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THE CHEMISTRY OF LIFE

Steven Rose has been Professor of Biology at the Open University since it started in 1969. He was trained as a biochemist at the University of Cambridge and has worked in Oxford and London. His laboratory research is about the ways in which experience - especially during early development - affects the properties of the cells of the brain, but he has also written and worked extensively on issues concerning the social framework and consequences of science. His books include The Conscious Brain (Penguin) and the edited collections Against Biological Determinism and Towards a Liberatory Biology (1982). With sociologist Hilary Rose he has written Science and Society (Penguin), The Radicalization of Science and The Political Economy of Science, and, with Leon J. Kamin and R. C. Lewontin, Not in Our Genes (Penguin). Cath Sanderson graduated from Newcastle upon Tyne in microbiology in 1972. She has worked in dental research at the University of Leeds, and was a research assistant in the Department of Physiology at Newcastle before joining the Brain Research Group at the Open University. At present she works in the Department of Biochemistry at the University of Leeds.

STEVEN ROSE WITH

CATH SANDERSON

THE CHEMISTRY OF LIFE Second Edition

PENGUIN BOOKS

Penguin Books Ltd, Harmondsworth, Middlesex, England Viking Penguin Inc., 40 West 23rd Street, New York, New York 10010, U.S.A. Penguin Books AUSlralia Ltd, Ringwood, Victoria, Australia Penguin Books canada Ltd, 2801 John Street, Markham, Ontario, Canada L3R I B4 Penguin Books (N.Z.) Ltd, 182-190 Wairau Road, Auckland 10, New Zealand

First published 1966 Reprinted 1968 Reprinted with revisions 1970 Reprinted 1971, 1972, 1974, 1975, 1976, 1m Second edition 1979 Reprinted 1980,1982,1983,1985

Made and printed in Great Britain by Hazell Watson & Viney Umited, Member of the BPCC Group, Aylesbury, Bucks Set in Monotype Times Roman

Except in the United States of America, this book is sold subject to the condition that it shall not, by way of trade or otherwise, be lent, re-sold, hired out, or otherwise circulated without the publisher's prior consent in any form of binding or cover other than that in which it is published and without a similar condit ion including this condition being imposed on the subsequent purchaser

CONTENTS Preface to the Second Edition (1979)

Acknowledgements

Introduction: What is Biochemistry?

1 Before We Start

2 The Small Molecules

3 Macromolecules

4 The Organization of the Cell

5 Work and Enzymes

6 Pathways of Metabolism

117

7 Energy-providing Reactions

127

8 Sources of Energy

141

The Synthetic Pathways

172

Synthesis of Proteins and Nucleic Acids

184

ContrQlling the Cell

207

12 The Cell in Action

246

13 The Unity of Biochemistry

267

14 Can Biochemistry Explain the World?

286

A Note on Further Reading

290

Index

291

DEDICATION

For Hilary, who insisted that biochemistry be made illfelligible

PREFACE TO THE SECOND EDITION (1979) The first edition of The Chemistry a/Life was published in 1966. I had only a few years since completed a 'classical' biochemical training at Cambridge, done a Ph.D in brain biochemistry in London and was just finishing off a postdoctoral period in Rome when I wrote the book in 1963-4. I was full of optimism at the time, a biochemical positivism which reflected the exuberant atmosphere of the advances in biochemistry of the '50s and '60s. Biochemical knowledge might not change the world, I thought, but it would help explain it, and I believed then, as I believe now, in the importance of opening up science and making it accessible to those outside the arcane elitism with which it surrounds itself. Today, I am more conscious of the problematic nature of the biochemical reductionism which the book exuded, and I find the relationship between my science and the social and philosophical framework within which it is embedded more complex than in the past. I never intended The Chemistry o/Life as a text book, but merely to convey some of the excitement of biochemistry to a Jay reader, by discussing its central concepts with a minimum of sheer fact. However, perhaps by default of alternatives, the book soon became used in schools and university and college ancillary biochemistry and first-year courses, and this use has become extended since the Open University adopted it as a set text for their Science Foundation Course. This left me in a dilemma, as I was aware that the explosive growth of biochemistry was rendering some areas il;l particular in urgent need of revision, and the direction of my own thoughts and research had led me to re-analyse some of the orientations that I had taken more easily for granted in the early l%Os. In the event, I have waited to take advantage of the fact that the Open University's Science Foundation Course was to be rewritten so as to have the freedom to reconstruct the book without creating problems for students who were using the earlier version. 7

Preface to the Second Edition (1979) I have giVe51 a lot of thought to the question of how far I should try to move the book in the direction of my more recent interests, but this would, I decided, have been a very different venture. Instead, I have tried to retain as much of the earlier structure and mode of analysis as possible - the central themes of biochemistry as analysis, as metabolism and control - but I have tried to bring them further into line with present-day thinking. This book, then. is still about the internal content of biochemistry. not cell biology, neurobiology or the social structure of the discipline. Its structure and organization has, I hope, benefited from the experience of the intervening years, and the continued feedback from several generations of Open University students and my colleagues in the Biology Department at the Open University. I could never have undertaken the task without being able to find someone to work with, and I was exceptionally fortunate in Cath Sanderson, a person whose vitality, enthusiasm. dedication and determination to argue for her interpretation of the world has been an essential ingredient of this new edition. Dr Anna Furth and Dr Irene Ridge, of the Biology Department at the Open University, kindly read and commented on sections of the revised text for me and I should also like to thank Peter Wright, at Penguin, for his ready acceptance of the need to prepare a thorough revision of the book, as well as Nancie Pugh for her impeccable translation of hieroglyphs into typed English. None but me, however. should be blamed for any inaccuracies or idiosyncrasies that may remain. Now, as to the structure of the new edition. Essentially, it follows the old in that Chapter I introduces the basic chemical terms to those who are unfamiliar with them, and Chapters 2 and 3 deal respectively with the properties of the small and giant molecules of which the cell is composed. In the earlier edition, the structure of the. cell itself was not discussed until much later; this time, and because of the importan~ we attach to showing some subcellular organelles as composed of higher order hierarchies of macromolecules, the account of the cell forms Chapter 4. This concludes the section on biochemistry as analysis, and we turn in Chapters 5 and 6 to work, enzymes and metabolism. The core of biochemical energetics and metabolism is in Chapters 7 and 8, and it is these

Preface to the Second Edition (1979) which may be heavy going for the less dedicated. Connoisseurs will note that SI units and chemiosmosis are 'in', 'energy-rich' bonds and the classical squiggle notation 'out'. As in the earlier editions, a bit of judicious skipping would be acceptable here for the less involved. Chapters 9 and 10 deal with biosynthesis, and because of the expansion in theoretical significance and sheer factual information now available on protein and nucleic acid synthesis, these form a chapter of their own. Like Chapter 10, Chapter lIon control mechanisms has been extensively rewritten to account for new knowledge, especially on hormone action and receptors, but I have rigorously curbed my desire to enlarge on 'The Cell in Action' (Chapter 12) and 'The Unity of Biochemistry , (Chapter 13). Perhaps because biochemistry has expanded and diversified so much in the period since the early 1960s, I should spell out yet again that the emphasis in the book is throughout on animal biochemistry, not cell or molecular biology. Lest this bring down upon my head the wrath of microbiologists and plant biochemists, let me apologize in advance for this deliberate bias; it reflects my own special enthusiasms, and implies no denigration of their favoured organisms.

August 1978

STEVEN ROSE

Acknowledgements The following acknowledgements are made for the use of photographs: to Dr M. G. Stewart, Ms P. Mullins and Mr D. Spears for Plates 1,3,6 and 7; to Dr J. A. Armstrong for Plate 2; to Dr H. Beaufay for Plate 4; to Dr H. Fernandez·Moran for Plate 8a; to Dr H. E. Huxley for Plate 5; and to Dr E. Kellenberger for Plate 8b. Dr Stewart, Ms Mullins and Mr Spears are members of the Brain Research Group of the Open U ni. versity.

INTRODUCTION

WHAT IS BIOCHEMISTRY? 'lJiochemistry is the study of the chemical constituents of living ",atter and of their functions and transformations during life processes' - the definition of biochemistry in almost any standard

'"tbook. .. One does not normally regard a kitchen as having much in common with a laboratory. Most sciences are associated with )leavy and elaborate machinery, the creation of high temperatures, \'1St pressures, or the concentration of great amounts of energy. The prototype of biochemical experiments, however, is the frying of an egg. Material from a biological source (the chicken) is taken ancl removed from extraneous surrounding substances (the shell), care being taken all the while not to disrupt its natural organized structure (by breaking the yolk). This partially purified biological material is then subjected, under carefully controlled and regulated eonditions. to a number of mild chemical and physical treatments (the egg is gently heated in fat, and pepper and salt are added). , Whether the ultimate product is fit for anything more than the dustbin depends entirely on the skill with which these separate operations are performed; the margin between a good breakfast and a charred and tasteless mess is very small. . This analogy is not as unlikely as might at first be imagined; it . is very probable that most good biochemists would also make good cooks, for, like cooks. they deal with fragile substances derived from living or recently dead animals and plants, which they must handle rapidly, gently and subtly in order to obtain m~n mgful results. As in cooking, the type of operation performed is .a1J..important; DO one would mistake a fried for a boiled or a tcrambled egg; the differences in procedure by which one is arrived at rather than another are small (heating in or out of the shell, with or without prior mixing), but they are critical to the end product. So with biochemistry. Small variations in experimental procedure, in the concentration of a reactant, in the acidity or alkalinity of the medium in which the. reaction is being 11

INTRODUCTION

WHAT IS BIOCHEMISTRY? 'Biochemistry is the study oj the chemical constituents oj living matter and oj their functions and transformations during life processes' - the definition oj biochemistry in almost any standard textbook. One does not normally regard a kitchen as having much in common with a laboratory. Most sciences are associated with heavy and elaborate machinery, the creation of high temperatures. vast pressures, or the concentration of great amounts of energy. The prototype of biochemical experiments, however, is the frying of an egg. Material from a biological source (the chicken) is taken and removed from extraneous surrounding substances (the shell). care being taken all the while not to disrupt its natural organized structure (by breaking the yolk). This partially purified biological material is then subjected, under carefully controlled and regulated conditions, to a number of mild chemical and physical treatments (the egg is gently heated in fat, and pepper and salt are added). Whether the ultimate product is fit for anything more than the dustbin depends entirely on the skill with which these separate operations are performed; the margin between a good breakfast and a charred and tasteless mess is very smalL This analogy is not as unlikely as might at first be imagined; it is very probable that most good biochemists would also make good cooks, for, like cooks, they deal with fragile substances derived from living or recently dead animals and plants, which they must handle rapidly, gently and subtly in order to obtain meaningful results. As in cooking, the type of operation performed is all-important; no one would mistake a fried for a boiled or a scrambled egg; the differences in procedure by which one is arrived at rather than another are small (heating in or out of the shell, with or without prior mixing), but they are critical to the end product. So with biochemistry. Small variations in experimental procedure, in the concentration of a reactant, in the acidity or alkalinity of the medium in which the reaction is being 11

Introduction: What is Biochemistry? carried out, or in its temperature, cause critical changes to occur in the behaviour of the highly complex chemical entities being studied. Whether these behavioural alterations make sense or nonsense will depend on both the experimental and the theoretical ingenuity of the biochemist. It is for this reason that biochemistry is so recent a science. The wealth of sophisticated techniques and concepts needed to make possible an approach to the understanding of the chemical order and functioning of the living cell is such that they could not have arisen except on the secure foundations of more elementary sciences. In essence, the medieval doctors speculating on the composition of blood, or devotedly distilling retorts filled with urine, were performing biochemical operations. So was the Italian abbot, Lazzaro Spallanzani, who, in 1783, fed hawks with pieces of meat enclosed in wire boxes, trained the bi rds to vomit up the boxes at various subsequent times, and observed that the action of the gastric juices on the meat was progressively to liquefy it. He deduced that the liquefaction was the result of chemical interaction between certain substances in the gastric juices and the meat; such active principles were later, under the name of enzymes, recognized as the cornerstone of modern biochemistry. Equally a biochemist was Friedrich Wohler, who, in 1828, synthesized the biological material urea from the non-biological cyanic acid and ammonia, thereby settling a debate as old as alchemy itself, by providing the first unassailable evidence that the substances present in living organisms are chemical entities which differ from those in the chemist's reagent bottles only in their complexity, and not by the introduction of any mysterious hypothesis of the 'nature of life '. These were pioneer steps indeed, and great strides forward were made in the hundred years between Wohler's synthesis of urea and the isolation of the first crystalline enzyme - also, by some strange chance, one related to urea, 'urease' - by Sumner in America in 1926. But the really explosive growth of biochemistry has had to wait on the consoIidationof chemical theory, and the pushing forward of the frontiers of biology to a region where the distinction between it and 'chemical physiology' became obscure. By the

Introduction: What

Biochemistry?

1930s, the time was at last ripe for the biochemists to take over. The first signs of the new biochemistry emanated from the German laboratories of Meyerhof and War burg, which in the 19205 and . early 1930s housed some of the most brilliant biochemists in the world. With the coming of the Nazis, many of these youngsters fled to England and America. They found refuge in the everhospitable laboratories of Frederick Gowland Hopkins at Cambridge, and in such Americaillaboratories as that of the Rockefeller Institute in New York. From this period, and until the late 19608, dates'the dominance of British and American biochemistry. From then on, the science of biochemistry has expanded at an immense rate, increasing more rapidly even than that of nuclear physics, itself no sluggard. It is still in the full flood of its growth; dramatic breakthroughs in hitherto little-understood areas are becoming so frequent as to be accepted by the practising biochemist almost with weary resignation, whilst, to cope with the tidal wave of learned papers and reports now appearing, publishers are being forced to produce weekly issues of journals which only a short while ago were monthlies or even quarterlies. In order to ensure rapid distribution of their findings, some researchers have now abandoned the journals and instead circulate duplicated copies of their results to a selected mailing list. Not content with this, biochemistry spills over into related disciplines, and biochemists have staged take-over bids for the journals of physiology and chemistry, to say nothing of such general magazines as Nature in Britain and Science in America. Concurrently with its expansion, biochemistry has itself begun to fragment, so that its practitioners refer to themselves as enzymologists, molecular biologists, chemical microbiologists, neurochemists, even mitochondriologists. To remain a plain biochemist is almost passe. Yet, despite the expansion and fragmentation of their subject, there has until now remained a common ethos amongst biochemists, a common method of approach to their problems that distinguishes them from their colleagues in other, adjacent disciplines. A biochemist (at least, the sort this book is about) is not just a physiologist applying chemical tools to living things, nor an organic or physical chemist who is interested in the properties of the chemical substances of the cell. Both such types of 13

Introduction: What is Biochemistry? scientist exist, and the biochemist will most likely get along very well with them; but at the same time they are recognizably different, asking different questions and demanding different standards frbm the answers they get. Biochemistry has arisen at the meeting point of many sciences, and has been fertilized and enriched by all, yet it is itself essentially unique. Faced with a living cell, or the tissue or organ which is composed of several million of such cells in close proximity to one another, the types of question the biochemist asks can be summarized under four major heads: (1) What is the composition of the cell, in terms of individual chemical compounds which can be recognized as functionally different· from one another and which cal) be separated by the techniques of chemical and physical fractionation? (2) What are the relationships between these chemicals, and how are they made and converted one into another by the cell ? (3) How are these chemical interconversions controlled and regulated within the cell so as to enable it to maintain its organized structure and activities? (4) What distinguishes the cell studied from those of other tissues, organs, or species? That is, in what way is its design related to the function that the cell performs within the living organism considered as a whole? These questions, of course, all interconnect. Although they are arran~ed on the page in order of increasing complexity, it is not always necessary, or easier, to answer (1) before (2) or (3) before (4). An attempt to solve (2) may provide clues to (3) instead. But, nonetheless, they do represent separate passages in the biochemist's mind, and generally a different approach is required for each. In fact, a case may be made that they are also those questions which have been asked, broadly speaking, at different stages in the development of biochemistry as a science. Thus, in the first phase of the history of biochemistry, the most important and pressing problem was the establishment of the nature and composition of the chemicals of the body. To this phase belong the work of Wohler and Sumner already referred to, and the massive development of the French and German schools of organic chemistry in the hands of such nineteenth-century 14

Introduction: What is Biochem istry? giants as Berthelot, Liebig, and Fischer, which resulted in the identification and subsequent synthesis of most of the simpler chemicals utilized by living organisms. The attack on the biological macromolecules (those with molecular weights of 10,000 upwards to a million or more), such as proteins, fats, carbohydrates, and nucleic acids, required a different order of techniques to those available a hundred years ago, despite the classical and painstaking analyses of such workers as Thudicum, who, in 1884, listed in his Treatise on the chemical constitution a/the brain some 140 individual constituents, many of them complex combinations of fats, proteins, and carbohydrates. isolated by methods of extraction involving prolonged subjection of the tissue to conditions so extreme in acidity, alkalinity or temperature as to curl the hair of a contemporary biochemist with horror. Indeed a long battle was fought, lasting through until the 1920s, against those who believed that there were such giant molecules, rather than merely loose associations, colloids, of smaller units. Today, the isolation and characterization of complex proteins has become a standardized operation. The first detailed molecular structure of a protein was worked out for the hormone insulin by Fred@rick Sanger in Cambridge in 1956, after nearly a decade of minute and laborious analysis which well deserved the Nobel Prize with which it was received; these procedures too have now become a routine piece of1abora tory technique. Important though many remaining problems may be, the great days of the age of biochemical analysis are now truly past. In this book, we discuss its findings in Chapters 1 to 3. Meanwhile, as the scale on which the biochemists could work moved up, that on which physiologists and microscopists were working moved down. The advent of the electron microscope for routine laboratory use in the 1950s united the two. It made possible magnifications of 100,000 times and more, and enabled the inside of the cell to be studied visually in great detail for the first time. so that biochemists could see in detail, as well as imagine, the inside of the cell whose properties they were investigating. As will be shown in Chapter 4, it became possible to deduce just where within the cell particular substances were located, and where particular reactions occurred. It became apparent that the

Introduction: What is Biochemistry? cell was not merely a bag of randomly distributed chemicals, but that each substance had its own place and position. To many, the cell now seems to resemble more closely the regularly patterned form of a complex, hierarchically ordered set of macromolecules than the primitive sack of 'protoplasm' that had been the nineteenth-century picture. The second phase of biochemistry was one of kinetics, of drawing a route-map setting out the major pathways by which chemical transformations occur within the living cell, and of understanding at a molecular level the mechanism of each individual chemical reaction. A primitive experiment of this sort was Spallanzani's, already described. The recognition in the early nineteenth century of the phenomenon of catalysis, in which chemical reactions are assisted and accelerated by substances (catalysts) which are themselves unaltered during the reaction, led to the assumption by the Swede Berze)ius, in 1836, that the vast range of chemical activities occurring in living tissues depended upon the existence of potent chemical catalysts within the cell. And so indeed it proved. The heated controversies which followed, and which found distinguished chemists lined up in bitter opposition over whether the chemical reactions characteristic of life could be performed in the test-tube in the absence of living organisms, were resolved by the brothers Buchner, who, in 1897, ground yeast with sand in amortar and extracted from the mixture a distinctly dead juice which was nonetheless able satisfactorily to ferment sugar to produce alcohol. The name enzyme was coined for the catalyst in the yeast-juice that performed this desirable function, and the properties of enzyme soon showed that it was protein in nature. As more and more catalysts were discovered and extracted from the cell, the name enzyme became accepted as a general one for the entire class of biochemical catalysts. It is now recognized that practically every chemical reaction that occurs within the body requires its specific enzyme to cata[yse it. Each enzyme catalyses only a single reaction, and the complete synthesis or degradation of a complex substance - for example, the breakdown of the starch in food to the sugar molecules of which it is composed during digestion in the gut, followed by the absorption of the sugar into the cells and its 16

Introduction: What is Biochemistry? synthesis there to glycogen ('animal starch ') or breakdown to carbon dioxide and water - requires a whole series of enzymes acting in sequence, one after the other. A reaction chain of this sort is called a metabolic pathway. The mapping of these pathways, for the synthesis and breakdown of sugars, fats, and amino acids (a few of which are outlined in Chapters 8 and 9), was the work of the generation of biochemists of the 1930s, and the names of Krebs, Embden, Meyerhof, Warburg, and Dickens stand high in this respect. One problem remained in the understanding of these metabolic chemical interconversions: that of the energy-balance of the reactions. Destructive reactions (sometimes called 'catabolic '), such as those of the breakdown of the sugar, glucose, to carbon dioxide and water, release considerable quantities of energy; synthetic (or 'anabolic') reactions, such as the manufacture of proteins or fats, are energy~requiring. It is necessary for the cell to strike a balance between energy-producing and energy-demanding reactions, for it cannot afford to run for long at either a profit or a loss. It was F. Lipmann, of New York, who showed, in 1941, that the cell runs a sort of energy-bank, which can trap and store the energy released by catabolism, and provide it again on demand for anabolism, and that this bank consists of the chemical adenosine triphosphate (ATP for short). The significance and properties of ATP, as they are at present envisaged, will be discussed in detail in Chapters 5 and 7. The 1950s saw a change in emphasis from the analysis of biochemistry-as-kinetics to that of biochemistry-as-information. The theoretical rationale for this transition was provided by the growth of the new'sciences associated with the development of computers. Theories of 'control', 'feedback', and 'information transfer' were collated in 1948 by the American engineer and mathematician Norbert Wiener under the name of 'cybernetics'. As more and more became known about the mechanisms of individual enzymic reactions, about their energy-requirements, and about the workings of series of enzymes in the harmony of metabolic pathways, biochemists seized on these new concepts in order to probe the ways in which the cell controlled and regulated its own metabolism; how. so to speak, it decided at anyone time

Introduction: What is Biochemistry? how much glucose to break down to carbon dioxide and water, or how much new protein to synthesize. And the triumph of biochemistry-as-information-fiow was of course the spectacular solution to the problem of the mechanics of the accurate replication of giant molecules such as DNA and of the translation of the genetic messages coded for in the DNA into the structure of the proteins themselves, undoubtedly one of the key scientific developments of this century (Chapter 10). This picture of the cell as a self-regulating mechanism, continually changing, yet continually unchanged, is one of the most important and significant results of the new biochemistry of the 1950s and 1960s (Chapters 11 and 12). In the mid nhieteenth century, the great French physiologist, Claude Bernard, had described the fundamental property oflife as that ofthe ability to 'maintain the constancy of the internal environment'. Living organisms responded to exterior events impinging upon them in such a way as to absorb the effects of these events into their systems as rapidly, and with as little disturbance, as possible. They needed constantly to renew themselves, to recreate from within those portions of themselves destroyed in the rough-and-tumble of existence. The pattern of their bodies was fixed, although its individual components were forever changing. For this process, the name 'homeostasis' - 'staying the same' - was invented. It has become the task of today's biochemistry to transfer this concept from the body as a whole to the working of each individual cell within it. Only when this has been done does it become possible to define in chemical and physical terms how the organism as a whole, as the sum of its constituent cells, and all their myriad interactions. with each other and with the external world, can function. Armed with such knowledge, we can go on to ask the most fundamental of all questions: 'What is life, and how did it arise 'l' And to answer this is the prime aim of biochemistry.

CHAPTER 1

BEFORE WE START In this book we shall be describing the biochemical make-up and behaviour of the Jiving organism in some detail. In order to do this, we need constantly to use certain chemical words, phrases, and ideas. To those who have studied chemistry, what follows here is familiar territory; they would do better to skip the rest of this chapter. But, for those who are unacquainted with its jargon, there are included here a few brief paragraphs of defini tion in the hope that, having been disposed of, they need not trouble us unduly later on. Chemists work with substances, which they attempt to purify one from another by making use of differences in their physical properties. For example, a mixture of salt an4 sand is separated because salt dissolves in water and sand does not; later, the salt can be recovered by boiling off the water. A substance which cannot be split by such physical methods into separate components is a compound. All chemical compounds, and there are many hundreds of millions of them, are formed by combination, in varying proportions of two or more, of a small number (about a hundred) of chemical elements; the elements can neither be converted into each other nor split into simpler substances by chemical means. The elements are represented by symbols, C for carbon, 0 for oxygen, Na for sodium, etc.; the compounds are indicated by a combination of these symbols. For example, common salt - sodium chloride - is NaCI. Atoms and molecules

The smallest particle of an element is an atom, although the atom itself has an internal structure made up of smaller particles, protons, neutrons, ~nd electrons. Neutrons and protons are packed together in the atomic nucleus. Neutrons are so named because they have no electrical charge, protons are positively charged, whilst electrons (sometimes written e-) have an equal though opposite charge to the protons, making the atom neutral. The 19

The Chemistry of Life electrons are distributed between very precisely defined orbits or energy levels at varying distances away from the positive nucleus (they can be thought of like planetary orbits around the sun). Each of the energy levels are known as valency shells, and there is a maximum number of electrons that can exist in each shell. Atoms can combine with each other in fixed proportions to form molecules and when this happens they do so in a way that attains this maximum number in their outermost valency shell, as this gives the atom greatest stability. The number of electrons a parti· cular atom has to lose or gain to achieve this stability is described as the valency of the atom. There are two ways this gaining or losing of electrons can be done: either by transferring electrons between atoms, or by sharing them. For example, the sodium atom, Na, achieves stability by losing an electron, thus becoming positively charged. The CI atom does it by gaining an electron to become negatively charged. Occasionally, two or more atoms of a single element may join together covalently to form a molecule of that element; thus the gases hydrogen (H) and oxygen (0) normally exist as molecules containing each two atoms, Hz, Oz. The terms atomic weight and mclecular weight are used to express the relative weights of atoms and molecules compared to the weight of hydrogen, which is the lightest element and whose weight is arbitrarily defined as 1. Thus the atomic weight of oxygen is 16, meaning that the oxygen atom is 16 times as heavy as the hydrogen atom. The molecular weight of water (H 20) is (2.x 1 + 16) or 18. In general, the more complex a molecule, the larger its molecular weight. Salt (NaC!) has a molecular weight of 58, the sugar glucose (C 6 H 12 0 6 ) ofI80, and some proteins ofarniIlion or more.

Ions. electro valent bonds. and buffers Charged atoms or molecules are called ions. If N a and CI are close together in solution, transfer of electrons between them easily occurs, and because of their opposing charges, they are mutually attracted, combining to form salt, NaG. The bond between them is described as electrovalent. In solution in water, electrovalent compounds such as NaCI tend to separate into their component ions, and to join together again only in the solid material, thus: NaO +t Na+ + a20

Before We Start The opposite-headed arrows of this equation indicate that the reaction may, depending on circ*mstances, proceed in either direction. It is said to be reversible. When it is going from left to right, NaCI is said to dissociate; in the opposite direction, Na and Cl are referred to as aSSOCiating to form NaCl. Other reactions - for example the oxidation (addition of oxygen) of coal gas (carbon monoxide) to form carbon dioxide, which occurs when we light a gas fire - are irreversible under normal circ*mstances; 2CO

2CO z

Compounds formed by the combination of two ions, such as NaCl, are called salts. Chemically, they are usually produced during the reaction of two other compounds, an acid and an alkali. Acids are substances containing the hydrogen ion, H+ (for example hydrochloric acid, HCl). Alkalies, on the other hand, contain a negative ion, the hydroxyl ion OH- , itselfa combination between hydrogen and oxygen. An example is sodium hydroxide (caustic soda), NaOH. Acids and alkalies react in solution to give a salt plus water, thus: NaOH

HCI

NaCl

H2 0

A further word should be said here about the concept of acidity and alkalinity, because although a full treatment of it properly belongs to text-books of physical chemistry, biochemists find they cannot get very far without taking it into consideration. An acid solution, as we know, contains hydrogen ions, an alkaline one, hydroxyl ions. Both hydrogen and hydroxyl ions are generated by the ionization of water: H~O

H+ +OH-

This is a reversible reaction, and in reversible reactions the relative concentrations of the reactants always tend towards a constant equilibrium point. Therefore the amounts of hydrogen and of hydroxyl ions present in any aqueous solution must always bear a constant relation to one another. If we know the number of hydrogen ions present, the number of hydroxyl ions follows automatically. Thus an alternative way of regarding the addition of hydroxyl ions is to look at it as a subtraction of hydrogen ions from C.O.L.-'2

The Chemistry of Life the solution. We can then define both acidity and alkalinity in terms of the concentration of hydrogen ions present in the solution; the more hydrogen ions. the more strongly acid the solution is. The hydrogen ion concentration of a solution is measured on a scale called the pH scale. The scale (which is logarithmic) runs from 0 to 14,0 being the acid, and 14 the alkaline, end of the scale. Midway between the two, at a pH of 7·0, represents neutrali ty. When hydrogen ions are added to a solution, the pH decreases; when they are removed (or hydroxyl ions added), the pH rises. But when certain substances are present in the solution, they will tend to combine with any H+ or OH- ions added, and, by removing them. they act so as to prevent the change in pH that would otherwise occur. Substances that act in this way are caned buffers. In their ability to mop up hydrogen or hydroxyl ions, buffers act as regulators of pH. Such regulation is extremely important to the delicately balanced living cell, where sharp fluctuation in acidity and alkalinity can easily spell disaster.

Covalent compounds But by far the largest number of substances with which we shall have to deal are not salts at all but compounds of the element carbon. Carbon compounds are so universally distributed amongst living organisms, and are so numerous, that their study has been split from those of other chemicals under the special name of organic chemistry. The carbon atom attains stability most easily by sharing its electrons with four atoms of a monovalent element or two atoms of a divalent atom. The simplest organic compound is thus methane (marsh gas) which has the formula CH4 • It is often useful when discussing carbon compounds, though, to abandon the simpler notation of inorganic chemistry imd to try to draw a picture of the molecule as it actually exists in space. Thus H

H-C-H I H 22

Be/ore We Start is a two-dimensional drawing of the three-dimensional methane molecule, demonstrating that the carbon atom in methane is in fact entirely surrounded by hydrogen atoms. Really, though, even this is only an approximation to the actual three-dimensional structure ofthe molecule, which, if one had a microscope powerful enough to see it, would appear to be not flat all, but pyramidshaped:

All the formulae we shall draw are, like that of methane, merely two-dimensional pictures of the real three-dimensional shape of the molecule. In deference to the fact that the rule says that carbon must always be linked to four other atoms, four linking lines (bonds) are always drawn stretching out from any carbon atom. In cases such as that of carbon dioxide, CO 2 , where the carbon atom is linked only to two atoms of oxygen, we draw the molecule as

o=c=o showing that it is linked to oxygen not by single but by double bonds.

This type of bonding, by sharing electrons, is called covalent. Once made, covalent bonds are hard to break, and the substances which contain them therefore tend to be rather durable. In certain circ*mstances one partner atom of a covalent molecule may tend to obtain a greater or lesser share of the electrons than otJ::ier partners. There is then a distribution of electric charge within the molecule which gives it a certain polarity, an important 23

The Chemistry of Life property, as we shall see in relation to macromolecules (electrovalent molecules are of course completely polarized). Drawing pictures of the molecules in space also reveals certain other characteristics about them. For instance, when we examine the substances corresponding tp the formula G3 H 6 0, we find that two possible structures exist, represented by

These two are quite different in properties: the first is propionaldehyde. formed by certain bacteria and a reactive, acrid-smelling substance; the second is the sweet-smelling acetone, familiar as the volatile base of nail varnish and aeroplane dope. The structure and arrangement in space of the molecules of organic compounds is thus critical to their behaviour. Substances (like proprionaldehyde and acetone) whose overall chemical composition is the same but whose structure and shape are different are called isomers. Isomerism is a common occurrence amongst the chemicals of the Jiving body, and we shall meet it frequently. Yet another property of carbon atoms is their ability not only to link together in long, straight chains such as those we have been drawing, but also to form branched chains:

Y H-r-H

H-C--C:--C-O-H

H H H CH a.CH(CH 3 )CH zOH Iso-butanol

Before We Start or even rings: H

H, Both DNA and RNA occur in the tissue in combination with yarying amounts of protein, and such protein, especially that found attached to DNA in the cell nucleus, has quite character-

.':

The Chemistry of Life istic properties; notably, it is very alkaline, containing large amounts of the amino acid arginine. Such proteins are known as histones, whilst the nucleic acid with protein attached is described as a nucleoprotein. In extracting the nucleic acid from the tissue, one of the problems is to remove the tightly bound protein without using methods so drastic as to harm the nucleic acids themselves. This problem is made easier by the fact that the nucleic acids are considerably more robust than proteins; they are stable to mild acids and alkalies, and to heating to almost one hundred degrees, procedures which rapidly wreck the delicate protein molecules. Thus RNA may be extracted from yeast by dissolving in a detergent solution at ninety degrees, whilst DNA can be prepared from thymus by extraction with alkali or salt solution followed by chloroform. The material that is obtained by these methods is a whitish, fibrous substance (DNA rather resembles asbestos in appearance), quite stable, which can be stored for longish periods without coming to much harm. Its molecules have a weight in the order of tens of millions and, like proteins or polysaccharides, have a primary structure consisting of a repeating series of simple units joined into a chain. Although, as we shall show, many different DNAs and RNAs exist, the problem of isolating one DNA from a mixture of others, so critical in protein biochemistry, scarcely exists. This is at least partIy because the physical properties of the different molecules do not differ sharply enough from one another to make it possible to exploit differences in solubility or stability as can be done with the proteins. Chromatography of DNA does reveal that the 'total' DNA obtained from any tissue can be split into a number of slightly differing fractions, but it has become customary to refer to the' total' DNA of any organ almost as if it were a single molecular species - so one talks of thymus DNA, liver DNA, and so forth. In fact the properties of calf 'thymus DNA' do differ slightly from those of calf' liver DNA'. and those of rat kidney RNA from guinea-pig kidney RNA. Protein consists only of amino acids, polysaccharides of sugars, but the nucleic acids contain purines, pyrimidines, sugars, and phosphoric acid. Thus the basic pattern of the repeating unit is less simple than that of the other macromolecules. It was early

Macromolecules apparent that the base, sugar, and phosphate were combined as nucleotides, where the purine or pyrimidine is linked through the sugar to the phosphate (see page 41). How were the nucleotides connected to each other, though? The early hypothesis was that four bases, sugar and phosphate groups were linked together in a so-called' tetranucleotide' repeating unit, but such a concept was found to conflict with the experimentally observed base ratios in DNA and RNA. Some relationship between the four bases must exist, though, because it was noticed by Erwin Chargaff in New York that for RNA the amount of (adenine plus cytosine) always equals (guanine plus uraci)), whilst for DNA an even more precise relationship holds, so that the amount of purines and pyrimidines present in any sample is always identical and the ratio of adenine to thymine and of guanine to cytosine present is exactly one. Also, DNAs tend to fall into one of two main groups - either one in which there is more adenine and thymine than guanine and cytosine, or the rarer one in which guanine and cytosine are the two main components. So it is clear that the nucleic acids are highly organized and well-ordered compounds, even if the pattern of arrangement is not immediately obvious. The layout of bases, sugars, and phosphates within the molecules was discovered during the 1930s and 19405. The proof was a difficult one, involving some complex feats of organic chemistry, but the conclusions are now well established. For RNA, each nucleotide can be drawn like this:

the

base

o I ®

where carbon atom l' of the sugar is attached to the base, and carbon atom 3' to the phosphate. When the nucleotides join to form RNA, the link is between the phosphate of one nucleotide and carbon atom 5' of the second sugar:

The Chemistry of Life base

CH10H

® I o I

CH 2

I ®

Thus the primary chain link runs through the sugar phosphates, and can be shown schematically: sugar-base

phosphate

sugar-base

phosphate

sugar-base

phosphate The variable in RNA molecules is then the order in which the four bases are arranged along the chain. Although there are only four bases, the number of available permutations down a chain of several hupdred is obviously very large, though smaller than for proteins. What knowledge there is of the actual arrangement found in RNAs that have been extracted from the tissues depends on the type of analyses we are now quite familiar with. Hydrolysis, which splits the RNA into its residual nuc1eotides, gives a modicum of information; partial hydrolysis, in this case using a variety of enzymes, including ribonuclease and also some potent nucleic

Macromolecules acid splitting enzymes from snake venom, is of more value. Ribonuclease from pancreas has an interesting specificity, for it seems preferentially to attack bonds between pyrimidine nueleotides and other bases. For example, the enzyme will split the nucleic acid chain pupupylpylpylpupylpupupy!pyl (writing pu for purines and py for pyrimidines), along the lines . drawn, to give 2 pupupy + pupy + 3py. But despite the many ingenious methods that have been devised, and the enormous interest that there now is in the whole problem of RNA and DNA in relation to their role in genetics and in protein synthesis, only a few molecules have yet had their sequence reliably and completely determined, although in the next few years progress is likely to prove a good bit more rapid. The situation is better for a group of relatively low-molecular-weight RNA molecules which occur in solution in the cell, the so-called transfer RNAs (see Chapter 10) with about 80 nucleotides each, whose structure was worked out at all levels by American researchers. The structure of the repeating unit of DNA is similar to that of RNA, with the simple replacement of ribose by deoxyribose. Thus, like RNA, the DNA chain is linked through a 3'-5' sugar phosphate bond, with the purine and pyrimidine bases tacked on to carbon atom l' of each deoxyribose residue (see diagram on page 10 and Figure 5). But why, if this is all, the peculiar regular ratios that seem to exist between the amounts of the different bases? And what are the aspects of the secondary and tertiary structure of DNA, whose fibrous character and behaviour in solution are those of a rigid, linear molecule, like a stiff rod? X-ray studies made by Rosalind Franklin and by Maurice Wilkinsdn London in the early 19505 indicated that there was a regular repeating sequence within the molecule every twenty-eight Angstrom units. Now the distance between two nucleotide residues, from phosphate to phosphate, is only seven A, so in some way there must be a strong repeating tertiary structure to the DNA. James Watson and Francis Crick, in Cambridge, solved the problem in 1953 (Wilkins, Watson and Crick were given the 1962 Nobel Prize for doing so, and Watson has written an entertaining,

The Chemistry of Life if disingenuous account of the affair in his book The Double Helix). They found the answer in a helix - at almost the same time as Pauling and Corey found that the a-helix also gave the answer to the keratin problem for proteins. The DNA helix as proposed by Watson and Crick turns once eyery ten nucleotides (proteins, it may be remembered, manage to make the turn in 3·7 amino acids), and it contains a double strand of two DNA chains twisted together about the same axis. All the way along, the two chains link together through their bases. Now when Watson and Crick came to make scale models of the structure they proposed, they found that the two chains could only fit together if every adenine on one chain was opposite a thymine on the other, and every guanine opposite a cytosine. Then, and only then, the two strands would latch together, and hydrogen bonds between each pair of bases would hold them firmly in place. By contrast, if one considers adenine and cytosine, which do not form base pairs, the three atoms necessary for H bond formation cannot be set into a straight line, and hence the bond cannot form. The secondary structure of DNA is very robust because of the additive effect of many H bonds between the base pairs, all orientated in the same way, i.e. stacked on top of each other. Figure 5a shows base pairing between adenine and thymine, and how it works for one portion of the double chain is shown diagrammatically in Figure 5b. The helical tertiary structure is made possible by the orientation in space of the flat aromatic rings of the bases. If the double chain twists into a helix a large number of weak bonds can be formed between each ring and others lying directly above or below it. Again the sheer number of these bonds greatly enhances stability of the molecule, but they are nevertheless individual, weak and easily broken - we shall see in later chapters how very important this is to nucleic acid function. In three dimensions, Watson and Crick drew their model as in Figure 5(c). The whole concept brilliantly accounted for the X-ray analysis and also for the strange constancy of the ratios of the amounts of the different bases that had been so puzzling. In the structure of DNA shown here, the amount of adenine inevitably equals that of thymine, and guanine that of cytosine, just as the chemical analysis had shown. Despite the restrictions placed on the base-order in such a 72

Macromolecules FIGURE

(a) Mortice-and-tenon arrangement of base-pairs. CH]

I N-H

HyN

... O=C

/C~

~ )N ........... H-~,c/~, II

/N -----\~N#

sugar

thymine

adenine

(b) Bonding of two chains in DNA helix.

sugar-A ..• T-sugar

phosphate

sugar-T ..• A-sugar

phosphate

sugar-G .,. C-sugar

phosphate

sugar-C ... G-sugar j I (c) Watson-Crick DNA helix.

model, the number of permissible isomers is, in fact, the same as that of RNA - that is, astronomically large. Finding the order of the bases presents similar problems to those faced with RNA, and, Hke that molecule, they have not yet been solved. Perhaps the most hopeful way forward will lie in the fact that, as we shall see

The Chemistry of Life when we come to discuss the mechanism of protein synthesis, given the structure of a protein, it is possible to predict the structure of the DNA and RNA which helped make it. But such a discussion properly belongs to a different phase of biochemistry. LIPIDS

We have left the lipids until last mainly because they do not conform to our concept of a macromolecule quite as well as the other three classes that have been discussed. They are a diverse group, with few points in common other than the property of being insoluble in water but soluble in organic liquids ,- a very operational definition! They are much smaller than the other macromolecules, having molecular weights in the hundreds instead of thousands or millions, and their inherent structural hierarchy is a little more difficult to define. This is because they are rarely found as individual molecules, because they have a tendency towards self.aggregation, and it is the aggregate that we must consider as the'macromolecule, the building blocks of the primary structure being the individual lipid molecules themselves, However, the building blocks in lipids are not held together by strong covalent bonds but by weak bonding due to interaction between their dominant non-polar regions. The absence of a strong backbone and the presence of only weak bonding in the structure of lipid aggregates means tbat the macromolecular property of flexibility is much more strongly emphasized, so that it can almost be described as fluidity, Lipids, as a group, include, as well as fats, esters, formed by the combination of an alcohol with an organic acid. The ester linkage is produced according to the equation R,COOH

R'.OH ?

R.COOR'

HzO

where Rand R' may represent any organic radical.

Fats and Oils The prototype fat is a combination of a straight chain fatty acid, sixteen or eighteen carbon atoms long, with glycerol, a sweet, sticky liquid containing three alcoholic groups which can combine

Macromolecules with one, two, or three fatty acid molecules, yielding a mono-, di-, or tri-glyceride. An oil differs from a fat only in being liquid at normal temperatures - that is, an oil is a fat with a low melting point. The typical fat therefore has the formula: CHzOOCR

CHOOCR'

CHz·OOCR'

R. R and R" need not necessarily be the same, and this general formula can cover a variety of possible permutations. But in practice eighty per cent or more of fats contain one or more of the three adds: I

palmitic [CH 3 (CH z)14COOH], stearic [CH 3 (CH 2 )u;COOH]. and oleic [CHiCHzhCH=CH(CHz),COOH]. These three are simple straight chain acids; oleic differs from the other two in having a double bond (see Table 1, page 26). The fats are named according to the acids present, so tbat tristearin has three stearic acids, whilst if one oleic and two palmi tics are found the fat is oleodipalmitin. In a naturally occurring fat a mixture of many of the possible combinations will be found, although the exact proportions of each will depend on the source of the fat, and are not purely random; some combinations are preferred to others. In plants, double-bonded acids such as oleic tend to predominate - olive oil is almost pure triolein (plants also have a higher proportion of the more unusual acids than do animals). In animals, on the other hand, the single-bonded acids are more frequent. As the melting point of the fat is directly related to the number of double bonds present in its constituent acids (the more double bonds, the lower the melting point) plant fats tend to be liquid at normal temperatures and are therefore called oils. If the double bond is saturated by hydrogenating the oil. a solidification ('hardening') occurs, an operation which is utilized in producing margarine by reducing vegetable oils with hydrogen. Conversely.

The Chemistry of Life a fat left exposed to the air undergoes oxidation along the fatty acid chain, resulting in double bonds and hence liquefaction, which is familiar as the first stage of the complex process whereby butter goes rancid. Butter is a mixture of animal fats, and the 'rancidification' is continued by·a slight hydrolysis of some of the esters present, releasing the free acids, amongst them the foul smelling butyric acid. Another familiar smell, that of burning fat, is provided by the heat-decomposition product of glycerol, acrolein. The main function of the fats is, like that of glycogen and starch amongst the polysaccharides, to serve as a food store. Surplus food taken in by the animal is laid down as fat (adipose) tissue to be used again when harder times come. Such subcutaneous fat has other functions, too - it serves as an insulator against heat loss by the body, and also as a cushion for such delicate internal organs as the kidneys, which are generously embedded in fat. Waxes, which are lipid esters which contain rather longerchain acids and alcohols than the fats, often have a role also as external protection - e.g. against water loss from the surfaces of insects, fruits, leaves, or petals. w

Phospholtpids So far we have discussed neutral fats, but often a monoglyceride, instead of combining with other fatty acids, will react with the strongly electronegative phosphate group to give either monoacyl or diacyl phospholipids (depending on the number of fatty acids present). It is the reactivity of the phosphate group that enables phospholipid aggregates to form complex structures with other molecules, most notably with proteins in membranes. How the lipid aggregates form higher order structures depends on several variables: the degree of fatty acid substitution in a phospholipid, the presence or absence of strongly polar terminal groups, the degree of saturability in the fatty acids and the relative proportions of lipid and water in the mixture are all important considerations. Diacyl phospholipid molecules are extremely well fitted for their role as membrane constituents mainly because they exhibit 76

Macromolecules directionality in their relationship to H 2 0 - they tend to aggregate into bilayers, their hydrophobic tails, composed of the long fatty acid chains, are held together by weak bonds, whilst the strongly polar phosphate heads face outwards, enabling them to interact with the water that surrounds the membranes. We shall consider further aspects of membrane structure in the next chapter. Neutral fats behave differently at similar concentrations mainly because they lack the strongly polar head so that their behaviour is determined by their non-polar residue and in solution they tend to form small round fat droplets - it is this particular feature that makes them so suited as a food store. Monoacyl phospholipids like lysolecithin tend to behave more like the neutral fats - they prefer to form micelles even at quite low concentration. This means that the insertion of just a few lysolecithin molecules into a lipid bilayer will greatly disrupt its organized structure.

Steroids Also included in the lipid group by reason of their solubility in Qrganic solvents is a group of substances which seems at first sight quite different from the fats and phospholipids. These are the 6(eroids. whose many members are produced by a series of minor modifications on a basic pattern of seventeen carbon atoms linked together as four interlocking carbon rings: 17

• ,

These rings, however. are simply the result of secondary folding of the long non-polar CH l chain, so the difference in structure is less great, though still important, than might at first appear. A typical steroid is cholesterol, containing twenty-seven carbon atoms and an alcoholic group at carbon atom 3 of the steroid nucleus:

The Chemistry of Life uCH

.....CH, M

V,CHJD

/1 16CH.CH1 .CH1 .CHz.CH CH u U u

HO Other steroids derived from cholesterol include certain v;tamiJ (e.g. Vitamin D), several drugs and poisons, the bile acids of dige tion, and many hormones, in particular the sex hormones (sc page 233), Derivatives of cholesterol itself circulate in the blool stream and form part of the walls of arteries and veins, and di orders in the body's use of cholesterol are widely believed to I associated with arterial diseases, such as coronary thrombosis. This completes our survey of the large molecules of which Jivir organisms are made; it is time to turn from them to a discussic of the structures which, in life, they compose and with which th' interact - the internal organization of the unit of life, the cell.

CHAPTER 4

THE ORGANIZATION OF THE CELL So far, we have treated living tissue as if it were merely to be regarded as a convenient source from which to extract various classes of chemical, using greater or lesser degrees of violence, but in any event, without regard for the structures f(om which those chemicals have been derived. There are three ways of considering the cell; the first is the chemists'. For them, it is merely the logical extension of those chemicals we have so far discussed; a hierarchy runs from atom to molecule, from molecule to macromolecule with its primary, secondary, and tertiary structures. The cell is merely a higher order of these, its components may be regarded as quaternary structures, delicately related ensembles of proteins, . lipids, polysaccharides, and nucleic acids, held in particular con.figurations by an appropriate ionic environment of water, sodium, . potassium, and chloride ions. Implicit in this understanding (and we will return to this) is the chemist's belief that the higher order .lItructures and their interactions are essentially predictable from ~~e lower order ones; that primary structure determines tertiary ..which in its turn determines cell structure. j' The second way of looking at the cell is the physiologists'; for ;,them, like other 'organismic' biologists, the cell is the smallest il1nit of which tissues and organs are composed - the lowest level ~Pf a new hierarchy; it is the interaction of cells which is of interest ~~ther than what goes on inside them. ( The third viewpoint is that of the biochemist or cell biologist, tIoncerned with both what the cell is made of and how it is main; ,tained; where the chemist's quaternary structures and the physioik>gist's smallest living unit meet. This is the terrain of the present ,chapter. In fact, in its earlier years, biochemistry got on by ignoring the ~II as we have done in the previous three chapters. The reasons ''were largely methodological. Un til the development of certain new :tools of research in the 1940s - the ultra-centrifuge and electron Jlucroscope - biochemists had no means of relating observations !"

(

The Chemistry of Life about the behaviour of chemicals within the cell to those of bio· logists, who for many years had been using microscopes to ma~ out the cell as a complex structure composed of many differen! subunits (or organefles, as they came to be generally called). BUI the time has now come when >,ye can legitimately stop pretendin~ that the cell is made of smooth paste like butter in a butter-dish, and can recognize that it is more like marmalade - full of small, solid lumps, often quite different in texture and composition from the surroundi ng jelly. The sciences long concerned with the microscopic study of the cell are histology and cytology; their relation to biochemistry is confirmed by the two new subjects histochemistry and cytochemistry. The techniques of histology and cytology, developed over the last two hundred years, involve cutting very thin sections of biological material, mounting them on a microscope slide, staining them with a variety of dyes that colour different parts of the specimen more or less intensely, examining them at various magnifications, and finally attempting to relate back the twodimensional picture seen down the microscope tube to the original three-dimensional living tissue. By such techniques the early microscopists were able to study plant and animal tissues and to show that they were divided into a set of box-like compartments separated one from the other by thin walls. The organ, or the body, they realized, was built of these boxes just as a wall is built of a set of individual bricks. They called the boxes, cells, and the thin walls, cell membranes (though plant cell membranes are themselves surrounded by a second line of fortification in the form of a more rigid cell wall of cellulose). The cell was not merely the smallest unit of living organisms like plants and animals; for many forms of life it was found to be the only unit. Moulds, such as yeast and bacteria, all manage to compress the whole range of their activities within the walls of one tiny cell often considerably smaller, even, than the less-versatile plant or animal cells. Another familiar example of a one-celled animal is the little pond-water creature studied at one time or another by all biology students with access to a microscope - the amoeba. Early microscopists suspected, but could not prove, the existence of 80

The Organization of the Cell even smaller organisms, the viruses. the detailed study of which depended on the advent of the electron microscope in the midtwentieth century. The virus completed the range of'living units', and is something of a special case, as we will see later. But average cells, from animals or plants, are all rather similar in both size and construction. They are likely to be about onethousandth of a centimetre in diameter, and to weigh up to about one ten-millionth of a gram. In a person weighing about 80 kilos (11 stone), there might be as many as 100,000,000,000 (or 1011) cells. Table 5 gives an idea of the range of sizes and weights with which one is dealing here. The cells from different species, say rats and humans, or algae and roses, differ from one another slightly in their properties, and so do the cells from the different organs of anyone species - thus liver cells are slightly different from kidney cells and both from brain or blood cells. , But the differences, as we shall see, are not nearly as great as the similarities, and for much of this book we shall be describing the biochemistry of an 'ideal' or' typical' animal cell, which might be that of most organs of most species. Only very much later will we 'come to consider in any detail how the biochemistry of the cells of different organs and species actually differ from one another. , According to the first theories, all cells were filled with a clear, jelly-like substance which was endowed with all the mystic properties of life, and was called protoplasm. By the 18305, it had become clear that all cells contained at their centre a large, oval body, darker than the surrounding protoplasm and occupying as much as one-third of the total cell area. This body was called the nucleus (and at a later date, of course, gave its name to the nucleic acids - see page 67). The nucleus was separated from the rest of the cell by a thin skin of its own - the nuclear membrane. By the 18805 and 1890s, the microscopists had shown that the . protoplasm surrounding the nucleus was itself far from being a clear jelly. Instead, it was lumpy and granular, filled with small specks of material which the highest-powered microscopes revealed as taking characteristic shapes as minute ovals, rods, and spheres. These specks were called mitochondr!a. But even the highest powered light microscope can enlarge only up to about 3,000 times, and objects half-seen or only suspected C.0.L·-5

The Chemistry of Life TABLE

Species

Hydrogen atom (H) Glycine molecule (CHzNHzCOOH) Typical protein molecule Typical virus Typical bacterial cell Typical anima I cell Human

5. Dimensions o/molecules and cells

Weight (gm.)

1·6 x 10- 24 1·2 x 10- 22

Atomic Weight

1 7S

3·2 X 10- 20 _ 2x 104 _ 1·6 x 10- '8 1 X 106 1 X 10- 16 4x 10 7 2 X 10- 15 ] X 109 8 x 10- 7 1 -10 X 10- 5 80,000

Length (longest axis) cm .

1 X 10- 8 2 X 10- 8 S-20x 10- 7

5 X 10- 6 1

10-4

1-5 X 10- 3

200

at this magnification could not be identified until, in the last years of the 1940s, the ordinary microscope could be supplemented by a new sort which used not light at all but a beam of electrons to examine specimens, and which could produce magnifications ranging from 10,000 up to one million or more. At once, a whole world of substructures unsuspected by the light microscopist became visible. The nuclei and mitochondria could be examined in fine detail to show a complex internal organization of their own, and the remaining' cytoplasm' (the term which by now had replaced 'protoplasm' for whatever clear jelly was still considered to exist within the cell) was seen to be criss-crossed with a rich network of twisting strands, membranes, and small groups of tiny linked particles, the ribosomes, till it looked like a lace tablemat. Today, the typical animal cell can be pictured as drawn in Figure 6 or as photographed in Plate 1. Table 6 shows the various particles that help compose the cell; the mitochondria are fifty to one hundred times smaller than the nuclei and the ribosomes up to sixty times smaller than the mitochondria. Very little remains of the pure protoplasmic jelly of 150 years ago in this modern picture. An appropriate question for the biochemist to ask at this stage 82

The Organization of the Cell FIGURE

6. storage

mitochondrion -------->''----{r'"

~-"'.------ vesicles

endopl as mic -----cl'--t'\. reticulum

I-------'>,--golgi complex \-,-.-J.---\---Iysosome

nudeolus--+-7.~~~i

nucleus an~d_+-J:.~.L-m4 chromatin

.~"---\\--W'----- nuclear

pore

ribosomes---\---'::':::~~.---

o--f-~ecretory

cell membrane---->o.

vesicles

Schematic diagram of a 'typical' animal cell is: what are the individual subcellular structures composed of and how do they maintain their structural individuality? Are there chemical differences and specializations which distinguish them? It might be expected that certain activities are located in one organelle, others in another, and this localization is significant to an understanding of how the cell co-ordinates and regulates its activity. But before the biochemist can test these hypotheses, there must be available some method of separating the organelles from one another, and obtaining on a relatively large scale (which means, for the biochemist, quantities in the order of tens of milligrams of material) purified preparations of nuclei, mitochondria, ,ribosomes and so forth. It is in the development of such methods that the techniques of . centrifugation have proved invaluable. If the cell membrane is gently ruptured, aU the internal organelles are released into suspension, like holding a cellophane bag full of sand and water under water and puncturing it. Now, jfthe resulting suspension of sand is shaken up and allowed to settle, the densest and heaviest grains fall to the bottom first, then the medium ones, whilst some Very fine grains remain suspended in the water even after several hours. The rate at which the particles fall depends on several factors: their weight, their density compared to the surrounding 83

The Chemistry of Life TAB LE

6. Sedimentation 0/ subcellular particles in a centrifuge

Sedimented after:

Shape

Particle

Time (mins,)

Gravitational K force

Nuclei

800

Mitochondria

12,000

25.000

105,000

Lysosomes

•••• :'... ••. J / . :::~.: . • ';.r~ _

Ribosomes membranes

~'";.r:r~:",,-.J,.,.

~.. ..'.. ~ .... "'" ,

solution ( a particle of cork, even a very large one, would not fall to the bottom however long one waited. as the density of cork is less than that of water, and it floats), and finally the gravitational force to which they are subjected. In Our ex.periment with sand and water, the gravitational force is constant whilst the beaker holding the suspension is standing still, and the particles may take several hours to sediment. But ways exist of increasing the force towards the bottom of the beaker. Accelerating it upwards in a lift or rocket would be one way, Another would be to whirl it round very fast at the end of a long arm, when centrifugal force will tend to

The Organization of the Cell drive all the particles towards the part of the beaker furthest from the centre of the circle round which it is being rotated. Such a principle, called centrifugation, is used to simulate the increased force of gravity during rocket acceleration when training fledgling spacemen. It is also the principle of the spin driers attached to most modern washing machines. In the hands of the biochemists, centrifugation is a technique enabling them to separate out particles from a suspension of biological material rapidly and efficiently. Modern laboratory centrifuges are capable of spinning tubes containing nearly a litre ofsuspension at speeds of 60 to 70,OOOr.p.m., which is the equivalent of applying a force of 500,000 times gravity to the particles in the suspension. By employing a carefully selected range of speeds and times, it is possible to sediment and remove, first the cell nuclei, the heaviest particles, then the mitochondria, the next . heaviest, and finally the ribosomes and membranes. The clear solution which remains after all the particles have been removed represents the soluble or cytoplasmic fraction of the cell. One such selection of speeds and times is shown in Table 6. A refinement of this method is density gradient centrifugation. Here the suspension is layered on top of a series of sucrose solutions of varying concentrations so that the density of the liquid increases from the top to the bottom of the centrifuge tube. Duringcentrifugation the cell organelles move through the gradient until they reach the sucrose concentration that has a density equal to their own, where they will pile up (see Figure 7). So at the end of the run, layers of sucrose solutions of increasing

c:l-sarr1ple to be separated '}it,;""" .

density

_ _- ; - ; ; ; _ - ; - _ +

1-----1

lightest

F~'"""'4-particles

centrifugation' heaviest particles "'==~-cell

FI G U R E 7. Sucrose density of gradient centrifugation 85

debris

The Chemistry of Life distinct bands can be seen at intervals down the tube, each band being a fairly hom*ogeneous sample of one particular organelle. The accuracy and resolving power of this method is remarkable. Particles of very similar size can be easily separated and it can be done with just one spin instead of the many necessary with the differential method. With this by way of background, we can now turn to a discussion of the individual subcellular components that can be seen under the electron microscope, isolated with the centrifuge, and studied chemically and biochemically. MEMBRANES

Perhaps the best place to begin with our description of the cell is at its boundary, its point of contact with its environment, the cell membrane. The membrane has two main functions to perform. It has to maintain the structural integrity of the cell by its strength, insolubility and protective nature and it has to act as a selective barrier to substances in the ex.ternal environment, allowing the passage of some but not of others. The membrane has the power to discriminate between harmful substances that would damage the cell if they gained entry, and therefore to prevent their entry, and substances that are essential nutritional requirements and must be allowed in to provide the cell with energy. The membrane is also responsible for helping maintain the correct internal ion concentration so that such variables as pH, electric potential (the different concentrations and charges on ions inside and outside the cell) and osmotic pressure remain constant, often against wildly different external ionic concentrations that would tend to unbalance the internal environment if it were not for the membrane barrier. Research into membrane properties is today one of the most active areas in biochemistry. However, despite this activity, knowledge of the precise mechanisms of selective permeability is still very poor. It has been known for some time that the membrane is predominantly lipoprotein in nature. We have already seen how lipid molecules, because of their hydrophobic nature, spontaneously organize themselves into a double layer or sandwich when placed in a watery environ86

The Organization of the Cell ment; their long non-polar tails line up together. forming the cheese inside the sandwich, whilst their polar heads face outwards, where they can readily bind to protein or glycoprotein components which represent the bread. The most widely accepted current theory of membrane structure is that of a core of lipid molecules arranged in a bilayer with its surface more or less covered with protein molecules, some of which penetrate right through the bilayer (see Figure 8). Because its secondary and tertiary structure is maintained by many weak bonds the lipid bilayer is very fluid and flexible; although again. because of the sheer number of internal hydrophobic bonds, it is at the same time strong and resistant to solvents. protein oT glycoprotein components

-""'hnlrl

FIG U R E 8.

bilayer

Fluid mosaic model 0/ membrane structure

There seem to be several ways for substances to enter the cell across the membrane. The simplest method, that works for the smaller molecules and ions, is one of passive diffusion, and occurs whenever their relative concentrations inside and outside the cell are different. Thus a chemical gradient in the case of non-electrolytes or an electrochemical gradient in the case of charged particles

The Chemistry of Life will exist across the membrane. Molecules will move across the membrane from the region of high concentration to the region of low concentration until an equilibrium is reached, when diffusion will cease. Diffusion is easier the more soluble the substance is in the lipid of the membrane, an9 may be further facilitated by the presence of holes or pores in the membrane, although this point is at present arousing a great deal of controversy in membrane research. Active transport, on the other hand, is the process by which cells accumulate substances against a concentration gradient When the internal concentration of the molecules is already quite high and the external concentration is low. The molecule must then be helped across the membrane and it is the protein components on the membrane surface which seem to be involved here. They have the power to scavenge molecules from the surrounding medium, to specifically recognize and bind them. The protein will then carry the substance across the membrane and deposit it on the other side. Alternatively, the protein may undergo a conformational change and expand to span the width of the membrane so that the substance to be transported can be carried on a sort of protein bridge. The first binding protein may even pass the substance on to another, carrier protein, which is free within the membrane, and it may be this 5e"'-------thyroxine < E : ( : - - - - - - -

• • • • • target cells (liver, muscle, etc.)

