Efraim Racker

[49] Preparation and assay of chloroplast coupling factor CF1

Publisher Summary This chapter discusses the preparation and assay of chloroplast coupling factor (CF1). Treatment of chloroplasts with a dilute EDTA solution liberates a soluble coupling factor. The active factor in the EDTA extract is identical to coupling factor CF1, which contains a latent Ca2+ dependent ATPase. An assay for CF1 with EDTA-treated chloroplasts or with subchloroplast particles obtained by sonication of chloroplasts in the presence of phospholipids (CP particles) is described in this chapter. Because EDTA-treated chloroplasts cannot be stored and the degree of resolution of CP particles is somewhat variable, an assay with stable subchloroplast particles, which are completely resolved with respect to CF1, is used. In addition to coupling activity, CF1 has also a latent Ca2+-dependent ATPase activity, which can be activated by heat treatment, trypsin digestion, or prolonged incubation at high concentrations of dithiothreitol. The ATPase activity provides a rapid and convenient assay of the enzyme and is especially useful in the course of purification. The activity of the enzyme is assayed by determining the amount of Pi liberated by the enzymatic hydrolysis of ATP.

The reductive pentose phosphate cycle. I. Phosphoribulokinase and ribulose diphosphate carboxylase

Abstract The preparations and some of the properties of phosphoribulokinase and RDP carboxylase from spinach leaves are reported. Methods of assay of these enzymes and of intermediates of the reductive pentose phosphate cycle are described. The possible function of these enzymes in photosynthesis is discussed.

The reductive pentose phosphate cycle. II. Specific C-1 phosphatases for fructose 1,6-diphosphate and sedoheptulose 1,7-diphosphate.

Abstract 1. 1. The purification procedure and the properties of a very active fructose 1,6-diphosphatase from spinach leaves are described. The enzyme acts optimally at pH 8.5 and has little activity at neutrality. It is dependent on Mg++ and is stimulated by EDTA. Since fructose 1,6-diphosphate, which is cleaved to fructose 6-phosphate and inorganic phosphate, appears to be its only substrate, the enzyme serves as a useful analytical tool. The enzyme is specifically inhibited by its antibody. 2. 2. An enzyme was obtained from bakers yeast which specifically cleaves sedoheptulose 1,7-diphosphate to sedoheptulose 7-phosphate and inorganic phosphate. 3. 3. Spinach leaves contain still another enzyme (or enzymes) which hydrolyze both fructose 1,6-diphosphate and sedoheptulose 1,7-diphosphate at a neutral pH. Chloroplast preparations were found to cleave FDP and even more rapidly SDP at neutral pH but had little activity at pH 8.5. Neither Mg++ nor EDTA was required for this activity. 4. 4. The possible role of the enzymes which hydrolyze FDP and SDP in the photosynthetic cycle is discussed.

The oxidative pentose phosphate cycle. I. Preparation of substrates and enzymes

Abstract The preparations of intermediates and enzymes of the pentose phosphate cycle suitable for microanalytical procedures are described.

[76] Reconstitution of membrane processes

Publisher Summary Reconstitution is the approach of classic biochemistry to the analysis of multienzyme systems. With soluble systems, such as glycolysis or the oxidative and reductive pentose phosphate cycles, this requires the isolation of all the participating catalytic components, which are then simply mixed in appropriate concentrations. The study of multienzyme systems, such as oxidative phosphorylation or photophosphorylation, which are embedded in membranes, is more complicated because (1) the firm associations between some of the proteins with each other and with phospholipids make the resolution more difficult and (2) reconstitution requires formation of a compartment with appropriate physical and chemical properties. The most important lesson to be learned from experience with resolution and reconstitution of membrane proteins is that the isolation of the components must be carried out under conditions that preserve their ability to integrate within the catalytic community of the membrane. Some membrane proteins that have been isolated in the past, based on an assay of either a catalytic or a spectral property, are reconstitutively active. Enzyme preparations isolated with cholate as detergent are reconstitutively active; those isolated with deoxycholate are poor or inactive in reconstitution experiments.

