Williams, G. R., 123, 167, 176, 218, 220, 236 Williams, J. N., 134, 180 Williams, J. P., 190, 234 Williams, S., 467, 473 Williamson, R., 414, 448, 467, 470 d71, 473 Willig, F., 285, 297, 313 Wilson, A. T., 209, 233 Wilson, C, 448, 470 Wilson, J. W., 249, 312 Winitz, M., 489, 506 Winkelman, J., 345, 406 Winkler, H., 256, 317 Wintersberger, E., 130, 158, 180 Wissig, S. L., 263, 270, 315 Wissler, R. W., 243, 288, 293, 295, 311 Withers, R. F. J., 245, 311 Wit-Peeters, E. M., 129, 777 Wittekind, D., 276, 319 Witter, R. F., 31, 96 Wittgenstein, Eva, 488, 508 Wittner, M., 257, 315 Woessner, J. F., Junr., 242, 255, 319 Wolfe, L. S., 321, 323, 377, 383, 389, 406 Wollman, S. H., 256, 266, 267, 184, 318, 319 Woodin, A. M., 282, 283, 196, 319, 3 9 406 546 AUTHOR INDEX Woods, J. E., 288, 319 Woods, J. F., 288, 319 Woods, M. W., 10, 25 Woods, P. S., 57, 67, 68, 100, 101, 102 Woodson, M., 489, 504 Woohsmann, H., 303, 319 Woolfolk, C. A., 500, 509 Work, T. S., 32, 99, 110, 113, 131, 137, 158, 179 Worthen, H. G., 341, 379, 403 Wright, C. S., 277, 312 Wu, C, 345, 370, 371, 406 Wu, R., 486, 509 Wust, C. J., 467, 473 Wynn, C. H., 245, 310 Wynn, J., 499, 509 Yamada, K., 345, 406 Yamana, K., 66, 100 Yang, L., 202, 234 Yankofsky, S. A., 59, 66, 102, 413, 473 Yarnell, M. M., 271, 278, 309 Yasuzumi, G., 37, 38, 102 Yermolaeva, L. P., 30, 32, 44, 45, 97 Yoneda, M., 497, 509 Young, E. M., 495, 508 Young, M. R., 276, 306 Zalokar, M., 57, 102, 476, 488, 509 Zamecnik, P. C, 2, 25, 417, 418, 428, 429, 470, 473, 502, 506 Zamir, A., 496, 506 Zatti, M., 245, 281, 315 Zbarsky, I. B., 30, 32, 40, 44, 45, 76, 78, 79, 80, 97, 102 Zech, A. C, 491, 509 Zeidenberg, P., 257, 271, 319 Zeitler, E., 17, 24 Zelitch, I., 221, 222, 223, 226, 237 Zetterquist H., 254, 316 Zeya, H. J., 250, 319 Zillig, W., 64, 97, 414, 439, 440, 466, 473 Zilversmit, D. B., 272, 319 Zink, M. W., 221, 235 Zinsser, H. H., 486, 505 Zipser, D., 430, 433, 436, 462, 473 Zivago, P. I., 41, 102 Zollinger, H. U., 268, 319 Zubay, G., 37, 40, 70. 102 Zucker, M., 185, 237 Zucker-Franklin, D., 243. 256, 266, 274, 312, 319 Zweifach, B. W., 250, 297, 311 SUBJECT INDEX (The numbers in brackets after certain enzymes refer to their Enzyme Com- mission numbers — see ^'List of Enzyme Numbers", at the end of the Index. These numbers are also given in the text, on the first occasion that a given enzyme is mentioned in each chapter). I I Acetabularia RNA synthesis, 58 Acetate conversion to CO2 by nuclei, 84 into fatty acids in chloroplasts, 225 Acetate activating enzyme (6.2.1.1.) (acetyl CoA synthetase) in chloroplast extracts, 222 Acetoacetate from 3-hydroxybutyrate in mitochon- dria, 116 in liver mitochondria, metabolism, 116 transacylation with succinate in mito- chondria, 116 Acetoacetyl CoA in mitochondria, 116 Acetyl cholinesterase (3.1.1.7.) in cytomembranes adrenal, 378 brain, 378 muscle, 380 in liver microsomal fraction, 343 linkage to brain cytomembranes, 382 Acetyl CoA and acetoacetate in mitochondria, 116-17 and P-oxidation, 114 Acetyl CoA acyl transferase (2.3.1.16.) in mitochondria, 114 Acetyl CoA synthetase (6.2.1.1.) (acetate activating enzyme) in chloroplast extracts, 222 N-Acetyl-P-glucosaminidase (3.2.1 .29.) cytochemical stain, 303 in lysosomes, 245, 303 Acid deoxyribonuclease cytochemical stain, 303 in lysosomes, 137, 245, 303 Acid hydrolases markers for lysosomes, 7 Acid phosphatase (3.1.3.2.) and bone resorption, 288 and phagolysosome enlargement, 295 and post-partum involution of uterus, 287 and regressio nof Miillerian duct, 286 cytochemical staining, 301 electrophoresis, 493 Gomori procedure, 301 in lipid decomposition by lysosomes, 285 in lysosomes, release from, 252 lipofuscin granules, 286 liver fractions, and, 7 lysosomal marker, 338-9 marker enzyme, 3 nuclei, absence from, 88 presence in: Amoeba, 257 autophagic vacuoles in Euglena, 257 brain lysosomes, 255 chondrioclasts, 288 cisternae of endoplasmic reticulum, 258 "dense bodies" of thymus, 256 endothelial cells, 272 fused lysosomes and phagosomes, 264 Golgi lamellae, 259 granules in ciliates, 257 547 548 SUBJECT INDEX Acid phosphatase (3.1.3.2) — cont. presence in: — cont, granules in fibroblasts, 257 granules in macrophages, 256, 257 granules in monocytes, 257 granules in planarians, 257 granules in tissue culture cells, 257 intestine, 254 kidney lysosomes, 254 cliver lysosomes, 253^^ V. ■ Jysosbmes, 24 1, 245 muscle, 247 osteoclasts, 288 osteoclast granules, 256 pinocytic vesicles, 258 plant spherosomes, 258 platelets, 256 prostate, 255 protozoa, 257 reticulo-endothelial cells, 256 ribosomesy456) uterine lysosomes, 255 -staining, 5 Acid phosphatases heterogeneity, 248 Acid proteinase in Amoeba, 257 in lysosomes, 285 in plant spherosomes, 258 Acid ribonuclease, heterogeneity, 248 in lysosomes, 245 in muscle, 247 in plant spherosomes, 258 "Acidic protein" in nuclei, 79 Acidic proteins in ribosomes, 414 Aconitase (4.2.1.3.) in chloroplast extracts, 222 in mitochondria and cell sap, 113 Acrylamide gel electrophoresis, 494 ACTH, and membrane-linked enzymes, 394 Actinomycin and RNA synthesis in mitochondria, 129-30 inhibition of RNA synthesis, 59 inhibition of R-RNA synthesis, 72 Activation of enzymes, 251, 367 Actomyosin in mitochondria, 136-7 Acylase in kidney cytomembranes, 379 Acyl CoA dehydrogenase (1.3.99.3.) in mitochondria, 114 Acyl CoA synthetase (6.2.1.3.) in intestinal cytomembranes, 379 in liver microsomal fractions, 345 in membrane fractions, 359 3-Acylglycerophosphoryl choline acyl transferase in liver microsomal fraction, 342 Adenosine deaminase (3.5.4.4.) electrophoresis, 493 in nuclei, 90-1 Adenosine-2'-monophosphate, 204 Adenosine triphosphatase (see ATPase) Adenosine triphosphate (see ATP) Adenosyl-methionine RNA methyl- transferase in nucleoli, 74 Adenylate cyclase in nuclei, 88 Adenylate kinase (2.7.4.3.) binding to mitochondria, 144 in chloroplasts, 227 in mitochondria, 140 in nuclei, 87 Adenylate-3 '-phosphatase absence from nuclei, 90 Adenylate-5'-phosphatase absence from nuclei, 90 ADP control of mitochondrial respiration, 2 in "non-aqueous" nuclei, 83 ADPase deoxycholate effect, 369 in cytomembranes, 332, 335 in liver microsomal fraction, 344 in membrane fractions, 357, 359 in plasma membrane, 335 Adrenal cytomembranes, 378 Adsorption by cells, 263 by ribosomes, 425 of enzymes, 480 of soluble components, 480 "Aggregated enzyme", 62 Aggregation of ribosomal proteins, 415 reversibility, 477 Agranular cytomembranes, 325-7 Alanine activation in chloroplasts, 225 SUBJECT INDEX 549 Ift I Alanine aminotransferase (2.6.1.2,12.) in mitochondria and SP, 132, 499 Albumin in membrane fractions, 354 radioactive, in membranes, 355 synthesis in membranes, 366 Alcohol dehydrogenase (1.1.1.1.) in ribosomes, 462 Aldolase (4.1.2.7.) in chloroplasts, 214 in nuclei, 82, 92 in plants, 217 ^ in ribosomes, 460, 462 Aleuroplasts, 188 Alkaline deoxyribonuclease in mitochondria, 137 Alkaline D-fructose-l ,6-diphosphatase in chloroplasts, 215 induction in plants, 230 Alkaline nucleases, absence from nuclei, 91 Alkaline phosphatase (3.1.3.1.) absence from nuclei, 88, 90, 91 and amino acid pools, 490 and phosphate transport, 390 in bacterial plasma membranes, 382, 383 n cytomembranes, 332 n Golgi cytomembranes, 376 11 hepatomas, 396 ^^.-'— ^ n intesti^pJ cytomembranes. THn n kidney cytomembranes, 379 -in liver microsomal fraction, 343 n lysosomes, 245 n mammary gland cytomembranes, 380 in membrane fractions, 358, 359 in pituitary cytomembranes, 381 in ribosomes, 456 release from spheroplasts, 445 solubilization, 382 "Alkaline protease" in pituitary cyto- membranes, 381" Alkaline ribonuclease in liver microsomal fraction, 343 in membrane fractions, 359 Alkylguanidines and mitochondria, 152 Allosteric change, 499 Amino acid activation and ribosomes, 427 Amino acid activation — cont. in chloroplasts, 224 in microsomal sub-fractions, 349 in isolated nuclei, 77 in SP, 496 Amino acid dependent ATP/PPi ex- change in nuclei, 77 Amino acid incorporation into protein (see also protein synthesis) in mitochondria, 130-1 in mitochondria, role of ATP, 127 in mitochondria, role of oligomycin, 127 in nuclei, 76 in ribosomes of nuclear sap, 78 stimulation by microsomal factor "X", 349 D-Amino acid oxidase (1.4.3.3.) in microbodies, 172 L-Amino acid oxidase (1.4.3.2.) in liver microsomal fraction, 341 Amino acids and cold hardening of wheat, 491 and osmoregulation, 491 as respiratory substrates, 490 automated assay, 484 in mitochondria, 1 37 in plants, 224 in SP, 486 metabolism in mitochondria, 132-5 micro-assay, 488 oxidation by mitochondria, 1 1 8 penetration into nuclei, 93 pools, 486-91 role in SP, 490 synthesis, in chloroplasts, 223-4 Amino acyl-sRNA and GTP, 427 formation in chloroplasts, 225 Amino acyl-sRN A synthetases (6.1.1.1.- 6.1.1.11.) intra-nuclear localization, 77-8 in nuclear sap, 77 in nuclei, 77 in ribosomes, 462, 454 pH 5 precipitation, 502 S-Amino-laevulinic acid in chlorophyll synthesis, 226 synthesis by mitochondria, 132 Aminopeptidases (3.4.1.2,3.) in membrane fractions, 358 550 SUBJECT INDEX Aminopeptidases (3.4.1.2.3.) — cont. in microsomal fractions, and necrosis, 297 Ammonium ions formation in chloroplasts, 227 uncouplers of photophosphorylation, 205 Amoeba acid phosphatase, 257 protein uptake, 271 AMP in nuclei, 83 AMPase in cytomembranes, 333 Amylase (3.2.1.1.2.) absence from nuclei, 91 in liver microsomal fraction, 343( in ribosomes, 431, 460, 462 solubilization, 382 synthesis in membranes, 366 a-Amylase (3.2.1.1.) and plastids, 219 p-Amylase (3.2.1.2.) and plastids, 219 Amylopectin, 218 Amyloplasts, 188 Amylose, 218 Anisotropic ATPase in oxidative phos- phorylation, 120, 121 Ankistrodesmus, photophosphorylation, 208 photosynthesis, 199 Antibody absorption interference by serum proteins, 280 Antibody formation, and lysosomes, 289 Anticodons, and protein synthesis, 420 Anti-P-galactosidase, 437 Antigen-antibody complexes, at cell surface, 293 and phagolysosomes, 291 uptake by leucocytes, 292 Antigens degradation by lysosomal enzymes, 284 degradation by phagolysosomes, 289 in membrane fractions, 357 in phagosomes, 304 in smooth membranes, 362 uptake, and opsonins, 277 Antimycin inhibitor of cyclic photophosphoryla- tion, 207 and mitochondria, 152 Areas of focal degeneration, 243,250, 288 Areas of focal degeneration — cont. and lysosome formation, 261 and potassium deficiency, 294 in pathological conditions, 294 Arginase (3.5.3.1.) in liver microsomal fraction, 343 in microsomal sub-fractions, 349 in nuclei, 90 release by cations, 371 Arsenate, and mitochondria, 152 Arsenite, and mitochondrial amino acid uptake, 127 "Arthrocytosis", 274 Arthus reaction, and phagocytic vacuoles, 292 Arylesterase (3.1.1.2.) and glucose-6-phosphatase marker, 346 and RNase, 370 in brain cytomembranes, 378 in liver microsomal fraction, 343 in membrane fractions, 354 in microsomal sub-fractions, 350 in muscle cytomembranes, 380 in plasma membrane fractions, 375 localization problems, 385 Aryl-4-hydroxylase (1.14.1.1.) absence from SP, 499 in liver microsomal fraction, 341 Aryl sulphatase (3.1.6.1.) cytochemical stain, 303 in lysosomes, 245, 303 in muscle, 247 in plant spherosomes, 258 Aryl sulphatase C in liver microsomal fraction, 343 release by detergents, 370 Ascaris lumbricoides, mitochondria, 167 Ascites cells, membrane fractions, 357 Ascorbate, and NADP photoreduction, 200 Ascorbic acid synthesis and SP, 499 in liver microsomal system, 341 in microsomal fractions, 391 in microsomal sub-fractions, 349 Asparagine, chloroplast activation, 225 Aspartate aminotransferase (2.6.1.1.) in chloroplasts, 221 isoenzymes in SP and mitochondria, 132,499 1 SUBJECT INDEX 551 Assimilatory power, chloroplast enzymes, 197-208 Astrocytes, mitochondrial helices, 149, 150 Atebrine, uncoupler of photophos- phorylation, 205 Atherosclerosis and fat uptake, 272 and phagosomes, lysosomes, 272 ATP and amino acid incorporation in mitochondria, 127 and ion balance, 483 formation, in non-cyclic photophos- phorylation, 198 formation, by pyruvate kinase in chloroplasts, 220 impermeability of nuclear membrane, 81 incorporation into mitochondrial RNA, 129 in extrusion of lysosomal enzymes, 296 in metabolism, 485 in "non-aqueous" nuclei, 83 in substrate level phosphorylation, 122 lability, 481 synthesis in nuclei, 127 ATP/ADP exchange enzyme, in mito- chondria, 144 ATPase (3.6.1.3.) adsorption on ribosome, 425 and "coupling factors", 136 and deoxycholate, 368, 369 anisotropic enzyme, 120, 121 binding to mitochondria, 145 in biliary obstruction, 397 in brain cytomembranes, 378 in chloroplasts, 227 in contraction, 389 in corneal epithelium, 267 in cytomembranes, 333 in epithelium of ciliary processes, 267 in hepatomas, 295, 396 in ion transport, 389 in kidney cytomembranes, 380 in liver microsomal fraction, 343 in lysosomes, 246 in membrane fractions, 356, 357, 359 in mitochondria, 135-7, 331, 386 in nuclei, 88 ATPase (3.6.13.)— cont. in plasma membrane, 335, 374 in protoplast membranes, 383 in ribosomes, 460, 462 in spleen cytomembranes, 381 linkage to cytomembranes, 382 solubilization, 381 Atractylate, and fat metabolism, 116 Atractylis gummiferuy and atractyloside, 159 Atractyloside inhibitor of: nucleotide transport, 140, 159 oxidative phosphorylation, 159 Autolysis, 286-9 and lysosomes, 288 Autolysosomes, 243 Autolytic vacuoles, 249 Autophagic vacuoles, 242, 288 in brown adipose tissue, 288 in Euglena, 257 in foetal rat duodenum, 288 in kidney, 289 in liver, 288 in regressing MuUerian duct, 288 Autophagy, 262, 286-9 Autoradiography of labelled nuclei, 76 of nuclear RNA, 57 Azide, inhibition of O2 evolution, 200 Azo-dye reductase (1.6.6.7.) (azo-ben- zene reductase) in liver microsomal fractions, 341 in membranes, 366 in membrane fractions, 354 in microsomal sub-fractions, 350 Azo-dye stain for acid phosphatase, 301 Azo-dyes, and cytomembranes, 396 B Bacteria degradation by phagolysosomes, 284 ingestion by cells, 264 in mitochondrial fractions, 1 10 plasma membranes, 382 RNase free, 449 Bacteriochlorophyll, 182 in chromatophores, 186 Ik 552 SUBJECT INDEX Balance sheets in cell fractionation, 17, 18 of SP components, 483 Barium phosphate, granules in mito- chondria, 150, 151 Basement membranes, as permeability barriers, 279 Basic dyes, lysosomal stains, 276 Basic proteins, in ribosomes, 414 Basophilia, and ribosomes, 408 Bence- Jones proteins, 269 Bentonite, and ribosomal RNase, 440 Benzoyl cholinesterase (3.1.1.9.) in liver microsomal fractions, 343 Betaine aldehyde dehydrogenase (1.2.1.8.) in mitochondria, 134 Bicarbonate in lipid synthesis in chloroplasts, 225 in plasma membrane isolation, 372 Bile canaliculi and plasma membranes, 372 enzyme content, 323, 333 Bile-duct ligation, membrane enzymes, 397 Bile salts, and cytomembranes, 390 Biliary epithelium, enzymes, 333 Blood vessel walls, enzymes, 333 Bone resorption, and phagolysosomes, 288 Bound amino acids, 487 Brain cytomembranes, 378 lysosomes, 254 stem, lysosomes, 255 Branching enzyme (see Q enzyme) "Bristle coat", of micropinocytic vesicles, 278 Brown plastids, 182 Brush border, vesicles in microsomal fraction, 376 n-Butanol, and microsomal enzymes, 370 Butyryl CoA, synthesis by mitochon- dria, 129 Caesium ions, binding to v«sicles, 362 Calcium ions and ribosomal sub-units, 41 1 and SP flocculation, 501 Calcium ions — cont. in mitochondrial granules, 150, 151 in SP, 483 nuclei, use in isolation of, 32 stabilization of nucleoli, 46 stimulation of chloroplast ATPase, 227 uptake by mitochondria, 128 Calvin cycle (see reductive pentose cycle) Carbohydrate metabolism and fat metabolism, 115 of chloroplasts, 217-20 2-Carbon acid metabolism, in chloro- plasts, 222, 223 Carbon starvation, in Euglena, 257 Carbon tetrachloride in chloroplast isolation, 193 liver injury and lysosomes, 297 Carbonyl cyanide w-chlorophenyl hydrazone, 205 Carboxydismutase (see also ribulose- 1,5-diphosphate carboxylase) in plants, 217 in reductive pentose cycle in chloro- plasts, 210 loss from chloroplasts, 195 Carboxylative phase, of pentose cycle, 209 Carboxylesterase (3.1.1.1.), in micro- somal fraction, 343 (see also Esterase) Carboxylic acids, in plant metabolism, 220 Carcinogens and cytomembranes, 395 Carnitine esters in mitochondria, 115 in mitochondrial fat metabolism, 115 Carotenes, 226 a-Carotene, in chromoplasts, 187 P-Carotene in chloroplasts, 186 in chromoplasts, 187 synthesis in chloroplasts, 226 Carotenoids biosynthesis, 226 in chromatophores, 186 Catalase (1.11.1.6.) absence of mitochondrial labelling, 131 absence from mitochondria, 1 10 SUBJECT INDEX 553 I Catalase (1.11.1 .6.) — cont. electrophoresis, 493 in chloroplasts, 228— '^ in liver microsomal fraction, 341 in mernbranelractions, 355 in microbodies, 172, 367 in nuclei, 90 latency in peroxisomes, 8 Catecholamine granules, 7 Cathepsin (3.4.4.9.) and regression of MuUerian duct, 286 in lipofuscin granules, 286 in lysosomes, 245, 284, 297 in mitochondria, 137 in muscle, 247 in prostate, 255 CDPase and cytomembranes, 335, 332 deoxycholate effect, 369 in liver microsomal fraction, 344 CDP coenzymes, 455 CDP-choline : 1 ,2-diglyceride choline phosphotransferase (see choline phosphotransferase) Cell constituents, classification, 477-81 Cell cycle, 54 Cell differentiation, 71 Cell disruption, by osmotic shock, 31 Cell fractionation analytical approach, 13-15 artifacts, 20 biochemical approach, 12 cell heterogeneity, problems, 8 control of measurements, 15, 16 criteria, 110 density distribution data, 23 disruption of tissue framework, 8 distribution of sedimentation co- efficients, 23 graphical representations, 22, 13-14, 24 homogenization, 6 interpretation, 17-21, 481-3 isolation media, 6 isolation of single cells, 8 morphological approach, 10, 16-17 non-aqueous media, 6 postulate of biochemical homo- geneity, 14, 18 postulate of single location, 21 A* Cell fractionation — cont. preparative approach, 10-13 presentation of results, 18, 24 rare particles, 12 sub-units, characterization, 18, 19 terminology, 21 (see Appendix) Cell membrane, 322 rat-liver, 11 Cell surface, immunology, 293 Cell volume, 482 Centrifugal fractionation, of mem- branes, 354-60 Centrifuging and sedimentation coefficients, 478 of particles, 8, 10 Cerebrosides, in lysosomes, 285 Ceroid granules, and lysosomes, 285 "Chase" experiments, 59 Chemi-osmotic theory, of oxidative phosphorylation, 120 Chitobiase (3.2.1.29.) (see A'-acetyl-p- glucosaminidase) Chlamydomonas, photo-oxidation of cytochrome /, 202 Chloramphenicol, and mitochondrial protein synthesis, 130 Chlorella, chloroplast, 182 Chloride, and water photo-oxidation, 199 p-Chloromercuribenzoate inhibitor of: carboxydismutase, 211 ferredoxin-NADP reductase, 204 phosphoribulokinase, 216 Chlorophyll biosynthesis, 225, 226 in chloroplasts, 186 in chromoplasts, 182 Chlorophyll a in chromoplasts, 182 in non-cyclic photophosphorylation, 198 in photo-oxidation of water, 200 Chlorophyll a^iz, in water photo-oxida- tion, 200 Chlorophyll aesa, in NADP photoreduc- tion, 200 Chlorophyll b in green plastids, 182 in non-cyclic photophosphorylation, 198 554 SUBJECT INDEX Chlorophyll Aeso, in water photo-oxida- tion, 200 Chlorophyllase (3.1.1.14.) in chloroplasts, 226 Chloroplasts chemical composition, 185, 186 definition, 182 from Elodea, 194, 195 enzymes, 197-230 enzymes for assimilatory power, 197-208 isolated, electron microscopy, 191, 192 isolation in aqueous media, 189 isolation in non-aqueous media, 189, 193, 194 lamellae, 163 localization of enzymes, 195-7 structure and shape, 182-6 Cholesterol in microsomal fraction, 340 in muscle cytomembranes, 381 in plasma membrane fractions, 375 linkage to cytomembranes, 382 metabolism, 395 Cholesterol esterase (3.1.1.13.), in microsomal fraction, 343 Cholesterol synthesis, in microsomal fractions, 350 Choline, oxidation by mitochondria, 118, 134 Choline dehydrogenase (1.1.99.1.), in mitochondria, 144 Choline phosphotransferase (2.7.8.2.) in microsomal fraction, 342 Cholyl-CoA synthetase (6.2.1.7.), in microsomal fraction, 345 Chondrioclasts, phagolysosomes in, 288 Chromatin, 68 Chromatography of soluble proteins, 494, 495 of viruses and cell particles, 10 Chromatophores, 182, 186, 187 Chromoplasts, 182 Chromosomes DNA synthesis, and, 52-6 in sub-nuclear fractions, 46 messenger RNA synthesis, and, 66 Chymotrypsin (3.4.4.5.) absence from nuclei, 91 and microsomal enzymes, 370 Chymotrypsinogen, in ribosomes, 431, 432, 465 Ciliates, acid phosphatase, 257 cw-Aconitate, in Krebs' cycle, 112 Cisterna, in cytomembranes, 325 Cistrons, 69 Citrate in Krebs' cycle, 112 oxidation by mitochondria, 222 Citrate synthase (4.1.3.7.), (condensing enzyme) in chloroplasts, 222 in mitochondria, 115 inSP, 113 Citric acid cycle (see also Tricarboxylic acid cycle, Krebs' cycle) in mitochondria, 111-14 in nuclei, 83, 84 "Clusters" of ribosomes (see Polysomes) CoA (see Coenzyme A) Cobalt ions, and carboxydismutase, 211 CO-binding pigment effect of drugs, 397 in membrane fractions, 340 Codon-anticodon interaction, 420 Codons, 420 Coenzyme A in lipid synthesis in chloroplasts, 225 in Krebs' cycle, 112 in oxoglutarate oxidation, 113 stimulation of protein breakdown, 285 Coenzyme Q, in cytomembranes, 365 Coenzyme Qg, in microsomal fraction, 340 Coenzyme Qio, in microsomal fraction, 340, 378 Coenzymes, moles per cell, 482 CO2 fixation cycle, in chloroplasts, 208-17 Collagen, in cytomembranes, 380 CoUagenase (3.4.4.19.), in lysosomes, 245 Colloidal particles, markers for lyso- somes, 304 "Colloid droplets", and lysosomes, 266 Complement fixation, and lysosomes, -293 Complexes I-IV, in mitochondria, 155-6 SUBJECT INDEX 555 II "Composite bodies", 243 and areas of focal degeneration, 294 and mitochondria, 249 Condensing enzyme (see also citrate synthase) in chloroplast extracts, 222 in microsomal fraction, 341 Conformational changes, 499 Conjugation processes, in cytomem- branes, 390, 391 Connective tissue, enzymes, 333 Constant proportion groups, in mito- chondria, 157 Contamination of mitochondrial fractions, 109-10 of nuclear fractions, 76, 91 Contractile proteins, in mitochondria, 136, 137 Copper and Hill reaction, 201 in chloroplasts, 186 in mitochondria, 156 in plastocyanin, 200 Corpora lutea, and lysosomes, 287 Cortisone and lysosomal membrane, 251 and protein uptake, 278 Coupled photophosphorylation effect of protein components, 208 in chloroplasts, 205 Coupling factors, in oxidative phos- phorylation, 136 in photosynthetic phosphorylation, 205 Coupling membrane, in oxidative phos- phorylation, 120, 121 Creatine kinase (2.7.3.2.), in mito- chondria, 140, 144 Cristae mitochondriales, 105, 106 Cristae space, 1 39 Crystalline bodies, in kidney, 250 CTP, metabolism, 485 CTPase deoxycholate effect, 369 in ribosomes, 462 Cupric ions, and phosphoribulokinase, 216 "Cuts", in microsomal fractions, 346 Cyanide inhibitor of: Cyanide — cont. carboxydismutase, 211, 212 Hill reaction, 201 Cyanide resistant NADH oxidase, 281 Cyclic photophosphorylation, 206 in chloroplasts, 197,^206-8 Cyclophorase, 12 Cysteine activation in chloroplasts, 225 activator of carboxydismutase, 211 and phosphoribulokinase inhibition, 216 Cysteinyl-glycine dipeptidase (3.4.3.5.) adsorption onto ribosomes, 425 in kidney cytomembranes, 379 in ribosomes, 457, 459, 463 Cystine reductase (1.6.4.1.) in kidney cytomembranes, 379 in liver microsomal fraction, 341 Cytidine triphosphatase (see CTPase) Cytochemistry of cytomembranes, 330-7 of lysosomal enzymes, 301-3 Cytochrome a and stalked bodies, 167 binding to mitochondria, 145 complex with structural protein, 162 in constant proportion group, 157 in mitochondrial respiratory assembly, 154 moles per mitochondrion, 155 Cytochrome as and stalked bodies, 167 binding to mitochondria, 145 in mitochondrial respiratory assembly, 154 Cytochrome b binding to mitochondria, 145 complex with structural protein, 162 in cytomembranes, 365 in mitochondrial respiratory assembly, 154 Cytochrome 62 (1.1.2.3.), and lactate dehydrogenase DNA, 148 Cytochrome Z>3, in plant membranes, 383 Cytochrome 65 and ion flux, 388 as contaminant in nuclei, 86 drug effects, 397 in brain cytomembranes, 378 in microsomal fractions, 340 556 SUBJECT INDEX Cytochrome be and ferredoxin, 207 in chloroplasts, 186, 208 in cyclic photophosphorylation, 206 re-oxidant