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.
D000818:Animals; D001051:Apoenzymes; D003080:Cold Temperature; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors*; D007700:Kinetics; D046911:Macromolecular Substances; D009132:Muscles / Q000201:enzymology*; D009243:NAD; D010710:Phosphates / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D012965:Sodium Chloride / Q000494:pharmacology
D001051:Apoenzymes; D046911:Macromolecular Substances; D010710:Phosphates; D009243:NAD; D012965:Sodium Chloride; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases