人红细胞17-羟基类固醇脱氢酶的分离与鉴定

Isolation and characterization of 17 -hydroxy steroid dehydrogenase from human erythrocytes.

作者信息

Mulder E, Lamers-Stahlhofen G J, van der Molen H J

出版信息

Biochem J. 1972 May;127(4):649-59. doi: 10.1042/bj1270649.

DOI:10.1042/bj1270649

PMID:4405572

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1178762/

Abstract

The 17beta-hydroxy steroid dehydrogenase was solubilized during haemolysis of erythrocytes and was isolated from the membrane-free haemolysate. Membrane preparations isolated in different ways did not contain 17beta-hydroxy steroid dehydrogenase activity. The 17beta-hydroxy steroid dehydrogenase activity in the haemolysate was concentrated by repeated ammonium sulphate precipitation and gel filtration on Sephadex G-150. The 17beta-hydroxy steroid dehydrogenase activity of the purified preparation per unit weight of protein was 350-3000 times higher than the activity of the crude erythrocyte haemolysate. The 20alpha-hydroxy steroid dehydrogenase activity was lost during this purification procedure. 2. The 17beta-hydroxy steroid dehydrogenase was NADP-dependent and had a pH optimum for conversion of testosterone between 8.5 and 10. For the molecular weight of the enzyme a value of 64000 was calculated from Sephadex chromatography results. 3. p-Chloromercuribenzoate inhibited the enzymic activity. The oxidative activity of the enzyme for the 17beta-hydroxyl group was only partly inhibited when a large excess of 17-oxo steroids was added. The catalysing activity of the enzyme was influenced by the NADP(+)/NADPH ratio. The oxidation of the 17beta-hydroxyl group in the presence of NADP(+) proceeded faster than the reduction of the 17-oxo group with NADPH. When both reduced and oxidized cofactors were present the oxidation of the 17beta-hydroxyl group was inhibited to a considerable extent. 4. The enzyme had a broad substrate specificity and not only catalysed the conversion of androstanes with a 17beta-hydroxyl group, or 17-oxo group, but also the conversion oestradiolleft arrow over right arrowoestrone. In addition the steroid conjugates dehydroepiandrosterone sulphate and oestrone sulphate were also converted. There were no indications that more than one 17beta-hydroxy steroid dehydrogenase was present in the partially purified preparation.

摘要

17β - 羟基类固醇脱氢酶在红细胞溶血过程中被溶解，并从无膜溶血产物中分离出来。以不同方式分离的膜制剂不含有17β - 羟基类固醇脱氢酶活性。溶血产物中的17β - 羟基类固醇脱氢酶活性通过重复硫酸铵沉淀和在葡聚糖凝胶G - 150上的凝胶过滤进行浓缩。纯化制剂每单位重量蛋白质的17β - 羟基类固醇脱氢酶活性比粗制红细胞溶血产物的活性高350 - 3000倍。在此纯化过程中，20α - 羟基类固醇脱氢酶活性丧失。2. 17β - 羟基类固醇脱氢酶依赖于NADP，在睾酮转化过程中，最适pH值在8.5至10之间。根据葡聚糖凝胶色谱结果计算，该酶的分子量为64000。3. 对氯汞苯甲酸抑制酶活性。当加入大量过量的17 - 氧代类固醇时，该酶对17β - 羟基的氧化活性仅部分受到抑制。酶的催化活性受NADP( + ) / NADPH比例的影响。在NADP( + )存在下，17β - 羟基的氧化比用NADPH还原17 - 氧代基团进行得更快。当还原型和氧化型辅因子都存在时，17β - 羟基的氧化受到相当程度的抑制。4. 该酶具有广泛的底物特异性，不仅催化具有17β - 羟基或17 - 氧代基团的雄烷的转化，还催化雌二醇⇌雌酮的转化。此外，甾体共轭物硫酸脱氢表雄酮和硫酸雌酮也被转化。没有迹象表明在部分纯化的制剂中存在不止一种17β - 羟基类固醇脱氢酶。