Racker Reconstitutions of Transporters, Receptors, and Pathological States

1. Auflage 1985
ISBN: 978-1-4832-1423-8
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: Adobe DRM (»Systemvoraussetzungen)

E-Book, Englisch, 288 Seiten

Reconstitutions of Transporters, Receptors, and Pathological States presents lectures about the reconstitutions of transporters, receptors, and pathological states. The book discusses the principles and strategies of the resolution and reconstitution of soluble pathways and membrane complexes; and lessons in the resolution and reconstitution after the natural structure of the membrane has been destroyed. The text then describes the analyses of reconstituted vesicles; the ATP synthetase of oxidative phosphorylation; and the ?1?2 pumps of plasma membranes. The ATP-driven ion pumps in organelles, microorganisms, and plants; the proton motive force generators, electron transport chains, and bacteriorhodopsin; and facilitated diffusion, symporters, and antiporters are also considered. The book further tackles plasma membrane receptors, as well as the reconstitutions of pathological states. Biochemists, molecular biologists, and cell biologists will find the book invaluable.

Autoren/Hrsg.

Racker, Efraim

Inhaltsverzeichnis

1;Front Cover;1
2;Reconstitutions of Transporters, Receptors, and Pathological States;4
3;Copyright Page;5
4;Table of Contents;6
5;Preface;10
6;Lessons;14
7;Abbreviations;16
8;Lecture 1. Resolution and Reconstitution of Soluble Pathways and Membrane Complexes: Overview of Principles and Strategies;18
8.1;I. Reconstitution of Soluble Pathways;18
8.2;II. Resolution and Reconstitution of Membrane Complexes;22
9;Lecture 2. Methods of Resolution and Reconstitution;35
9.1;I. Solubilization and Purification of Membrane Proteins;35
9.2;II. Purification of Membrane Proteins;41
9.3;III. Methods of Reconstitution;43
10;Lecture 3.
What Can We Learn from Resolution and Reconstitution after the Natural Structure of the Membrane Has Been Destroyed?;57
10.1;I. What Are the Protein Components of the System and What Are Their Functions?;57
10.2;II. What Are the Phospholipid Components and What Are Their Functions?;60
10.3;III. What Is the Role of Asymmetry? How Do We Achieve It in Reconstitution? How Do We Measure It?;63
10.4;IV. How Do We Measure the Extent of Scrambling during Reconstitution?;64
11;Lecture 4. Analyses of Reconstituted Vesicles:
Pitfalls and Obstacles;68
11.1;I. Analyses of Reconstituted Vesicles;68
11.2;II. Pitfalls and Recommended Cautions;72
12;Lecture 5.
The ATP Synthetase of Oxidative Phosphorylation;82
12.1;I. F1 (Mitochondrial MF1, Chloroplast CF1, and Bacterial BF1);84
12.2;II. The Stalk, OSCP, and F6;92
12.3;III. The Hydrophobic Sector;95
12.4;IV. Mechanism of Action of F1;102
13;Lecture 6. The .1.2 Pumps of Plasma Membranes;104
13.1;I. The Na+,K+ Pump and Na+,K+-ATPase;104
13.2;II. The Ca2+ Pump and Ca2+-ATPase;113
13.3;III. The H+,K+-ATPase of the Gastric Mucosa;118
14;Lecture 7.
ATP-Driven Ion Pumps in Organelles, Microorganisms, and Plants;121
14.1;I. The Ca2+ Pump of Sarcoplasmic Reticulum and Related Organelles;121
14.2;II. ATP-Driven H+ Fluxes in Organelles;132
14.3;III. ATP-Driven Ion Pumps of Microorganisms and Plants;136
15;Lecture 8. Proton Motive Force Generators,
Electron Transport Chains, and Bacteriorhodopsin;141
15.1;I. Reconstitution of the Mitochondrial Electron Transport Chain;141
15.2;II. Reconstitution of Photosynthetic Electron Transport Pathways;154
15.3;III. Bacteriorhodopsin;157
15.4;IV. Halorhodopsin and a Bacterial Na+ Pump;160
16;Lecture 9. Facilitated Diffusion, Symporters, and Antiporters;162
16.1;I. Transporters of Plasma Membranes;162
16.2;II. Transporters of Mitochondria;184
16.3;III. Transporters of Other Organelles;192
17;Lecture 10. Plasma Membrane Receptors;197
17.1;I. RGC Receptors;201
17.2;II. Polypeptide Signal Receptors;204
17.3;III. Channel Receptors;206
17.4;IV. Transport Receptors;211
17.5;V. Drug and Toxin Receptors;212
18;Lecture 11. Reconstitutions of Pathological States;216
18.1;I. About the Artificiality of Cancer Research;218
18.2;II. Two Approaches to Cancer Research;219
18.3;II.. The Scenic Route from ABC to X;224
19;Lecture 12. Glimpses into the Future of Reconstitutions: Hypotheses,
Speculations, and Fantasies;238
19.1;I. Methods of Reconstitution;239
19.2;II. Orientation-Directed Reconstitution and Co-Reconstitutions;241
19.3;III. Mechanisms and Regulations;242
19.4;IV. Incorporations of Cellular Components into Cells;243
19.5;V. Reconstitution of Organelles, Cells, Organs, etc.;245
19.6;VI. Reconstitution of Pathological States;247
20;Bibliography;254
21;Index;282

Leseproben

Lecture 1

Resolution and Reconstitution of Soluble Pathways and Membrane Complexes: Overview of Principles and Strategies

Publisher Summary

This chapter discusses resolution and reconstitution of soluble pathways and membrane complexes providing an overview of principles and strategies. Many biochemical pathways have been reconstituted by the combinations of isolated enzymes participating in the formation and degradation of purines, pyrimidines, fatty acids, and amino acids, and in the biosynthesis of macromolecules. A great deal has been learned from the studies of reconstituted multienzyme systems. The soluble multienzyme pathways are catalyzed by water-soluble enzyme systems that do not require a compartment for function. The chapter presents a broad outline of the various methods that have been used in the resolution and reconstitution of membrane components. It describes why membrane complexes are resolved and reconstituted and presents approaches to problems in the field of transport that are susceptible to attack by reconstitution. The chapter further describes reconstitution of membrane complexes starting with intact organelles or vesicles and resolution and reconstitution of membrane complexes with detergents.

What a good thing Adam had—when he said a thing he knew nobody had said it before.

Mark Twain

I Reconstitution of Soluble Pathways

Resolution and reconstitution is a classical approach of biochemists to the mysteries of intact cells. In 1927 Otto Meyerhof added a fractionated yeast extract to a crude muscle extract. Neither preparation alone fermented glucose to lactate; together they did (Fig. 1-1). The muscle extract contained the enzymes that fermented glycogen; the yeast fraction contained an enzyme which “activated glucose.” This is how hexokinase was discovered, and this is how hexokinase was first assayed in a reconstituted system. It illustrates the two purposes of reconstitution: a method of assay and an approach to the analysis of the whole from its parts. During the subsequent years, one glycolytic enzyme after another was separated, and studied in isolation. Finally, highly purified glycolytic enzymes and the required cofactors were put together and shown to catalyze steady-state glycolysis, provided an ATPase was added (Gatt and Racker, 1959). The insight into the role of ATPase in glycolysis was again a contribution by Otto Meyerhof, who in 1945, as a German refugee in Philadelphia, performed some of his last experiments (Meyerhof, 1945). He added a partially purified potato enzyme that hydrolyzed ATP to an extract of dried yeast that fermented glucose poorly. If the appropriate amount of ATPase was added, steady-state fermentation was observed, but if either too little or too much ATPase was added, the utilization of glucose ceased after a burst of activity. Meyerhof concluded that the ATPase must be “in step” with the hexokinase. For each molecule of glucose that was phosphorylated by hexokinase, two molecules of ATP must be hydrolyzed to deliver the appropriate amount of ADP and Pi required for the oxidation of glyceraldehyde 3-phosphate (Fig. 1-2).

Fig. 1-1 The first reconstitution experiment.

Fig. 1-2 “ATPase” is a glycolytic enzyme.

In the same year it was observed (Racker and Krimsky, 1945) that a homogenate of mouse brain did not glycolyze in the presence of Na+ because of an excess of ATP hydrolysis. Glycolytic activity was restored by slow infusion of ATP or by addition of phosphocreatine (Table 1-1). Thus, a heterologous ATP-generating system was introduced into a biological pathway to rectify the imbalance caused by excessive ATPase activity.

