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.