Racker A New Look at Mechanisms in Bioenergetics

1. Auflage 1976
ISBN: 978-1-4832-6842-2
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: Adobe DRM (»Systemvoraussetzungen)

E-Book, Englisch, 212 Seiten

A New Look at Mechanisms in Bioenergetics is a collection of lectures given at the Robbins Lectures at Pomona College in April, 1973. The lectures contained in the book are based on laboratory works on oxidative phosphorylation. The text starts by defining oxidative phosphorylation, and after which, began a series of discussions on the biochemical approach to the problem of photophosphorylation; functions and structure of membranes and the mechanism of phosphorylation coupled to electron transport; and the basic principle of the control that governs both glycolysis and oxidation. Biochemists and biologists will find the book interesting and amusing.

Autoren/Hrsg.

Racker, Efraim

Inhaltsverzeichnis

1;Front Cover;1
2;A New Look at Mechanisms in Bioenergetics;4
3;Copyright Page;5
4;Table of Contents;6
5;Preface;10
6;Abbreviations;14
7;Lecture 1. Troubles Are Good for you;16
7.1;How It All Started;16
7.2;What Is Oxidative Phosphorylation?;19
7.3;How Do We Measure Oxidative Phosphorylation?;22
7.4;First Approaches to the Resolution of the Membrane;23
7.5;Allotopic Properties of F1;26
7.6;Electron Microscopy;28
7.7;Isolation of Resolved Particles;30
7.8;Reconstitution of Oxidative Phosphorylation;36
8;Lecture 2. Photophosphorylation;42
8.1;On the Origin of Life;42
8.2;Electron Pathway in Photophosphorylation;43
8.3;Further Similarities with Mitochondria;46
8.4;Resolution of a Coupling Factor;48
8.5;Proton Movements in Chloroplasts;53
8.6;Asymmetric Assembly of the Chloroplast Membrane;55
8.7;Reversal of Photophosphorylation;57
9;Lecture 3. Functions and Structure of Membranes and the Mechanism of Phosphorylation Coupled to Electron Transport;60
9.1;General Comments;60
9.2;Function and Structure of Membranes;61
9.3;Mechanism of Phosphorylation Coupled to Electron Transport;66
9.4;Experimental Approaches;74
10;Lecture 4. The Coupling Device;82
10.1;Partial Reactions and Components of the Coupling Device;83
10.2;The ATP-Driven Proton Pump;101
11;Lecture 5. The Oxidation Chain and the Topography of the Inner Mitochondrial Membrane;104
11.1;Analysis of the Oxidation Chain;105
11.2;Isolation of Complexes and Individual Catalysts;106
11.3;The Three Segments of the Oxidation Chain;108
11.4;The Topography of the Oxidation Chain;115
12;Lecture 6. Resolution and Reconstitution of Oxidative Phosphorylation;122
12.1;General Comments;122
12.2;Reconstitutions of Mitochondrial and Chloroplast Membrane Functions;124
12.3;Reconstitution of the Proton Pump of Halobacterium halobium and of Rhodopsin-Catalyzed Photophosphorylation;139
13;Lecture 7. Reconstitution and Mechanism of Action of Ion Pumps;142
13.1;General Comments;142
13.2;Reconstitution of Ion Pumps;143
13.3;What Can We Learn from Resolution and Reconstitution Experiments?;153
14;Lecture 8. Control of Energy Metabolism;168
14.1;Oxidation Control;168
14.2;The Pasteur Effect;171
14.3;The Competition Mechanism;172
14.4;High Aerobic Glycolysis in Tumor Cells (The Warburg Effect);177
14.5;ATPases in Tumor Cells;179
14.6;Repair of Ion Pumps in Tumor Cells;182
15;Bibliography;192
16;Index;204