TABLE 1-1

Stimulation of Glycolysis of Brain Homogenates in the Presence of Na+ by an ATP-Regenerating System

None

-11.3

+21.1

Phosphocreatine (5.5 m)

-15.3

+24.9

NaCl (40 m)

-0.5

+2.4

NaCl + phosphocreatine

-12.4

+24.1

The first reconstitution of a complete pathway was based on the work of Horecker and our own group on the enzymes that participate in the reductive pentose phosphate cycle (Racker, 1955). Glycolytic enzymes isolated from yeast and muscle were combined with enzymes of the pentose phosphate cycle isolated from yeast and spinach leaves. In the presence of ATP and NADPH2 the mixture fixed CO2 and converted it to hexose.

Reconstitution experiments performed by combining enzymes from muscle, yeast, and spinach, can hardly be called physiological. We should designate Otto Meyerhof as the father of unphysiological reconstitutions. What is a daydream to the biochemist may be a nightmare to the physiologist. But the only way we can find out whether the whole is the sum of the parts is by putting the pieces together again and learning how they work. It is the task of the physiologist to help the biochemist by pointing out what is missing. It was the physiologist Otto Meyerhof who knew what sources of enzymes to use to achieve the best reconstitution of a pathway.

The next task was to see whether physiological control mechanisms can be observed in reconstituted systems. We added mitochondria to a reconstituted glycolytic pathway and observed phenomena resembling those first described by Pasteur and Crabtree (Fig. 1-3). We found (Wu and Racker, 1959) that glycolysis is inhibited when mitochondrial oxidative phosphorylation competes for ADP and Pi (the Pasteur effect) and that respiration is inhibited when glycolysis dominates and deprives the respiratory chain of ADP and Pi (the Crabtree effect). These experiments convinced us of the importance of Pi and ADP as rate-limiting factors in bioenergetics (Racker, 1965, 1976), a mechanism of regulation that was first recognized by M. Johnson (1941) and, independently, by F. Lynen (1941). Both in mitochondrial respiration and glycolysis the generation of ADP and Pi can be a rate-limiting step. In mammalian cells the ATP-generating machineries are present in excess, geared to energy utilization. What an ingenious and simple way of food economy! Energy is generated only as it is needed.

Fig. 1-3 Reconstitution of the Crabtree and Pasteur effects.

The experiments on the Pasteur effect were extended (Uyeda and Racker, 1965). The role of the allosteric inhibition of phosphofructokinase by ATP and hexokinase by glucose 6-phosphate was demonstrated in reconstituted systems of glycolysis. These important control mechanisms regulating the utilization of sugar do not change the Meyerhof stoichiometry. For each mole of lactate that is formed, one mole of ATP is generated and must be hydrolyzed to maintain steady-state glycolysis. To understand the driving force that propels glycolysis, the ATP-hydrolysing processes, referred to broadly as ATPases, must be identified.

It will be shown in Lecture 11 that the major contribution to ATP hydrolysis in tumor cells takes place in membranes. Thus, glycolysis, which is dependent on the availability of ADP and Pi, is a membrane-dependent process. A membrane-free cell extract does not glycolyze unless an ATPase is added (Racker 1984). It is a reflection on the individuality of cells that the relative contributions to ATP utilization by the plasma membrane and organelle membranes vary considerably and are related to the specific function of the cell. We are just beginning to gain an insight into the balances of budgetary expenditures in bioenergetics.

Many biochemical pathways have been reconstituted by combinations of isolated enzymes participating in the formation and degradation of purines, pyrimidines, fatty acids, and amino acids, as well as in the biosynthesis of macromolecules. A great deal has been learned from studies of reconstituted multienzyme systems.

1. Are the known components sufficient to catalyze the overall pathway?

2. What are the kinetic interactions between functional neighbors?

3. How do manipulations of individual enzyme concentrations change the multiple rate-limiting steps and the susceptibility of the pathway to inhibitors?

4. Which enzymes are controlled by allosteric regulators and how do they affect the operation of the overall pathway at different enzyme concentrations?

The pathways mentioned above are catalyzed by water-soluble enzyme systems that do not require a compartment for function. The next task was to explore membrane-bound pathways. In this lecture I shall present a broad outline of the various methods that have been used in the resolution and reconstitution of membrane components and recapitulate what we have learned from these...