title: Studies on the Enzymatic reduction of C-nitroso compounds. II. Multiple forms of liver C-nitrosoreductase and the identity with alcohol dehydrogenase.
abstract: Investigations were made of the multiplicity of the major C-nitrosoreductase in porcine liver cytosol catalyzing NADH-dependent reduction of p-nitrosophenol (p-NSP) to p-aminophenol (P-AmP). A partially purified preparation prepared by precipitation with ammonium sulfate, gel filtration with Sephadex G-100, and ion-exchange chromatography on DEAE-Sephadex A-50 showed two or more apparent Km values for p-NSP and also for NADH. This preparation could be resolved into at least four subfractions having different Km values by affinity chromatography on 5'-AMP-Sepharose, Even a more purified preparation, which was obtained from the Sephadex G-100 gel filtrate by affinity chromatography followed by ion-exchange chromatography, could be resolved into multiple subfractions by isoelectric focusing in polyacrylamide gel. All nitrosoreductase subfractions extracted from the polyacrylamide gel showed NADH-aldehyde reductase (alcohol dehydrogenase [EC 1.1.1.1]) activity and, except for a few minor subfractions, the two activities were in parallel. A commercially supplied, crystalline preparation of equine liver alcohol dehydrogenase also showed a similar multiplicity and the multiple subfractions had the both enzymatic activities, although the pattern of isoelectric focusing was different from those of the porcine liver preparations. Different heat inactivation curves were observed with various preparations, but in each preparation the curve for nitrosoreductase activity always agreed with that for aldehyde reductase activity. The nitrosoreductase preparations and the alcohol dehydrogenase preparation showed very similar pH-activity curves in catalyzing NADH-dependent reduction of p-NSP. Furthermore, ethanol inhibited the NADH-p-NSP reductase reaction competitively and p-AmP inhibited the NADH-aldehyde reductase reaction competitively. These results clearly indicate that the major C-nitrosoreductase in porcine liver was present in multiple forms having different Km values and the enzyme was identical with liver alcohol dehydrogenase.
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D002846:Chromatography, Affinity; D003600:Cytosol / Q000201:enzymology; D006736:Horses; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009603:Nitroso Compounds / Q000378:metabolism; D013552:Swine
chemical_list: D009603:Nitroso Compounds; D000429:Alcohol Oxidoreductases; C021880:C-nitrosoreductase (NADH)