of photoreduced ferre- doxin, 203 Cytochrome 6 ~ I2, 123 Cytochrome c absence of mitochondrial labelling, 131, 158 as contaminant in nuclei, 86 complex with phospholipid, 161 in constant proportion group, 157 in mitochondrial extracts, 144 photo-oxidation of reduced form, 201 site of synthesis, 104 Cytochrome c -^ I3, 123 Cytochrome c oxidase (see also Cyto- chrome oxidase) absence from chloroplasts, 229 Cytochrome c photo-oxidation, 229 Cytochrome ci and stalked bodies, 167 binding to mitochondria, 145 complex with structural protein, 162 in constant proportion group, 157 in mitochondrial respiratory assembly, 154 Cytochrome/, 201, 202 in chloroplasts, 186 in chromatophores, 187 in cyclic photophosphorylation, 206, 207 in non-cyclic photophosphorylation, 198 oxidation in light, 229 Cytochrome m (see Cytochrome 65) Cytochrome P-450 in adrenal cytomembranes, 378 in microsomal fraction, 340 Cytochrome oxidase (1.9.3.1.) in complex IV, 155 in erythrocyte nuclei, 85 in mitochondria, 117 in mitochondrial membrane, 154 liver fractions, and, 7 marker for mitochondria, 7, 117, 118 nuclei, absence from, 85 site of synthesis, 104 terminal oxidase, 4, 117, 118 Cytolysomes, 243, 288 Cytomembranes, 323 and buffers, 362 and carcinogens, 395 and diet, 393 biochemical study, 328-30 classification, 325-7 chemical constituents, 365 cytochemistry, 330-7 development, 391-3 enzymes, 365, 331-7 glucose-6-phosphatase, 332 hormonal influences, 394, 395 nucleoside diphosphatases, 332 nucleoside triphosphatases, 333 5 '-nucleotidase, 333 pathology, 395 phospholipids, 365 RNA, 364 summary of properties, 397-9 thiamine pyrophosphatase, 333 thickness, 322 a-Cytomembranes, classification, 325 P-Cytomembranes, classification, 325-7 Y-Cytomembranes, classification, 325-7 Cytopempsis, 267 Cytoplasmic RNA, nuclear origin, 57 Cytosegresomes, 243 Cytoskeleton, 327 Cytosomes, and lysosomes, 243 Cytosis, 243 D "Dark" ATPases, in chloroplasts, 227 Dark reactions, in non-cyclic photo- phosphorylation, 198 deCDP-Choline-1 ,2-diglyceride choline phosphotransferase in liver microsomal fraction, 342 deCDP Choline synthesis adsorption, 339 in microsomal fractions, artifact, 371 Demethylases, in microsomal fractions, 337, 345, 355 Dense granules, in mitochondria, 150-3 Deoxycholate and membrane bound enzymes, 368 and microsomal enzymes, 369 and Mg2+ ions, 366 and nucleolar ribosome isolation, 46 as natural activator, 390 SUBJECT INDEX 557 Deoxycholate — cont. effect on cytomembranes, 346 effect on microsomal fractions, 361 effect on mitochondria, 141 effect on plasma membrane fractions, 375 release of microsomal enzymes, 366 Deoxycytidylate deaminase, in SP, 497 Deoxyribonuclease (see DNase) Derepressors, 71 Desmosomes, 323 Desmosterol reductase, in liver micro- somal fraction, 341 Desorption of nucleotides, 480 of soluble components, 480 Detergents, and microsomal enzymes, 370 Development, of cytomembranes, 391-3 Dextran in cell fractionation, 337 lysosomes, effect on, 10 uptake by liver, 253, 275 Diabetes, and membrane enzymes, 394 Diaphorase in chloroplasts, 229 in microsomal fraction, 341, 387 in mitochondria, 140 Dichlorophenolindophenol, in NADP photoreduction, 200 Dicumarol, and nuclear phosphoryla- tion, 127 Didymium nigripes, mitochondrial DNA, 148 Differentiation and cytomembranes, 392 of nuclei, 90 Digestive vacuoles, 242 Digitonin, and chloroplasts, 201 Diglyceride acyltransferase (2.3.1.20.), in microsomal fraction, 342 Dihydrofolate dehydrogenase (1.5.1 .4.), at pH 5, 502 Dihydrofolate reductase (see Dihydro- folate dehydrogenase) Dihydrolipoate dehydrogenase (see Lipoyl dehydrogenase) Dihydrouracil, in RNA, 70 Dihydroxy-acetone phosphate in chloroplasts, 214 in non-aqueous nuclei, 83 2,4-Dinitrophenol, in oxidative phos- phorylation, 135 o-Diphenol oxidase (1.10.3.1.)(see Poly- phenol oxidase) Diphosphoglycerate, in chloroplasts, 212 DNA as primer in RNA polymerase, 62, 63 biosy ithesis (duplication, autorepro- c'uctijn), 49-55 in chlorjp lasts, 224 in mitoch ndria, 106, 145-50 in isolated nucleoli, 46 in RNA synthesis, 66-72 single stranded, 52-4 synthesis in SP, 496 uptake by fibroblasts, 273 9-X174 DNA, 65 DNA dependent RNA polymerase, 60 (see also RNA polymerase) in mitochondria, 130 in SP, 497 DNA fibrils, 69 in mitochondria, 146 DNA-like RNA, 66 DNA nucleotidyl transferase (see DNA i polymerase) ■ DNA polymerase (2.1.1 J.) and nucleoside triphosphates, 50 in animal tissues, 50 in bacteria, 50 inhibition by DNases, 51 inhibition by histone, 55 in nuclei, 51 in nuclei, non-aqueous, 29 in oocyte nuclei, 51 in ribosomes, 453, 454, 462 in SP, 51,497 in unwinding of DNA strands, 54 single-stranded primer, 51 DNA protein, degradation by phagoly- sosomes, 273, 274 DNA replication and cell cycle, 54 semi-conservative mechanism, 52, 53 unwinding of strands, 54 DNase (3.1.4.5.) absence from non-aqueous nuclei, 53 adsorption, 431, 432 and phage infection, 453 binding to ribosomes, 450-2, 453 558 SUBJECT INDEX DNase (3.1.4,5.)— cont. fibrils in nuclei, and, 37 in Escherichia coli, 452 inhibition of nuclear glycolysis, 82 inhibition of nuclear protein syn- thesis, 76 inhibition of nuclear RNA poly- merase, 60 in plant spherosomes, 258 in ribosomes, 462, 450-3 separation of DNA strands, 53 DNase I, absence from nuclei, 90 DNase II (3.1.4.6.), in nuclei, 90 DNA synthesis in bacteria, 52 in chromosomes, 52-6 DNP-fibrils, 37, 40 Double staining, of lysosomes, 253, 254 Double stranded RNA, 64 DPN (see NAD) "Droplet" fraction, and kidney lyso- somes, 245, 254, 299 Drug metabolism in membrane fractions, 354, 391, 397 in microsomal sub-fractions, 350 in regenerating liver, 393 "DT diaphorase", in membrane frac- tions, 355 Dye-inclusion bodies, 276 E EAA (extractable amino acids), 478 EDTA, activator of RNase, 441 in microsomal fraction isolation, 337^ in plasma membrane isolation, 372 ^ release of microsomal enzymes, 366 "Effectors", of allosteric transition, 499 Egg white, and lysosomes, 9 Elaioplasts, 188 Electron microscopy of DNA replication, 53 of isolated chloroplasts, 195-6 of microsomal fractions, 377 of nucleus, 35-44 quantitative aspects, 16 Electron transport chain absence from nuclei, 85 during development, 392 Electrophoresis fractionation of homogenates, 10 Electrophoresis — cont. of SP proteins, 492-4 Elementary particles, in mitochondria, 154, 165-9 Elementary photosynthetic particles (see Quantasomes) Elodea, chloroplasts, 194, 195 Encephalomyocarditis virus, and NAD pyrophosphorylase, 459 "End addition" enzyme (see Terminal deoxyribonucleotide transferase) Endocytosis, 243 Endonucleases, 438, 452 Endoplasmic reticulum and synthesis of lysosomal enzymes, 258 and synthesis of secretory enzymes, 282 in autophagic vacuoles, 288 definition, 323 electron microscopy, 409 properties, 387 Endothelial cells, 280 Endotoxin, and lysosomal enzymes, 251 Energy carrier "I", 123 Energy metabolism, enzymes in nuclei, 81-6 Energy-rich intermediates, 125-7 Energy supply, in membranes, 387 Enolase (4.2.1.11.) (2-phospho-D-gly- cerate hydrolyase) in chloroplasts, 220, 230 in mitochondria, 114 Enucleation, and RNA synthesis, 58 Enzyme cytology definition, 3 historical significance, 1 objectives and methods, 3-5 Enzyme localization, in chloroplasts, 195-7 Enzymes assay in mixtures, 15 in cells, control, 499, 500 in chloroplasts, 197-230 in ribosomes, 421-6 of SP, 496-500 of SP, interpretation, 497-9 of SP, reviews, 483 precursors in ribosomes, 430 plurality, 386 problems of localization, 499 Eosinophil leucocytes, lysosomes, 256 SUBJECT INDEX 559 Epididymis, 255, 358 Epithelial cells alkaline phosphatase in cytomem- branes, 332 lysosomes, 253 Epoxysteroid lyase, in microsomal frac- tion, 345 Ergastoplasm, 323 Ergosomes (see Polysomes) Erythroblasts, ferritin uptake, 270 Erythrocytes nuclei, oxidative enzymes, 85 stroma, structural protein, 163 D-Erythrose-4-phosphate, in chloro- plasts, 214 Escherichia coli, ribosomal RNase, 440-7 Esterase, 301-2 (see also Carboxyl esterase) and cytomembranes, 335 and lipid breakdown by lysosomes, 285 and lipofuscin granules, 286 electrophoresis, 493 in granules in macrophages, 256 in kidney lysomes, 254 in liver microsomal fraction, 343 in lysosomes, 245 in microsomal sub-fractions, 350 in nuclei, 90 in plant spherosomes, 258 in protozoa, 257 in smooth membranes, 362 in uterine lysosomes, 255 staining by indoxyl acetate, 303 Etiolated plants, glycollate oxidase, 222 Ether (aromatic) cleaving system, in microsomal fraction, 345 ETP, NADH oxidation, 161 Euglena, chloroplasts, 196, 211 Exchange-diffusion system, in oxidative phosphorylation, 120, 121 Exocytosis, 243 Exonucleases, 438, 452 Exoplasmosis, 243 Extractable amino acids (E AA), 487, 489 Extramitochondrial oxidation, and peroxisomes, 173, 174 FAA (free amino acids), 487 FAD, in ferredoxin-NADP reductase, 204, 205 Fasting and glycogen levels, 340 and RNA synthesis, 365 Fat metabolism and carbohydrate metabolism, 115 and diet, 393, 394. Fat transport, 267 Fat uptake by intestine, 272, 273 by liver, 273 by reticulo-endothelial cells, 272 Fatty acids activation in mitochondria, 140 oxidation by mitochondria, 118 synthesis by sarcosomes, 129 synthesis in mammary gland cyto- membranes, 380 synthesis in SP, 497 transfer across mitochondrial mem- branes, 115 Fatty acid synthetase, electron micros- copy, 12 Fi coupling factor, 167 Feedback inhibition, 500 Ferredoxin, 202, 203 and nitrite reduction, 203, 227 and o-phenanthroline, 203 in chloroplasts, 195 in cyclic photophosphorylation, 207 induction, 230 in non-cyclic photophosphorylation, 198 in photophosphorylation, 203 loss from chloroplasts, 195 Ferredoxin-NADP reductase as a diaphorase, 207 in chloroplasts, 203-5 induction, 230 Ferricyanide, reduction by ferredoxin- NADP reductase, 204 Ferritin, in "light microsomes", 348 as marker for phagosomes, 304 uptake by erythroblasts, 270 uptake by kidney cells, 269 uptake by nerve cells, 270 uptake by tissue culture cells, 271 Ferritin-labelled antibody, 292 . "Fex", 391, 395 Fibrils, in nuclei, 37 560 SUBJECT INDEX Fibroblasts acid phosphatase granules, 257 DNA in mitochondria of, 146 lysosomes in tissue culture, 257 uptake of particles, 280 Ficoll, in plasma membrane isolation, 374 Fixatives, for cytomembranes, 331 Flavin mononucleotide, in cyclic photo- phosphorylation, 206 Flavin nucleotides, in lysosomes, 250 Flavoproteins, in chloroplasts, 186 Flocculation, of SP, 500 "Fluffy layer", 339 Fluoracetic acid, inhibitor of nuclear phosphorylation, 84 Fluorescence, of lysosomes, 250 Fluorescent-labelled proteins as markers for lysosomes, 304 uptake by kidney slices, 269 Fluoromalate, and malate dehydro- genase, 140 /7-Fluorophenylalanine, and jS-glucosi- dase synthesis, 434 FMN, and ferredoxin-NADP reduc- tase, 204 Foetal tissues, enzymes, 392 Food vacuoles, and phagososomes, 266 Formaldehyde, inhibitor of phospha- toses, 249 Formaldehyde-Ca2+, fixative, 331 Fraction I protein, carboxydismutase activity, 211 Fragility, of lysosomes, 252 Free amino acids (FAA), 487 a-Fructofuranosidase (3.2.1.26.), in membranes, 359, 379 D-Fructose, and sucrose synthesis, 219 D-Fructose-1 ,6-diphosphatase (3.1.3.11.), in chloroplasts, 214 Fructose-1 ,6-diphosphatase (alkaline), 217 D-Fructose-1 ,6-diphosphate in chloroplasts, 214 in non-aqueous nuclei, 83 D-Fructose-6-phosphate conversion to glucose- 1 -phosphate in chloroplasts, 217 in chloroplasts, 214 in sucrose synthesis in chloroplasts, 219 Fucoxanthin, in plastids, 182 Fumarase (4.2.1.2.) (fumarate hydratase in chloroplast extracts, 222 in liver microsomal fraction, 345 in mitochondria, 113 release by ribonuclease, 371 Fumarate, in Krebs' cycle, 112 G Galactolipids, in chloroplasts, 186 jS-Galactopyranosyl diglycerides, in chloroplasts, 186 P-Galactosidase (3.2.1.23.) biosynthesis, 430 in lysosomes, 245 in muscle, 247 in plant spherosomes, 258 in polysomes, 436 in ribosomes, 434-7, 462 in ribosomes, effect of antisera, 435 isoenzymes in SP and lysosomes, 499 mRNA half-life, 450 nascent enzyme, 434-7 polymerization, 437 GDPase deoxycholate effect, 369 in cytomembranes, 332 in microsomal fraction, 344 GDP coenzymes, in metabolism, 485 Gel electrophoresis, of acid phospha- tases, 249 Gene expression, and nuclear mRNA, 80 Genetic apparatus, duplication, 49 "Ghost" fractions, 364 Globulins, in nuclei, 45, 92 a-Glucan phosphorylase (2.4.1.1.) in SP, 497 release during starvation, 497 Glucono-i3-lactone-6-phosphate, in chloroplasts, 228 Glucose, in SP, 486 Glucose- 1,6-diphosphate, in chloro- plasts, 217 Glucose-6-phosphatase (3.1.3.9.) and deoxycholate, 368, 369 and diet, 393 and nucleoside diphosphatase in membranes, 367 binding. 