肝微粒体偶氮还原酶活性的连续分光光度法测定及其对细胞色素P-450的依赖性。
A continuous spectrophotometric determination of hepatic microsomal azo reductase activity and its dependence on cytochrome P-450.
作者信息
Mallett A K, King L J, Walker R
出版信息
Biochem J. 1982 Mar 1;201(3):589-95. doi: 10.1042/bj2010589.
Abstract
- A continuous spectrophotometric determination of rat hepatic microsomal anaerobic azo reductase activity has been developed. 2. The addition of soluble flavins (riboflavin, FMN or FAD) greatly increased this NADPH-dependent activity towards a number of azo substrates. 3. Investigations with amaranth as substrate gave an apparent Km of 34 microM and Vmax. of 4 nmol/min per mg of microsomal protein. The inclusion of a fixed concentration of FMN increased Vmax. and greatly decreased Km, the magnitude of these changes reflecting the concentration of flavin present. 4. Investigations using a fixed amaranth concentration over a range of flavin concentrations gave biphasic double-reciprocal plots with two apparent Km and Vmax. values. 5. Pretreatment of animals with cobaltous chloride, 2-allyl-2-isopropylacetamide, carbon tetrachloride, phenobarbitone and 3-methylcholanthrene altered azo reductase activity in parallel with changes in cytochrome P-450 content. 6. The significance of these results is discussed in terms of the electron-transfer components present in the hepatic microsomal fraction.
摘要
- 已开发出一种连续分光光度法测定大鼠肝微粒体厌氧偶氮还原酶活性。2. 添加可溶性黄素（核黄素、黄素单核苷酸或黄素腺嘌呤二核苷酸）极大地增强了这种对多种偶氮底物的依赖烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的活性。3. 以苋菜红为底物进行的研究得出表观米氏常数（Km）为34微摩尔，最大反应速度（Vmax）为每毫克微粒体蛋白4纳摩尔/分钟。加入固定浓度的黄素单核苷酸可提高最大反应速度，并极大地降低米氏常数，这些变化的幅度反映了黄素的存在浓度。4. 在一系列黄素浓度下使用固定的苋菜红浓度进行的研究得出双相双倒数图，有两个表观米氏常数和最大反应速度值。5. 用氯化钴、2-烯丙基-2-异丙基乙酰胺、四氯化碳、苯巴比妥和3-甲基胆蒽对动物进行预处理，会使偶氮还原酶活性发生改变，且与细胞色素P-450含量的变化平行。6. 根据肝微粒体部分中存在的电子传递成分对这些结果的意义进行了讨论。
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