Leseproben

Lecture 1

Troubles Are Good for You

Publisher Summary

This chapter describes oxidative phosphorylation and how to measure it. Substrates such as pyruvate enter the Krebs cycle and donate hydrogen to DPN. The reduced nucleotide is oxidized by the mitochondrial oxidation chain in discrete steps that permit conservation of energy of oxidation and formation of ATP. There are three classes of compounds that interfere with oxidative phosphorylation: (1) inhibitors of the oxidation chain such as cyanide or antimycin, (2) uncouplers such as 2, 4-dinitrophenol or carbonyl-cyanide trifluoromethoxyphenylhydrazone that abolish the conservation of energy and give rise to a dissipation of the oxidation energy into heat, and (3) energy transfer inhibitors such as oligomycin or rutamycin that prevent the conversion of the oxidation energy into ATP. There are three ATP molecules generated for each DPNH that is oxidized by molecular oxygen. There are several methods available for measuring oxidative phosphorylation. The older methods of manometric determination of oxygen uptake have been replaced by the polarographic method.

If you have built a perfect demonstration do not remove all traces of the scaffolding by which you have raised it.

Clark Maxwell

How It All Started

Before going into the complex details of the structure and function of mitochondrial and chloroplasts membranes, I would like to transmit to you some of the general lessons I have learned in doing research in the field of bioenergetics. When you read excellent scientific articles in popular journals such as or even in professional journals, you will be much impressed. You are exposed to lucid expositions, sometimes brilliant experiments, important developments, and visions of an even more interesting future. The data are usually unambiguous and the conclusion convincing. The writer may even succeed in transmitting some of the excitement of the laboratory and you appreciate it. But almost invariably certain aspects of the work will be missing. How did the discovery really come about? How much sweat and trouble was there in its making? How much of it was thought out beforehand; how much was accidental? Since these lectures are primarily addressed to students I would like to transmit, at least in the first one, a picture of research life which I believe is somewhat closer to reality. It contains large shares of troubles, doubts, serendipity, and, last but not least, interaction between different investigators. I believe that we should prepare our students for these aspects, not only to avoid disappointment, but to convey to them the concept that troubles and doubts are seeds of the future. They can lead us, if we follow them, into unknown territory and challenging problems. To paraphrase Goethe with equal exaggeration: For the true scientist nothing is more difficult to bear (and indeed suspect) than an uninterrupted series of beautiful experiments. Or as Piet Hein says in a grook: “Problems worthy of attack prove their worth by hitting back.”

I want to give you first a personal account of how I got to the problem of oxidative phosphorylation and to tell you some of the things that happened on the way to the palace or to the castle. You have a choice here depending on whether you consider my account a fairy tale or a Kafka nightmare.

Let us start out with the first basic question. How does a biochemist choose a problem to work on? More specifically, what is a nice M.D. doing in oxidative phosphorylation rather than working on a relevant problem in medicine?

When I was a medical student almost 40 years ago, I wanted to be a psychiatrist, I wanted to understand mental diseases, I wanted to cure and heal the psychotic mind. This was a relevant problem, pregnant with economical and social implications. Having been raised in Vienna and put to sleep by the lullably of the Oedipus complex, I first turned toward the teaching of Freud, but could not find a firm footing. I was soon plagued by doubts fortified by a statement of Freud that psychosis is a child of the night, and cannot be cured through the mind. In fact, he believed in the organic genesis of psychosis.