In studying the finer aspects of the regulation of the cell, we found it necessary to take into consideration what is, for the cell, the out· side world of the body as a whole. Now that we have come so far as to begin to build up a biochemical picture of the whole body, it becomes necessary for us to take one final step. For the cell, the external environment is provided by the body. a homeostatic system which maintains the constancy of its internal composition and thus enables the cell to survive as part of an integrated whole. 244

Controlling the Cell For the ceIl, departure by very much from the norms of this uniform environment spells speedy death. But the body also has an external environment, and this, far from being the warm dark womb in which it cradles its own cells, is a tough, highly changeable, and often dangerous jungle. It is a jungle of rapid alterations in temperature, humidity. and consistency. in which food, instead of being wafted effortlessly along a swift-flowing bloodstream, must be hunted down ruthlessly and captured by skill. The body needs to maintain homeostasis, but in order to do so it has to convert the non-homeostatic systems of the world around it. It has to act on this world in order to mould it into favourable contours. If temperature changes, the organism must organize itself so as to reverse these changes, by warming its surroundings or moving to more sheltered ones. It must be constantly in search of food, of air, of water, in order to meet the insatiable demands of its own interior. And in this search it must inevitably conflict with other life forms which are driven by the same internal urges to hunt for the sume ends (for instance, organisms which are bent on devouring it). This need to act upon the world around it demands a biochemical and physiological integration of the body at the highest level, and it is to that which we must now turn if we are to achieve the biochemist's goal of describing the total behaviour ofliving organisms in physical and chemical terms.

O.O.L·-14·

24S

CHAPTER 12

THE CELL IN ACTION For the organism to act on the ~orld around it, three abilities are required. In the first place, it must have the means of receiving stimuli from outside which will enable it to assess its position in space and its relationship to other objects; it must be able to see, hear, scent, and feel, food and danger; i.e. it must have sense receptors, eyes, ears, nose. Second, it must be able to recognize, coordinate, and respond in the correct way to incoming sense stimuli. This is the job of the brain and central nervous system. Third, it must have a means of achieving purposeful movement in order to obtain a given goal, whether the capture of food or the avoidance of harm ; this movement is produced by muscles. We can sum up this three-cornered relationship in a simple diagram (Figure 32). A considerable percentage of the human body is taken up by these specialized organs - sensory receptors, limbs, and brain - and most human life at the conscious level is lived only in their terms; we are content to leave our internal homeostasis to operate by means of nerves, hormones, and regulators below or at the fringe of consciousness. The specialized organs demand an equally specialized biochemical composition in order to achieve their tasks. Most of this specialized biochemistry is concerned with methods for the conversion and transmission of energy and information. The sensory receptors have to convert information arriving as light, heat. or sound waves into forms which can be transmitted through the .nerves to the brain. The brain has to process this information so as to arrive at two objectives: one an irnmediate set of instructions to be dispatched to the muscles, the other a permanent or memory store which will be of subsequent use in helping determine later actions. Finally, the muscles have to recognise and respond to the information arriving at them from the brain by contraction or relaxation: in contracting they have to perform mechanical work, and, to do so, they need a source of energy. By now, we should have no difficulty in framing the biochemist'S 246

The Cell in Action FIGURE

32. Muscle,

nervlf!,~.

and brain

sensory receptors

effector organs question: what special chemical characteristics can we observe in these organs to account for their physiological role? This question has indeed existed for as long as biochemistry itself, and is, at one stage or another, even if it must be confined to their off-duty moments, on the lips of every biochemist. Ultimately, we only recognize life by its power to act upon the inanimate world around it, and our biochemical analysis cannot stop short merely at showing how the body, left to-itself at a suspended interval oftime, is composed and functions as an internally self-regulating homeostat; in the long run, we must also hunt for the biochemical mainspring of the body's relationship with the external world. A good deal is in fact known of the actual workings, biochemical as weIl as physiological, of nerve, muscle, and brain - much more than we have space to consider here. We must reluctantly leave out, for example, any description ofthe biochemistry of the special receptor cells of sense organs, although a study of each would

247

The Chemistry of Life reveal an exquisite interplay of biochemical and structural specialization which enables incoming information, in the form of pressure, vibration (sound), light. or indeed chemical composition, in the context of taste or smell, to be transduced into the universal language of the nerves, the passage of electrical impulses. * Here, we will begin first by 'considering the biochemistry of muscle, and then some aspects of the special biochemistry of nerve and brain. I. MUSCLE

The typical skeletal (or voluntary) muscle, which is responsible for the movement of limbs and body, consists of a bundle of long fibres, each up to 0'01 centimetres in diameter and running the entire length of the muscle, joined at each end by tendons to the bone or organ they are responsible for moving. Movement takes place as a result of the contraction of these fibres, which may shorten by as much as sixty per cent of their resting length, pulling the bone with them as they do so. Each fibre, in turn, may be seen under the microscope to consist of a set of long parallel fibrils, each about 0·0001 centimetres in diameter. These fibrils contain the contractile material of the muSCle. Like other cells, they also have a nucleus, mitochondria, microsomes, and cell membrane, but when examining them under the microscope one is scarcely conscious of these; one sees the fibril primarily as a long, thin, striped tube. These stripes are the most remarkable thing about the skeletal muscle fibril; they run across it at right angles at regular intervals, like the black and white markings on the pole of a belisha beacon. What is more, the stripes of adjacent fibrils all run in parallel, so that the stripes appear to continue right the way across the entire muscle fibre. It is this striped quality that particularly characterizes skeletal muscle, as opposed to certain types of smooth muscle which are not normally under voluntary control, but are responsible, for instance. for intestinal or stomach movements.

* It would be less than human of me if I did not refer those interested in this theme to my book The Conscious Brain (Penguin, 1976) for further exciting details I

248

The Cell in Action Viewed under a higher powered microscope, the stripes may be resolved into alternate light and dark bands, forming the complex but regular patterns of Figure 33. The microscopists refer to these FIGURE

33. Tire muscle fibres

whole muscle

(a,

10-10011 (b)

1 muscle fibre 1.2{

(c)

isolated myofibril ~I-band~(

A-band

)I+-I.band~

H-zone ,.-,

Z-line

·.ld)

Muscle at different magnificatIOn. (Rabbit psoas muscle, after H. E. Huxley)(l,u = 1O- 4 cm.)

bands as the 1- (light) and A- (dark) bands, the H-zone (lighter area in the middle of a dark A-band) and Z-Iine. The one A-band, two half I-bands, and one H-zone between each pair of Z-Iines represents a single unit, called a Sarcomere. Each single unit is no more than 0'0002 centimetres in length, so to each fibril there are several thousand sarcomeres joined end to end. When the magnification is increased stilI further, using the electron microscope,

249

The Chemistry of Life the A- and I-bands break down into a set of filaments running parallel to the axis of the fibril. The A-band is dark because the filaments within it are thick; the I-band, on the other hand, consists of much finer filaments. Careful examination shows that the slender fiJaments of the I-band run into the region of the A-band, filling the space between the thick A-band filaments. They run into it so as to form, in cross section, a regular pattern, each thin I-band filament being surrounded by three thick A-band filaments, whilst each A-filament has surrounding it six thin I-filaments. The I-filaments stop before they reach the centre of the A-band, so that the lighter-coloured H-zone of the A-band is in fact an area of the band which contains only thick A-filaments (Figure 34). Very close examination reveals that each I-filament, where it runs into the A-band, is connected with the A-filaments by fine, hairlike, cross-linkages (Plate 5). FIGURE

34. Musclefibrils at high magnification A I

•

--•--.---

•• • • •• •• • • •• • •• • •• • • •••

•· •

• B• •

•

-,)

I I

• • •

• • • • • • • • • • • • • • C• •

---..~

.......

--.•...•

• ...... . '. • • •

••

•

• •• • • •• • • ••

• B• •

•

The arrangement of the filaments in the muscle fibrils. A: Longitudinal view. Band C: Cross sections showing thick and thin filaments. (After H. E. HUXley.) Now when contraction of the muscle occurs, the sequence of events seen under the microscope is that the fiJaments themselves do not change length. but that the thin I-filaments slide in between the thick A-filaments until all the H-zone is filled up; when this happens the normal limit of contraction is reached. Thus the effect is that the I-bands seem to disappear whilst the A-bands

250

The Cell in Action remain unaltered, and the two Z-lines defining the limit of the sarcomeres are drawn in towards the central region. The mechanism of muscular contraction, then, seems to demand the existence of a set of two types of interlocking filaments, contraction occurring when the filaments are slid together, relaxation as they move farther apart. In essence, muscular contraction is more like the shutting of a telescope than the tautening of an elastic band. What can the biochemist add to this microscopist's and physiologist's view of muscle? [t was early recognized that the contractile elements of the fibre were almost entirely composed of protein. At first, it was believed that only one type of protein was present, and it was calIed myosin. It is now known that there are at least four components, myosin, actin, tropomyosin, and troponin. Myosin is the largest of the proteins, with an estimated molecular weight of 460,000 and constituting the greatest proportion (about fiftyfour per cent) of the total protein. Actin constitutes about twenty per cen t of the total protein but it is a much smaller molecule wi th a molecular weight of only 41,000. X-ray diffraction patterns for myosin show it to be a fibrous (l;-protein; that is, it is composed of two polypetide chains twisted in a helix for ninety per cent of its length. In addition myosin can be split by enzymic digestion into two subfractions known as light and heavy meromyosin. Actin on the other hand is a globular protein. The proteins can be separately extracted from muscle, and both types of molecule can be shown to be long and fibrous in shape. In addition, myosin has the remarkable property of being capable of being extruded through a nozzle to form long artificial fibres (a process analogous to the formation of artificial silks such as rayon). When actin and myosin are extruded together, they form a complex fibre, actomyosin, and it was found by F. Straub and A. Szent-Gyorgyi in Hungary in 1942 that, if this actomyosin fibre is ·placed in a solution containing ATP and suitable salts, it will contract spontaneously, simultaneously dephosphorylating the ATP. Neither myosin nor actin by themselves, however, would behave like this. N ow it had already been known for some years that ATP and creatine phosphate were broken down during extended muscular

251

The Chemistry of Life contraction to release ADP and inorganic phosphate. Indeed it was these observations, mflde with muscle in the early 1930s, that first led to the recognition of the role of organic phosphates in biochemical reactions. In 1933, V. A. Engelhardt in Moscow was able to show that, in the presence of myosin, ATP was rapidly split to ADP and inorganic phosphate. Thus myosin seemed to function as an ATP-ase enzyme. It was clear that these observations must be related in some way to the explanation of muscular contraction. The situation was further complicated though by the observation that the injection of a tiny amount of Ca + + ions into a single muscle in vivo will produce a local contraction although the purified actomyosin complex does not require Ca++ for its contraction. However, it required the coming of the electron microscope and the techniques of X-ray crystallography to be able to link decisively the biochemical "and microscopical observations, and these later interpretations were to a large extent the work of H. E. and A. F. Huxley and their co-workers in England. According to the present conception (at least in its simplest form), the two major proteins, myosin and actin, correspond to the two different types of filament in the muscle fibril. The thick filaments of the A-band are composed of myosin, the thin I-band filaments are actin. The myosin molecules in the thick filaments form rods composed of two chains. Each chain has a thickened portion at one end giving the rod a double-headed appearance. It is these •heads' that come into contact with the actin molecule and they are composed of heavy meromyosin which is the ATP-ase carrier. The direction of the meromyosin heads reverses in the centre of the thick filament so that each filament is bipolar with a central bare zone, visible under very high magnification, containing no meromyosin heads. As we have said, actin is a globular protein and the thin filaments are formed by two strings or chains of actin molecules wound loosely together like two strings of beads. It is only recently that the association of troponin and tropomyosin with the actin filaments has heeri clarified. Tropomyosin is a fibrous protein which seems to lie in the groove formed by the winding of the two actin chains. Troponin is arranged at regular intervals along the tropomyosin molecule.

252

The Cell in Action With this structurai information, we are now ready to consider the contractile process, at least as far as current understanding will allow us. The signal for contraction in the type of muscle we have been considering is the release of a chemical transmitter from nerve endings at the junction between nerve and muscle, which we will shortly consider in more detail. This leads to a depolarization of the muscle cell membrane, which in turn results in the release of a large number of Ca + + ions into the muscle fibre - the amplifi~ cation process. It is these Ca+ + ions that act as the second messenger and react with the effectors, that is, the muscle proteins, to bring about contraction. Thus the initial stimulus has been coupled to the contractile process by the Ca++. It has been suggested that Ca + + combines with the troponin and this in some way causes the tropomyosin in the same region to shift slightly. exposing a pre~ viously hidden part of the actin molecule. Now both the actin and myosin molecules carry strongly negative charges on their surfaces - indeed it is this electrochemical repulsion that helps maintain the structural integrity of the whole muscle. The minimum potential, that is, the most positive region, exists at the mid point between the two filaments and it is this region that is occupied by the meromyosin projections containing the multivalent and electronegative ATP system. The effect of Ca + + binding to the actin filament will be to make the surface of the filament temporarily electropositive over a very localized area. The ATP complex will be attracted towards it and will interact. The ATP is then broken down to ADP with the resultant release of energy. Cessation of the nervous impulse causes an immediate reuptake of the calcium ions from the fibrils, the meromyosin/ATP complex is released back into the inter-filament space and the . tropomyosin/troponin component shifts back into the actin groove, but in a slightly different position, relative to its original alignment with the myosin filament. Thus the next site of attachment of the meromyo,sin molecule will be to a troponin molecule farther along on the actin filament, and the process is repeated. During contraction then the muscle shortens not by any change in length of the filaments but by the filaments sliding past each other, gradually filling the H zone. The process is summarized in Figure

35. 253

The Chemistry of Life FIGURE

35. Structure o!myofilaments and stimulus

contraction coupling meromyosin projections

meromyoSIn

This model of muscle action incorporates many of the observable facts, but there are also many more questions that remain to be answered, particularly the exact involvement of Ca + +. Nevertheless muscle cells provide a prime example of specialization of structure to fulfil a specific role - to enable the organism to respond quickly and efficiently to a changing external environment. II. NERVE AND BRAIN

The great mass of cells of the central nervous system is concerned with the co-ordination of the millions of incoming messages arriving by the second from the internal and external sense organs of the peripheral nervous system, the control of the motor activities of the myriad of muscles, and the continued matching and comparing of the messages and responses which represent the

254

The Cell in Action present moment of existence of the organism with the accumulated wisdom of its past history, relating this to present needs and possible outcomes of actions. or all the things that might be said of the biochemistry of nerve and brain at this point, we will confine ourselves here, by a process of tunnel vision, to just two. We wish to describe how the structures of the nerve cells are specialized to enable them to perform their functions, and what the biochemist can say about two fundamental properties of cell nerve cells, their capacity to conduct messages (impulses) over relatively long distances, and to communicate the import of these messages either to other nerve cells, or, in the last analysis, to effector organs such as the muscle. Like the muscle itself, the nerve trunk is made up of a number of smaller nerJle fibres; in what follows, we will use the word nerve to apply to one single such fibre. The nerve fibre, then, is basically a long tube which connects the top end of the nerve cell, generally located in the brain or spinal cord, with the muscle. Wrapped round this tube, or axon, which is in fact part of a longdrawn-out single cell, is a layer of fatty material (myelin), which provides it with a protective sheath, in appearance exactly like the insulating rub ber round the metal core of an electric cable, and, as we shall see, serving much the same function. At the top of the nerve axon is a large, diamond-shaped swelling which is the nerve cell body (neuron), which contains all the conventional apparatus of the cell; nucleus, mitochondria, and microsomes. The cell body is so large in comparison with the axon that it sits at the top of it not unlike a toffee-apple at the end of its stick. Where the axon, at its lower end, comes into contact with the muscle cell, it again spreads out slightly, so as to form a sort of broad foot which treads on the muscle cell, and, as a result, touches it over a quite considerable area. This region of contact between the two cells is called the neuromuscular junction. The neurophysiologist's name for such ajunction is synapse. If, as frequently happens, the distance between the brain or spinal cord and the muscle is too long for one axon to stretch the whole way, a break is made half way between them. The nerve leading from the brain then forms a synapse with the cell body of a second nerve, whose axon can then reach the remaining distance to the muscle. Such a synapse between two nerve cells, as 255

The Chemistry of Life well as being a means of sending a signal over long distances, also, as we shall see, provides a point at which the message of command, descending from the brain, can be modified to take additional factors into consideration. The message corping from the brain crosses the synapse between the first nerve and the second and continues down the second nerve to the neuromuscular synapse where it causes the muscle to contract. In general, whenever it becomes necessary to transmit a message from one part of the nervous system to another, it is done by means of such a synapse. A similar system carries messages /rom sensory organs such as eyes and ears to the brain, and yet further synapses interconnect the large number of cells within the brain itself. Thus messages can be rapidly transmitted over considerable distances and to a large number of different nerve cells or muscles (Figure 36). FIGURE

arrives

36.

nerve cell body

to muscle

synapse to 2nd nerve

with other modifying synaptic contacts The highest region of the brain, the cerebral cortex, contains an astronomic number of such nerve cells (something like 10,000,000,000 in humans) each one of which may make ten thousand or more synaptic contacts with others. The richness of such interconnections accounts for the tremendous ability of the brain to co-ordinate, compare, and control the activities of the sense organs and muscles, and to learn from experience. En masse, this huge array of interconnecting nerve cells within the brain acquires characteristics which transcend the behaviour of any

256

The Cell in Action individual cell, just as the activity of a computer transcends the properties of anyone of its transistors or magnetic tapes. The essential functions of the nerve cell are three. At the head of the toffee-apple, the cell-body has to receive and respond to the incoming messages arriving from other nerve cells which synapse with it. These messages must be passed swiftly down the axon, and finally, at the bottom, must be transmitted across the cell membrane to the nerve or muscle cell at the other side .of the synapse. Over the last ten years, a good deal of the biochemical mechanism of each of these three steps has been unravelled. The simplest part of the process to understand is the way the messages pass down the nerve axon itself. Here our earlier description of the nerve fibre, surrounded by its fatty sheath like an insulated electric cable, comes strikingly into its own. In fact, messages pass down the nerve axon in the form of electric currents which begin where the axon springs out of the nerve cell body and travel down the fibre at a rate of about twenty metres a second. Two sorts of job are done by more familiar electric currents. A steady current flowing to an electric light bulb provides a constant source of power for the bulb; if the flow of electricity alters, the bulb flickers. A door-bell, on the other hand, must ring briefly and then stop. A short burst of electricity is wanted here, to act as a signal. The currents flowing in the nerve fibre are signals, not power supplies. Thus if we record the passage of electricity down a nerve we find not a steady reading, of say four volts, such as one might get from a torch battery, but instead, at anyone point along the nerve, the voltage suddenly fluctuates, rises to a peak, and declines again as the electric signal arrives, passes the recording point, and is gpne. A walle of electricity has passed down the fibre. Ifwe draw a curve plotting the rise and fall of voltage at a particular point on a nerve fibre during the passage of an impulse down it, we get the picture shown in Figure 37. What is the mechanism of this remarkable phenomenon? In the sort of electricity produced by a generator or battery, the current running through the cables is in fact carried by a flow 0/ electrons moving down the wire from the negative to the positive terminal. In animal-generated electricity, the current is still carried by a flow 257

The Chemistry of Life FTG U'I! E 37,

Voltage changes as impulse passes dawn nerve fibre

action potential

] 1 8

j ----. ~ - """""5

__"restmg potential'

. ..

tune In milliseconds

of charged particles, but, instead of the negatively-charged elec trons, the carrieJ;'s are positiveIy-charged ions. That this flow is possible depends on a fact we have already commented on On more than one occasion - that the ionic composition of the inside of a cell is quite different from that of the outside (see page 88, for instance). Inside, there is a high concentration of potassium, but little sodium. Outside, there is a great deal of sodium but only a small amount of potassium. Nerves resemble most other cells in this. Sodium and potassium, as we know, exist in solution as their charged ions - each carries one positive charge, and we have become accustomed to writing them Na+, K+. Of course, the cell contains many other charged ions as well: magnesium and cal dum (Ma ++, Ca++) amongst the positive ions, and chloride and phosphate amongst the negative ions (Cl-, HP04 --). In general,there are about as many negative as positively charged ions within the cell, so that the overall net charge is zero, and the same is also true of the fluid in which the cell is bathed. But the fact that the concentrations of sodium and potassium are different either side of the cell membrane results in certain peculiarities despite this overall electrical neutrality. For, in a non-living system, we would expect that if we separated a solution of potassium chloride from one of sodium chloride by a thin, permeable membrane - say a cellophane sheet - potassium ions 4

258

The Cell in Action would pass through the barrier in one direction, and sodium in the other, until the concentration of both ions was equal on either side . of the membrane. That this does not happen in living cells is because the cell membrane behaves, as we discussed in relation to the general properties of the cell membrane in Chapter 4, as if it were impermeable to sodium. It permits potassium to enter the cell but not to leave, but does not let sodium enter at all. This is not entirely a passive refusal of admittance, for if an experiment is made in which sodium is injected into the cell, it is quickly pumped out through the cell membrane again against the sodium concentration gradient. The membrane is thus a dynamic system capable of selectively distinguishing between sodium and potassium ions. What is the effect of these differences in concentration ~ If potassium is not to flow out of the cell down a concentration gradient, it must be in response to some other force acting inwards. This force is provided by the positively-charged sodium ions, which line up on the outside of the membrane so as to provide a barrier of positive charge which repels the positively-charged potassium ion. At the same time the sodium ions tend to attract negatively-charged ions such as phosphate or chloride across the cell membrane. The net effect is that we can draw the axon as shown in Figure 38a with a line of positive charges down its outside surface and of negative charges on its inside. A potential difference thus exists between the two sides of the membrane - if they were shorted by a wire running between them they would register a voltage. This can, in fact, be done, if a tiny glass microelectrode is inserted into the axon, and connected through a voltage recorder (galvanometer) to the outside surface of the nerve. It is also possible to calculate a theoretical voltage which should arise in a system in which a membrane permeable to potassium but not sodium exists - a comparatively simple piece of mathematics. Fortunately, the theoretical and experimental results agree; the voltage recorder indicates that the inside of the cell maintains a potential sixty-five to ninety-five millivolts negative to the external surface. The existence of such a voltage is absolutely identical in principle to the voltage produced by the dry chemical battery of a torch or the wet battery of an accumulator. Now consider what happens if for some reason at one point 259

The Chemistry FJOUR~·

0/ Life

3 8 ~ Nervous tral'.sm!ssion

(a) At rest, charged ions at either side of nerve membrane provide a potential difference. out

out

+ + +

+ +

(b) A local depolarization at A results in current flowing, and the message passes down the nerve fibre.

