Enderle-Schmitt U, Neuhaus C, Aumüller G
Institut für Anatomie und Zellbiologie, Marburg, F.R.G.
Biochim Biophys Acta. 1989 Dec 11;987(1):21-8. doi: 10.1016/0005-2736(89)90450-1.
delta 4-Steroid-5 alpha-reductase (3-oxo-5 alpha-steroid:NADP+ delta 4-oxidoreductase, EC 1.3.1.22), is a membrane-bound enzyme. In the ventral prostate of the rat, its activity is found within the nuclear envelope. Solubilization of this enzyme can only be achieved in the presence of detergents. We studied the inhibitory effect of various detergents on 5 alpha-reductase activity as a function of detergent concentration, of pH, of incubation time, of salt concentration and of additives to the buffer system. Four detergents (Lubrol WX, CHAPS, L-alpha-lysophosphatidylcholine and octyl D-glucopyranoside) were selected for subsequent solubilization studies. The overall recovery of solubilized enzyme activity was about 30% when compared to 100% of 5 alpha-reductase activity found in freshly prepared nuclei. Up to 20-30% of the nuclear proteins were extracted during the solubilization procedure. Among the various treatments tested, a concentration of 3 mg/ml L-alpha-lysophosphatidylcholine per 10 mg/ml of nuclear protein in the presence of 5 mM MgCl2, 0.1 M KCl, 0.1 M sodium citrate and 5 mM NADPH yielded the maximal enzymic activity of 56%, 15% of the nuclear proteins being solubilized in an active and stable form. The activity in these extracts could be kept stable for 2 days at 4 degrees C with a recovery of 75% of enzymic activity. A 3-fold increase of specific 5 alpha-reductase activity was obtained during solubilization under optimal conditions.
δ4-甾体-5α-还原酶（3-氧代-5α-甾体：NADP +δ4-氧化还原酶，EC 1.3.1.22）是一种膜结合酶。在大鼠腹侧前列腺中，其活性存在于核膜内。只有在洗涤剂存在的情况下才能实现这种酶的溶解。我们研究了各种洗涤剂对5α-还原酶活性的抑制作用，该作用是洗涤剂浓度、pH、孵育时间、盐浓度和缓冲系统添加剂的函数。选择了四种洗涤剂（Lubrol WX、CHAPS、L-α-溶血磷脂酰胆碱和辛基-D-葡萄糖苷）用于后续的溶解研究。与新鲜制备的细胞核中发现的100%的5α-还原酶活性相比，溶解酶活性的总体回收率约为30%。在溶解过程中提取了高达20 - 30%的核蛋白。在测试的各种处理中，在5 mM MgCl2、0.1 M KCl、0.1 M柠檬酸钠和5 mM NADPH存在下，每10 mg/ml核蛋白中3 mg/ml的L-α-溶血磷脂酰胆碱浓度产生了最大酶活性的56%，15%的核蛋白以活性和稳定的形式溶解。这些提取物中的活性在4℃下可保持稳定2天，酶活性回收率为75%。在最佳条件下溶解过程中，5α-还原酶的比活性提高了3倍。
