Effect of NADH-X on Cytosolic Glycerol-3-phosphate Dehydrogenase

• Published in: Archives of biochemistry and biophysics 1998-12, Vol.360 (2), p.195-205
• Main Authors: Prabhakar, Prakash, Laboy, José I., Wang, Jianbo, Budker, Tatyana, Din, Zafeeer Z., Chobanian, Michael, Fahien, Leonard A.
• Format: Article
• Language: English
• Subjects: Adenosine Diphosphate Ribose - metabolism; Animals; Chromatography, High Pressure Liquid; cytoplasmic glycerol-3-phosphate dehydrogenase; Cytosol - enzymology; Dialysis; Enzyme Activation; Glyceraldehyde-3-Phosphate Dehydrogenases - metabolism; Glycerol-3-Phosphate Dehydrogenase (NAD+); Glycerolphosphate Dehydrogenase - antagonists & inhibitors; Glycerolphosphate Dehydrogenase - metabolism; Hydrogen-Ion Concentration; Islets of Langerhans - enzymology; Kinetics; Ligands; Liver - enzymology; Muscle, Skeletal - enzymology; NAD - metabolism; NAD - pharmacology; NADH-X; NADP - metabolism; Oxidation-Reduction; rabbit skeletal muscle; Rabbits; Rats
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1998.0939

At pH 7.05 NADH-X prepared by incubating NADH with glyceraldehyde-3-phosphate dehydrogenase (E.C. 1.2.1.12) was a potent noncompetitive inhibitor, with respect to coenzyme, of NADPH oxidation by pure rabbit muscle cytosolic glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8) and also a potent inhibitor of NADPH oxidation catalyzed by this enzyme in a rat pancreatic islet cytosolic fraction. It was a much less potent inhibitor of NADPH oxidation catalyzed by this enzyme in a rat liver cytosolic fraction and of NADH oxidation catalyzed by this enzyme from all three sources. Glycerol-3-phosphate dehydrogenase 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 polypeptide chain. A deproteinized supernatant of this enzyme contained these three ligands and produced the same type of inhibition of the enzyme described above for prepared NADH-X with aKi, in the reaction with NADPH at pH 7.05, in the range of 0.2 μM with respect to the total concentration of ligands ([ADP-ribose] + [NAD] + [NADH-X] = 0.2 μM). 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 aKiconsiderably higher than 0.2 μM. 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 bound NADH-X, NADH-X might become attached to the enzymein vivowhere it could play a role in regulating the ratio of NADH to NADPH oxidation of the enzyme.