Edenharder R, Pfützner M, Hammann R
Institute of Hygiene, University of Mainz, F.R.G.
Biochim Biophys Acta. 1989 Mar 14;1002(1):37-44. doi: 10.1016/0005-2760(89)90061-1.
A lecithinase-lipase-negative Clostridium sp. 25.11.c., not fitting in any of the species of Clostridia described so far as judged by morphological, physiological, and biochemical data, was shown to contain NADP-dependent 3 beta-, 7 alpha- and 7 beta-hydroxysteroid dehydrogenases. The three hydroxysteroid dehydrogenases could be demonstrated in the supernatant and in the membrane fraction after solubilization with Triton X-100, suggesting enzymes which were originally membrane bound. The 3 beta-hydroxysteroid dehydrogenase was synthesized constitutively, and the specific enzyme activity was significantly reduced by growth medium supplementation with 3-keto bile acids and trisubstituted bile acids. A pH optimum of 7.5 and a molecular weight of approx. 104,000 were estimated by molecular sieve chromatography. The enzyme reduced the 3-keto group of bile acids; an oxidation of a 3 beta-hydroxyl function could not be demonstrated. The lowest Km values were found for disubstituted bile acids, trisubstituted and conjugated bile acids having higher Km values. 7 alpha-Hydroxysteroid dehydrogenase, but not 7 beta-hydroxysteroid dehydrogenase, was already present in uninduced cells. The specific activities, however, were greatly enhanced when cells were grown in the presence of chenodeoxycholic acid or 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid. Ursodeoxycholic acid with its 7 beta-hydroxyl group was ineffective as an inducer. Molecular weights of approx. 82,000 and 115,000 were found for the 7 alpha-hydroxysteroid dehydrogenase and the 7 beta-hydroxysteroid dehydrogenase, respectively. In contrast to the in vivo situation, the reaction could only be demonstrated in the reductive direction in vitro. Here, the pH optimum for the overall reaction was 8.5-8.7. 3 beta-, 7 alpha- and 7 beta-hydroxysteroid dehydrogenase activities were readily demonstrated for at least 48 h when preparations were stored at 4 degrees C, but were found to be heat-sensitive.
一株卵磷脂酶 - 脂肪酶阴性的梭菌属菌株25.11.c.，根据形态学、生理学和生化数据判断，它不属于目前已描述的任何梭菌属物种，该菌株被证明含有NADP依赖性的3β -、7α - 和7β - 羟基类固醇脱氢酶。在用 Triton X - 100溶解后，在上清液和膜组分中均可检测到这三种羟基类固醇脱氢酶，这表明这些酶最初是膜结合型的。3β - 羟基类固醇脱氢酶是组成型合成的，当生长培养基中添加3 - 酮胆汁酸和三取代胆汁酸时，其比酶活性显著降低。通过分子筛色谱法估计其最适pH为7.5，分子量约为104,000。该酶可还原胆汁酸的3 - 酮基；未检测到3β - 羟基功能的氧化反应。二取代胆汁酸的Km值最低，三取代和共轭胆汁酸的Km值较高。7α - 羟基类固醇脱氢酶在未诱导的细胞中就已存在，但7β - 羟基类固醇脱氢酶不存在。然而，当细胞在鹅去氧胆酸或3α - 羟基 - 7 - 酮 - 5β - 胆烷酸存在下生长时，其比活性会大大提高。具有7β - 羟基的熊去氧胆酸作为诱导剂无效。7α - 羟基类固醇脱氢酶和7β - 羟基类固醇脱氢酶的分子量分别约为82,000和115,000。与体内情况相反，该反应在体外仅能在还原方向上检测到。在此，整个反应的最适pH为8.5 - 8.7。当制剂在4℃储存时，3β -、7α - 和7β - 羟基类固醇脱氢酶活性至少在48小时内很容易检测到，但发现它们对热敏感。
