Kuwada M, Horie S, Ogura Y
J Biochem. 1980 Sep;88(3):859-69. doi: 10.1093/oxfordjournals.jbchem.a133040.
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.
对猪肝细胞溶质中催化对亚硝基苯酚（p-NSP）依赖NADH还原为对氨基酚（P-AmP）的主要C-亚硝基还原酶的多样性进行了研究。通过硫酸铵沉淀、Sephadex G-100凝胶过滤和DEAE-Sephadex A-50离子交换色谱制备的部分纯化制剂，对p-NSP和NADH均显示出两个或更多个表观Km值。通过5'-AMP-Sepharose亲和色谱，该制剂可被分离成至少四个具有不同Km值的亚组分。即使是通过亲和色谱随后离子交换色谱从Sephadex G-100凝胶滤液中获得的更纯的制剂，通过聚丙烯酰胺凝胶等电聚焦也可被分离成多个亚组分。从聚丙烯酰胺凝胶中提取的所有亚硝基还原酶亚组分均显示出NADH-醛还原酶（醇脱氢酶[EC 1.1.1.1]）活性，并且除了少数次要亚组分外，这两种活性是平行的。市售的马肝醇脱氢酶结晶制剂也显示出类似的多样性，并且多个亚组分具有两种酶活性，尽管等电聚焦模式与猪肝制剂不同。不同制剂观察到不同的热失活曲线，但在每种制剂中，亚硝基还原酶活性曲线总是与醛还原酶活性曲线一致。亚硝基还原酶制剂和醇脱氢酶制剂在催化NADH依赖的p-NSP还原反应中显示出非常相似的pH-活性曲线。此外，乙醇竞争性抑制NADH-p-NSP还原酶反应，对氨基酚竞争性抑制NADH-醛还原酶反应。这些结果清楚地表明，猪肝中的主要C-亚硝基还原酶以多种具有不同Km值的形式存在，并且该酶与肝醇脱氢酶相同。