368 SUBJECT INDEX 561 Glucose-6-phosphatase cont. diffuse distribution, 335 drugs, effect on, 397 during development, 392 electron-microscope histochemistry, 335, 336 histochemistry, 384, 385 hormonal effects, 394 in adrenal cytomembranes, 378 in cytomembranes, 332, 497 in cytomembranes, fixation, 331 injhepatomas, 395 in kidney cytomembranes, 379 in liver microsomal fraction, 344 in membrane fractions, 340, 354, 358, 359, 362, 366 in micosomal sub-fractions, 350 in nuclei, low amounts, 91 in regenerating liver, 392, 393 liver fractions, and, 7 localization, 386 reaction product diffusion, 331 release by detergents, 370 release from membranes, 366 standard microsomal marker, 341-5 Glucose- 1 -phosphate and phosphorylase, 218 and UDPG synthesis, 219 in chloroplasts, 217 Glucose-6-phosphate in chloroplasts, 217 in non-aqueous nuclei, 83 oxidation in chloroplasts, 228 Glucose-6-phosphate dehydrogenase (1.1.1.49.) in chloroplasts, 228 in nuclei, 83 muscle cytomembranes, absence from, 380 Glucose phosphate isomerase (5.3.1.9.), in chloroplasts, 217 Glucose-1 -phosphate uridyl transferase (see also UDPG pyrophos- phorylase), in nuclei, 88 Gludosidase, and glycogen accumula- tion, 286 a-Glucosidase (3.2.1.20.), in lysosomes, 245 P-Glucosidase (3.2.1.21.) in intestinal cytomembranes, 379 in plant spherosomes, 258 P-Glucosidase (3.2.1.21.)— con/. in ribosomes, 434, 463 latency of nascent enzyme, 437 nascent enzyme, 434 solubilization, 382 p-Glucuronidase (3.2.1.31.) electrophoresis, 493 in "dense bodies" of thymus, 256 in liver microsomal fraction, 344 in lysosomes, 245, 303 in muscle, 247 in plant spherosomes, 258 in ribosomes, 460, 463 localization problems, 386 D-Glucuronolactone, and microsomal ascorbate synthesis, 349 Glucuronolactone reductase (1.1.1.20.) in microsomal fraction, 341 Glucuronyl transferase, in endoplasmic reticulum, 387 Glutamate from oxoglutarate in chloroplasts, 222 oxidation by mitochondria, 118 transamination, in chloroplasts, 221 Glutamate dehydrogenase (1.4.1,2.) in chloroplasts, 223 in mitochondria, 133, 140, 143 in ribosomes, 460, 463 ratio to mitochondrial glycerol-1- phosphate dehydrogenase, 157 Glutamate dehydrogenase (NAD?) (1.4.1.3.4.) in mitochondria, 134 Glutamine synthetase (6.3.1.2.) cumulative feedback inhibition, 500 . effect of RNase, 370 in brain cytomembranes, 378 in liver microsomal fraction, 345 release by hypotonic saline, 371 Glutaraldehyde and nucleotide dephosphorylation, 331 and thiamine pyrophosphatase, 333 Glutathione activator of carboxydismutase, 21 1 hydrolysis by kidney cytomembranes, 379 Glutathione reductase (1.6.4.2.), elec- trophoresis, 493 562 SUBJECT INDEX D-Glyceraldehyde-3-phosphate, in chloroplasts, 214 Glyceraldehyde-3-phosphate dehydro- genase (see Triose phosphate dehydrogenase) Glycerol, and nuclear isolation, 33 Glycerol-l-phosphate, in non-aqueous nuclei, 83 Glycerolphosphate acyltransferase (2.3.1.15.) in liver microsomal fractions, 342 Glycerol-3-phosphate dehydrogenase (1.1.1.8., 1.1.99.5.) in muscle mitochondria, 157 in nuclei, 82 ratio to mitochondrial glutamate dehydrogenase, 157 a-Glycerophosphatase cytochemical marker for lysosomes, 253 in lysosomes, 248 Glycine, in 8-aminolaevulinic acid synthesis, 226 Glycogen and UDP glucose-glycogen glucosyl transferase, 370 in heavy microsomal fraction, 347 metabolism in SP, 497 under microsomal pellet, 339, 340 Glycolipids, in lysosomes, 250 GlycoUate oxidase (1.1.3.1.) in chloroplasts, 222 induction during illumination, 230 GlycoUate reductase, in chloroplasts, 222, 223 Glycolysis assay of intermediates, 484, 486 as energy source for pinocytosis, 281 in nuclei, 81-3 in SP, 497 "Glycolytic complex", 383 Glycolytic enzymes, and plasma mem- branes, 375 Glycoproteins, in plasma membranes, 375 Glyoxylate from glycollate, in chloroplasts, 222 to glycine, in chloroplasts, 226 Glyoxylate cycle, in plants, 223 Glyoxylate reductase (1.1.1.26.), in chloroplasts, 223 Gold-DNA-protein, complex in phagosomes, 266 Golgi complex and phsophate transport, 390 and rat-liver, 1 1 function, 386 properties, 387 Golgi cytomembranes alkaline phosphatase, 332 classification, 325-7 development, 392 in zonal ultracentrifuge, 367 thiamine pyrophosphatase, 333, 335 Golgi cytomembrane fractions, 376 Golgi zone assembly of secretory granules, 282 and pinocytosis, 282 and site of assembly of lysosomal en- zymes, 259 and secretory enzymes, 259 Gomori procedure, 242 for acid phosphatase, 242 Grana, of chloroplasts, 184 Granular cytomembranes, 325 Granules, in nucleus, 41 Green plastids, 182 GTP in Krebs' cycle, 112 in metabolism, 485 in protein synthesis, 426, 427 in substrate level phosphorylation, 122 into mitochondrial RNA, 129 GTPase and protein synthesis, 427 deoxycholate effect, 369 in ribosomes, 463 Guanosine triphosphatase (see GTPase) L-Gulonolactone, in microsomal ascor- bate synthesis, 349 H Haem, mitochondrial synthesis, 132 Haemagglutinin, and lysosomes, 257 Haemoglobin and phagosome lysosome fusion, 264 in nuclei, 90 marker for phagosomes, 304 readsorption by kidney, 241 I- I SUBJECT INDEX 563 Haemoglobin — cont. synthesis in nuclei, 78 synthesis in vitro, 428 uptake by kidney cells, 269 Haemorrhagic shock, and lysosomes, 297 Haem protein Fex, in cytomembranes, 365 H-2 antigens, in membrane fractions, 356 "Heavy" microsomal fraction, 347 chemical constituents, 348, 349 electron microscopy, 348 Helices, in mitochondria, 150 Heparin, in cell fractionation, 337 Hepatectomy, and pinocytosis, 273 Heterologous protein, clearance rate, 278 Hexokinase (2.7.1.1.) and Pi in SP, 486 and transport, 388 in chloroplasts, 229 in nuclei, 82 Hexosamine, in membrane fractions, 356 Hexose monophosphate shunt, in nuclei, 83 Hill reaction, 199 in chloroplasts, 195 inhibition by cyanide, 201 Histamine, and colloid transport, 279 Histidine, activation in chloroplasts, 225 L-Histidine ammonia lyase (4.3.1.3.), 430 Histones and ribosomal proteins, 414 chemical heterogeneity, 70 inhibition of DNA polymerase, 55 inhibition of RNA polymerase, 69 inhibition of synthesis by puromycin, 79 in nuclear extracts, 45 Holocrine secretion, and lysosomes, 287 Homogenization and cell fractionation, 6 and microsomal fraction isolation, 337 Homograft antigens, in plasma mem- branes, 374 Homograft antigens — cont. homologous protein, clearance rate, 278 Homopolynucleotides, in nucleus, 75 Hormones, release by phagolysosomes, 226, 267 Horse-radish peroxidase, injection ex- periments, 240 "Hyaline droplets", 269 "Hyaline inclusions", 343 Hyaluronic acid, in lysosomes, 250 Hyaluronidase (4.2.99. 1 .), in lysosomes, 245 Hybrids of nucleic acids, 59, 68 Hydrogen ions, and mitochondrial ion uptake, 151 Hydrolases, in mitochondria, 135-8 Lo-Hydroxyacid dehydrogenase, in microbodies, 4, 174 3-Hydroxyacyl-CoA dehydrogenase (1.1.1.35.), in mitochondria, 114 Hydroxyadipaldehyde, 331 Hydroxyapatite, in mitochondria, 150 activation by lecithin, 161 D(-)-3-Hydroxybutyrate dehydrogenase (1.1.1.30) in mitochondria, 116, 140, 161 Hydroxylamine, inhibition of O2 evolu- tion, 200 3-Hydroxy-3-methylglutaryl-CoA lyase (4.1.3.4.) in mitochondria, 116, 117 3-Hydroxy-3-methylglutaryl-CoA re- ductase (1.1.1.34.) in liver microsomal fraction, 341 in microsomal fractions, 116, 117 3-Hydroxy-3-methylgutaryl-CoA syn- thase (4.1.3.5.) in mitochondria, 116, 117 "I", in oxidative phosphorylation, 123 IDPase deoxycholate effect, 369 in cytomembranes, 332 in liver microsomal fraction, 344 ^311-labelled antigen, uptake by macro- phages, 279 Iminodipeptidase (3.4.3.6.), in ribo- somes, 437, 463 Immune cytolysis, 293 564 SUBJECT INDEX Immune processes and lysosomes, 289-93 and phagosomes, 289 Indoxyl acetate, stain for esterase, 303 Induced enzyme formation, in chloro- plasts, 230 Inflammation, and lysosomes, 297 Ingestion, and lysosomes, 261-85 Initial clearing rates, of proteins, 278 Inner compartment, of mitochondria, 105, 139 Inorganic phosphate, uptake by mito- chondria, 128 Inorganic pyrophosphatase (3.6.1.1.) in liver microsomal fraction, 344 in nuclei, 89 Inosine, in microsomal fraction, 340 Inositol phosphatide, in phagocytosis, 281 Insect flight muscle mitochondria enzymes, 157 labelling of insoluble proteins, 158 Insulin and cytomembranes, 394 and pinocytosis, 278 Intestine cytomembranes, 379 epithelium, protein uptake, 270 fat uptake, 272-3 lysosomes, 254 mucosa, membrane fractions, 358 Intracellular digestion, 283 Intracellular pools, of amino acids, 489, 490 Intracellular transport, and phago- somes, 267 Invertase (see also jS-fructofuranosidase) in membrane fractions, 359 Iron-dextran, uptake by liver, 275 lodoacetamide, inhibitor of phosphori- bulokinase, 216 lodoacetate, inhibitor of: ferredoxin-NADP reductase, 204 nuclear glycolysis, 83 phosphoribulokinase, 213 triose phosphate dehydrogenase, 213 Ion flux, and cytochrome 65, 388 Ions in mitochondria, 483 in SP, 483 Ion transport, 389 in mitochondria, 128, 129, 159, 160 Iron, in chloroplasts, 186 Iron-dextran, uptake by liver, 253 Ischaemic necrosis, and lysosomes, 297 Isocitrate, in Krebs' cycle, 112 Isocitrate dehydrogenase (1 . 1 . 1 .41 .) Isocitrate dehydrogenase (1.1.1,41.) — cont. in mitochondria, 143 in mitochondria and SP, 113, 118 in nuclei, 83 Isocitrate dehydrogenase (NADP) (1.1.1.42.) in chloroplasts, 222 in mitochondria, 140 Isocitrate lyase (4. 1.3.1 .), and plants, 223 Isocitrate oxidation in mitochondria, 118, 222 problems in localization, 118 Isoenzymes, 498 in SP and particles, 499 Isoleucine activation, in chloroplasts, 225 Iso-octane, in release of ribosomes, 362 Isopentenylpyrophosphate, in terpene formation, 226 ITPase, deoxycholate effect, 369 K Keilin-Hartree preparation, 113 3-Keto-acid CoA transferase (2.8.3.5.), in mitochondria, 116 a-Ketothiolase (see acetyl CoA acyl- transferase) Kidney cytomembranes, 379 lysosomes, 254, 240, 299 mitochondria, 152 peroxisomes, 174 protein uptake, 269 Kidney slices, fluorescent labelling, 269 uptake of peroxidase, 269 Krebs' cycle assay of intermediates, 486 enzymes in chloroplasts, 221, 222 in mitochondria. 