The preparation of alcohol dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase from baker''s yeast

A procedure has been developed for the preparation of alcohol dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase from the same sample of baker's yeast. The two enzymes were obtained in good yield in a pure crystalline form. The method minimizes the work involved in preparing the two enzymes and would be of particular advantage for preparing the enzymes in radioactive form from yeast grown in a radioactive medium.     

Glyceraldehyde-3-phosphate dehydrogenase from human erythrocyte membranes. Kinetic mechanism and competitive substrate inhibition by glyceraldehyde 3-phosphate

Glyceraldehyde-3-phosphate dehydrogenase (GAPD) was isolated from human erythrocyte ghosts by a simple procedure utilizing ammonium sulfate precipitation and affinity chromatography on NAD⁺-Sepharose 4B. The purified enzyme had a specific activity of 98 units/mg protein. The kinetic mechanism of GAPD was studied by product and deadend inhibition using NADH, α-glycerophosphate, nitrate, and 2,3-diphosphoglycerate. The results indicated that the human erythrocyte GAPD-catalyzed reaction follows an ordered ter bi mechanism characterized by the sequential addition of NAD⁺, glyceraldehyde 3-phosphate (GAP), and phosphate to the enzyme and the sequential release of 1,3-diphosphoglycerate and NADH from the enzyme. This contrasts with the mechanism (rapid equilibrium random ter bi) proposed by Oguchi (1970, J. Biochem. (Tokyo)68, 427–439) who based his conclusion on the initial rate data alone. Since the Michaelis-Menten kinetics were not applicable to this enzyme because of the competitive substrate inhibition by GAP, we devised a new kinetic approach for determining the parameters of the GAPD-catalyzed reaction. Results of this study indicate that the GAPD-catalyzed reaction is regulated by both ATP and GAP. We propose that GAP acts as an “amplifier” for the feedback inhibition effect of ATP. We discuss the effect this may have played in causing controversy over the regulatory role of this enzyme in glycolysis.     

Half-of-the sites reactivity and negative co-operativity: the case of yeast glyceraldehyde 3-phosphate dehydrogenase

Covalent modification of two of the four cysteine-149 residues of yeast glyceraldehyde 3-phosphate dehydrogenase, at pH 8.5, decreases the reactivity of the remaining two cysteine-149 residues and essentially inactivates the protein. The structure of the modifying reagent has only a secondary influence on this half-of-the-sites effect. Reactivity studies, together with the existing X-ray and sequence studies, suggest that the four active sites are initially functionally identical both in activity and in cysteine reactivity. The half-of-the-sites effect therefore arises in part or in whole from ligand-induced negatively co-operative conformational changes. A detailed kinetic study with iodoacetamide gives relative values of two rapidly reacting groups, a third more slowly reacting, and a fourth very slowly reacting group. These data, added to the existing data on cytidine triphosphate synthetase and alkaline phosphatase, suggest that the half-of-the-sites phenomena in many enzymes may be explained by ligand-induced negative co-operativity triggered by binding or covalent bond formation or both.     

Activation properties of the redox-modulated chloroplast enzymes glyceraldehyde 3-phosphate dehydrogenase and fructose-1,6-bisphosphatase

Rapid changes of enzyme activity are obtained by post-translational modification of cysteine residues of some chloroplast enzymes. Individual fine-tuning is achieved by specific factors acting upon the redox cycle. In order to study the regulatory properties of these enzymes, they are purified from leaves or in a recombinant form from Escherichia coli . The various factors acting upon the enzyme in vivo can be simulated in vitro. However, in these studies, some subtle technical problems can be encountered. Two cases are presented in this article, and an attempt is made to explain some previous, seemingly contradictory results. The Calvin-cycle enzyme glyceraldehyde 3-phosphate dehydrogenase in its less active A8B8 form can be dissociated and thereby activated in vitro simply by diluting out the protein. On the other hand, activation requires the presence of reduced thioredoxin (Td) and an increase in ionic strength when performed at a high protein concentration, as present in vivo. Chloroplast fructose-1,6-bisphosphatase (FBPase) is purified from E. coli as an enzyme similar to that purified from leaves. However, using the standard protocol for lysis of the bacteria leads to a form with some unusual properties as changed isoelectric point, lack of Ca2+/fructose-1,6-bisphosphate (FBP) dependency of reductive activation, and lack of activity at high pH and high Mg2+ concentration. These observations are used in order to better understand the characteristics of the activation/inactivation process.