睾丸酮假单胞菌17-β-羟基类固醇脱氢酶活性的表征及3-α-羟基类固醇脱氢酶的亲和标记

Characterisation of an associate 17-beta-hydroxysteroid dehydrogenase activity and affinity labelling of the 3-alpha-hydroxysteroid dehydrogenase of Pseudomonas testosteroni.

作者信息

Battais E, Terouanne B, Nicolas J C, Descomps B, Crastes de Paulet A

出版信息

Biochimie. 1977;59(11-12):909-17. doi: 10.1016/s0300-9084(78)80706-8.

Abstract

The 3-alpha-hydroxysteroid dehydrogenase and the 3-beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni were purified to homogeneity by polyaerylamide gel electrophoresis using the following stages: DEAE cellulose chromatography, affinity chromatography on oestrone-aminocaproate sepharose and Sephadex gel filtration. The pure 3-alpha-hydroxysteroid dehydrogenase was completely devoid of 3-beta-hydroxysteroid dehydrogenase activity but could oxidize estradiol 17-beta at an appreciable rate. This activity accounts for about 40 per cent of the total 17-beta-estradiol dehydrogenase of the crude bacterial extract. Affinity labelling of pure 3-alpha-hydroxysteroid dehydrogenase was carried out using 5-beta-pregnane 3,20-dione-12-alpha-iodoacetate and 5-alpha-androstane 3-one-17-beta-bromoacetate. With both reagents, inactivation was obtained only in the presence of coenzyme, the substrate protected against inactivation and the enzyme was fully inhibited with covalent binding of 1 mole of reagent per mole of subunit suggesting an active site directed inhibition. Histidine and methionine were identified as the labelled aminoacid residues.

摘要

通过以下步骤，利用聚丙烯酰胺凝胶电泳将睾丸酮假单胞菌的3-α-羟基类固醇脱氢酶和3-β-羟基类固醇脱氢酶纯化至均一状态：二乙氨基乙基纤维素色谱法、雌酮-氨基己酸琼脂糖亲和色谱法和葡聚糖凝胶过滤法。纯化后的3-α-羟基类固醇脱氢酶完全没有3-β-羟基类固醇脱氢酶活性，但能以可观的速率氧化17-β-雌二醇。该活性约占粗细菌提取物中总17-β-雌二醇脱氢酶的40%。使用5-β-孕烷3,20-二酮-12-α-碘乙酸盐和5-α-雄甾烷-3-酮-17-β-溴乙酸盐对纯化后的3-α-羟基类固醇脱氢酶进行亲和标记。使用这两种试剂时，只有在辅酶存在的情况下才会出现失活现象，底物可保护酶不被失活，并且每摩尔亚基与1摩尔试剂共价结合时酶会被完全抑制，这表明是活性位点定向抑制。已鉴定出组氨酸和甲硫氨酸为被标记的氨基酸残基。