111 Kupffer cells, acid phosphatase granules, 256 lysosomes, 298 phagosomes, 264 SUBJECT INDEX 565 I ^jM Lactate dehydrogenase (1.1.1.27.) allosteric isoeniymes, 500 in nuclei, 82 in pinocytosis, 281 in ribosomes, 433, 434, 463 isoenzymes, 434 nuclear and cytoplasmic enzymes, 92 Lactate dehydrogenase (cytochrome 62) (see Cytochrome 62) Lactic acid from fructose-l-6-diphosphate in nuclei, 82 from glucose, in nuclei, 82 in non-aqueous nuclei, 83 Lactonase, in cytomembranes, 379 Lamellae, of cWoroplasts, 182-4 chemical composition, 186 in isolated chloroplasts, 190-3 "Lampbrush" chromosomes, 68 tency of enzymes and lysosomes, 8^^242, 251 binding to sub-units, use in study of, 19 cell fractionation, use in, 14 DNase in ribosomes, 453 membrane RNase, 366 mitochondrial enzymes, 134 nascent enzymes, 437 ribosomal RNase, 441, 442 Leaf proteins, 211 Lecithin activator of 3-hydroxybutyrate de- hydrogenase, 161 hydrolysis by microsomal enzymes, 370 in lysosomes, 250 synthesis in membrane fractions, 355 Leucine, activation in chloroplasts, 225 Leucine aminopeptidase (3.4.1.1.), in ribosomes, 456, 457, 464 Leucocidin, 296 Leucocytes enzymes, 359 lysosomes, 256 membrane fractions, 359 Leucoplasts, 187, 188 conversion to chromoplasts, 187 L-Leucyl-o-naphthyl amidase, in mem- brane fractions, 358 "Light-dependent" ATPase, in chloro- plasts, 227 "Light microsomal" fractions, 330 chemical constituents, 348, 349 electron microscopy, 348 RNA, 364 Light reactions, in photophosphory- lation, 198 "Light-triggered" ATPases, in chloro- plasts, 227 Lipase (3.1.1.3.) absence from nuclei, 90, 91 in lysosomes, 245, 285 in plant spherosomes, 258 release of enzymes, 371 *^ Lipid-amino acid complexes, 489 Lipid breakdown, by lysosomes, 285 Lipid micelles, in membranes, 323 Lipid peroxidase, in microsomal frac- tion, 341, 391 Lipids in chloroplasts, 185, 186 in lysosomes, 250 in mitochondrial membranes, 160 in SP, 495 uptake and phagolysosomes, 271-3 Lipid synthesis in chloroplasts, 225 in mitochondria, 129 Lipid transport, 273 Lipoamide dehydrogenase (1.6.4.3.), 122 Lipoate acetyl transferase (2.3.1.12.), in mitochondria, 156 Lipoate reductase transacetylase, 156 Lipofuscin granules, and lysosomes, 285 Lipolytic enzymes, and lysomes, 285 Lipoprotein lipase, in microsomal frac- tions, 344, 350 Lipoproteins in chloroplast lamellae, 186 in SP, 495 Lipoyl dehydrogenase (1.6.4.3.), 157 binding to mitochondria, 145 Liver fat uptake, 273 injury, and lysosomes, 297 lysosomes, 253 microsomal fraction, 340-6 parenchymal cells, 11, 269, 270 Lubrol W, and microsomal enzymes, 369 andjibosomes, 370 566 SUBJECT INDEX Lymphocytes acid phosphatase granules, 257 cytomembranes, 381 lysosomal enzymes, 260 Lysine, and cation balance, 491 Lysolecithin, and lecithin membranes, 169 Lysolecithinase (3.1.1.5.) (lysophos- pholipase) in liver microsomal fraction, 344 in microsomal sub-fractions, 350 Lysosomal enzymes and muscular dystrophy, 287 and regression of MuUerian duct, 286 and tail regression, 287 as contaminants, 249 cytochemical reactions, 301-3 digestion of viruses, 274 distribution patterns, 247-50 extrusion, 296, 297 increase after K+ deficiency, 260 index of lysosomal purity, 299 in virus infection, 298 origin and formation, 25Sz^ release in vivo, 250, 296 release by streptolysin, 296 stability to fixation, 301 Lysosomal membrane, 251, 252 esterase activity, 245 fragility, 248 Lysosomes acid phosphatase stain, 5 and autophagy, 288 and ingestion, 261-85 and pericanalicular bodies, 20 and post-partum involution of uterus, 287 and regression of ovaries, 287 as digestive vacuoles, 283 as "suicide bags", 296 ''development, 392 enzymes, absence from nuclei, 91 extrusion, 296, 297, 282 fragility during complement fixation, 293 from Kupffer cells, 298 fusion with phagosomes, 264-7 heterogeneity, 246, 247 history, 240-3 in brain, 254 in corpora lutea, 287 Lysosomes — cont. in different species, 257 in epididymis, 255 in epithelial cells, 253 in holocrine secretions, 287 in inflammation, 297 in intestine, 254 in kidney, 254, 240 in liver, 253 in microsomal fractions, 338, 339 in necrosis, 297 in oestrous cycle, 287 in plants, 258 in prostate, 255 in reticulo-endothelial cells, 256, 257 in shock, 297 in tissue culture cells, 257 in uterus, 255 in virus infection, 298 life cycle, .246, 247 methods of isolation, 298-303 non-enzymic components, 250, 251 occurrence, 253-8 origin and formation, 258-61 pathological changes, relation to, 294-7 swelling by Triton WR 1339, 172 terminology, 242, 243 M Macroglobulins, in SP, 491 Macromolecules, in SP, 478, 491-6 Macrophages acid phosphatase granules, 256, 257 and antibody formation, 291 esterase granules, 256 lysosomal enzymes, 260 lysosomes in muscular dystrophy, 287 particle uptake, 280 Magnesium, in chloroplasts, 186 Magnesium ions activators of: alkaline fructose-l,6-diphospha- tase, 215 car boxy dismutase, 21 1 phosphoribulokinase, 216 and binding of polynucleotide phos- phorylase, 455 and deoxycholate on membranes, 366 and DNase binding to ribosomes, 451-2 SUBJECT INDEX 567 Magnesium ions — cont. and ribosome isolation, 410 and ribosome sub-units, 411-13 and SP flocculation, 501 binding to vesicles, 362 in cell fractionation, 338 in dense granules in mitochondria, 151 in SP, 483 stabilizer of ribosomes, 439 stimulation of chloroplast lipid syn- thesis, 225 uptake by mitochondria, 128 Malate from oxalacetate in chloroplasts, 221 in Krebs' cycle, 112 in non-aqueous nuclei, 84 Malate dehydrogenase (1.1.1.37.) absence of labelling by mitochondria, 131, 158 extraction from mitochondria, 141, 143 in chloroplasts, 221, 222 in constant proportion group, 157 in liver microsomal fraction, 341 in mitochondria, 140 in mitochondria and SP, 113 in nuclei, 83 Malate synthase (4.1.3.2.), and plants, 223 "Male" protein, in SP, 494 Malonate, into fatty acids in sarco- somes, 129 Malonyl Co A, synthesis by sarcosomes, 129 Mammary gland cytomembranes, 380 lysosomes, 7 synthesis of fatty acids, 129 Manganese ions in chloroplasts, 186 and oxygen evolution, 199 stimulation of lipid synthesis, 225 a-Mannosidase (3.2,1.24.), in lyso- somes, 245 Marker enzymes cytochrome oxidase for mitochond- ria, 117, 118 in cell fractionation, 7, 8 in isolation of chloroplasts, 197 Melanin, in lipofuscin granules, 286 Membrane-bound enzymes, 387, 395 "Membrane complex", from B. mega- terium, 382 Membrane flow, 252 Membrane formation, role of SP, 503 Membrane fractions characteristics, 354-60 enzymes, 354-60 from plants, 383 in micro-organisms, 382, 383 invertebrate, 382-4 in yeast, 383 "Membrane-RNA", 165, 366 Membrane systems biochemistry, 337-84 definitions, 322 summary of properties, 397-9 Membrane vesiculation, 263 Membranes, 321-5 Mercuric chloride, inhibitor of: carboxydismutase, 211 ferredoxin-NADP reductase, 204 phosphoribulokinase, 216 Mercuric sulphide, uptake by carcino- ma, 275 Mesomolecular subfraction, of SP, 478 Mesomolecules, in SP, 484-91 Messenger RNA (see mRNA) Metabolites in SP, 485, 486 limiting molarity, 481, 482 Metachromatic vacuoles, in Amoeba, 257, 276 Methaemoglobin reducing factor, 202 (see Ferredoxin) Methionine activation in chloroplasts, 225 and RNA methylation, 454 6-Methyl adenine, in sRNA, 74 Methylases, of RNA in ribosomes, 454 Methylated glycines, oxidation by mitochondria, 134 Methylation in nucleus, 71 of RNA in Chironomus, 74 1 -Methyl guanine, in sRNA, 74 Mevalonic acid, in carotenoid synthesis, 226 "M" fraction, 365 and smooth membranes, 361 568 SUBJECT INDEX Mg-protoporphyrin, 226 Microbodies, 172 Micromolecular subfraction, of SP, 477 Micromolecules, in SP, 483, 484 "Micro-organelles", 477 Micropinocytic vesicles, 263 nucleoside phosphatase activity, 267 Microsomal "cuts", 353 Microsomal enzymes activation, 367 release, 370 solubilization, 367 Microsomal fractions and vesicles, 328 criteria in analysis, 377 electron microscopy, 353, 328 in Neurospora crassa, 383 in peas seedlings, 383 liver chemical constituents, 340 enzymes, 340-6 methods of isolation, 337-40 morphology, 340 nomenclature, 330 structural protein, 163 "Microsomes", nomenclature, 330 Microtubules, 264 Microvilli, 323 Mitochondria anabolic reactions, 129-32 and ion balance, 483 and protein uptake, 267, 268 as contaminants of chloroplasts, 222 cristae, 105 dense granules, 150-3 DNA, 106, 145-50 evolution, 113 fatty acid oxidation, 114-17 in autophagic vacuoles, 288 in "autolytic vacuoles", 249 inclusions, 145-53 in composite bodies, 249 inner compartment, 105 ion transport, 128, 129 lysosomes, relationship with, 241 matrix, 105, 106 membrane, 106 membrane system, 153-69 metabolic activity, 107-38 morphology, 104-7 outer compartment, 105 Mitochondria — cont. oxidative phosphorylation, 119-28 respiratory chain, 117-19 sap, 139^5 structural basis of function, 138-69 tricarboxylic acid cycle, 111-14 Mitochondrial contraction, 152 Mitochondrial enzymes, fixation, 301 Mitochondrial fraction, lysosomal con- tamination, 249 Mitochondrial membrane, 1 53-72 and DNA, 149-50 chemical composition, 160-5 integrated structure, 169-72 labelling of proteins, 131 RNA, 165 transfer of fatty acids, 1 15 Mitochondrial respiration, ADP con- trol, 2 Mitochondrial sap, 139-45 Mitotic apparatus, proteins, 502 "Mixed-function" oxidases, 391 Monoamine oxidase (1.4.3.4.), in mito- chondria, 118 Monocytes, acid phosphatase granules, 257 mRNA and chromosomes, 66 and RNase, 448, 449 association with 30S particles, 71 hybridization with DNA, 68 inactivation by RNase, 449 in membranes, 365, 384 in nuclear fibrils, 45 in nuclear protein synthesis, 80 in RNase-free bacteria, 449, 450 intracellular site of synthesis, 66, 67 synthesis, 65 transfer to cj^oplasm, 71 mRNA codons, 420 Mucopolysaccharides in cell surface of Amoeba, 250 in lysosomes, 250 Mullerian duct, regression, 286 Multivesicular bodies, 259 Muscle cytomembranes, 380 Muscular dystrophy, lysosomal en- zymes, 287 Myoglobin, absence from nuclei, 90 SUBJECT INDEX 569 Myoinositol, in microsomal fraction, 342 N NAD in chloroplasts, 204 in nuclei, 84 in oxoglutarate oxidation, 113 reduction by succinate, 124 synthesis in nuclei, 87, 88 NADH-Coenzyme Q reductase Complex I, 155 in mitochondria, 161 NADH-cytochrome b^ reductase, 1.6,2.2., in microsomal fractions 341 NADH-cytochrome c reductase (1.6.2.1.; 1.6.99.3.) absence from nuclei, 84, 85 and deoxycholate, 368, 369 in hepatomas, 395 in liver microsomal fraction, 341 in lysosomes, 245 in membranes, 366, 387, 354, 359 in microsomal sub-fractions, 350 in muscle cytomembranes, 380 in plant membranes, 383 in prostate cytomembranes, 381 in spleen cytomembranes, 381 localization problems, 386 relationship to NADH dehydro- genase, 119 NADH dehydrogenase (1.6.99.1.) and NADH-cytochrome c reductase, 119 in mitochondria, 156 in mitochondrial respiratory assem- bly, 154 NADH diaphorase, in membrane frac- tions, 357 NADH - Ii, 123 NADH oxidase and phagocytosis, 281 in kidney cytomembranes, 380 in muscle cytomembranes, 380 in spinach mitochondria, 222 phospholipids, in mitochondria, 161 NADH-ubiquinone reductase, and NADH dehydrogenase, 119 NAD(P) transhydrogenase (1.6.1.1.) (transhydrogenase) and ferredoxin-NADP reductase, 204 in chloroplasts,'204 in mitochondria, 140, 134, 154 in respiratory assembly, 154 NAD nucleosidase (3.2.2.5.) in brain cytomembranes, 378 in liver microsomal fraction, 344 in muscle cytomembranes, 380 NADP in chloroplasts, 204 in microsomal fraction, 340 in non-cyclic photophosphorylation, 198 in reductive pentose cycle, 212 photoreduction and salicylaldoxime, 200 photoreduction by water, 199 photoreduction (photoreaction A), 200 reduction by ferredoxin, 203 stimulation of chloroplast lipid syn- thesis, 225 NADPH-cytochrome c reductase (1.6.2.3.) and ferredoxin-NADP reductase, 204 drugs, effect of, 397 in adrenal cytomembranes, 378 in brain cytomembranes, 378 in liver microsomal fraction, 342 in membrane energy supply, 387 in membrane fractions, 355 in prostate cytomembranes, 381 NADPH-diaphorase, in chloroplasts, 204 NADPH-linked lactate dehydrogenase, in pinocytosis, 281 NADPH oxidase drugs, effect of, 397 in membrane fractions, 354 NADPH peroxidase (1.11.1.2) in liver microsomal fraction, 342 NADP reducing factor (see Ferredoxin) NAD pyrophosphatase (3.6.1.9.) in liver microsomal fraction, 344 in membrane fractions, 356 NAD pyrophosphorylase (2.7.7.1.) in nucleus, 459, 87 in ribosomes, 463, 457-9 in viral infection, 459 570 SUBJECT INDEX Nascent enzymes, in ribosomes, 426, 429-38 Nascent polypeptide, and puromycin, 428 N-Deacylase, in microsomal fractions, 343 Necrosis enzyme changes, 297 role of lysosomes, 297 Negative staining, of mitochondria, 106 Nerve cells, ferritin uptake, 270 Nerve endings, isolation, 7 Neuraminidase (3.2.1.18.) and plasma membranes, 375 viral, in ribosomes, 432 Neurospora crassa, mitochondrial RNA polymerase, 130 mitochondrial DNA, 146 Neutral proteases, 285 and necrosis, 297 Nickel ions, and carboxydismutase, 21 1 Nicotinamide deamidase, in micro- somal fractions, 344 Nicotinamide phosphoriboxyltrans- ferase (2.4.2.12.) in mitochondria, 87 nuclei, absence from, 87 Nitrate, metabolism in chloroplasts, 226, 227 Nitrate reductase (1.6.6.2.), 227 Nitrite, metabolism in chloroplasts, 227 Nitrite reductase, (1,6.6.4.), 227 Nitrite reduction, and ferredoxin, 203 Nitrophenyl phosphate, phosphatase substrate, 249 Non-cyclic photophosphorylation in chloroplasts, 197-206 in lipid synthesis in chloroplasts, 225 Non-haem iron in chromatophores, 187 in ferredoxin, 202 in respiration, 119 Non-photosynthetic chromoplasts, 182, 187 Nuclear envelope (nuclear membrane) contaminant of plasma membrane fraction, 372 definition, 323 fine structure, 35-7 impermeability to ATP, 81 permeability to