+ +

A f

+ +++ I

+ +1+++++++

-1'- - - --

- - -+ ++++++++ -dU~tionofm~~ge:--------------------------------~ + +

+ +++

along the nerve axon the set of charges is reduced to zero, for example, by the application of an electric shock. This condition is referred to as the depolarization of the nerve, and its results are indicated in the diagram Figure 38(b). The results of depolarization are to cause at a certain point on the nerve a reversal of the set of p Ius and minus charges wi thin and wi thout the membrane. Thus, at the outside of the membrane, one region, A, becomes negative with respect to a region farther along, B, and, correspondingly, within the membrane B becomes negative with respect to A. A tiny local circuit is completed between A and B and current flows between them. But the effect of the arrival of the current at B is to depolarize B in its tum, and hence render it negative to a point still farther along the axon; another local current then begins to flow and the depolarization is repeated. Thus a depolarization at one end of the nerve axon results in the establishment of a series of local circuits, which, moving like a wave over the surface of the axon, will carry the depolarization along its length. Provided the initial stimulus is large enough to set the circuits going, it is carried 260

The Cell in Action rapidly down the nerve fibre. This method of propagation of nerve impulses, suspected for many years, was finally verified experimentally by A. L. Hodgkin, R. D. Keynes, and their collaborators in Cambridge. It was Hodgkin, too, who provided a satisfactory explanation of the phenomenon in biochemical terms. We have seen that the resting potential of the nerve is maintained by the difference in concentration and permeability of sodium and potassium. Suppose, Hodgkin argued, that the effect of depolarization is to cause a change in the permeability of the membrane so that it becomes for a brief while very permeable to sodium, what will happen? Sodium will flow rapidly into the nerve axon down the concentration gradient, entering faster than potasSium can leave. As this happens the membrane potential will drop still further, as positive charges are transferred from outside to within. As the membrane potential drops further, it will become still more permeable to sodium, and the result will be a positive feedback system in which the entry of sodium is self-stimulating (Figure 39). This inrush of sodium continues until the membrane potential of eighty to ninety millivolts negative is reduced to zero and finally converted to one of some twenty to thirty millivolts positive. This represents the' spike' of the wave of Figure 37. Sodium entry now ceases, as it is having to enter uphill against a potential gradient. At the same time, according to the Hodgkin model, the potassium permeability of the axon increases, and potassium leaks out down its concentration gradient until the membrane potential falIs to its old value of about eighty-five millivolts negative and the electrical wave has passed on down the axon. In fact, potassium exit goes on slightly longer than is strictly necessary, and the potential slightly overshoots its original value, resulting in the second 'trough' of Figure 37. During this period, where the potential is some milli· FIGURE

39.

increase in sodium peimeability

~di so :umentry

membrane depolarization

261

The Chemistry of Life volts below the normal resting level, the nerve becomes incapable of carrying a second message. Within a few thousandths of a second, however, the old value has been restored once more and the nerve is again excitable and ready for action. Hodgkin's theory has been amply borne O).1t by experiments. Studies using radioactive sodiUm and potassium have been able to demonstrate the changes in entry and exit of these ions across the membrane during nervous stimulation, whilst it has long been known that,just as the theory would predict, nervous conduction becomes impossible if all the external sodium is removed. For his part in this work, Hodgkin shared the 1963 Nobel Prize for medicine. The changes in membrane potential are achieved at the expense of the sodium and potassium levels which the cell membrane normally maintains. The changes we have described as occurring at the membrane result only in rapid local changes of concentration, so that they hardly affect the overall differences in sodium and potassium levels inside and outside the cell. Despite the local changes, these concentration differences remain so great that, in a large nerve, upwards of a million impulses could be carried before the decline in internal potassium and rise in sodium became great enough to inactivate it. Nonetheless. ultimately, the nerve must set to work to rectify the changes in concentration and restore the old differentials between sodium and potassium. It may be noted that, until now, processes we have described have not demanded the output of energy by the nerve; the changes in membrane perme. ability instead utilized the potential energy latent in the differences in sodium and potassium concentration across the membrane. The work of the nerve consists in establishing this potential energy once more. Again, the critical experiments which demonstrated the mechanisms of this process were performed by Hodgkin, by Keynes, and by A. F. Huxley. They used in their experiments the biggest nerve they could find - the giant axon of the squid, which is so wide, being up to a millimetre in diameter, that it is relatively easy to inject test substances down inside it and to study their effects. They first showed that nervous conduction is dependent on energy metabolism. Nerves poisoned with cyanide, which prevents oxida-

262

The Cell in Action tive phosphorylation, rapidly lose their ability to conduct impulses. But if ATP is injected into the poisoned nerve, it becomes active once more, and can go on conducting until all the ATP is broken down once more. Thus, just as in muscle, the ultimate energy on which the cell draws for its ability to act is the energy of ATP. Later work has been able to show that the ATP is in fact utilized in order to maintain the sodium and potassium levels of the nerve. ATP is broken down during the extrusion of sodium and the entry of potassium. The entry and exit of these ions are found to be linked processes, and, in general. one molecule of ATP is broken down for about every three ions of sodium and potassium transported across the membrane. The exact mechanism whereby the energy of ATP is utilized in this way is still unknown, but it presumably involves phosphorylation of membrane proteins so as to induce conformational changes in carriers, or open pores through which ions can pass, according to the general principles discussed earlier in relationship to membrane properties, but the full details of this process are even less well-understood than the fine mechanism of the coupling of ATP breakdown to muscular contraction, even though, just as with that problem, one is tantalizingly close to the solution.

Transmission ojimpu/ses at synapses The nerve impulse flows down the axon in a wave of depolarization of the membrane. But at the synapse a different problem arises. The membrane of the nerve cell or muscle fibril at the other side of the junction is not continuous with that of the incoming nerve. A means must be found of bridging the gap between the two cells. Essentially, this demands that the arrival of the depolarizing wave at the end of one nerve axon acts as the trigger or signal for the start of a similar wave in the nerve cell and axon of the second nerve. This triggering action depends on the existence of chemical transmitters. The arrival of the nerve impulse at the synapse stimulates the release from the nerve endings of a chemical which can diffuse through the mem brane of the first nerve to arrive at the cell body of the second (Figure 40). There, it causes depolarization of the membrane and triggers an impulse in the second nerve. The chemical nature of these transmitter substances differs in different

263

The Chemistry of Life

2nd nerve

1st nerve

synaptic vesicles

receptor Sl[ell'V'

Acetylcholine. released from synaptic vesicles by incoming stimulus, diffuses across synapse to receptor sites in second nerve, where it is destroyed by cholinesterase. depolarizing second nerve in process. parts of the nervous system. Some are simple amino. acids, like glutamate. Others are amines, substances' related to the hormone adrenaIin described in the previous chapter. Still others - a whole new, and recently discovered class of transmitters - are small peptide molecules. But one of the commonest is the substance acetylcholine. It seems to be the case that different pathways in the brain and nervous system, in which many nerve cells are involved, utilize different transmitters; thus in some of the brain pathways involved in emotional and attentional responses, amines seem to be the transmitters; some of the smaIl peptide transmi tters may be Involved in the transmission of pain sensations (there was milch excitement in the mid-1970s when one of these transmitters was found to be chemically rather similar to the artificial pain-reliever morphine). Many of the synapses between nerve and voluntary muscle are mediated by acetylcholine, and we can look at its mode of action in a little more detail. Acetylcholine is manufactured by the acetylation of the fat choline with acetyl-CoA, and the responsible enzyme, choline acetylase, is present in the nerve endings. Both it, and the acetylcholine itself. are held in a series of particles, in many ways similar to the lysosomes, and which have been called by their discoverers, Victor Whittaker in Cambridge and George Grey in London, 264

The Cell in Action • veSic,es. • I T1. • 1 ... 'buted through t h e nerve synaptic ~ "CSC particles, uistn ending but not found elsewhere, contain the acetylcholine in a bound, inactive form. Additional acetylcholine, however, is found free in the cytoplasm of the synaptic vesicle. There is a school of thought which maintains that the vesicles are simply reserve stores of the transmitter. However, probably the majority of people working in the area believe that the effect of the arrival of the nerve impulse is to cause many of the vesicles to move towards the membrane, fuse with it and empty their contents into the gap between the two cells, the synaptic cleft, above which they can diffuse to the second nerve cell or to the muscle. On the postsynaptic side, there are specific receptors located in the membrane on to which the transmitter binds, in a similar way to the type of hormone-receptor interaction proposed in the previous chapter. (It is also possible that cAMP is involved in the postsynaptic response to some transmitters.) The transmitterreceptor results in a change in the postsynaptic membrane structure. If the receptor is an excitatory one, this may result in an influx of Ca++ ions large enough for the postsynaptic membrane to become depolarized. If a sufficient number of synapses transmit excitatory messages to the postsynaptic nerve at around the same time, the result will be a general depolarization, and the second nerve will 'fire' or the muscle contract. On the other hand, there are other transmitters which, working in the same way, produce a hyperpolarization of the postsynaptic nerve (i.e. make it more, rather than less negative) - thus making it harder to fire. These are inhibitory transmitters and are of equal importance to the excitatory ones (nerve cells need to be able to say No as well as Yes!). Once it has exerted its effect, the transmitter must be destroyed to prevent its presence from interfering with further messages. In the case of acetylcholine, this is achieved by the enzyme acetyl-cholinesterase. which. present in the postsynaptic membranes, hydrolyses it again to choline and acetic acid. Other transmitters are reabsorbed after use, either into the presynaptic cells or into a further class of cell, the glial cells, which are present in nervous tissue in large numbers, surrounding most of the nerve cells and 0.0.L·-'5 265

The Chemistry of Life ..

+1. .. Sl(l"g~S"S (Sn~ ot+kol3. n·o~k o~m" 0."-- 1... ~,..._ .... +""" ...... ! ....... 1-. .... 1__ .... H.L"" J"""Y '"' 1 \ vuu... ~ LU .... YV J.. • I.. U:1 WJ.J l(l.UVI.C:UUJ.)' UJ LUC J4::iL

decade has been concerned with the relationship between the biochemistry of neurons and glial cells and ofthese complex questions of factors determining nerve cell transmission.) The whole process of synaptic transmission is s?ffi111arized diagrammatically in Figure 40. The neurotransmitters like acetylcholine establish contact between two cells across the gap which separates them. It is not surprising to find that this is a very vulnerable link in the chain of command between nerve and muscle. Anything which interferes with the diffusion of the transmitter across this space will prevent the message being passed on, and many drugs which interfere either with transmitters, the receptors, or their subsequent destruction by postsynaptic enzymes, thus block synaptic transmission and result in a sort of chemical paralysis. For acetylcholine, nicotine is one such drug; another is the poison which Central American Indians used to dip their arrows into and which has since become beloved of crime-writers, the mysterious curare. In more recent times another class of substances which affect cholinesterase have achieved more sinister significance, though. Originally a product of German wartime research, a group of organophosphorus compounds which act in this way were developed as the nerve gases. An even more toxic variant (a milligram or so absorbed through the skin is said to be lethal) was developed in the mid 1950s at Porton, Wiltshire, the so-called V-agents. They are amongst the most poisonous chemicals known. If the internal working of our cells depends ultimately on ATP, it may be maintained that our ability to control and command our body to act is similarly dependent on the neurotransmitters. Many other specialized drugs and pharmacological agents interfere with transmitter substances. Some of those used in the control of mental illness, antidepressants and tranquillizers, work this way. and indeed there are proposals that some of the mood- . affecting psychedelic agents, like cannabis (marijuana) and LSD, exert their effect by interacting in some subtle way with particular central nervous system transmitters. There is no doubt that the area of brain biochemistry has been amongst the most fertile of recent years, and will be of increasing excitement in the future.

266

CHAPTER 13

THE UNITY OF BIOCHEMISTRY In what has gone before, we have tried to describe the behaviour, in chemical and physical terms, of the biological unit called • the cell'. But when speaking of this cell, we have in fact nearly always been referring to, not the cell of anyone of the millions of different species of living things that inhabit the earth, but the cell of one particular group of animals - the mammals. Although on rare occasions we have digressed into the fields of plant and microbial biochemistry, such trespasses have nearly always been brief, and then normally intended to illustrate a point about the mammalian cell. Indeed, we are not even being very fair in extending ourselves as far as even a description of ' the mammalian cell '. Most laboratory work, and hence the experiments we have been describing, is done on only a very small number of different species. Rats and guineapigs, rabbits and hamsters are studied by biochemists in their tens of thousands; dogs, cats, and domestic animals such as sheep, cows, and pigs have also provided starting material for research. Occasionally experiinents are made with birds such as chickens and pigeons. Always, attempts are made to relate this biochemistry of laboratory animals back to humans. But these species form only a very small fraction of the widely differing groups of known mammals. And when we come to plants, insects, or micro-organisms, the number of whose known species overwhelmingly outnumbers the total of the mammalian kingdom, our biochemical studies seem to fade into insignificance. Yet we have confidently described' the cell', its enzymes, metabolism, and behaviour almost as if unaware of the existence of so many other different sorts of cell remaining to be examined. Have we not been grotesquely over-confident in doing so? Fortunately, ,we are fairly secure in saying no. The differences in appearance and overall behaviour between humans and yeast are indeed vast. Yet it is astonishingly. almost at first sight unbelievably, the case that most of the chemicals that compose the 267

The Chemistry of Life two are practically identical; ahnost without exceptioil, their proteins are made of the same twenty amino acids, their nucleic acids of the same four purine and pyrimidine bases, their carbohydrates of the same or similar sugars. And even where slight differences may be pinned down between, say, the amino acid sequences of individual enzymes, the metabolic pathways catalysed by these same enzymes remain, in yeast and humans, identical over large regions. The pathway of glucose breakdown, studied originally in yeast as the fermentation of sugar to alcohol, was subsequently found to be identical with the route by which the muscle cell converted glucose to pyruvic acid. The only difference lay in the subsequent fate of the pyruvic acid. Similarly. it is possible to purify an enzyme from one organism and use it without apparent difficulty to study a reaction in another quite different one. Ribosomes from a micro-organism and soluble fraction from a rabbit or duck will contentedly collaborate to synthesize protein. Even though the biocheJllical behaviour of only a minute percentage of the different forms of life 00 earth have been examined, one may still predict with a good chance of success that the general conclusions may be extrapolated to COver all other forms as well. The basic mechanisms of carbohydrate, fat, and protein catabolism and synthesis are the same in all forms of life now existing. This is a fact at first sight so unexpected and so surpriSing when one thinks of the manifold differences of form which life manifests, that it demands almost a positive effort of will to accept it. Its implications become all the more startling when one comes to consider the undoubted biochemical differences that do exist between different life forms. The most profound of these differences lie in the sources from which the organism obtains the energy it requires in order to remain alive. Animals, fungi, viruses, and most bacteria rely on the existence of preformed organic compounds. We have dwelt at some length on the way in which animals burn glucose to carbon dioxide and use the energy sO released to synthesize ATP. Deprived of glucose, or its rather ineffective substitutes in fat and protein, an aniinal rapidly wastes away and dies. In the presence of glucose and certain essential amino acids and vitamins, it can synthesize all the thousands of 268

The Unity of Biochemistry other chemicals it requires. Many bacteria are less demanding; they can survive on simpler 2- or 3-carbon organic acids, and some of them do not need amino acids but can make their own by transamination reactions with ammonia. Yet even these rather cleverer bacteria and fungi can normally exist as well (or better) in the presence of glucose and amino acids as they do in their absence. Most of them have the enzymic ability to deal with these substances just as animals can, even if the bacteria can do without if times get hard. They are just rather less specialized than the animals, and can therefore live rougher. A second major difference lies between those organisms which require oxygen to act as an 'energy-sink' and oxidize their foodstuffs, and those 'anaerobic' bacteria which either never use oxygen, or can do without it at a pinch. Yet even such differences are more apparent than real. Those micro-organisms which obtain their energy entirely by fermentation do so by pathways of metabolism similar to the routes taken in animals for the initial steps of glucose breakdown. The difference lies only in the fact that the fermentative organisms are unable to complete the process and oxidize the end-products of fermentation, and instead resort to a variety of tricks to extort the maximum energy from their excreta before finally discarding them as alcohol, acetaldehyde, lactic acid, or other essentially half-digested substances. In this case the animals possess oxidative abilities that the fennentative microorganisms do not. But their basic biochemistry is not greatly different, only more efficient. A different class of anaerobes are those which can do without oxygen, not because they do not oxidize their substrates, but !)e. cause they find an alternative hydrogen acceptor and ultimate 'energy-sink'. Typically, they use either sulphate or nitrate as their acceptors; the nitrate-reducing bacteria, for example, convert nitric acid, HN0 3 • to nitrous acid, HN0 2 , and in doing so gain an atom of oxygen to which they can pass hydrogens and reduce to water. But the most fascinating thing about these nitrate-reducing bacteria, seemingly operating on such different principles from the animal world, is that the electron-transport pathways by which they pass their hydrogen to nitric acid are identical with those of the animals which use the oxygen of the air. The substrate yields its

269

The Chemistry of Life hydrogen to a dehydrogenase which passes it on to the intermediate hydrogen carriers which are cytochromes. The only difference is that, in the final stage, instead of using cytochrome oxidase to take the hydrogen from the cytochrome to oxygen, the nitrate-reducing bacteria oxidizf; their cytochromes with a different enzyme, nitrate reductase, which converts nitric to nitrous acid and water (Figure 41). FIGURE

41.

