Akao T, Akao T, Kobashi K
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University.
J Biochem. 1987 Sep;102(3):613-9. doi: 10.1093/oxfordjournals.jbchem.a122095.
7 beta-Hydroxysteroid dehydrogenase (7 beta-HSD) was produced by Ruminococcus sp. PO1-3 obtained from among human intestinal bacteria. The enzyme was purified from a crude extract by ammonium sulfate fractionation, and Butyl-Toyopearl 650M, Sephadex G-150, Matrex Red A and Octyl-Sepharose chromatographies. The purified enzyme was obtained as a single band on polyacrylamide gel electrophoresis with enzyme activity staining and as one band corresponding to a molecular weight of 30,000 on SDS-polyacrylamide gel electrophoresis. On gel filtration, its apparent molecular weight was estimated to be 60,000. The enzyme had a sulfhydryl group(s) in its active site. Substrate specificity studies revealed that the enzyme showed absolute specificity for the beta-configuration of a hydroxyl group at the 7 position of bile acids, and required NADP+ and NADPH as cosubstrates. The Km values for ursodeoxycholic acid, 7-k etolithocholic acid, NADP+, and NADPH were 5.0, 8.5, 7.7, and 24 microM, respectively.
7β-羟基类固醇脱氢酶（7β-HSD）由从人体肠道细菌中分离得到的瘤胃球菌属PO1-3产生。该酶通过硫酸铵分级沉淀、丁基- Toyopearl 650M、葡聚糖凝胶G-150、Matrex Red A和辛基-琼脂糖凝胶色谱法从粗提物中纯化得到。经聚丙烯酰胺凝胶电泳酶活性染色后，纯化的酶呈现为单一条带，在SDS-聚丙烯酰胺凝胶电泳上呈现为一条对应分子量为30,000的条带。在凝胶过滤中，其表观分子量估计为60,000。该酶的活性位点含有巯基。底物特异性研究表明，该酶对胆汁酸7位羟基的β构型具有绝对特异性，并且需要NADP⁺和NADPH作为共底物。熊去氧胆酸、7-酮石胆酸、NADP⁺和NADPH的Km值分别为5.0、8.5、7.7和24μM。