proteins, 92, 93 Nuclear envelope — cont. permeases, 93, 94 Nuclear fraction, contamination, 249 Nuclear membrane (see Nuclear envelope) Nuclear oxidative phosphorylation, 86 Nuclear RNA, labelling, 57 Nuclear sap glycolysis, 82 similarity to SP, 92, 45 "Nuclear sap fraction", 45 Nuclear transplantation, 494 Nuclear volume, 493 Nucleases, alkaline and nuclei, 91 Nuclei adsorption of proteins, 268 and pH, 502 in bacteria, 60 incorporation of nucleosides, 57 isolation methods, 28 aqueous media, 31-5 artifacts, 28 calcium ions, and, 32 concentrated sucrose, and, 32 homogenization methods, 31 mechanical methods, 46-9 organic solvents, and, 29-31 polyribonucleotide polymerase, 454 RNase in ribosomes, 466 separation from cytomembranes, 337 subfractionation, 49 Nucleic acids enzymes for synthesis, in SP, 496, 497 uptake by cells, 273, 274 Nucleohistones DNP-fibrils in, 40 melting temperature, 70 Nucleoli effect of deoxycholate, 46 in methylation reactions, 47 in protein synthesis, 80, 81 methods of isolation, 46-9 oocytes, 35 presence of R-RNA, 72 ribosomes, NAD pyrophosphorylase, 459 sRNA, 47 stabilization by Ca^^ ions, 46 Nucleolo-chromosomal complex in protein synthesis, 79-81 and ribosomal RNA synthesis, 72 SUBJECT INDEX 571 Nucleolonemata, 41 Nucleolus in synthesis of ribosomal RNA, 72-4 in synthesis of sRNA, 72-4 Nucleonemata, and RNA synthesis, 73 Nucleoside diphosphatase (3.6.1.6.) absence from S.P., 499 and glucose-6-phosphatase, 367 deoxycholate effect, 369 in cytomembranes, 335, 332 in liver microsomal fractions, 344 in membrane fractions, 371, 355 localization problems, 384 possible function, 390, 391 solubilization, 368 Nucleoside diphosphate kinase (2.7.4.6 in mitochondria, 140 in substrate level phosphorylation, 122 Nucleoside-51-monophosphatase (see 51-Nucleotidase) Nucleoside nephrosis, 254 Nucleoside phosphatase, lysosomal marker, 304 Nucleoside pyrophosphorylase (see Purine nucleoside phosphorylase) Nucleoside triphosphatases absence from cytomembranes, 335 deoxycholate effect, 369 during development, 392 in liver microsomal fraction, 344 in membrane fractions, 355 in plasma membranes, 335 in ribosomes, 460 localization problems, 384 5'-Nucleotidase (3.1.3.5.) activation, 390 and deoxycholate, 368, 369 and glucose-6-phosphatase, 346 cobalt activation, 335 during development, 392 electron microscope histochemistry, 335, 336 histochemistry, 384, 385 in adrenal cytomembranes, 378 in bacterial membranes, 382, 383 in biliary obstruction, 397 in cytomembranes, 333 in hepatomas, 395, 396 in liver microsomal fraction, 344 5 '-Nucleotidase (3 . 1 .3.5.)— cont. in membrane fractions, 356-9 in plasma membrane, 335, 374 in plasma membrane fragments, 367 in regenerating liver, 393 localization problems, 384 reaction product diffusion, 331 release from membranes, 366 Nucleotides, and nuclear envelope, 93 in SP, 485 metabolism in nuclei, 87-9 molecules per cell, 482 Nucleotide translocase, in mitochon- dria, 140 Nucleus chemical fractionation, 42, 44-6 fine structure, 35-44 globulins, 494 metabolic activities, 27-8 methods of fractionation, 28 ribosomes, polynucleotide phos- phorylase, 459 site of RNA synthesis, 57-60 structure, 35-44 uptake of DNA, 273 o Oenethera, chloroplast proteins, 185 Oils, in elaioplasts, 188 Oleate synthesis in chloroplasts, 225 in microsomal fraction, 342 Oligomycin and amino acid incorporation in mitochondria, 127 in oxidation phosphorylation, 124 site of action, 135 Oocytes, protein uptake, 270 Operational terms, in cell studies, 21 Opsonins and antigen uptake, 277 and "self" recognition, 294 Organic solvents in isolation of: chloroplasts, 193-5 nuclei, 29-31 Orotate, in membrane RNA, 364 Orthophosphate, incorporation into RNA, 364 Osmium tetroxide, and nucleotide dephosphorylation, 331 572 SUBJECT INDEX Osmotic shock, 31 Osteoclasts acid phosphatase granules, 256 phagolysosomes, 288 Ouabain and membrane ATPase, 357 and plasma membrane ATPase, 374 Outer compartment, of mitochondria, 105, 139 Oxalacetate from pyruvate in Chloroplasts, 221 in Krebs' cycle, 112 to malate in chloroplasts, 221 Oxalosuccinate and malonyl Co A in sarcosomes, 129 in Krebs' cycle, 112 Oxidases, in protoplast membranes, 383 P-Oxidation of fatty acids in mitochondria, 114 of palmityl CoA in mitochondria, 115 Oxidations, extramitoChondrial, 117 Oxidative phosphorylation, 119-28, 231 anisotropic ATPase, 120-1 and photosynthetic phosphorylation, and protein synthesis in mito- chondria, 158 chemi osmotic theory, 120-1 inhibitors, 125 in nuclei, 127, 85-6 in spinach mitochondria, 222 minimum hypothesis, 123-4 partial reactions, 135-6 reversal, 124-5 2-Oxoglutarate from phosphoenolpyruvate in chloro- plasts, 226 in Krebs' cycle, 112 in non-aqueous nuclei, 84 to glutamate in chloroplasts, 223 to succinate in mitochondria, 122 transamination in chloroplasts, 221 2-Oxoglutarate dehydrogenase (1 .2.4,2.) enzyme complex, 477 in mitochondria, 113 2-OxogIutarate oxidase, in mitochond- ria, 156,222 Oxygen evolution in photo-oxidation, 199 uptake in phagocytosis, 281 Palade particle, 330 Palmitate synthesis in chloroplasts, 225 to phosphatides in mitochondria, 1 29 Palmityl-carnitine, in mitochondria, 1 1 5 Palmityl-CoA, in mitochondria, 115 Palmityl-CoA : carnitine palmityl trans- ferase, in mitochondria, 1 1 5 Pancreas cytomembranes, 381 enzymes in ribosomes, 431, 432 Paneth cells, 259 lysosomes, 254 Para-aminohippuric acid, mitochon- drial synthesis, 131 Paramecium aurelia, mitochondrial DNA, 146 Parenchymal cells, alkaline phospha- tase, 332 "Pars amorpha", in nucleus, 41 Passive accumulation, of lysosomal enzymes, 261 Penicillinase (3.5.2.6.), in ribosomes, 463 Pentose phosphate cycle, in SP, 497 Peptidases, in ribosomes, 456, 457, 463, 464 Pericanalicular dense bodies, 20, 242 Permeases and nuclear membrane, 93, 94 in mitochondria, 159 Peroxidase (1.11.1.7.) and egg-white, 280 as marker for phagosomes, 304 catabolism after injection, 284 in Kupffer cells, after injection, 256 in ribosomes, 460, 464 intravenous injection, 264 uptake by: kidney slices, 269 liver lysosomes, 253 tumour cells, 271 Peroxisomes, 172-5 catalase, presence of, 110 electron microscopy, 174 resolution of, 7 role, 173, 174 "Petite" mutants, 114 pH, of SP, 484, 501,502 SUBJECT INDEX 573 "pH 5" enzymes, in SP, 496, 502 Phage T4r, 453 Phagocytic vacuoles, 264 I and lysosomes, 243 and virus uptake, 274 markers, 303-5 Phagocytosis, 262, 277-81 phagolysosomes, 262 I and dye-inclusion bodies, 276 I and Golgi vesicles, 259 ' and protein uptake, 267 and "watery vacuoles^ 295 extrusion of foreign materials, 282 in Kupffer cells, 256 in thyroid, 266 mechanism of enlargement, 295 methods of isolation, 299 hagosomes, 243, 262 ^ and lysosomes, 242, 243 and pathology, 294-7 and transport, 26Y^ fusion with lysosomes, 264-7 Phenanthroline, and ferredoxin, 203 henazine methosulphate, 206 'henobarbital, and cytomembranes, 396 henylphosphate: cytidine phospho- transferase, in microsomal fractions, 342 henyl-urethane, 200 hosphatases _^ in nucleiy 88-9 in ribosomes, 464, 456 Phosphate incorporation by nuclei, 57 in SP, 486 nuclear RNA labelling, 57 Phosphatidate phosphatase (3.1.3.4.) in cytomembranes, 380 in lysosomes, 245 in membrane fractions, 359 hosphatides, mitochondrial synthesis, 129 hosphatidic acid, and phagocytosis, 281 hosphatidylethanolamine, in lyso- somes, 250 hosphatidylglycerol, in chloroplasts, 186 'hosphatidylinositol kinase, in mem- brane fractions, 342, 379 Phosphatidylibositol phosphate, mito- chondrial labelling, 129 Phosphatidylserine, inphagocytosis, 281 Phosphodiesterase I (3.1.4.1.) in kidney cytomembranes, 379 in liver microsomal fraction, 344 in membranes, 366 in plasma membrane fractions, 375 in ribosomes, 455, 464 Phosphoenol pyruvate from phosphoglycerate in chloro- plasts, 220 in nuclei, 83 to oxalacetate in chloroplasts, 221 to oxoglutarate in chloroplasts, 226 Phosphoenol pyruvate carboxykinase (see also Phosphopyruvate car- boxylase) in mitochondria, 138 isoenzymes in mitochondria and SP, 499 Phosphofructokinase (2.7.1.11.) and Pi in SP, 486 in nuclei, 82 Phosphoglucomutase (2.7.5.1.) in chloroplasts, 217 inSP, 4^7 6-Phospho-i>-gluconate, oxidation in chloroplasts, 228 in chloroplasts, 228 6-Phosphogluconate dehydrogenase (1.1.1.43.) in nuclei, 83 « in plants, 230 Phosphogluconate dehydrogenase (de- carboxylating) (1.1.1 .44.) Phosphoglyceraldehyde, in reductive pentose cycle, 212 Phosphoglycerate kinase (2.7.2.3.) in nuclei, 82 in plants, 212, 217 in reductive pentose cycle, 212 D-2-Phosphoglycerate from D-3-phosphoglycerate, in chloroplasts, 220 to phosphoenol pyruvate, in chloro- plasts, 220 D-2-Phospho-D-glycerate hydrolase (4.2.1.11.) (enolase) in chloroplasts, 220 574 SUBJECT INDEX 3-Phospho-i>glycerate, in chloroplasts, 220 Phosphoglyceratemutase (5.4.2.1.), in chloroplasts, 220 3-Phosphoglyceric acid in nuclei, 83 in reductive pentose cycle, 209, 212 Phosphohexoisomerase (see also Glu- cose phosphate isomerase) in muscle cytomembranes, 30 Phosphohistidine in oxidative phosphorylation, 123 in substrate level phosphorylation, 122, 123 Phosphoketopentose epimerase (see also Ribulose phosphate 3-epimerase) in plants, 217 Phospholipase (3.1.1.5.), release of lysosomal enzymes, 252 Phospholipase C, D (3.1.4.3.4.), in plastids, 229 Phospholipids complex with respiratory enzymes, 161 complex with structural protein, 162 extraction from mitochondria, 141 in brain cytomembranes, 378 in chromatophores, 186 in cytomembranes, 365 in kidney cytomembranes, 379 in liver microsomal fractions, 340 in microsomal sub-fractions, 348 in mitochondria: and membrane, 161 and NADH oxidation, 161 and succinoxidase, 161 in muscle cytomembranes, 380 in plasma membrane fractions, 375 in reticulosomes, 365 synthesis by mitochondria, 129 synthesis and phagocytosis, 281 "Phospholipo-glycoprotein core", 375 Phosphopentoisomerase, in plants, 215, 217 (see also Ribose phosphate isomerase) Phosphopentokinase, in plants, 216, 217 (see also Phosphoribulo- kinase) Phosphoprotein phosphatase (3. 1 .3. 1 6.), in lysosomes, 245 Phosphoproteins, in mitochondria, 122 Phosphopyruvate carboxylase (4.1.1.31,32.) in chloroplasts, 221 in mitochondria, 140 Phosphoribulokinase in chloroplasts, 216 inhibition by iodoacetate, 213 Phosphorylase, in chloroplasts, 2. (see also o-Glucan phos- phorylase) Photo-oxidation of cytochrome /, 201, 202 of ferrocytochrome c, 201 of water, 198-200 Photophosphorylation cyclic and non-cyclic, 197 in chromatophores, 1 87 Photoreaction A, 200 Photoreaction B, 199 Photosynthetic CO2 fixation, 216, 217 Photosynthetic phosphorylation, 231 comparison with oxidative pho phorylation, 230-2 "Photosynthetic pyridine nucleotic reductase", 202, 207 (see Fern doxin) Phycocyanin, in plastids, 182 Phycoerythrin, in plastids, 182 Phytic acid, in aleuroplasts, 188 "Piggy-back" pinocytosis, 278 Pigment metabolism, in chloroplast 225, 226 Pigment P700, 200 Pinocytic vacuoles, 243, 303-5 Pinocytic vesicles, 258 Pinocytosis and hepatectomy, 273 and lysosomes, 262 and uptake of antigen-antibody, 29 energy supply, 281 in Amoeba, 257 influencing factors, 277-81 Pituitary, cytomembranes, 381 Plants membrane fractions, 383 RNases, 441 Plasma cells, antibody formation, 291 Plasma membranes, 322 biochemistry, 374 enzymes, 335 fractions, 371-6 SUBJECT INDEX 575 Plasma membranes — cont. in plants, 393 in zonal ultracentrifuge, 367 methods of isolation, 371-4 properties, 387 re-formation, 503 thickness, 322 Plasma membrane sheets, 372 Plastocyanin, 200, 201 and photophosphorylation, 198, 207 in chloroplasts, 186 Plastoquinone and Hill reaction, 199 in chloroplasts, 186 in photophosphorylation, 198, 206 Platelets, acid phosphatase, 256 Polyadenylate nucleotidyl transferase (2.7.7.19.), in nucleo, 75 Polyadenylate synthetase, in nuclei, 75 Polyamines, and RNA synthesis, 71 Polyanions, and SP flocculation, 500 Polymorphonuclear leucocytes, lyso- somes, 256 Polynucleotide phosphorylase (2.7.7.8.) in nuclei, 75 in ribosomes, 455, 464 in RNA breakdown, 75 Polypeptides release by puromycin, 428 synthesis, 419-20 Polyphenol oxidase (1.10.3.1.), in chloroplasts, 229 