[Use of immobilization for the study of glyceraldehyde 3-phosphate dehydrogenase. Immobilized dimers of the enzyme]. / Ispol'zovanie immobilizatsii dlia izucheniia glitseral'degid-3-fosfatdegidrogenazy. Immobilizovannye dimery fermenta.

The immobilized dimers of glyceraldehyde 3-phosphate dehydrogenase have been obtained after dissociation of the tetrameric enzyme molecule linked by one of the subunits with Sepharose 4B. The catalytic parameters (V, Km and pH-dependence of activity) of the immobilized dimers and tetramers of the enzyme are identical. The immobilized dimers are capable of reassociating with the enzyme subunits from solution and possess a higher stability a compared to soluble dehydrogenase. The type of the dimer interaction with the reagents causing the "half-of-the-site reactivity" effect suggests that the immobilized dimers of the yeast and rat skeletal muscle enzymes possess the catalytic activity and, besides, are capable to express cooperative interactions between the active centers of the both subunits. The immobilized dimers of yeast dehydrogenase are able to form hybrids with soluble modified dimers of the yeast enzyme and with those from other sources. A procedure for the enzyme isolation from tissue extracts based on hybridization between the immobilized and soluble dimers of homologous dehydrogenases have been developed.

Cold inactivation of glyceraldehyde-phosphate dehydrogenase from rat skeletal muscle.

Inactivation of apo-glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase(phosphorylating) (EC 1.2.1.12) from rat skeletal muscle at 4 degrees C in 0.15 M NaC1, 5 mM EDTA, 4 mM 2-mercaptoethanol pH 7.2 is a first-order reaction. The rate constant of inactivation depends on protein concentration. With one molecule of NAD bound per tetrameric enzyme, a 50 per cent loss in activity is observed and the rate constant of inactivation becomes independent of the protein concentration over a 30-fold range. Two moles of NAD bound per mole of enzyme fully protect it against inactivation. NADH affords a cooperative effect on enzyme structure similar to that of NAD. Inactivation of 7.8 S apoenzyme is reflected in its dissociation into 4.8-S dimers. In the case of enzyme-NAD1 complex, no direct relationship between the extent of inactivation and dissociation is observed, suggesting that these two processes do not occur simultaneously; we may say that dissociation is slower than inactivation. A mechanism in which the rate-limiting step for inactivation is a conformational change in the tetramer occurring prior to dissociation and affecting only the structure of the non-liganded dimer, is consistent with the experimental observations. Inorganic phosphate protects apoenzyme against inactivation. Its effect is shown to be due to the anion binding at specific sites on the protein with a dissociation constant of 2.6 plus or minus 0.4 mM. The NaC1-induced cold inactivation of glyceraldehyde-phosphate dehydrogenase is fully reversible at 25 degrees C in the presence of 20 mM dithiothreitol and 50 mM inorganic phosphate. The rate of reactivation is independent of protein concentration. Inactivated enzyme retains the ability to bind specific antibodies produced in rabbits, but diminishes its precipitating capability.