Okuda K, Masumoto O, Ohyama Y
Department of Biochemistry, Hiroshima University School of Dentistry, Japan.
J Biol Chem. 1988 Dec 5;263(34):18138-42.
5 beta-Cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylase (5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol, NADPH:oxygen oxidoreductase (26-hydroxylating), EC 1.14.13.15) was purified from female rat liver mitochondria based on its catalytic activity. The final preparation of the enzyme showed a single major band on the sodium dodecyl sulfate-polyacrylamide gel electrophoretogram. The content of purified enzyme was 12 nmol/mg of protein, and the specific activity was 431 nmol/min/mg of protein. The molecular weight of the enzyme was determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis as 52,500. The absorption spectra of the purified enzyme and that of the dithionite-reduced CO complex showed peaks at 417 and 450 nm, respectively, indicating the enzyme belongs to the cytochrome P-450 family. Upon reconstitution with the electron-transferring system of the adrenal (adrenodoxin and NADPH-adrenodoxin reductase), the enzyme showed high activity hydroxylating 5 beta-cholestane-3 alpha,7 alpha-12-triol at position 27 with a turnover number of 35.5 min-1 and Km of 6.3 microM. The enzyme activity was completely lost when the electron-transferring system was replaced by that of microsomes (NADPH-cytochrome P-450 reductase purified from rat liver microsomes), confirming that the P-450 enzyme was of the mitochondrial type, but not of the microsomal. The omission of cytochrome P-450, adrenodoxin, or NADPH-adrenodoxin reductase resulted in complete loss of enzyme activity. The specific activity toward 5 beta-cholestane-3 alpha, 7 alpha-diol was less than one-half that toward cholestanetriol and that toward cholesterol was about one-fiftieth. The enzyme showed no activity toward xenobiotics such as benzphetamine, 7-ethoxycoumarin, and benzo[a]pyrene. Its activity was not inhibited by metyrapone and slightly inhibited by aminoglutethimide. The enzyme activity was markedly lowered in an atmosphere of CO/O2/N2, 40/20/40.
基于其催化活性，从雌性大鼠肝线粒体中纯化出5β-胆甾烷-3α,7α,12α-三醇27-羟化酶（5β-胆甾烷-3α,7α,12α-三醇，NADPH：氧氧化还原酶（26-羟化），EC 1.14.13.15）。该酶的最终制剂在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳图谱上显示出一条主要条带。纯化酶的含量为12 nmol/mg蛋白质，比活性为431 nmol/min/mg蛋白质。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定该酶的分子量为52,500。纯化酶及其连二亚硫酸盐还原的CO复合物的吸收光谱分别在417和450 nm处出现峰值，表明该酶属于细胞色素P-450家族。在用肾上腺的电子传递系统（肾上腺铁氧还蛋白和NADPH-肾上腺铁氧还蛋白还原酶）重组后，该酶在27位羟基化5β-胆甾烷-3α,7α-12-三醇表现出高活性，周转数为35.5 min-1，Km为6.3 μM。当电子传递系统被微粒体的电子传递系统（从大鼠肝微粒体中纯化的NADPH-细胞色素P-450还原酶）取代时，酶活性完全丧失，证实该P-450酶是线粒体类型，而非微粒体类型。省略细胞色素P-450、肾上腺铁氧还蛋白或NADPH-肾上腺铁氧还蛋白还原酶会导致酶活性完全丧失。对5β-胆甾烷-3α,7α-二醇的比活性小于对胆甾烷三醇的比活性的一半，对胆固醇的比活性约为对胆甾烷三醇比活性的五十分之一。该酶对诸如苄非他明、7-乙氧基香豆素和苯并[a]芘等外源性物质无活性。其活性不受美替拉酮抑制，而受氨鲁米特轻微抑制。在CO/O2/N2（40/20/40）气氛中，酶活性显著降低。