CARBOHYDRATE METABOLISM IN ASCITES TUMOR CELLS

About six years ago, I stated in a lecture that carbohydrate catabolism is a field in biochemistry in which we are on the solid ground of enzymology, and that nearly ail fundamental problems have been solved. What remained to be explored were problems of hormonal control and highly specialized problems of comparative carbohydrate biochemistry. This lecture, fortunately, was never published. I t seems that during the past six years we have unlearned a great deal about carbohydrate metabolism, and I must admit today that our knowledge is so fragmentary that we have little accurate information about the fate of carbohydrates while and after they enter specific cells. What has brought about this change in attitude? The major reason is that we have come to recognize the existence of alternate pathways, and that we are just beginning to learn how to evaluate their relative quantitative significance. The approach that Doctor Chance has just described represents a new phase in these studies. With ingenious methods of his own design he has studied metabolic events in intact cells and has analyzed electron transfer and phosphorylation processes in the steady state of intracellular metabolism. He has thus permitted us to glance behind the iron-porphyrin curtain of intracellular oxidations. Another fruitful approach to the fate of carbohydrates in intact cells is being made with C14-labeled glucose, which Doctor Wenner has discussed. I should like to point out again the need for caution in the use of isotope incorporation studies for the quantitative evaluation of alternate pathways. Doctor Brown has made some very pertinent comments in regard to the occurrence of exchange reactions in nucleic acid synthesis, Since many biologists do not seem to be fully aware of the existence of these reactions, which may completely invalidate quantitative estimates of alternate pathways, or of the net synthesis of proteins, Rh-A or DNA deduced from incorporation studies only, I shall mention just one of many examples of exchange reactions. A rapid incorporation of C402 into oxalacetate is catalyzed by purified preparations of oxalacetate decarboxylase from Micrococcus lyodeiclicus, although no net synthesis of oxalacetate from pyruvate and COz can be detected. Thus, this process of isotope incorporation cannot be considered to be a synthetic pathway, in spite of the very rapid rate of labeling that takes place. In our laboratory, Doctor Kvamme has studied the inhibitory effect of glucose on the respiration of Ehrlich ascites tumor, which was described by Kun et a1.l and by McKee et aL2 We have confirmed and extended some observations made by these investigators. As shown in TABLE 1, it was found that sugars that serve as substrates for hexokinase (fructose, glucose, and mannose) inhibit oxygen uptake, while nonfermentable sugars (sucrose, ribose, and galactose) had no effect. An exploration of the concentration of glucose required

The oxidative pentose phosphate cycle. II. Quantitative determination of intermediates and enzymes

Abstract The analytical procedures used for the determination of the intermediates and enzymes of the oxidative pentose phosphate cycle are described.

Stable phosphorylating submitochondrial particles from baker's yeast.

Abstract The genetic control and the enzymic and morphological variability of mitochondria from bakers yeast ( Saccharomyces cerevisiae ) have been extensively studied (1–4). Little is known, however, about oxidative phosphorylation in these organelles. In mammalian systems, studies of oxidative phosphorylation have been greatly facilitated by the availability of phosphorylating submitochondrial particles which are less complex and in general also more stable than mitochondria. The preparation from bakers yeast of phosphorylating mitochondria (5–7) as well as of non-phosphorylating submitochondrial particles (8) has been reported, but submitochondrial particles catalyzing oxidative phosphorylation have not been described. The present communication outlines a procedure for the isolation of stable phosphorylating submitochondrial particles from commercially grown bakers yeast. In addition, evidence is presented that the mitochondria as well as the submitochondrial particles as isolated by the present procedure lack the phosphorylation site between DPNH and cytochrome b.

The oxidative pentose phosphate cycle. III. The interconversion of ribose 5-phosphate, ribulose 5-phosphate and xylulose 5-phosphate☆

Abstract The purification of ribose 5-phosphate isomerase from spinach leaves and of xylulose 5-phosphate epimerase from rabbit muscle is described. These enzymes have been used for the analysis of the individual sugar phosphates in equilibrium mixtures. The findings obtained by these methods, which are in disagreement with some other reports, are discussed.

Studies on a phosphoprotein phosphatase derived from beef spleen.

1. 1. A modification of the purification procedure of Sundararajan andSarma for beef spleen phosphoprotein phosphatase is described which yields a highly active soluble and stable enzyme preparation. 2. 2. The enzyme cleaves phosphate from nucleotides and from aromatic phosphate esters as well as from phosphoproteins. Other substrates include phosphorylenol pyruvate, phosphoamide, 3-phosphoglyceric acid and fructose-I,6-diphosphate. 3. 3. Enzyme activity is considerably enhanced by reducing agents, ascorbic acid and ferrous ion being the most effective activators. No other metal was found to stimulate the enzyme. Enzyme activity is inhibited by low concentrations of molybdate, I,IO-o-phenanthroline, α, α′-dipyridyl and 8-hydroxyquinoline. 4. 4.The enzyme is inactivated by short exposure to reducing agents in the absence of substrates. Ferrous ion protects against this inactivation. A hypothesis to explain these observations is presented.