Kudo K, Amuro Y, Hada T, Higashino K
Third Department of Internal Medicine, Hyogo College of Medicine, Japan.
Biochim Biophys Acta. 1990 Aug 28;1046(1):12-8. doi: 10.1016/0005-2760(90)90088-f.
The NADPH-dependent 3 alpha-hydroxysteroid dehydrogenases (peaks 1, 2 and 3) acting on 3-keto-5 beta-cholanoic acid separated by carboxymethyl-cellulose chromatography from human liver cytosol were purified to homogeneous protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using Affi-Gel blue, phenyl-Sepharose CL-4B, TSKgel G3000 SW chromatography and chromatofocusing. The overall purifications of the enzymes from cytosol were 316-fold (peak 1), 232-fold (peak 2) and 345-fold (peak 3) and the recoveries of the enzymes were 0.4% (peak 1), 7.1% (peak 2) and 3.7% (peak 3). The isoelectric points of the enzymes were found to be 7.34, 7.46 and 7.88 by chromatofocusing. The molecular weights of the enzymes were similar and estimated to be about 32,000 by size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymological properties were nearly identical among the three forms. The reaction was reversible and the optimum pH of the enzymes for oxidation was about 8.4 and that for reduction was about 7.4. The enzymes could not reduce 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid, 7 alpha-hydroxy-5 beta-cholestan-3-one and 7 alpha, 12 alpha-dihydroxy-5 beta-cholestan-3-one to the corresponding 3 alpha-hydroxysteroids, whereas the enzymes could reduce 3,7-disubstituted 3-keto bile acids. Thus, the enzymes purified in this study were found to have a stereospecific character for some 3-ketosteroids. The enzyme activity was inhibited by p-chloromercuribenzoate; however, the inhibition was prevented by addition of dithiothreitol to the reaction mixture, indicating that the enzyme required a sulfhydryl group for activity.
通过羧甲基纤维素色谱法从人肝细胞溶胶中分离得到的、作用于3-酮-5β-胆烷酸的NADPH依赖性3α-羟基类固醇脱氢酶（峰1、峰2和峰3），利用Affi-Gel蓝、苯基琼脂糖CL-4B、TSKgel G3000 SW色谱法和色谱聚焦法，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上纯化至均一蛋白。从细胞溶胶中纯化得到的这些酶的总纯化倍数分别为316倍（峰1）、232倍（峰2）和345倍（峰3），酶的回收率分别为0.4%（峰1）、7.1%（峰2）和3.7%（峰3）。通过色谱聚焦法发现这些酶的等电点分别为7.34、7.46和7.88。通过尺寸排阻色谱法和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计这些酶的分子量相似，约为32,000。这三种形式的酶学性质几乎相同。该反应是可逆的，这些酶氧化的最适pH约为8.4，还原的最适pH约为7.4。这些酶不能将7α,12α-二羟基-3-酮-5β-胆烷酸、7α-羟基-5β-胆甾烷-3-酮和7α,12α-二羟基-5β-胆甾烷-3-酮还原为相应的3α-羟基类固醇，而这些酶可以还原3,7-二取代3-酮胆汁酸。因此，在本研究中纯化得到的这些酶对某些3-酮类固醇具有立体特异性。该酶活性受到对氯汞苯甲酸的抑制；然而，通过向反应混合物中加入二硫苏糖醇可防止这种抑制，这表明该酶的活性需要一个巯基。
