title: Effect of nicotinamide adenine dinucleotide on the membrane-associated reduced nicotinamide adenine dinucleotide oxidase of Mycobacterium phlei.
abstract: NAD+ had a biphasic effect on the NADH oxidase activity in electron transport particles from Mycobacterium phlei. The oxidase was inhibited competitively by NAD+ at concentrations above 0.05 mM. NAD+ in concentrations from 0.02 to 0.05 mM resulted in maximum stimulation of both NADH oxidation and oxygen uptake with concentrations of substrate both above and below the apparent K-M. Oxygen uptake and cyanide sensitivity indicated that the NAD+ stimulatory effect was linked to the terminal respiratory chain. The stimulatory effect was specific for NAD+. NAD+ was also specific in protecting the oxidase during heating at 50 degrees and against inactivation during storage at 0 degrees. NAD+ glycohydrolase did not affect stimulation nor heat protection of the NADH oxidase activity if the particles were previously preincubated with NAD+. Binding studies revealed that the particles bound approximately 3.6 pmol of [14C1NAD+ per mg of electron transport particle protein. Although bound NAD+ represented only a small fraction of the total added NAD+ necessary for maximal stimulation, removal of the apparently unbound NAD+ by Sephadex chromatography revealed that particles retained the stimulated state for at least 48 hours. Further addition of NAD+ to stimulated washed particles resulted in competitive inhibition of oxidase activity. Desensitization of the oxidase to the stimulatory effect of NAD+ was achieved by heating the particles at 50 degrees for 2 min without appreciable loss of enzymatic activity. Kinetic studies indicated that addition of NADH to electron transport particles prior to preincubation with NAD+ inhibited stimulation. In addition, NADH inhibited binding of [14C]NAD+. The utilization of artificial electron acceptors, which act as a shunt of the respiratory chain at or near the flavoprotein component, indicated that NAD+ acts as at the level of the NADH dehydrogenase at a site other than the catalytic one resulting in a conformational change which causes restoration as well as protection of oxidase activity.
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D002462:Cell Membrane / Q000201:enzymology; D003486:Cyanides / Q000494:pharmacology; D004579:Electron Transport; D007700:Kinetics; D009161:Mycobacterium / Q000201:enzymology*; D009243:NAD / Q000494:pharmacology*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D010101:Oxygen Consumption; D011485:Protein Binding; D013696:Temperature
chemical_list: D003486:Cyanides; D009243:NAD; D009247:NADH, NADPH Oxidoreductases