The difference between humans and nitrate-reducing bacteria substrate

dehydrogenase

cytochromes

cytochrome oxidase

nitrate reductase

HNO) ~ HN02 + H20 nitrate-reducers

;> H20

human

AUTOTROPHES AND HETEROTROPHES

But the real dividing line in the biochemical world comes between those groups we have just discussed, all of which depend on the existence of preformed organic compounds for survival, and those living things which can make all their own organic substances from simple inorganic materials. Such organisms are called autolrophes, indicating that they are self-sufficient by comparison with the heterotrophes, like yeast and humans, who have to be cushioned by the existence of sugars and amino acids against the harsh realities of the inorganic world. Autotrophes do not obtain their energy by burning ready-made fuel, but cast about to find an alternative energy-source. The paradigm case, of course, is the green plant, which avoids the heterotrophe's dilemma by trapping the light energy pouring

270

The Unity of Biochemistry down on to the earth from the sun, and using it to •fix' carbon dioxide as organic carbon and, ultimately, to synthesize sugars and starches, a process known as photosynthesis. The 'higher' green plant is not alone in performing photosynthesis; both algae and a· group of photosynthetic bacteria can perform similar reactions. Another group of autotrophes (the chemo-autotrophes as opposed to 'photo-autotrophes ') use the energy latent in certain inorganic chemicals for carbon dioxide fixation instead by the oxidation, for example, of sulphur, ammonia, or hydrogen. Although biochemically fascinating, these chemo-autotrophes seem to form an evolutionary backwater, an essay in chemical versatility that did not quite come off, and most attention has, not unnaturally, been focused on the photosynthesizing organisms, particularly as it was at one time believed that their self-sufficiency might provide the clue to the origin of life. THE MECHANISM OF PHOTOSYNTHESIS

The pro blems faced by the plant are in essence identical to those of the animal. They may be summed up as the need to trap energy and obtain a source of primary building blocks so they may carry out the biochemical synthesis of more complex molecules. Both forms of life obtain their precursors from essentially the same source- breakdown of glucose to CO 2 and H 2 0 via glycolysis and the citric acid cycle- the routes and the enzymes used are identical. The real difference lies, as we shall see, in the source of the glucose. Both plants and animals use ATP as their energy reservoir and in both it is formed by linking its synthesis to the passage of electrons along a chain of carriers. Again, the real difference lies in the source of the energy that is trapped in this way. Animals obtain this energy from glucose, and we have gone into some detail concerning the mechanism of ATP synthesis linked to the respiratory process. However, in green plants, in the light, glucose breakdown is not the main source of ATP. Any ATP formed is a result of substrate level phosphorylation at particular stages during the glycolytic and citric acid cycle pathway. Nevertheless, given glucose and a nitrogen source, plants can live for a very long time in the dark, though they are clearly much happier in the presence 271

The Chemistry of Life of sunlight. So even an autotrophic organism ,vi!! live !ike a heterotrophe given the right circ*mstances. Plants obtain the energy which is trapped into the ATP molecule by transforming the light they receive from the sun. During this process electrons are passed from H 2 0 to NADJl, releasing O2 and giving reducing power in the forin of NADPH 2 • So the net result is a reversal of the energy conservation process in animals, where NADH2 is oxidized to NAD and O 2 is reduced to H 2 0. But the essential feature is that a stepwise electron transfer pathway is again involved and is again linked to ATP formation. It is the ATP and NADPH z synthesized during this light reaction that is subsequently used to make glucose from the simple inorganic substances CO 2 and H 2 0, and it is this set of reactions which make up the process of photosynthesis. CO 2 fixation has been called the dark reaction because, if the plant already has adequate supplies of ATP and NADPH z, it can indeed take place in the dark, but normally, during daylight hours, both reactions take place together. The overall reaction then can be written: 6C0 2

6H 2 0

light energy

C6 H120 6

60 2 (1)

In this way plants provide themselves, and the heterotrophic world as well, with the only ultimate source of organic compounds at present available in the world outside the chemist's synthetic test tubes. The total' fixing' of CO 2 that occurs by photosynthesis is prodigious, providing as much as 100,000,000,000 tons of organic carbon a year. At the same time it continually renews the oxygen of the atmosphere and removes the carbon dioxide accu· mulated during respiration, providing a turnover so rapid that every molecule of CO 2 in the atmosphere gets incorporated into glucose by photosynthesis once every 200 years, and every oxygen molecule every 2,000 years or so. Between them, the photosyn· thetic mechanisms of the plant and the respiratory system of heterotrophes provide for the regular revolution of the 'carbon cycle' which takes so prominent a place in every child's biology book. The analogies between the photosynthetic light reaction and hydrogen transport in animals are not merely chemical or mechanistic, for photosynthesis takes place in a subcellular structure 272

The Unity of Biochemistry called the chloroplast which has a striking structurai simiiarity to the mitochondrion (Figure 42). Like the cristae of the mitochondrion, the grana and lamellae of the chloroplast provide the sites for ATP synthesis and hydrogen (electron) transport, and have a FIGURE

4Z. A chloroplast

grana

lipoprotein structure similar to that already described for the mi tochondrion. In the trapping of light energy. the first and most critical of the steps of photosynthesis, the substance chlorophyll (which gives plants, and, according to the advertisers, some toothpastes, their green colour) is all important. Although chlorophyll is by no means the only photosynthetic pigment, it is the only essential one. The molecule has a hydrophobic hydrocarbon tail by which it becomes firmly embedded in the chloroplast lamellae. The polar head part of the molecule is in fact very similar in design to that of the haem of the cytochromes and haemoglobin (see page 135). Like haem, it consists of a linked series of four carbon-andnitrogen containing rings C' pyrrolle rings') joined together to form a sort of doughnut with a hole in the middle. This hole is tilled in haem by the metal iron; in chlorophyll on the other hand 273

The Chemistry of Life the jam in the doughnut is made of magnesium. The ring structures contain a series of alternating double and single bonds,and the absorption of a given small amount of light (a quantum) of a particular wave-length causes a sort of vibration, or resonance around these bonds. Because of the close packing and stable orientation of the pigment molecules withiti the lamellae, this resonance energy can be transferred from one pigment molecule to another until it is eventually channelled into a slightly different chlorophyll. molecule from which it cannot escape. This final energy trapping type of chlorophyll receives an input from over 300 of the standard chlorophyll molecules. The energy from the light is thus very highly concentrated at a single site, giving the second molecule the ability to transfer an electron to a non-pigment receptor which in turn passes it, via an intermediate set of carriers, to NADP. The chlorophyll at the central site, which has thus become oxidized, is converted to its original state byaccepting electrons from the hydroxyl group of water. The protons from water are used to reduce NADP to NADPH 2 and oxygen is released in the process: 2H 2 0

+ oxidized chlorophyll ~ reduced chlorophyll + O2 + 4H+ 2(H) . + NADP ~ NADPHz

Recent research work has shown that there are in fact two different such reaction centres each receiving energy from two different pigment systems. The situation is further complicated by the fact that flow of electrons from H 2 0 to NADPH z involves the co-operation of both pigment systems in a sequence of events worked out by Hill and Bendall in 1960 and known as the Z scheme. The Z scheme bears, once again, a remarkable similarity to the respiratory electron transport chain, although it is eVen more complex and still poorly understood in parts, particularly the identity of many of the electron carriers. It is known, however, that they include plastoquinone, two cytochromes (f and b) and ferredoxin. The photosynthetic phosphorylation of ADP to ATP occurs if the difference in redox potential between the electron , carriers is sufficient to allow this endothermic reaction to take

274

The Unity of Biochemistry place, as with oxidative phosphorylation in the mitochondrion; but the exact mechanism by which this is achieved is still being investigated. Given the other similarities with respiratory phosphorylation the mechanism may well involve the pumping of protons across membranes as in chemiosmosis (page 138) and indeed in some of the original demonstrations of the existence of trasmembrane, proton gradients were carried out with chloroplasts. Let us return to the so-called dark reaction of photosynthesis. In it, both the NADPH 2 and the ATP formed in the light reactions are consumed in the fixation of CO 2 • The fixation reactions were charted by Melvin Calvin and his co-workers in Berkeley, Cali· fornia (for which Calvin received the Nobel Prize for 1961), with the use of radioactive carbon dioxide. During these reactions, CO 2 is made to combine with a pentose (S-carbon) sugar, ribulose diphosphate, to give an unstable 6-carbon intermediate which breaks down to two molecules of the 3-carbon phosphoglyceric acid.

noulose carbon dioKide

intermediate

pbospnogiyceric acid

Phosphoglyceric acid lies (see page 150) on the well-mapped pathway of glucose metabolism; some of it can be used to manufacture fructose and glucose phosphates (seepage 170) whilst some of the other molecules of phosphoglyceric acid are recombined through a maze of interlocking reactions to resynthesize ribulose phosphate once more. ATP is used, finally, to rephosphorylate ribulose phosphate to ribulose diphosphate, and the cycle can start up again. Most of the enzymes concerned are precisely those of the pentose phosphate pathway and the glycolytic pathway that we have already discussed, the exception being that enzyme which actually initially fixes the CO 2 , ribulose diphosphate carboxylase, which is a large complex allosteric molecule with a molecular weight of around 300,000, subject to inhibition and activation by many different substances. Needless to say, this particular enzyme is closely concerned with the control of the Calvin cycle,

275

The Chemistry of Life the metal ion Mg++ playing a cruciai part in the controi process. But the essential point to note is that, with the exception of the apparatus responsible for the splitting of water and hence providing the primary energy source, all the reactions of photosynthesis, fixation of carbon dioxide, and synthesis of sugars follow pathways with which we are already familiar in the biochemistry of the animal cell. Once again, we find that what at first sight appeared to be a major difference in biochemical systems, between photosynthetic green plants and heterotrophic animals, is in fact more startling for its similarities. COMPARATIVE BIOCHEMISTRY AND BIOCHEMICAL EVOLUTION

We have argued in this way without intending to deny the very real evidence of interesting biochemical distinctions between various species. And it is possible to interpret such differences in terms of a biochemical evolutionary process. Humans are probably right to feel themselves 'more advanced than anaerobic bacteria in that they can oxidize their food all the way to carbon dioxide and water, which demands more biochemical finesse and subtlety than the micro-organism, which can only tap off a small portion of the potential energy of glucose before being obliged to discard as refuse such energetically potent substances as alcohol or lactic acid. Similarly, very primitive organisms contain in their cells neither nucleus nor mitochondria, whilst more advanced ones have evolved them as an obvious contribution to the stability and efficiency of the cell. One of the most interesting of such examples of biochemical evolution in action has been demonstrated by Ernest Baldwin in his study of the nitrogen-elimination mechanism. It will be remembered that we have described the problem faced by the mammal in disposing of the nitrogen produced during the breakdown of protein; this nitrogen exists in the form of ammonia, and ammonia is extremely poisonous even in very small quantities. Thus the animal, in order to avoid building up lethal quantities of ammonia, COnverts it instead into urea and excretes it in the urine. Not all

276

The Unity of Biochemistry animal species dispose of their ammonia in this manner, however. Bony fishes, for example, are content to excrete ammonia intact and without further conversions. whilst reptiles and birds instead produce not the soluble urea but the highly insoluble uric acid, which is excreted as solid nodules.

ammonia

urea

llric acid

H, ......H

HN""'C'c _ ~

':;c=O

.pC, /C-N

N H

Why this difference in metabolism? Baldwin showed that it could be related to the availability of water to the different species. In fresh-water fishes, water is constantly available and continually diffuses into and out of the fish. Under these circ*mstances the ammonia formed will be washed out of the bloodstream as a very dilute solution and carried away into the surrounding water, without there being time for toxic concentrations to aCcumulate. Bony sea-fishes are in a similar position, complicated by the fact that the water in which they live contains salt at a higher concentration than that in the blood, and they therefore have to take steps against either too great an influx of these saIts from the sea, or too great a loss of water into the sea. So they can only excrete pari of their nitrogen as ammonia, but have to convert the remainder (about one-third) into other less poisonous substances. Land animals, though, are in a quite different position. They have to conserve water to avoid dying of dessication. So they cannot afford to dilute ammonia down with large amounts of water before getting rid of it; instead, they..turn it into the far Jess dangerous urea. The most interesting example of this adaptation is provided by the frog, which whilst a tadpole excretes nitrogen as ammonia, but, on changing into its adult, land-based form, also rearranges its internal biochemistry so as to make urea instead. Finally, the birds and reptiles. The ioung of these species are 277

The Chemistry of Life hatched from eggs, but unlike the water-borne eggs of fish, the eggs of birds and reptiles are laid on dry land. In order to avoid complete drying out while waiting to hatch, they therefore have hard shells which are impermeable to water. But this impermeability means that they have nq way at all of ,disposing of their waste nitrogen in solution. This clearly rules out making nitrogen into ammonia, and even disposing of it as urea would mean that concentrations of urea would begin to build up in solution towards the time when the egg is due to hatch, and these concentrations could be quite unpleasantly high (enough, as it has been described, to give the embryo a rather bad headache, at the least). So instead, the ammonia is converted to the quite insoluble uric acid, which is harmless and can be disposed of, not as a solution, but as small solid nodules. The whole story provides a fascinating example of the operation of natural selection at the biochemical level. But despite these examples of biochemical evolution and modification in progress, to which more are being added as the science of comparative biochemistry matures, it remains true that the biochemical composition and organization of all the forms of life now present On earth demonstrate unity; a unity quite at variance with their more obvious differences in gross structure and behaviour. Most of us would hesitate to compare ourselves with fish, typhoid bacteria, cancer cells, or even oak trees, yet the fact is that we have very much more in common with them than we might have guessed. Why? The forms of life that we know today have evolved over several thousands of millions of years, in the course of which they have branched out into such a host of diverse directions that their outward resemblances are remote. Yet during the whole of this evolution, their biochemical forms have remained remarkably constant. The only convincing explanation of this is that these biochemical parameters were established and fixed before the species began to evolve along the different pathways that biologists have traced. Insofar as the biochemical forms are identical, it must be the case that all existing species had a commOn ancestor about whose external shape and form we can only guess but about whose biochemistry we may be ~rtain that it was very similar to the bio-

278

The Unity of Biochemistry chemistr; of living tr.Jngs today. \Ve can express this relationship thus: humans and other animals

~Plants

primordial living organism =-

--~ b~cteria viruses

THE ORIG IN OF LIFE It is such an analysis that has led biochemists into one of the most exciting of their present-day hunts - that for a convincing description of the origin oflife 00 earth. If all current-day manifestations of life can be accounted for in strictly chemical and physical terms, as every biochemist is of course convinced, it follows that it ought to be possible also to describe the origins of these presentday life forms in chemical and physical terms as well. The alternatives would be either to assume that the primordial ancestor of both beast and human was set up by some non-chemical and physical intervention and then left to go along under its own chemical and physical steam for ever afterwards; or to maintain that life in one form or another has always existed, for as long as the universe itself, and therefore had no need to arise specifically anywhere. Neither of these possibilities is intellectually satiifying, nor are either of them at all necessary provided we can demonstrate a convincing way in which life could possibly have arisen without violating chemical and physical principles. If we can provide such an account, in broad terms, it does not matter unduly if some of the details later have to be amended in the light offurther scientific advances; the important thing is to show that, even in our presentday limited scientific state, we can nonetheless provide an explanation which is logically satisfactory. It is up to those who wish to mystify the nature of life then to criticize our hypotheses, if they 279

The Chemistry of Life are determined to try to show thai Hfe couid not have arisen in the way we propose. For many years the principle of spontaneous generation of everything from barnacles to micro-organisms was implicitly believed by the vast bulk of mankind. It was the rigorous investigations of Pasteur in France in the middle of the nineteenth century that conclusively demonstrated that life as we now know it could not have arisen spontaneously from non-living matter. Every living thing, said Pasteur, has arisen from another living thing. Nor should this surprise us, for even the simplest of present-day living organisms are highly complex, highly improbable molecular structures, whose chance assembly from their elements would involve odds of such astronomic unlikelihood that we may regard it, for practical purposes, as impossible. The chemicals which compose present life forms require to be synthesized by specifiCally catalysed reactions, and these specific catalysts are themselves the product of the living organism and cannot arise spontaneously. If we are to seek for the origins of the complex of attributes that we regard as life today, we must assume that these attributes evolved only slowly over the thousands of millions of years of blank history that separate the origin of the earth from even the earliest living form whose fossilized traces we can now observe. In order to provide some account of the way in which these attributes may have arisen, we need to be able to make plausible suggestions about the conditions that existed on earth at that remote time. The first rigorous and systematic attempt to do this was made in the 1930s by A. I. Oparin in the Soviet Union, and in its broad form his thesis is still the most satisfactory, though the advances of knowledge over the last thirty years have demanded many amendments in detail. Before Oparin, difficulties had arisen because it was assumed that the earth's primitive atmosphere was largely oxygen and that the first organisms to evolve must have been capable of performing photosynthesis in order to trap energy and synthesize the organic substances they needed. Yet photosynthesis, as we have seen, is a highly complex process clearly only possible to already well-developed and highly-skilled organisms. This dilemma was resolvedwhen Oparin was able to point out that the atmosphere of the primitive earth, far from being oxygen-rich, 280

The Unity of Biochemistry must have resembled that of the other planets, containing vast quantities of hydrogen, methane (marsh-gas, CH4 ), ammonia, and carbon dioxide. The present-day atmosphere has replaced this primitive one precisely because of many millions of years of life, turning methane and carbon dioxide into organic chemicals, and, by way of photosynthesis when it finally evolved, releasing oxygen. We may picture the primitive earth as containing huge warm oceans in which were dissolved a variety of salts derived from rocks, and over which hung an atmosphere of gases which would rapidly be lethal to any presently living organism. Under these conditions, a number of organic compounds would have begun to be formed and scattered in solution throughout the sea. The formation of these compounds would have depended precisely on the reducing atmosphere and the steady influx of energy in terms of light and ultra-violet radiation from the sun, for in those circ*mstances CO 2 , H 2 0, CH 4 , and NH3 can react to give a mixture of products including amino acids, urea, and many other substances. An interesting experimental verification of this statement has been provided by Stanley Miller in America, who passed an electric charge through a gas mixture ofhydmgen, methane, and ammonia in a closed water bath for periods of twenty hours or more. At the end of this time the products were analysed and found to contain more than eight different amino acids and seven monocarboxylic and dicarboxyIic acids, all of which are amongst the basic building blocks of present-day organisms. Similar experiments have even been able to demonstrate the synthesis of ATP and of small proteins under completely non-biological conditions of this type. Thus the primitive ocean must have steadily increased in organic content. These substances would have interacted with one another to form a whole range of new substances. The surface of the rocks and clays of the beds of the shallow seas, containing iron, magnesium, and copper, would have provided catalytic surfaces on which the organic substances would have begun to collect and to polymerize. As a result, short-chain peptides and nucleic acids, and possibly carbohydrates as well, would also have begun to accumulate, both bound on to mineral surfaces and free in solution in the seas. What followed was the most critical stage in the process. It has

281

The Chemistry of Life '-no--"-' t' __ - - - - - --ears 'h-' -0"":0-- ~~-+~:~:-~ l~~"D ~ole Al WlJ. JUi llH1UY Y' u en ~ 1UU U~ \,.VllLQ.lllJU& 1(:&.16..... I..... cules, such as the polymers of amino acids or carbohydrates, have a remarkable tendency to break up into small droplets containing the polymers in concentrated form, leaving the surrounding water comparatively free of dissolve~ substances. Salts and low-molecular-weigti.t organic substances present in the solution also tend to be sucked into these droplets together with the polymers. This phenomenon is called coacervation, and has a perfectly logical, though somewhat involved, explanation in physical laws. Such coacervate drops may be formed from mixtures containing, for example, gelatin or gum arabic, and have been extensively studied in the branch of physics known as colloid science. Oparin argued that, in the primitive oceans containing polymeric organic compounds, just such coacervate drops would have begun to be formed - the organic material would all have tended to coalesce into small, highly concentrated droplets. Within the droplets, the different compounds which had collected would have begun to interact with one another because of their new proximity. In some of the coacervate drops the results of these interactions would have been to make the drops unstable - by changing the pH, for example - and they would have broken up once more. Others, though, would have remained stable for longer periods, and with the passage of time have begun to grow as they collected into themselves more chemicals. But coacervate drops have an optimum size and, if they grow beyond it, they split into two or more smaller fragments, the composition of each of which will be similar to that of the parent drop. And so the process would have continued. Unstable coacervates would have broken down and their organic material have become available once more for incorporation into stable ones. Stable coacervates would have grown and divided. Within them, more and more complex polymers would have been formed. Metal ions acting as catalysts for favoured reactions, and coenzymes such as nucleotides, would have become more active as they became bound to the peptide polymers which were the forerunners of proteins, thus formingprolo-enzymes. Over the course of many hundreds of millions of years, the oceans would have become peopled with these stable, reproducing, primitive, semi·living droplets. At some t..~ __

