Horie S, Watanabe T, Ogura Y
J Biochem. 1980 Sep;88(3):847-57. doi: 10.1093/oxfordjournals.jbchem.a133039.
The subcellular distribution of NADH-p-nitrosophenol (p-NSP) reductase activity at pH 6.0 in porcine liver was studied by spectrophotometric assay. About two-thirds of the activity was found in the cytosol fraction and the pH optimum of this fraction was about 5.5. The activity at pH 5.8 of cytosol fractions from various tissues of rats, quails, frogs, carp, and scallops was also studied. All these fractions showed more or less NADH-p-NSP reductase activity but the activity of NADH-aldehyde reductase (alcohol dehydrogenase [EC 1.1.1.1]) was detected only in these from liver and a few other tissues. Supernatants from sonicated cells of Bacillus subtilis and Escherichia coli also showed C-nitrosoreductase activity but were devoid of aldehyde reductase activity. The major C-nitrosoreductase of porcine liver cytosol was purified 20- to 30-fold by fractionation with ammonium sulfate, gel filtration, and ion-exchange chromatography. The pH optimum of this preparation was 5.5 and activity was strongly inhibited by p-chloromercuribenzoate (p-CMB). The enzyme preparation was stable at 5 degrees C for at least a week in the presence of NADH at pH 8.4. High concentrations of ammonium sulfate also stabilized the enzyme. An equilibrium between monomeric and dimeric forms of the enzyme was found and the molecular weight was estimated to be about 83,000 and 160,000 daltons for the monomeric and dimeric forms, respecively. The enzyme utilized NADH almost specifically and 2 mol of NADH were consumed per mol of p-NSP reduced to p-aminophenol. Nitrosobenzene and aldehydes could also serve as the electron acceptor. The aldehyde reductase activity became concentrated roughly in parallel with the C-nitrosoreductase activity during the course of the purification and these two activities could not be separated even after further purification by 5'-AMP-Sepharose affinity chromatography. N-Nitroso compounds were not affected by this enzyme.
采用分光光度法测定了猪肝中pH 6.0时NADH-对亚硝基苯酚（p-NSP）还原酶活性的亚细胞分布。约三分之二的活性存在于胞质溶胶部分，该部分的最适pH约为5.5。还研究了大鼠、鹌鹑、青蛙、鲤鱼和扇贝不同组织胞质溶胶部分在pH 5.8时的活性。所有这些部分或多或少都显示出NADH-p-NSP还原酶活性，但仅在肝脏和其他一些组织的部分中检测到NADH-醛还原酶（乙醇脱氢酶[EC 1.1.1.1]）的活性。枯草芽孢杆菌和大肠杆菌超声破碎细胞的上清液也显示出C-亚硝基还原酶活性，但没有醛还原酶活性。通过硫酸铵分级分离、凝胶过滤和离子交换色谱法，将猪肝胞质溶胶中的主要C-亚硝基还原酶纯化了20至30倍。该制剂的最适pH为5.5，对氯汞苯甲酸（p-CMB）强烈抑制其活性。在pH 8.4存在NADH的情况下，酶制剂在5℃至少可稳定一周。高浓度的硫酸铵也能使酶稳定。发现该酶存在单体和二聚体形式的平衡，单体和二聚体形式的分子量分别估计约为83,000和160,000道尔顿。该酶几乎特异性地利用NADH，每还原1摩尔p-NSP生成对氨基苯酚消耗2摩尔NADH。亚硝基苯和醛也可作为电子受体。在纯化过程中，醛还原酶活性大致与C-亚硝基还原酶活性平行浓缩，即使通过5'-AMP-琼脂糖亲和色谱进一步纯化后，这两种活性也无法分离。N-亚硝基化合物不受该酶影响。