In 1938 when a mass psychosis invaded Vienna I left for England and joined the laboratory of Dr. Quastel who had written a fascinating article on the relationship of mental disorders and amines (Quastel, 1936). I became interested in mescaline, benzedrine (better known as speed) and other analogues of biogenic amines, which in large doses produce illnesses resembling psychoses. Thus I became in 1938 a biochemical hippy. Coming to the United States in 1941, I found no interest in biogenic amines, but there were funds for studies in applied research in the field of poliomyelitis. My research was supported by the “March of Dimes” and I had the impressive salary of 12,000 dimes per year. In exploring the effect of polioviruses on brain metabolism I observed a defect in glycolysis which I traced to an inactivation of glyceraldehyde-3-phosphate dehydrogenase (Racker and Krimsky, 1948). In examing this key enzyme of glycolysis more closely I discovered that the popular hypothesis of its mode of action was incorrect. Warburg and Christian (1939) had proposed a simple and ingenious mechanism based on a chemical model system shown in Mechanism I of Table 1-1. The first step is the formation of an adduct between the aldehyde and inorganic phosphate which is directly oxidized to 1,3-diphosphoglycerate. Warburg’s influence on the biochemical society was so great that his formulation was not only blindly accepted in all textbooks but an enterprising firm in New York City sold the hypothetical adduct diphosphoglyceraldehyde for about $1000 per 100 mg. Nevertheless, we could show without ambiguity (Racker and Krimsky, 1952) that the enzyme catalyzes the two steps shown in Mechanism II, Table 1-1. The first step is an oxidation of the aldehyde–enzyme complex to an acyl enzyme intermediate which we obtained in crystalline form (Krimsky and Racker, 1955). Phosphate enters in a second step by cleaving the thiol ester of the enzyme yielding 1,3-diphosphoglycerate. Phosphoglycerate kinase transfers the phosphate in the 1-position of this compound to ADP yielding ATP. Warburg never accepted this formulation since his chemical model was simpler and better. Let us look at the first two lessons we can learn from this account.

TABLE 1-1

Glyceraldehyde-3-phosphate Dehydrogenase

Chemistry is good, nature is better.

If there is no money available for fundamental research start working on a project of applied research. If you proceed logically you will soon be doing basic research.

You can see now how I became interested in oxidative phosphorylation. My turning to these fundamental problems was not caused by a loss of interest in biogenic amines, but by a clear realization that without the fundamentals of biochemistry we cannot understand either physical or mental disorders. Actually I have started recently to work again with biogenic amines (McCauley and Racker, 1973). Having fled in my youth from a mass pychosis in Germany, facing at present a drugged society blinded by acute pains, I cannot think of a more relevant problem to work on.

What Is Oxidative Phosphorylation?

Let us look at the processes that take place in the inner mitochondrial membrane. As shown in Fig. 1-1 the basic reaction is oxidation. Substrates such as pyruvate enter the Krebs cycle and donate hydrogens to DPN. The reduced nucleotide (DPNH) is oxidized by the mitochondrial oxidation chain in discrete steps which permit conservation of energy of oxidation and formation of ATP. In fact, the formation of ATP is compulsorily coupled to the oxidation process so that respiration ceases when no ADP and Pi are available. We shall return to this important regulatory mechanism called “respiratory control.” There are three classes of compounds which interfere with oxidative phosphorylation. There are (1) inhibitors of the oxidation chain such as cyanide or antimycin; (2) uncouplers such as 2,4-dinitrophenol or carbonylcyanide -trifluoromethoxyphenylhydrazone which abolish the conservation of energy and give rise to a dissipation of the oxidation energy into heat; and (3) energy transfer inhibitors such as oligomycin or rutamycin (Lardy , 1958) which prevent the conversion of the oxidation energy into ATP. These energy transfer inhibitors block the formation of ATP coupled to oxidation as well as the breakdown of ATP by mitochondrial ATPase. As will be elaborated later, the oligomycin-sensitive ATPase activity represents a partial reversal of the process of oxidative phosphorylation.

Fig. 1-1 Oxidative phosphorylation in mitochondria.

There are three ATP molecules generated for each DPNH which is oxidized by molecular oxygen and we speak of a P:O ratio of 3, an expression of the efficiency of the process. It was shown many years ago (Ochoa, 1943) that for each molecule of pyruvate oxidized there are 15 molecules of ATP formed from ADP and inorganic phosphate. However, pyruvate has only 4 hydrogens to donate to 2 oxygens and with a P:O ratio...