282

The Unity of Biochemistry stage during this period in their deveiopment. the nucieic acids and proteins must have arisen as interdependent and mutuallysynthesizing molecules, to form the forerunners of the DNARNA-protein complex which is today responsible for genetic transfer. Meanwhile, as the coacervates continued to accumulate into themselves the organic substanccs in solution in the ocean, the availability of these substances must have steadily diminished. An evolutionary period must have arisen when there were not enough preformed organic molecules to go round. At this stage, natural selection would inevitably have favoured those coacervates which could make use of inorganic energy sources such as hydrogen sulphide - for a brief period, such autotrophes must have flourished more extensively than they do today. It would seem likely that it was at this stage in evolution that the mechanism ofphotosynthesis developed. Once the photosynthetic and autotrophic organisms had evolved, though, the conditions of existence for other organisms must have changed for the better - oxygen would have begun to appear in the atmosphere and the stock of preformed organic material in the ocean have risen sharply once mo~. Ultimately, a self-regulating 'carbon cycle' between heterotrophes and autotrophes would have come into play, and the era of life as we know it today would have opened. Such, in barest outline, is the Oparin hypothesis as it has been developed over the last thirty years by biochemists in Russia and elsewhere. Obviously, it raises innumerable questions and problems. Some are complex chemical issues, such as that of the origins of the universal existence in living organisms, but not in non-living nature, of' assymetric' molecules like those of the amino acids or sugars (see page 33). Nor are there yet very satisfactory accounts of the polymerization steps which produced proteins, nucleic acids, and so on from their more primitive ancestors. In addition, alternative hypotheses to Oparin's place the synthesis of prebiotic molecules in the hot, dry atmosphere of early volcanoes rather than the oceans (there are some laboratory ~xperiments showing that peptides and small proteins can be lbiotically synthesized under these conditions). Some still maiD:ain, like the astronomer Fred Hoyle, that the earth was 'seeded' 283

The Chemistry of Life with preformed macromolecules present on comets. Indeed, since the development of space probes and the' possibility of seeking for life forms on other planets, a whole new area - part experiment, part theory, called xenobiology - has grown up around the dis, cussion of such ideas. Other questions are more theoretical and philosophical. Just when, for example, in this evolution of living from non-living, can we be said to have stepped across the border between the two? What, in fact, is the definition of living as opposed to non-living? There are those who try to bring to bear rigorous tests on these questions - many biochemical geneticists, for ex:ample, are convinced that a system can only be described as living when it contains a nucleic acid-protein complex capable of precise selfreplication and mutation. Clearly, a key feature of today's living organisms is their capacity accurately to reproduce themselves. When and how did this evolve? Is the present genetic code an evolved form from some primordial ancestra[, simpler version? Did proteins once copy each other? These questions are not merely scholastic; even though they are not open to clear experimental answer, they remain deeply fascinating. However, to draw a hard and fast line between the living and the non-living is probably not a useful attempt. Clearly some thingsdogs, flowers, yeast cells - are alive. Others - such as molecules of salt, urea, or amino acids - are not. Between the two extremes lies an uncertain half-world filled with coacervate drops, viruses, and some biochemical preparations like isolated mitochondria or nuclei. There is no hard and fast dividing line between living and non-living, any more than there is between a fertilized ovum in the womb and a full-grown adult, or between a raw and a hard-boiled egg. The two ex:tremes are quite different, but the one is converted to the other by an infinite series of small steps. and it is only at the ex:tremes that one can be very precise. Such issues have raised heated debates at the various congresses on the origin of life that have been held over recent years. But they are debates which have been fought out within the framework laid down by physical and chemical theory and its applicabili ty to complex systems. This is not to say that life reduces to' mere' chemistry and physics. There are biological principles which express the 284

The Unity of Biochemistry . . re I'~' 'I orgamzlng atlonSulps between macromoJecu es," celiS, organisms, and which include within them an understanding of historicity; biological systems have to be understood in temporal as well as molecular terms if their development and evolution are to become meaningful. Such principles, however, are materialistic; to understand the ex:istence and origins of life, and of humans, needs no recourse to principles outside those of the material world.

C.o.L.-16

285

CHAPTER 14

CAN BIOCHEMISTRY EXPLAIN THE WORLD? In this book, we have moved rapidly across the-field covered by modern biochemistry. We have tried to do it fairly systematically. to separate the various stages in thought, experiment, and theory which characterize the biochemical approach to life, and at the same time to show how biochemists now believe that they can draw up a general balance-sheet of life which can account, in more or less broad terms, for those aspects of living behaviour we can study. Aspects of many fundamental life processes can now be described in quite precise molecular terms, analogous to those in which the chemist writes equations for the reactions of simple acids and alkalies, or the physicist for the quantum energies of the electrons of the reacting molecules. For many other qualities that go to make life we are far from bei ng able to do this; our under~ standing is still too superficial. Furthermore, many respects there are still fundamental theoretical problems in knowing what a 'complete' biochemical descrip tion of a living system would be. There are some molecular biologists and biochemists who believe that a total description of the physics and chemistry of the cell would readily extrapolate to a total description of the organism. For example, the entire nucleotide sequence of at least one simple virus is now known. But does such a specification say all there is to say abou t the properties of the virus? Or are there other things to say which. as I believe, can only be specified in terms of the history of the virus as an organism, its relationship to its environment and to its host or potential host cells. Today's molecular biologists. in their insistence on a rigid genetic. molecular reductionism, a total explanation of all there is to say in molecular terms, are, it seems to me, committing a philo~ sophical and scientific error akin to that of the physicist Kelvin in the nineteenth century who argued that a 'complete' physics was only possible when all phenomena could be reduced to mechanical

286

Can Biochemistry Explain the World? anaiogues - ciockwork models. This mechanical molecular materialism, which underlies the crudity of the 'centr\Ll dogma' of molecular biology that we discussed in Chapter 10, will need to give way to a much richer understanding of the need to interpret the phenomena of life at a series of levels, from the molecular to that of the population, and that we must never see any given level as either fundamental or static. All life has a history, a biochemical as well as an evolutionary and developmental history, and the task of the biochemist becomes that of understanding living processes at just one of these levels, and of coila bora ting in the discovery of the translation rules that relate biochemistry on the one hand to physics and chemistry, on the other to physiology, psychology, ecology. The account of biochemistry which has been given in these pages has perhaps falsely given the impression that this biochemistry represents the inevitable conquering march of science out of an ignorant error-ridden past into the glorious light of modern understanding. Such an impression would be quite wrong. Certainly the advance of biochemistry in the last half-century has been phenomenal, and we are still in the thick of this progress; there is no sign yet of slackening off. Biochemistry is still a young science, and many things only half-understood or mistakenly believed today will have to await a second and a third generation of researchers from now before they can be fully comprehended. Then those of us who have the temerity to publish our theories and concepts as those of the triumphant biochemistry of the 1970s will either be forgotten in the inex:orable advance of science or at best halfremembered as those whose insights were later verified or disproved. If we have emphasized in this book recent results and modern work, it should not permit us to forget that all of us now working in biochemistry stand on the shoulders of the chemists and physicists of the nineteenth century, and on those of the pioneers who created biochemistry in the early 1900s as a science where none existed before. Too many graduate biochemists and postdoctoral researchers seem to work on the assumption that what wasn't published in the most recent issues of Nature, Science, or one of the biochemical weeklies is archival, and what happened 287

The Chemistry of Life before 1960 prehistoric. In the last few years oniy, there have been some first steps towards the creation of a history of biochemistry; more of us need to study it. It is not only that we have to keep in mind how much is still not known, some of which we hav~ deliberately hinted at in the last few chapters; it is also that experiments, facts, descriptions which seem complete in one context may, with newer and greater understanding, need to be reshuffled and reinterpreted. Biochemistry has not yet been through the convulsions of the transition from Newtonian gravitational theory to Einsteinian relativity, that shook the foundations of physics in the early part of the century; it has certainly been through a pre- and a post-Copernican phase. The disputes over energy transport and oxidative phosphorylation are pro bably only a foretaste of the way thinking will have to change as biochemistry moves into the last decades of the twentieth century. The mechanisms of control processes and the regulation of the cell, the intimate details of the biochemistry of cell structure, the functioning of the cell as part of tbe organism as a whole, the biochemical mechanisms of genetic reproduction, hormonal control, and memory, to say nothing of the application of biochemical understanding to the prevention and cure of illness, disease, and malnutrition - these are all problems which seem to us today as large and in many ways as difficult and intractable as the determination of protein structure or the elucidation of the citric acid cycle did to oller biochemical elders. All of these are soluble problems, granted support, time, and a theor~tical approach which avoids an arid molecular reductionism. And such solutions are of importance not only to biochemistry itself, but outside too, for the practical application of results based on biochemical technology could be of great significance. So far, biochemistry has been associated with relatively little derived technology. Biochemists are to be found in the wine and food industries. (Pasteur was perhaps the first.) The modern pharmaceutical industry swallows the talents of others, although the biochemical rationale for the mode of action of nearly all drugs is still lacking and much of the work of devising new agents a sort of molecular roulette imposed by market demands rather than hwnan needs. Industrial microbiology is a growing field, as more 288

Can Biochemistry Explain the World? and more compiex substances are being produced by allowing the enzymes within micro-organisms free rein, rather than by chemical synthesis. The proportion of biochemistry graduates who go into industry, at least in Britain, is steadily increasing. But the breakthroughs likely in the next few years portend far more than this: potentially revolutionary medical technology; the advance of molecular biology toward the vexed field of genetic engineering; the application of understanding of brain mechanisms as techniques of mind control by precisely tai lored molecu les. Such possibilities have been actively canvassed. Will they be beneficial? Above all, this depends on the shape of the society which permits or denies their application. Some have their doubts. The development of anxieties over both the theoretical prospect of biological warfare and the actual use of chemical warfare do little to allay them. The recent furore over genetic engineering, in which molecular biologists themselves attempted to raise the question of the actual and potential hazards of techniques that were currently under development, has highlighted some of these problems. It is not a question of biochemists, or molecular biologists, as gods in white coats threatening, or sensitively refraining from threatening, the future of the rest of humanity ; that depends m0re on the structure of our society than the structure of our science, though each helps determine the other. However, biochemistry is •special 'knowledge, to which access is limited. Those who have it, have also the responsibility of spreading this knowledge, helping it to serve, not oppress the people, and learning the limitations of their own special knowledge in the process. To answer the question which forms the title of this final chapter: biochemistry by itself alone is not enough to explain the world or even the human portion thereof. However, it is an essential part of the totality of that explanation which is the goal of true science..

289

A NOTE ON FURTHER READING There are several good biochemistry and cell biology text-books available which could be read without difficulty by anyone who has got so far through this book. Amongst them we could include Comprehensible Biochemi3fry by M. Yudkin and R. Offord (Longman!>, 1973) and Biological Chemistry and Basic Biological Chemistry by H. R. Mahler and E. M. Cordes (Harper and Row, 2nd ed., 1971); Biochemistry by A. L. Lehninger (Worth, 2nd ed., 1975) IS excellent but expensive. A more general biological text is Cell Structure and Function by A. G. Loewy and P. Siekewitz (Holt Rinehart & Winston, 2nd ed., 1970). Special topics are enlarged upon in, for instance, Molecular Biology of the Gene by J. D. Watson (Benjamin, 3rd ed., 1976), Membranes and Iheir Cellular Function by J. B. Finean et al. (Blackwell, 1974), Hormone Action by A. M. Malkinson (Chapman & Hall, 1975) and Basic Neurochemistry by R. W. Albers, A. J. Siegel, R. Katzman and B. W. Agranoff (Little, Brown, 2nd ed., 1976). There are a.number of reprints of SCIentific American articles on cell biology and biochemistry published by Freeman, which are excellent value for money, and so too are the course units of a number of relevant Open University courses, especially S202 (Biology: a functional approach, Open University Press, forthcoming) and S322 (Biochemistry and Molecular Biology, Open University Press, 19n). These are available from university and general booksellers. The history of biochemistry too has begun to be a topic in its own right and J. S. Fruton's Molecules and Life (Wiley, 1972) is well worth reading. So is The History of Cell Respiration and Cytochrome by D. Keilin (Cambridge, 1966) and The Palh to the Double Helix by R. Olby (Macmillan, 1974). Finally, for the interested reader wanting to keep abreast of the most sigoificant new developments in biochemistry and related sciences, there is no substitute for one of the popular. science magazines such as SCientific American or New Scientist, which carry articles often by leading specialists in the field. These are frequently brilliant and not obtainable elsewhere and a subscription to one or both would pay off handsomely.

290

INDEX synthesis, 136-40 yield from f1-oxidation, 164 Adenylation, 127 Adenylate cyclase, 229-31 Adipose tissue, 76-182 Adrenal gland, 227,236 Adrenalin, 227, 237, 239 Agar, 55 Agonist, 228 Alanine, 38, 56,187 Alcohol, 16,28,30,74,102,116, 143,269 alcohol dehydrogenase, 116 Aldolase, 145 Aldose, 33 Aldosterone, 227, 241 Alkalies, alkalinity, 21,22,63 Alkaptonuria, 122 Allostery, 114-15. See also Enzyme Amino acids, 38-9,55-66,281 activation of, 195 AMP complex, 197 analyser 60,94 breakdown, 240 essential, 174 glucogenic,167 ketogenic, 167 oxidation of, 167 in protein synthesis, 185-206 Amino acyl synthetase, 195, 197 Amino acyl t-RNA, 201 p-Amino benzoic acid, 113, 114 Amino group, 39,56,169,202 Ammonia, 12, 166, J67, 168, 175, 271,276,277, 28t Amoeba, 80,141 Amylase, 107, 141 Amylopectin, 54 Amylose, 54 Amylo-transglycolase, 177

Abderhalden, E., 57 Aeetabularia, 191 Acetaldehyde, 161. 269 Acetamido group, 52 Aceticacid,25,31,151, 162, 179 Acetoacetic acid, 161, 162, 164, 180 Acetone,24,161 Acetylcholine. 264. 265 Acetyl CoA. 152.153.154,156, 163,164,170,178,180,264 ea rboxylase, 180 Acids and acidity, 21, 22, 63 Acid phosphatase, 104 Aeon itase, 154 Aeon itic acid, 154 Acrolein, 76 Actin.251-3 Activators, see Enzyme Active centre, see Enzyme Act ivc transport, 88, 222 Actomyosin, 251, 252 Acyl carrier protein, 180 Acyl CoA, 162, 163, 179, 181, 182 Adenine.41, 67, 69, 72,151,195 Adenosine diphosphate (ADP), 136-40,146,155, 173,212 Adenosine monophosphate (AMP). 137,162,229 cyclic AMP, 229-31,232,237, 265 Adenosine triphosphate (ATP), 17, 41,127,141. 144, 146,161, 164. 173,179,180,182,185,195, 207,212,223,235,251,253, 263,281 ATPIADPrations, 213-14 ATP-asc ,138,212,214 and membrane transport, 263 and rllotosynthesis, 271 production, 157 291

Index Anaboiislil, ji, i2i Anaemia (sickle cell), 43,58 Angstrom, 63 Antagonist, 228 Antibiotic, lOS, 114 Antibody, 119 Anticodon, 200, 201 Araban, 52 Arabinose. 52,54 Argenine, 39, 60, 68, 175 Aspanate semi-aldehyde, 217, 218 Aspanic acid, 39, 175,217 Aspanokinase, 218 . Astbury, W., 64 Atom; 19-20 Atomic weight, 20, 97n. Autotrophe, 270, 283 chemoautotrophe, 271 photoautotrophe, 271 Axon, 255, 257, 263

uuubie, 23, is, j02 eiectrovalent,23 hydrogen,46,52,61,63,64,72, 191,195.221 hydrophobic, 87 peptide, 56"60,185,195,200, 201,203 phosphate, I3 7, 148, 197 saturation, 33, 75 weak,46,47,61,63,64,72,191, 195,221 Brain, 123, 169,225,236,243,246, 254,256,264 Brenner, S., 199 Buchner brothers, 16, 102, 143 Buffer, 20, 94, 124 Butyric acid, 76

Calcium, 28, 30, 124, 223, 232, 252-3,265 gate, 232 phosphate, 28, 241 Calvin, M., 121,275 Capillary, 55, 128, 225 Carbohydrate, 37,47,59. See also Polysaccharide Carbon, 19, 22,28 burning of, 91, 100, 120 cycle, 283 . Carbon dioxide, 17, 152,281 as a catalyst, 179-80 fixation, 98, 121, 156, 175,271, 272,275 Carbonmonoxide,21,212 Carboxyl group, 25, 39, 56, 197,202 Carnitine, 162 Cascade system, 237,238 CatabOlism, 17, 127, 173 Catalysis, catalyst, 16, 102-4,282 Catechol, 129 Cell, 27, 79-93,222,207 division, 189,232 muscle cell, 175, 248 nerve cell, 255 wall,80 Cellophane, 52

Bacteria, 80, 105, 113, 143, 156, 172,202,205,217,218,219, 221,269 anaerobic, 269 nitrate reducing, 269 photosynthetic, 271 Baldwin, E., 276 Banting, F., 236 Base pairing, 41,72,295 Basic metabolic rate (BMR), 234, 235 Benzene, 25 Bendall, F., 274 Bernard, C.,18, 94 Berthelot, M., 15 Berzelius, J., 16, 55 Best, C., 236 Bile acid, 78 Biotin, 180 carboxybiotin, 180 Bond,23 bond breaking enzymes, see Lyase, 116 C-C bond formation, 153 covalent, 23, 46, 56, 74, 197

292

Index Cellulose, 43,48,51-1,54,63,80 Cent ral dogma, 206 Centrifugation, 44,83-6 density gradient, 85, 125,228 differential,92 ultra, 59, 79 Chance. B., 132 Chargaft', E., 69 Chemical gradient, 87 Chemiosmosis, 138-9.212, 274 Chitin, 51, 55 Chlo ride, 3D, 79 Chloroform, 68 Chlorophyll,273-4 Chloroplast, 273, 275 Cholesterol,77 Choline, 264 acetylase, 264 Chondroitin sulphate, 55 Chromatin, 89,233. Chromatography,44,58,.59,68,228 Chromosome, 90, 189-91 Chymotrypsin, 61, 141 Chymotrypsinogen, 62 Citrate-condensing enzyme, 154 Citric acid, 31.153,154 cycle, 153, 158,165, 175,215,271 Coacervation. 282-3 Codon, 200. 201,203,205 CoenzymeA.127,151,152,155,162 Coenzyme Q, see Quinone Collagen, 63, 184 Colloid, 15 Compartmentation,222 Compound, 19 Con[Jective tissue, 63 Co-operativity, 114. See also E[Jzyme Corey, R., 64, 72 Coronary thrombosis, 77 Corticosterone, 237 Cortisol, 211 Cortisone, 211 Covalency, ~O Creatine phosphate, 127, 139 Crick, F., 71, 72,191,199,206

293

Cristae, 90 Curare, 266 Cyanic acid, 12 Cyanide, 135,212,262 Cybernetics, 17, 186,201,208,244 Cysteine, 61, 110, 112, 175, 185 Cytochemistry, 80 Cytochrome, 134-6,270,273,274 oxidase, 135 Cytology, 80 Cytoplasm, 82,89-90,93,110, 162, 221 Cytosine, 39,67,69, 72 triphosphate CTP, 218 de Duve, C., 91 Denaturation, 47,66 Deoxyribonucleic acid (DNA), 18, 37,61,67-74,89,190,206,233 chain opening, 195 conformational change, 195,232 DNA-ase, 191 initiation point. 192 operator region. see Operon polymerase, 192 primer, 193 promotor site, 195,219,220 replication, 191-3 structure, 73 transcription, 194-5 Diabetes, 160, 164, 236 Dialysis, 112, 131 Dickens. F., 17,158 Diffusion, 88 facilitated, 88,222 passive, 87 Dihydrolipoic acid, 152 Dihydroxyacetone,33 phosphate, 145, 161 Diphosphoglyceric acid, 146 Disaccharide, 47 Disulphide bridge, 61-2,66, 109 Elasticity, elastin, 55, 63 Electric charge, 19,23,38,86, 110, 113,209,281

