Scheer H, Robaire B
Biochem J. 1983 Apr 1;211(1):65-74. doi: 10.1042/bj2110065.
Epididymal delta 4-steroid 5 alpha-reductase (cholestenone 5 alpha-reductase), the enzyme that catalyses the conversion of testosterone into the biologically active metabolite dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one), is a membrane-bound enzyme found in both nuclear and microsomal subcellular fractions. In order to characterize epididymal delta 4-steroid 5 alpha-reductase, it was first necessary to solubilize the enzymic activity. Of the various treatments tested, a combination of 0.5% (w/v) Lubrol WX, 0.1 M-sodium citrate and 0.1 M-KCl maintained enzymic activity at control values and solubilized 66% of total epididymal delta 4-steroid 5 alpha-reductase activity in an active and stable form. The sedimentation coefficient of solubilized delta 4-steroid 5 alpha-reductase, as determined in continuous sucrose density gradients, was greater for the microsomal than for the nuclear enzyme (11.6S compared with 10.1S). Although the apparent Km values of the enzyme for testosterone were similar in nuclear and microsomal subcellular fractions (range 1.75 x 10(-7) - 4.52 x 10(-7)M), the apparent Km of the enzyme for NADPH was about 30-fold greater for the microsomal enzyme than for the nuclear enzyme. The apparent Km of the enzyme for either substrate was not significantly altered after solubilization. The relative capacity of steroids to inhibit the enzymic activity, the pH optima and the effects of Ca2+ and Mg2+ were similar for membrane-bound and solubilized delta 4-steroid 5 alpha-reductase in both the nuclear and the microsomal fractions. The results reported demonstrate that epididymal delta 4-steroid 5 alpha-reductase can be solubilized in an active and stable form with no significant changes in the kinetic characteristics of the enzyme after solubilization; furthermore, kinetic and molecular-size differences observed for the nuclear and the microsomal forms of the enzyme suggest that there may exist at least two forms of epididymal delta 4-steroid 5 alpha-reductase.
附睾δ4-甾体5α-还原酶（胆甾烯酮5α-还原酶）可催化睾酮转化为具有生物活性的代谢产物二氢睾酮（17β-羟基-5α-雄甾烷-3-酮），是一种存在于细胞核和微粒体亚细胞组分中的膜结合酶。为了对附睾δ4-甾体5α-还原酶进行特性分析，首先必须使酶活性溶解。在测试的各种处理中，0.5%（w/v）Lubrol WX、0.1M柠檬酸钠和0.1M氯化钾的组合可使酶活性维持在对照值，并以活性和稳定的形式溶解了66%的附睾δ4-甾体5α-还原酶总活性。在连续蔗糖密度梯度中测定，微粒体溶解的δ4-甾体5α-还原酶的沉降系数大于细胞核酶（分别为11.6S和10S）。尽管该酶对睾酮的表观Km值在细胞核和微粒体亚细胞组分中相似（范围为1.75×10−7 - 4.52×10−7M），但该酶对NADPH的表观Km值，微粒体酶比细胞核酶大约高30倍。溶解后，该酶对任何一种底物的表观Km值均无显著变化。在细胞核和微粒体组分中，膜结合的和溶解的δ4-甾体5α-还原酶在甾体抑制酶活性的相对能力、最适pH以及Ca2+和Mg2+的影响方面相似。所报道的结果表明，附睾δ4-甾体5α-还原酶可以以活性和稳定的形式溶解，溶解后酶的动力学特性无显著变化；此外，观察到的该酶细胞核和微粒体形式的动力学和分子大小差异表明，可能存在至少两种形式的附睾δ4-甾体5α-还原酶。
