Effect of NADH-X on cytosolic glycerol-3-phosphate dehydrogenase

At pH 7.05 NADH-X prepared by incubating NADH with glyceraldehyde-3-phospha te dehydrogenase (E.C. 1.2.1.12) was a potent noncompetitive inhibitor, wit h respect to coenzyme, of NADPH oxidation by pure rabbit muscle cytosolic g lycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8) and also a potent inhibito r of NADPH oxidation catalyzed by this enzyme in a rat pancreatic islet cyt osolic fraction. It was a much less potent inhibitor of NADPH oxidation cat alyzed by this enzyme in a rat liver cytosolic fraction and of NADH oxidati on catalyzed by this enzyme from all three sources. Glycerol-3-phosphate de hydrogenase purified from muscle cytosol contains tightly bound NADH-X NAD, and ADP-ribose, each in amounts of about 0.1 mol per mole of enzyme polype ptide chain. A deproteinized supernatant of this enzyme contained these thr ee ligands and produced the same type of inhibition of the enzyme described above for prepared NADH-X with a K-i, in the reaction with NADPH at pH 7.0 5, in the range of 0.2 mu M with respect to the total concentration of liga nds ([ADP-ribose] + [NAD] + [NADH-X] = 0.2 mu M). However, only the NADH-X component could account for the potent inhibition because NAD, ADP-ribose, and the primary acid product (which can be produced from NADH-X) each had a Ki considerably higher than 0.2 mu M. Although at pH 7.05 NADH-X inhibited NADPH oxidation considerably more than NADH oxidation, the reverse was the case at pH 7.38. Since the enzyme purified from muscle contains tightly bo und NADH-X, NADH-X might become attached to the enzyme in vivo where it cou ld play a role in regulating the ratio of NADH to NADPH oxidation of the en zyme. (C) 1998 Academic Press.