Index Electric charge - cont, electronegativity, 30, 39, 46 electro positivity , 39 in muscle, 253 in nerve transmiSsion, 257-66 Electrochemical gradient, 87, 232 Electron, 19, 129 artificial donors, 138 carrier, 129-36 transport chain, 146. 213, 214, 272,274 Electron microscope, 15,52,79,81, 82,89,190,235,249 Electrophoresis, 45, 58, 59,218 electrophoretic mobility, 45 gel,45 Electrovalency,20 Element,19 Embden, G., 17,143,151 Embden-Meyerhofpathway. see Glycolysis Endocrine gland, 225 Endoplasmic reticulum, 89, 92, 181, 194 End-product inhibition, 217 Energy, 88, 91, 95-100 activation, 100,101,103 coupling, 128 for DNA synthesis, 193 during glucose oxidation, 157 law of conservation of, 97, 99 light, 98, I 72, 270. See also Photosynthesis potential, 96, 100,101, 130 resonance energy, 274 trapping, 274 Engelhardt, V. A.,252 Engels, F., 66 Enolase, 147 Enolpyruvate, 148 Enzyme, 12, 16.51,60,66,9(, 100-116,118-19,209 activator(coenzyme), 112, 149, 173,209 activecentre,I09,110, 112,113. 187.209.114

294

affinity, lOS allosteric, 114, liS, 192,209,214, 229,275 classification, liS conformational change, 214 dehydrogenase, 129, 131, 146, 215,270 esterase, 104, 112.115,116,181 heat sensitivity, 107 induction, 219 inhibition, 113-14,209 isoenzyme, 218 kinetics, 105-9 lipase, 141-61 mechanism ofaction,I09-12 mufiisubstrate enzyme, 114, liS, 146 oxidase, 129, 168 pH sensitivity. 101 phosphatase, 112, 170 prosthetic group, 112 protoenzyme, 282 repression, 219 substrate complex, 106, 109, 110 transaminase, 168, 240 Equilibrium, S8,I03, 176, 180,209, 226 Ester, 74, 104 esterification, 183 ester linkage, 74, 161 Evolution, 55, 159,275-85 Exoskeleton, 55 Fat, 74-6, 77 synthesis. 181,216 Fatty acid, 31-3, 74, 76,159,181 breakdown, 160 synthesis, I 78 Fatty liver, 161,227,236 Feedback, negative, 210, 217, 223,

243 positive, 210, 223, 261 Fermentation, 16. 102, 143, 268, 269 Ferredoxin, 131, 132,274 Fibroin, silk, 63, 64

Index Fischer, Eo, 15, 57 Flame photometer, 28 Flavin, 132-3 adenine dinucleotide FAD, 133, 152 mononucleotide FMN, 133 Flavoprotein, 133, 162, 164 Fluoride, 148 Foder, A., 57 Follicle Stimulating Hormone (FSH~243 Formic acid, 27 Formyl methionine, 202 Fructose, 34, 36, 54 1,6, diphosphate, 144, 148, 170, 214,224 6, phosphate, 144, 214,224 Fumarase, 156 Fumaric acid, 156 Galactosamine, 37, 47 Galactose, 34, 54, 176,219 Galacturonic acid, 55 Gamow, G., 199 Gelatin,282 Gel filtration, 45,58 Genes, 188, 190,219 Genetics, 71, 186 code, 202,284 defect, 122 GHa,265 GlUcagon, 227, 236,239 Glucocorticoid, 233, 239-40 Glucosaminoglycan,55 Glucose, 20,34,36,48-52,98, 117, 123,170 burning of, 99,217 metabolism, 141-9 oxidation, 142, 149-59 phosphorylation, 143-4 6-phosphate, 158,144, 159 I-phosphate, 159, 176 polymerization, 176 Glutamic acid, 39, 123, 168, 169. 175.264 dehydrogenase GDH, 168

Glutamine, 185 synthetase, 218 Glutathione, 185 Glyceraldehyde, 33, 39 phosphate, 145, 146, 147 Glycerol, 74, 76,161,182 phosphate, 161, 181 Glycine. 38, 56, 82.175,185,187 Glycogen, 17,48,51,54.76,123, 141,236 breakdown. 159-60,237 phosphorylase, 159, 175 synthesis, 175 synthetase, 177, 237 Glycolysis, 142-9,223,271 anaerobic, 142, 149 reversal of, 170 Glycosidic linkage, 36,49, SO, 56 / Goitre. 234 Goigi body, 89, 225 Grey, G., 264 Group transfer molecule, 127, 139, 151,152,154, 155,218 Growth hormone, 241, 242 Guanine, 41,67,69,72 trinucleotide phosphate GTP, 155,194,203,206,230 Gum arabic, 282 Haemoglobin, 42, 128, 135, 142, 184, 190,273 Haldane, F., 28, 288 Harden, A .• 1~3 a-Helix, 64. 65, 72 Heterotrophe, 270, 283 Hevesy, G., 95 Hexanoic acid. 163. 164 Hexokinase. 144. 159. 160, 170, 223 Hexose, 34, 145 Hill, A. V.• 274 Histidine, 218 Histochemistry. 80 Histology, 80 Histone, 68. 89,190,221,232,233 Hoagland. M., 197

295

Index Hodgkin, A. L., 261, 262 Homeostasis, 18,95,224,245,246 hom*ogenate, 28, 91,125 hom*ogentisic acid, 122 hom*opolysaccharide,50 Hopkins, F., 13 Hormone, 66, 61, 78, 216, 221, 224-5 general properties, 225 kinetics of bin ding, 228. 229 mechanism of action, 228-32 peptide hormones, 57, 225, 228-32 prohormone, 225 receptor interaction, 228, 232, 233 secretion, 89, 225 sex hormone, 78, 216, 227, 233 steroid hormone, 225, 221, 233 subunit, 230 trophic hormone, 243 Hoyle,-F., 283 Huxley, A., 262 Hyaluronic acid, 55 Hydrogen,20,28,82,271 carrier, 129-36,212,215,270 donor, 173-4. See also Nicotinamide adenine dinucleotide (NAD) ion, 21-2 sulphide, 283 Hydrolase, 116 Hydrolysis, 43n., 50,60,70, 104 Hydrophobic, 32, 86, -139,214,233,

273 Hydroquinone, 134 Hydroxybutyrate, 104, 161 Hydroxyl ion/group, 21, 22, 177, 192,191,274 Hyperthyroidism, 234 Hypothyroidism, 233

competitive, 113, 114,119 non-competitive, 115 uncompetitive,114 See also Enzyme Initiation factor, 201. See also Protein, Synthesis Insulin, 15. 62. 125,227,236.239, 240,241,242 Iodine, 234 Ion. 20, 86 bicarbonate. 178.241 concentration in blood, 240-41 cuprous, cupric, 135 electrode, 28 ferrous, ferric, 129, 135 gradient, 88, 259, 261 ionic environment, 205, 209, 240 ionic interaction, 66 metal, 105, 110, 112. 113,282 and nerve transmission, 258-66 pump, 88 Ionization, 11 Ion binding resin, 43 Islets of Langerhans, 236 Isobutanol.24 Isocitric acid, 154 dehydrogenase, ISS Isoleucine, 175 Isomer, Isomerism, 24, 33, 57, 73, 145 isomerase, 116 optical isomer, 34 stenio isomer, 36 Isotope, see Radioisotope Jacob, F., 218 Joule, 97n. Keiiin, D., 132, 161 Kelvin, 286 Keratin, 63, 64 Ketose, 33, 163 ketone bodies, 161 ketone grouP. 155, 169 Ketothiolase, 163, 179, 180 Keynes, R., 261 , 262

Imino acid, 167--8 Immunology, 119 In formation theory, 184. See also Cybernetics Inhibition, 118, 122

296

Index Kidney, 240-41 Kin.etics, 16,96,105,125 Knoop, F.,161,162 Krebs, H., 17, 143,167. See also Citric acid cycle Lactic acid, 104, 142, 149,269 dehydrogenase,l04, IDS, 129,149 isomer, 105 Law of mass action , 208 Le Chatelier, H.,94 Lehninger, A., 161 Leloir, L., 176 Leucine, 175 Liebig, J. von, 15 Ligand, 228 Ligase, 116 Lipid, 31,43, 59, 74,87,88 bilayer, 77, 86-8, 90,139 fluidity, 74, 87, 88 lipoprotein,86,130 Lipmann, F., 17, 51 Lipoic acid, 152, 155 reductase, 152 Liver, 123, 141,167, 169,175,227. 234,236,23~240,242

Luteinizing hormone, 243 Lyase, 116, 162 Lynen, F., 161 Lysine, 39, 60,175,217 Lysolecithin, 77 Lysosome, 91, 222 Macromolecule, 15, 16,24,29,39, 42-78,94,95 purification and structure, 44 structural hierarchy, 46 Magnesium, 30, ItO, 124, 147, 194, 205,209,230,274,275 Malic acid, 32, 156, 170 dehydrogenase, 156 Malonic acid, 179 Malonyl CoA, 179-81 Maltase, 116 Maltose, 36, 116 Manganese, lIO, 180

297

Mannose, 34, 3S Matthaei, H., 200,203 Membrane, 76,86-9,110 carrier protein, 88, 89 cell membrane, 80, 83, 94,222 depolarization, 263 fluidity, 231, 232 mitochondrial, 138, 139, 162,222, 223,235 muscle cell membrane, 258-9 nuclear, 81,90,195 pore, 232 potential, 259, 261 pump, 88, 232 selective permeability, 86, 222. 232,259 Mendel, G., 186 Mercury, 113 Meromyosin, 251-3 Metabolism, 17, 117-26, 127,268 Methane, 22, 281 Methionine, 175, 202, 211 Meyerhof, 0.,13,11,143 Micelle, 52, 77 Michaelis constant, 108 Michaelis-Menten equation, 108 Microenvironment, 46, 47 Miescher, F.,67 Miller, S., 281 Mineralocorticoid, 233, 241 Mitchell, P., 138 Mitochondria, 81, 82, 90-91,130, 162,212,223,234 inner membrane, 133-9 Molecules and molecular weight, 19,21,45 Monod, F., 218 Monoglyceride, 182, 183 transacylase, 182 Monolein, 182 Morphine, 264 Mulder, G., 55 Multi-end product inhibition, 218 Muscle, 30, 141,143,175,234,237, 246,248-54 contraction, 140, 232, 253-4, 256

Index Oil,74-6 Okazaki, 192 Oleic acid, 75, 162 Oligosaccharide, 48,50 Oparin, A., 280, 282, 283 Operon, 120. Organic acid, 31 Orthophosphoric acid, 30 Osmotic pressure, 86 Ovary, 227 Oxaloacetic acid, 32, 154, 156, 165, 171 decarboxylase, 156 Oxalosuccinic acid, 155 Oxidase,see Enzyme, classification Oxidation, 21,43, 104, 128-9,136, 173,212,234 p-oxidation, 162 Oxidative decarboxylation,lSI, 155,170 Oxidative phosphorylation, 136-9, 212-14,235,263 a-Oxoglutaric acid, 154, 155,168, 175,240 Oxygen, 19,28,96-100,131,135, 283

Muscle - cont. structure, 148-50,252 Mutation, 122 Myelin, 255 Myosin, 251-3 ~erve,30,

140,225,226,254,255 depolarization, 260, 265 hyperpolarization, 265 transmission, 257-66 Neuromuscular junction, 255 Neuron, 255 Neurotransmitter, 263~ Neutron, 19 Nickel, Raney, 102 Nicotinamide adenine dinucleotide (NAD), 131, 132, 133, 136, 137, 146,147,148, 152, ISS, 161, 178,213 NAD/NADH z ratio, 21S phosphate, 131, 155, 159,174, 178,181,216,272,274 Nicotine, 266 Nirenberg, M., 200,203 Nitrate, 269 reductase, 270 Nitrate acid, 269 Nitrogen, 28, 38,98,122 balance, 122,166,240,241 excretion, 275-8 Nitrous acid, 269 Nucleic acid, 15,29,39,67-74,81, 199,283 as template for protein synthesis, 188-91,205 Nuclein, 67 Nucleolus, 90 Nucleosome,190 Nucleotide, 37, 41, 69,105,112,197, 199,281 phosphate, 41, 127, 192 Nucleus, 67, 81, 82,89,195,221 Ochoa, S., 143, 201 Oestrogen, 227,243 Ogslon, A., 143

Palmitic acid. 75. 162 Pancreas, 227,236 Parathormone, parathyroid, 227, 241 Pasteur, L., 34. 223, 280 Pasteur effect, 223-4 Pauling, L., 64, 72 Pectin, 54 Pentose, 37, 52, 158 phosphate shunt, 158, 175, 275 Pepsin, J08, 115,141, 185 Peptide, 56. 57,58,59,282 Peptidy1transferase, 203, 205 Perfusion, 123 Peters, R., 152 pli,22,44,47,66,86,94,96,IOO, 107, 110, 149,210,282 Phage, 200 Phenotype, 190

298

Index Phenylalan ine, 61, 175, 201 Phenylketonuria, 122 Phosphatase, 170, 182 Phosphate, 3D, 59, 69, 76,93, 136, 146,159,176,192,212 amide, 31 amin 0 acid phosphate, 31 ester, 30 hydroxyacid phosphate, 31 nucleotide,31,41,192 salt,30 sugar phosphate, 31, 176 Phosphatidic acid,.I82 ph osphatase, 170, 182 Phosphatidyl inositol, 231 Phosphodiesterase, 232 Phospheonolpyruvate, 147 Phosphofructokinase, 144, 170,214 Phosphoglucomutase, I I 6, 160 Phosphoglyceric acid, 145, 147,275 Phosphoglyceromutase,I47 Phosphohexoisomerase, 144 Phospholipid,76-7 Phosphoric acid, 67-8 Phosphorylase, see Glycogen, phosphorylase Phosphorylase kinase, 237, 238 Phosphorylation, 127, 136, 143, 175, 18J,182,214,223,231,237-8 photosynthetic, 274 substrate level, 146, 155,271 See a/so Oxidative phosphorylation Photosynthesis, 121,271-6,280 Pituitary, 227, 241, 242 Polarity, 23, 74,76,88 Polymerization, 36, 43 Polynucleotide phosphorylase, 200, 201 Polyphenoloxidase, 129 Polysaccharide, 29,36,37,38,47, 48,49,54,55 . Polysome, 92, 205 Potassium, 30, 79, 93, 124. 194.205, 222,258, 261 Precursor, 121

299

Progesterone, 233 Proline, 175 Proprionaldehyde,24 Prosthetic group, 112, 130, J35. See also Enzyme Protein, 38, 55-66, 87 acidic chromatin protein, 221. 232,233 as energy source, 165-6 carrier protein, 88-9 elongation factor, 203 fibrous, 63, 65 flavoprotein, 133 globular, 63. 66 glycoprotein, 87,228 lipoprotein, 86,90.130 metalloprotein, 59 mucoprotein, 59 nucleoprotein, 68 phosphoprotein, 59 plasma protein ,225 regulation of metabolism, 241-2 repressor protein, 219, 220 ribos()mal protein, 92 structural protein, 63 synthesis, 184-235 Protein kinase, 231, 237 Protoeglycan, 52 Proton, 19, 30, 31, 138 gradient, 139,212,275 Protoplasm, 81 Purines, 39--41,67-74,175 Pyrimidine, 39--41, 67-74, 175 Pyrrole, 273 Pyruvic acid, 104, 142, 143, 148, ISS, 156, 165, 170 decarboxylase, 148 decarboxylation, 151,212,268 phosphokinase, 148 Quinone, 133-8 plastoquinone, 274 Radioisotope, 95, 120 labelling, 120,217,228 Rancidification, 76

Index Reaction, endergonic, 98,128,137, 152,162,174,274 equilibrium, 179 exergonic, 97,127,137,152,174 irreversible, 21 rate, rate-limiting, 106-8,209-11,. 215,235 reversible, 21, 103,208 Receptor, 228, 265 Redox potential, 130, 274 Reduction, 104, 148, 173 Reductionism, 286 Replication, see Deoxyribonucleic acid Reproductive cycle, 243 Respiration, see Oxidative phosphorylation rho factor, 195 Ribonuclease, 62, 70, 71 Ribonucleic acid (RNA), 61,67-74, 89,90 polymerase, 194,219, 22Q, 232, 233 primer for DNA synthesis, 193 in protein synthesis, 193 m-RNA, 194, 198,200,205,219, 233,240,242 r-RNA,92, 194, 205 in transcription, see DNA Ribose,37,41, 67,71,137,158,192, 194 5, phosphate, 175 Ribosome, 82,90,92. ]04,194, 198, 201,202,203,205,268 Ribulose-5-phosphate, ISS carboxylase, 275 dipDosphate,275 Salt, 19, 21, 113 balance,' 240 Sanger, F., 15,61 Sarcomere, 249 Schoenheinler, R" 95, 121 Second messenger, 229 Sense receptor, 246 Serine, 57, 112,175,231

Sodium, 19,20,30,79,93,124,222, 258,261 dodecyl sulphate SOS, 45 fluoride, 147 hydroxide, 21 Somatomediri,242 Spallanzani, L., 12, 16 Spectrophotometry, 109 Spinal cord, 225 Squid, giant axon, 262 Standard free energy, 98, 127, 174 Starch, 16,48,52,76,98 Stearic acid. 75, 162 Steroid, 77, 159.239 Stimulus-contraction-coupling, 232, 253 Stimulus-division-coupling, 232 Straub, F., 251 Structural gene, 219 Substrate, 105, 106, 110, 113. 114, 117.209 substrate level phosphorylation, 146 Succinic acid, 115, 133, 136, 138, 154 dehydrogenase, 115, 133, 156,235 Succinyl CoA, 155 Sucrose,36,48,85,125 Sugar, 16, 33-8 Sulphanilimide, 114 Sulphur, sulphates. 28. 271 Sulphydryl group, 112, 113, 146, 151,180 Sumner, J., 12, 14, 105 Synapse. 255. 256,263-6 Synaptic vesicle, 265 Synge, R., 57 Szent-Gyorgyi, A., 143, 151,251 Template, 188, 199,200 Testis, 227 Testosterone, 227 Thermodynamics, 100, 103,209 Thiamine, pyrophosphate, 152, 15S Thioesterification, 162 Thiokinase, 162

300

Index Thiolysis, 163, 179 Threonine, 175,218 Thudicum,J.,15 Thymine, 39, 67,72,195 Thymus gland, 67 Thyroid gland, 214, 227. 233 Thyroxine, ]25, 214, 227, 233, 239, 243 Tissue slice, 124 Tracers, 120--21, 158,164. See a/so Radioisotopes Transamination, 168 Transcription, 194,219. See a/so Deoxyribonucleic acid (DNA) Transducer, 230--31 Transferase, 116 Transhydrogenase, 216 Tricarbox.ylic acid cycle, See Citric acid cycle Triglyceride, 75, 182 Triose phosphate isomerase, 145 Trisaccharide, 48 Trophic hormone, 243 Tropomyosin, troponin, 251-3 Trypsin, 60, 11.5, 141, 185 Tryptophan, 175,218 Tswett, M., 60 Tyrosine, 61,123, 187,234 Umbarger, E., 127 U ncoupl ing, 136, 214, 235 Uracil, 39,62,69,176,195,200 Urea, 12,27, 166, 167,240,241, 276 Urease, 12, 105 Uric acid, 277, 278 Uridine diphosphate, UDP, 176, 178 galactose, 219 glucose, 176, 177

U ridine triphosphate, 176, 177 Urine, 27, 122, 161,166,276 V-agent, 266 Valency, 20 Valine, 175 Vasopressin, 241 Virus, 61, 81, 199,284 Vitamin, 172, 268 B I, 152 Biotin, 180 D,78 E.134 K, 134, 172 Vm,I06 Wakil, B. J., 179 Warburg, 0., 13, 17, 132, 1.58 Warburg-Dickens pathway, see Pentose, phospha te sh u n t Water, 17,20,47,79,154,20.5,214, 272,274 Watson, J., 71,72,191,206 Wax, 76 Weiner, N., 17 Whittaker, V., 264 Wilkins M., 71 Wohler, F., 12, 14 Work,94-IOO X-ray, 122 crystallography, 63 diffraction, 45, 63 Xenobiology, 284 Xylan, xylose, 52 Yeast, 16, 80, 102, 143, 268 . Young, N., 143 Z scheme, 274

Biochemistry has been at the centre of a vast expansion of biological understanding_ Its methods and flndings unify our knowledge of the mechanisms of life: biochemical theories approach questions ofthe origins of life on earth; biochemical techniques help to explain the interactions of viruses with their hosts, and to tailor-make the vital drugs in the pharmacologist's armoury. And the pace of biochemical research shows no sign of slackening, from the determination ofthe structures of the complex molecules of life to the myriad of chemical reactions that drive the workings of the cell. Today, biochemists are beginning to discover where individual molecules are located within the cell, how cells of different organs and organisms resemble and differ, how a specialized biochemistry underlies the workings of brain and muscles, and how a coordinated series of reactions results in the development of the cell and the organism. Written with his customary authority and clarity, this second edition of Professor Rose's well-known work is an indispensable companion for anyone interested in this field.

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