Polyphosphatases, in nuclei, 89 Polyribonucleotide synthesis, in ribo- somes, 464 Polyribosomes (see Polysomes) Polysaccharides granules in Euglena, 196 in muscle cytomembranes, 381 Polysomes, 421 electron microscopy, 422-4 p-Galactosidase, 436 Pores in membranes, 323 in nuclear envelope, 325 Porphyridium cruentum, photo-oxida- tion in, 202 "Post-microsomal" fractions, 330 chemistry, 348, 349 electron microscopy, 348 Post-partumi nvolution of uterus, and lysosomes, 287 Postulate of biochemical homogeneity, 14 Potassium deficiency and areas of focal degeneration, 294 and lysosomal enzymes, 254, 260 Potassium-dependent ATPase, in plas- ma membranes, 374 Potassium ions in cell fractionation, 338 in SP, 483 uptake by mitochondria, 128 "Potocytosis", 296 Procaine, hydrolysis in microsomal fraction, 350 Procarboxypeptidase, adsorption, 431, 432 Proline activation in chloroplasts, 225 oxidation by mitochondria, 134 Proplastids, 187 Prostate cytomembranes, 381 lysosomes, 255 Proteases in cytomembranes, 381 in ribosomes, 460, 464 zymogens in ribosomes, 431, 465 Protein catabolism, 285 denaturation, 283 dye-complexes, in lysosomes, 275 secretion, from membranes, 366 synthesis and GTPase, 427 and ribosomes, 417-21 enzymes of, in ribosomes, 465 enzymes of, in SP, 496 in chloroplasts, 224, 225 in cytomembranes, 391 in membranes, 165, 366 in mitochondria, 158, 171 in nucleoli, 79-81 in nucleus, 76-81 in ribosomes of nuclear sap, 77 ribosomal enzymes, 426-9 transport, in cells, 267 uptake and mitochondria, 267, 268 and phagolysosomes, 267-71 ft 576 SUBJECT INDEX Protein uptake — cont. by Amoeba, 271 by embryonic cells, 270 effect of dose, 279 in tissues, 268-70 specificity, 278 Proteins adsorption by nuclei, 268 by ribosomes, 425 in aleuroplasts, 188 in chloroplasts, 186 in mitochondrial inclusions, 152 in ribosomes, 413-15 in SP, 491-5 storage in kidney mitochondria, 152 Proteolysis, in mitochondria, 137 Prothrombin in microsomal sub-fractions, 349 synthesis, in microsomal fractions, 345, 350 Protolysosomes, 243 Protoplast membranes, 383 Protoporphyrin, formation by mito- chondria, 131, 132 Protozoa acid phosphatase, 257 esterase, 257 Pseudocyclicphotophosphorylation, 207 Pseudo-uridine, in nucleolar sRNA, 74 "Puffs", in Z)ra5c»/>A//a chromosomes, 67 Purine nucleoside phosphorylase (2.4.2.1.) electrophoresis, 493 in nuclei, 87, 91 Purine nucleosides, in nuclei, 57 Puromycin, and protein synthesis, 79, 80, 428 Puridine nucleotides in chloroplasts, 186 in chromatophores, 187 in lysosomes, 250 in mitochondria, 155 in nuclei, 57 Pyrophosphate, and nuclear enzymes, 89 Pyrophosphate: glucose phosphotrans- ferase, in microsomal fractions, 342 ji_Pyrroline-5-carboxylate, in mito- chondria, 134 Pyrroline-5-carboxylate reductase (1.5.1.2.), in mitochondria, 134 Pyruvate from phosphoenolpyruvate, in chloroplasts, 220 oxidation by mitochondria, 1 1 1 to CO2 in nuclei, 84 Pyruvate dehydrogenase (1.2.4.1.), 477 Pyruvate kinase (2.7.1.40.) in chloroplasts, 220 in nuclei, 82 Pyruvate oxidase in constant proportion group, 157 in mitochondria, 1 1 3, 1 56, 222 in plants, 221 sub-units, 157 Q enzyme (2.4.1.18.) (branching fac- tor), in chloroplasts, 219 Quantasomes and photo-oxidation of water, 198 in chloroplasts, 184 "Quencher", in photosynthesis, 199 Quinones, in chloroplasts, 186 R Rana pipiens, mitochondrial protein crystals, 153 Red enzyme (see Ferredoxin) Red plastids, 182 Reductive pentose cycle, 210, 217 in chloroplasts, 209 Regenerating liver, lysosomal enzymes, 249 Regression in endometrium, and lyso- somes, 287 Regulatory effectors, 485 Repressors, and histones, 70, 71 Residual bodies, 242 and phagolysosomes, 285 "Residual chromosomes", 42, 45 and nucleonemata, 45 "Residual" proteins in nuclei, 79 in sub-cellular fractions, 162 Respiration, and ion uptake, 128 Respiratory assemblies, in mitochon- dria, 154, 165-9 SUBJECT INDEX 577 Respiratory chain, 118-19 inhibitors, 125 in mitochondria, 117-19 reversal, 124, 125 Respiratory deficiency, in yeast, 164 Respiratory enzymes, in mitochondrial membranes, 154o8 "Reticulochrome" (see Cytochrome P-450) Reticulocytes, ribosomal RNase, 448 Reticulo-endothelial cells fat uptake, 272 lysosomes, 297, 262 uptake of: particles, 277 proteins, 268, 269 Reticulosomes, 362 and cytomembrane development, 392 phospholipids, 365 Reversed Arthus reaction, 292 Reversed pinocytosis, 282, 283 "Reversible microsome system", 392 Rhodanese (2.8.1.1.) electrophoresis, 493 in mitochondria, 134 Rhodospirillum haem protein, 187 Ribonuclease (see RNase) Ribose-5-phosphate, in chloroplasts, 215 Ribosephosphate, isomerase (5.3.1.6.), in chloroplasts, 215 Ribosomal RNA, site of synthesis, 72^ Ribosomes, 478 binding of enzymes, 455, 456 deoxycholate and nucleoli, 46 detergents, and, 370 electron microscopy, 412 enzymes, 421-6, 450-61 function, 417-21 history, 408-10 in chloroplasts, 185, 224 in "light microsomes", 348 in membranes, 456 in mitochondria, 145 in nuclei : in nuclear sap, 77, 78 in nucleoli, 46 isolation, 45 instability, 439 isolation techniques, 410, 411 Ri bosomes — cont. nascent enzymes, 429-38 nomenclature, 330 properties, 411-13 proteins, 413-15 protein synthesis, 2, 79, 80 RNA, 413 RNase, 438-50 structure, 415, 416 sub-units, 411-13, 415, 457, 496 terminology, 408 Ribosome-sRNA complex, 419 Ri bulose- 1 ,5-diphosphate in chloroplasts, 216 in reductive pentose cycle, 209 Ribulose-1 ,5-diphosphate carboxylase (4.1.1.39.) in chloroplasts, 209, 210 induction, 230 Ribulose-5-phosphate from 6-phosphogluconate, in chloro- plasts, 228 in chloroplasts, 215 Ribulose phosphate-3-epimerase (5.1.3.1.), in chloroplasts, 216 RNA in brain cytomembranes, 378 in chloroplasts, 185, 224 of cytomembranes, 364 in Golgi cytomembranes, 376 in "heavy" microsomal fractions, 347 in invertebrate membranes, 384 in membrane fractions, 354-60 in membranes, 393 in microsomal subfractions, 349 in mitochondria, 141, 165 in muscle cytomembranes, 380 in nucleoli, 46 in prostate cytomembranes, 381 release from membranes, 366 in ribosomes, 413 in SP, 496 in thymus cytomembranes, 381 species, 496 RNA-amino acid complexes, 489 RNA biosynthesis, 56-75 and enucleation, 58 and fasting, 365 asymmetrical and symmetrical, 65 during morphogenesis, 68 in bacterial "nucleoids", 60 2B 578 SUBJECT INDEX RNA biosynthesis — cont. in chloroplasts, 59 in "puffs" of chromosomes, 67 intracellular localization, 56-60 transcription of DNA strand, 64 RNA breakdown and polynucleotide phosphorylase, 75 in nuclei, 75 RNA:DNA hybrids, 383 RNA methylases, in ribosomes, 454, 467 RNA nucleotidyl transferase (2.7.7.6.) (see RNA polymerase) RNA polymerase (2.7.7.6.), 60-6 and mRNA synthesis, 65 binding to DNP, 62 competition with DNA polymerase, 54 complex with RNA and DNA, 62, 63 DNA primer, and, 62, 63 electron microscopy, 64 inhibition by histones, 69, 70 in mitochondria, 130, 158 inSP, 497 intracellular localization, 60, 62 intranuclear localization, 61 molecular weight, 64 sub-units, 64 RNase (2.7.7.16,17.) adsorption, 431, 432, 425 and diet, 393 and mitochondrial amino acid incor- poration, 130 and mitochondrial RNA, 165 and mRNA turnover, 448 and regression of MuUerian duct, 286 and SP flocculation, 501 binding to ribosomes, 444 biological function, 447-50 cytochemical stain, 303 definition, 439 effect on membrane RNA, 365 in E. coli ribosomes, 440-7 in nuclei, 75 in ribosomes, 431, AAl-A, 439-40, 438-50, 465-7 in plasma membrane fractions, 375 latency in membranes, 366 marker for pinocytosis, 304 mechanism of action, 438 release from spheroplasts, 445 RNA templates, and RNase, 448 RNP particle, nomenclature, 330 Rotenone, and mitochondria, 152 Rough surface cytomembranes and albumin synthesis, 366 classification, 325 in microsomal fractions, 340 in zonal ultracentrifuge, 367 "R-RNA", 66, 72, 73 i Saccharomyces cerevisiae, mitochon- drial DNA, 146 Salicylaldoxime, and photophosphoryl- lation, 200, 207 Sarcoplasmic reticulum, 323 Sarcosomes, fatty acid synthesis, 129 Satumiid moths, protein uptake, 270 Scenedesmus, chlorophyll deficient mu- tants, 207 Sebaceous glands, lysosomes, 287 Secretion, and reversed pinocytosis, 282, 283 Secretory enzymes and Golgi region, 259 synthesis, 282 Secretory granules, 258, 282 Sedimentation coefficients, of particles, 478 Sedoheptulose- 1 ,7-diphosphate in chloroplasts, 214 inhibitor of CO2 fixation, 213 and triose phosphate dehydro- genases, 213 Segresomes, 243, 276 "Self" and "non-self", in protein up- take, 278 "Self "recognition, duringphagocytosis, 293 Sephadex, analysis of SP, 479 Serotonin, 279 Serum albumin in microsomal sub-fractions, 349 factors, and reticulo-endothelial sys- tem, 277 proteins, and areas of focal degenera- tion, 294 proteins, breakdown, 283 SUBJECT INDEX 579 Shock, and lysosomes, 297 Sialic acid, 263 in membrane fractions, 356 in mitochondria, 165 in plasma membranes, 375 Single-stranded DNA, 52-4 Smooth-surfaced cytomembranes and albumin synthesis, 366 and "M" fractions, 361 classification, 325-7 function, 386, 387 in zonal ultracentrifuge, 367 RNA content, 165 "Smooth vesicles", 361 Snail digestive gland, lysosomal en- zymes, 257 Sodium-dependent ATPase, 374 Sodium fluoride, inhibitor of nuclear glycolysis, 83 Sodium ions in amino acid incorporation in nuclei, 76 in nucleus, 325 in SP, 483 Soluble phase (see SP) Soluble phase amino acids (SPAA), 487 Soluble proteins in chloroplasts, 195 in nuclear sap, 78 Soluble RNA (see SRNA) SP (soluble phase) analysis on Sephadex, 479 composition, 483-96 contamination with lysosomal en- zymes, 249 definition, 475, 476 DNA polymerase, 51 enzyme patterns, 498 isolation medium, 484 mesomolecules, 484-91 physiology, 500-3 RNase, 442 sub-fractions, 477, 478 SPAA (soluble phase amino acids), 487 Specific proportion groups, in mito- chondrial enzymes, 1 57 Spermine, and ribosomal enzymes, 432, 440 Spheroplasts alkaline phosphatase loss, 445 ribosomal RNase, 445 Spherosomes in autolytic degradation, 258 in plants, 258 lysosomes, comparison with, 258 Sphingomyelin, in lysosomes, 250 "Speicfiening", 275 and lysosomes, 262 Sphingosine, synthesis in cytomem- branes, 378 Spirogyra, chloroplasts, 182 Spleen cytomembranes, 381 lysosomes, 7 Squalene, synthesis in microsomal frac- tions, 350 sRNA (soluble RNA) and nascent enzymes, 430 and ribosomes, 448 6-Methyladenine, in, 74 methylation, and nucleolus, 74 1-Methylguanine, in, 74 in nuclei, 44 in nucleoli, 47 in protein synthesis, 419, 420 in SP, 496 site of synthesis, 72-4 30S sub-particles, of ribosomes, 411 50S sub-particles, of ribosomes, 411 Staining techniques acid phosphatase, for lysosomes, 5 use in enzyme localization, 5 Stalked bodies, in mitochondria, 165-9 Starch in amyloplasts, 188 in chloroplasts, 185, 196 in plants, breakdown, 219 synthesis, 218, 219 Starch-gel electrophoresis, 494 of ribosomal RNase, 443 Starch granules, phagocytosis, 281 Stearate dehydrogenation, in micro- somal fractions, 342 Steroid reductase, in microsomal frac- tions, 342 Steroid synthesis, in cytomembranes, 384, 390 Sterols, release of lysosomal enzymes, 251 Storage granules, 242 Storage protein, in aleuroplasts, 188 2B» 580 SUBJECT INDEX Streptolysin, release of lysosomal en- zymes, 251, 296 Stroma, of chloroplasts, 184 Strontium ions, uptake by mitochon- dria, 128 Structural protein and mitochondrial replication, 164 in mitochondria, 131, 1 62 labelling in mitochondria, 158 of ribosomes, 414 Sub-mitochondrial particles, 158 Substrate-level phosphorylation, 122 Substrate shuttles, 388 Succinate in Krebs' cycle, 112 in nuclei, 84 Succinate-coenzyme Q reductase, 155 Succinate-cytochrome c reductase, in mitochondria, 161 Succinate dehydrogenase (1.3.99.1.) as mitochondrial marker, 7 chloroplasts, absence from, 222, 226 in constant proportion group, 157 in mitochondria, 113, 140, 156 in mitochondrial respiratory assem- bly, 154 in nuclei, possible presence, 83 in yeast, 1 14 petite mutants, absence from, 1 14 Succinoxidase in mitochondria, 161, 222 nuclei, absence from, 84, 85 Succinic thiokinase, in mitochondria, 140 Succinyl CoA in S-aminolaevulinic acid synthesis, 226 in chloroplasts, 226 in Krebs' cycle, 112 in mitochondria, 140 in substrate- level phosphorylation, 122 transacylation in mitochondria, 116 Succinyl CoA deacylase (3.1.2.3.), and Krebs' cycle, 112 Succinylation, of structural protein, 165 Sucrose and lysosomes, 10 microsomal fraction, isolation in, 337 nuclei, isolation in, 32 Sucrose-6-phosphate, in chloroplasts, 219 Sucrose synthesis, in chloroplasts, 219 Sugan;, automated assay, 486 "Suicide bags", lysosomes, 296 Sulphite oxidase (1.8.3.1.), in micro- somal fraction, 342 Sulphodeoxyglycosyl diglyceride, in chloroplasts, 186 Sulpholipid, in chloroplasts, 186 Sulphur, in ferredoxin, 202 Surface-active agents, and microsomal enzymes, 370 Svedberg equation, 1 9 T2 phage DNA, and RNA polymerase, 64 Telolysosomes, 243 Terminal deoxynucleotide transferase, _ in SP, 497 fl Terminal oxidase, in mitochondria, 4 * Terminology (see Appendix) of lysosomes, 242, 243 operational and cytological terms, 21 Tetrahymena pyriformis, mitochondrial DNA, 130, 146 peroxisomes, 174 Tetrazolium, and dehydrogenases, 4 Thiamine pyrophosphatase, in cyto- membranes, 333-5 Thiamine pyrophosphate, in oxoglu- tarate oxidation, 113 Thiobacillus denitrificans, carboxydi- mutase in, 211 Thiosulphate sulphur transferase (see Rhodanese) Thorium oxide, uptake by kidney, 275 Threose-2,4-diphosphate, 213 Thymidine incorporation into chromosomes, 49 into DNA, 54, 130 into mitochondrial DNA, 130 into SP DNA, 497 Thymidine diphosphate kinase, in SP, 497 Thymidine kinase, in SP, 496 Thymidylate kinase, in SP, 496 Thymidylate synthetase, in SP, 497 SUBJECT INDEX 581 Thymus cytomembranes, 381 "dense bodies", 256 nuclei, 70 Thyroglobulin, 477 and phagolysosomes, 284 Thyroid, phagolysosomes, 266 Thyroid hormone and membrane linked enzymes, 394 and mitochondrial amino acid in- corporation, 131 and mitochondrial hypertrophy, 170 and oxidative phosphorylation, 170 Thyroxine degradation, absence from SP, 499 formation by phagolysosomes, 284, 266 Tissue culture cells lysosomes, 257 protein uptake 271 Tocophorylqu in one,inchloroplasts, 1 86 Toxic liver damage, and membrane linked enzymes, 397 TPN (see NAD?) "Transaminase" route, in mitochon- dria, 133 Transaminases in chloroplasts, 222, 223 in mitochondria, 133 "Transcriptase" (see RNA polymerase) Transcription, 56 Transfer enzymes, 428 Transfer RNA (see tRNA) Transketolase (2.2.1.1.) in chloroplasts, 215, 230 in plants, 217 "Translation" of genetic information, 56, 76-81 Transmethylases, in microsomal frac- tion, 343 Transport systems in mitochondria, 1 58-60 and phagosomes, 267 Tricarboxylic acid cycle (see also Citric acid cycle) intracellular localization, 111-14 in mitochondria, 111-14 Triose phosphate, in chloroplasts, 209 Triose phosphate dehydrogenase _ (1.2.1.12,13.) ■ in nuclei, 82 I I I Triose phosphate dehydrogenase (1.2.1. 12,13.)-co/7/rf. in plants in chloroplast fractions, 213 during illumination, 230 in ribosomes, 433, 467 in SP, 486 Triose phosphate dehydrogenase (NADP), in chloroplasts, 212 Triose phosphate isomerase (5.3.1.1.) in chloroplasts, 214 in nuclei, 82 "Triton" detergents and lysosomes, 9, 172 and mitochondria, 141 and ribosomes, 370 tRNA, 419 (transfer RNA) in SP, 496 "True" cholinesterase, in microsomal fractions, 343 Trypsin, and microsomal enzymes, 370 "Trypsin-activatable protease", in ribo- somes, 465 Trypsinogen absence from nuclei, 91 adsorption, 431, 432 in ribosomes, 645 Tryptophan synthase (4.2.1.20.), bio- synthesis, 433, 467 TSH, and phagolysosomes, 266 Tumour cells, protein uptake, 270 Tyrosine activation, in chloroplasts, 225 Tyrosine aminotransferase (2.6.1.5.), in mitochondria, 133 u Ubiquinone, 119 UDP, in nuclei, 87 UDPase in cytomembranes, 332 deoxycholate effect, 369 in membrane fractions, 344, 357 UDP coenzymes, 485 UDP glucose in nuclei, 88 in plant metabolism, 218 in sucrose synthesis, in chloroplasts, 219 582 SUBJECT INDEX UDP glucose-fructose glucosyl trans- ferase (2.4.1.13.), in chloro plasts, 219 UDP glucose-fructosephosphate glu- cosyl transferase (2.4.1.14.) in plants, 219 UDP glucose-a-glucan glycosyl trans- ferase in SP, 497 release during starvation, 497 UDP glucose-glycogen glucosyl trans- ferase (2.4.1.11.) and glycogen particles, 370 in microsomal fraction, 343 UDP glucuronyl transferase (2.4.1.17.) in cytomembranes, 390 in microsomal fractions, 343 UDPG pyrophosphorylase, (2.7.7.9.), in chloroplasts, 219 Ultracentrifuging, of SP proteins, 491 "Ultra-microsomal" fractions, 330 UMPase, in cytomembranes, 333 "Unit of electron transfer", 154 Unit membrane, 322, 250 Urate oxidase (1.7.3.3.), in micro- bodies, 172 Urea and ribosomal DNase, 451 and ribosomal RNase, 451 Uridine triphosphatase (see UTPase) Uronolactonase (3.1.1.19.) in microsomal fraction, 345 possible artifact, 371 Uterus, lysosomes, 255 UTP in metabolism, 485 in mitochondria, 129 in nuclei, 87 UTPase deoxycholate effect, 369 in cytomembranes, 333 in plasma membrane, 335 in ribosomes, 467 Valine, activation in chloroplasts, 225 Van der Waals forces, and membrane- bound enzymes, 367 "Vesicles", in microsomal fractions, 328, 340 Viral neuraminidase, in ribosomes, 432 Viruses infection, and lysosomes, 298 separation, 10 structure, 415 uptake, in phagocytic vesicles, 274 Vital dyes, uptake, 275-7 Vitalism, 171 Vitamin A and extrusion of lysosomal enzymes, 296 and lysosomal enzymes, 251, 296 Vitamin A esterase (3.1.1.1 2.), in micro- somal fraction, 343 Vitamin D, and lysosomal enzymes, 251 Vitamin E, and lysosomal enzymes, 251 Vitamin K and lysosomal enzymes, 251 in chloroplasts, 186 Vitamin K3 and ferredoxin-NADP reductase, 204 in cyclic photophosphorylation, 206 Vitamins, moles per cell, 482 w W~, 124 Waring Blendor, 31, 337 Water uptake, by mitochondria, 160 "Watery vacuoles", and phagolyso- somes, 295 Xanthine oxidase (1.2.3.2.), in plant membranes, 383 Xanthophylls, 226 in chloroplasts, 186 synthesis, 226 "X" factor, in microsomal sub-frac- tions, 349 XM, 123 and Ca2+ uptake by mitochondria, 159, 160 X-P, 123 and Ca2+ uptake by mitochondria, 195, 160 and oligomycin, 124 Xenopus laevis, ribosomal RNA syn- thesis, 73 X-rays, and nuclear phosphorylation, 86 Xylulose-5-phosphate, in chloroplasts, 215,216 SUBJECT INDEX 583 Y Yeast lactate dehydrogenase, and DNA, 148 mitochondria, 130, 158 succinate dehydrogenase, 114 Yellow plastids, 182 Yolk protein, 270 "Young" lysosomes, and secretory granules, 282 Zinc, in chloroplasts, 186 Zonal ultracentrifuge, 361, 367, 478 Zygnema, chloroplast, 182 Zymogen granules and diet, 393 and lysosomes, 258 Zymogens, and ribosomes, 431, 432 LIST OF ENZYME NUMBERS {This is a list of enzymes cited in the book, arranged according to their num- ber in ''Enzyme Nomenclature", Recommendations (1964), of the International Union of Biochemistry on the Nomenclature and Classification of Enzymes, Elsevier Publishing Company, 1965. Details of the enzymes may be found by referring to the main Subject Index). 1.1.1.1. Alcohol dehydrogenase 1.1.1.8. Glycerol-3-phosphate dehy- drogenase 1 . 1 . 1 .20 Glucuronolactone reductase 1 . 1 . 1 .26 Glyoxylate reductase 1 . 1 . 1 .27 Lactate dehydrogenase 1.1.1.30 3-H ydroxybutyrate dehydro- genase 1 . 1 . 1 .34 Hydroxymethylglutaryl-CoA reductase 1.1.1.35 3-Hydroxyacyl-CoA dehydro- genase 1,1.1.37 Malate dehydrogenase 1.1.1.41 Isocitrate dehydrogenase 1.1.1.42 Isocitrate dehydrogenase (NADP) 1.1.1.43 Phosphogluconate dehydro- genase 1.1.1.44 Phosphogluconate dehydro- genase (decarboxylating) 1 . 1 . 1 .49 Glucose-6-phosphate dehydro- genase 1.1.2.3 Lactate dehydrogenase (cyto- chrome b^ 1.1.3.1. Glycollate oxidase 1.1.99.1 Choline dehydrogenase 1.1.99.5 Glycerolphosphate dehydro- genase 1.2.1.8 Betaine aldehyde dehydro- genase 1.2.1.12 Triosephosphate dehydrogen- ase 1.2.1.13 Triosephosphate dehydrogen- ase (NADP) 1.2.3.2 Xanthine oxidase 1.2.4.1 Pyruvate dehydrogenase 1.2.4.2 Oxoglutarate dehydrogenase 1.3.99.1 Succinate dehydrogenase 1.3.99.3 Acyl-CoA dehydrogenase 1.4.1.2 Glutamate dehydrogenase 1.4.1.3 Glutamate dehydrogenase (NAD(P)) 1.4.1.4 Glutamate dehydrogenase (NADP) 1.4.3.2 L-Aminoacid oxidase 1.4.3.3 D-Aminoacid oxidase 1.4.3.4 Monoamine oxidase 1.5.1.2 Pyrroline-5-carboxylate reduc- tase 1.5.1.4 Dihydrofolate dehydrogenase 1.6.1.1 NAD(P) transhydrogenase 1.6.2.1 NADH cytochrome c reduc- tase 1.6.2.2 NADH cytochrome b^ reduc- tase 1.6.2.3 NADPH cytochrome c reduc- tase 1.6.4.1 Cystine reductase 1.6.4.2 Glutathione reductase 1.6.4.3 Lipoyl dehydrogenase 1.6.6.2 Nitrate reductase (NAD(P)) 1.6.6.4 Nitrite reductase 1.6.6.7 Azobenzene reductase 1.6.99.1 NAD(P)H dehydrogenase 1.6.99.3 NADH-cytochrome c reductase 1.7.3.3 Urate oxidase 1.8.3.1 Sulphite oxidase 1.9.3.1 Cytochrome oxidase 1.10.3.1 o-Diphenol oxidase 1.11.1.2 NADPH peroxidase 1.11.1.6 Catalase 1.11.1.7 Peroxidase 1.14.1.1 Aryl 4-hydroxylase 2.2.1.1 Transketolase 2.3.1.12 Lipoate acetyltransferase 2.3.1.15 Glycerolphosphate acyltrans- ferase 2.3.1.16 Acetyl-CoA acyltransferase 2.3.1.20 Diglyceride acyltransferase 2.4.1.1 a-Glucan Phosphorylase 2.4.1.11 UDPglucose-glycogen gluco- syltransferase 2.4.1.13 UDPglucose-fructose gluco- syltransferase 2.4.1.14 U DPglucose- fructose-phos- phate glucosyltransferase 2.4.1.18 Q-enzyme, branching factor 585 586 LIST OF ENZYME NUMBERS 2.4.2.1 Purine nucleoside phosphory- 3.1.4.5 Deoxy ri bonuclease lase 3.1.4.6 Deoxyribonuclease II 2.4.2.12 Nicotinamide phosphoribo- 3.1.6.1 Arylsulphatase syl-transferase 3.2.1.1 a-Amylase 2.6.1.1 Aspartate aminotransferase 3.2.1.2 |3-Amylase 2.6.1.2 Alanine aminotransferase 3.2.1.18 Neuraminidase 2.6.1.5 Tyrosine aminotransferase 3.2.1.20 a-Glucosidase 2.6.1.12 Alanine-ketoacid aminotrans- 3.2.1.21 /3-Glucosidase ferase 3.2.1.23 i3-Galactosidase 2.7.1.1 Hexokinase 3.2.1.24 a-Mannosidase 2.7.1.11 Phosphofructokinase 3.2.1.26 i8-Fructofuranosidase 2.7.1.40 Pyruvate kinase 3.2.1.31 /3-Glucuronidase 2.7.2.3 Phosphoglycerate kinase 3.2.2.5 NAD nucleosidase 2.7.3.2 Creatine kinase 3.4.1.1. Leucine aminopeptidase 2.7.4.3 Adenylate kinase 3.4.1.2 Aminopeptidase 2.7.4.6 Nucleosidediphosphate kinase 3.4.1.3 Aminopeptidase 2.7.5.1 Phosphoglucomutase 3.4.3.5 Cysteinyl-glycine dipeptidase 2.7.7.1 NAD pyrophosphorylase 3.4.3.6 Iminodipeptidase 2.7.7.6 RNA polymerase 3.4.4.5 Chymotrypsin 2.7.7.7 DNA polymerase 3.4.4.9 Cathepsin 2.7.7.8 Polynucleotide phosphorylase 3.4.4.19 Collagenase 2.7.7.9 UDPG pyrophosphorylase 3.5.2.6 Pencil linase 2.7.7.16 Ribonuclease 3.5.3.1 Arginase 2.7.7.17 Ribonuclease 3.5.4.4 Adenosine deaminase 2.7.7.19 Polyadenylate nucleotidyl- 3.6.1.1 Inorganic pyrophosphatase transferase 3.6.1.3 ATPase 2.7.8.2 Cholinephosphotransferase 3.6.1.6 Nucleosidediphosphatase 2.8.1.1 Thiosulphate sulphurtransfer- 3.6.1.9 NAD pyrophosphatase ase 4.1.1.31 Phosphopyruvate carboxylase 2.8.3.5 3-Ketoacid CoA-transferase 4.1.1.32 Phosphopyruvate carboxylase 3.1.1.1 Carboxylesterase 4.1.1.39 Ribulosediphosphate carboxy- 3.1.1.2 Arylesterase lase 3.1.1.3 Lipase 4.1.2.7 Aldolase 3.1.1.5 Lysophospholipase 4.1.3.1 Isocitrate lyase 3.1.1.7 Acetylcholinesterase 4.1.3.2 Malate synthase 3.1.1.9 Benzoylcholinesterase 4.1.3.4 Hydroxymethylglutaryl-CoA 3.1.1.12 Vitamin A esterase lyase 3.1.1.13 Cholesterol esterase 4.1.3.5 Hydroxymethylglutaryl-CoA 3.1.1.14 Chlorophyllase synthase 3.1.1.19 Uronolactonase 4.1.3.7 Citrate synthase 3.1.2.3 Succinyl-CoA deacylase 4.2.1.2 Fumarase 3.1.3.1 Alkaline phosphatase 4.2.1.3 Aconitate hydratase (aconi- 3.1.3.2 Acid phosphatase tase) 3.1.3.4 Phosphatidate phosphatase 4.2.1.11 Enolase 3.1.3.5 5 '-Nucleotidase 4.2.1.20 Tryptophan synthase 3.1.3.9 G lucose-6-phosphatase 4.2.99.1 Hyaluronidase 3.1.3.11 Fructose 1 ,6 diphosphatase 4.3.1.3 Histidine ammonia-lyase 3.1.3.16 Phosphoprotein phosphatase 5.1.3.1 Ribulosephosphate 3-epimcr 3.1.4.1 Phosphodiesterase ase 3.1.4.3 Phospholipase C 5.3.1.1 Triosephosphate isomerase 3.1.4.4 Phospholipase D 5.3.1.6 Ribosephosphate isomerase LIST OF ENZYME NUMBERS 587 5.3.1.9 Glucosephosphate isomerase 6.2.1.1 5.4.2.1 Phosphoglycerate mutase 6.2.1.3 6.1.1.1- 6.2.1.7 6.1.1.11 Aminoacyl-sRNA synthetases 6.3.1.2 AcetylCoA synthetase AcylCoA synthetase Cholyl-CoA synthetase Glutamine synthetase 4092 QP 601 R76 ERIN Roodyn , D. B . , cd . Enzyme cytology.