Isolation of 3 beta,20 alpha-hydroxysteroid oxidoreductase from sheep fetal blood.
Isolation of 3 beta,20 alpha-hydroxysteroid oxidoreductase from sheep fetal blood.
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DOI：
10.1016/0039-128x(87)90089-4
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发表时间：
1987-06-01
期刊：
影响因子：
2.7
通讯作者：
Sweet, F
中科院分区：
文献类型：
--
作者：
Chen, Q X;Nancarrow, C D;Sweet, F
采用硫酸铵分离、磷酸钙凝胶吸附、亲和层析和快速液相层析等2370倍纯化方案，从羊胎血中分离到3 β，20 α -羟基类固醇氧化还原酶。描述了一种新的高效液相色谱法测定20 α还原酶活性的方法。该酶是一个分子量为35000的单体，使用NADPH作为还原酶活性的辅助因子。将孕酮还原为4-孕酮-20 α -醇-3 -1或5 α -二氢睾酮还原为5 α -雄酮-3 β，17 β -二醇，其动力学特性分别为Km = 30.8 microM, Vmax = 0.7 nmol min-1（酶）-1或Km = 74 microM, Vmax = 1.3 nmol min-1（酶）-1。5 α -双氢睾酮竞争性抑制20 α -还原酶活性，Ki值为102微米。
3 beta,20 alpha-Hydroxysteroid oxidoreductase has been isolated from ovine fetal blood by a 2,370-fold purification scheme of ammonium sulfate fractionation, calcium phosphate gel adsorption, affinity chromatography, and fast performance liquid chromatography. A new high performance liquid chromatography-based assay for measuring 20 alpha-reductase activity is described. The enzyme is a monomer with a molecular weight of 35,000 and uses NADPH as a cofactor for reductase activity. It reduces progesterone to 4-pregnen-20 alpha-ol-3-one or 5 alpha-dihydrotestosterone to 5 alpha-androstan-3 beta,17 beta-diol with kinetic characteristics of Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1 or Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1, respectively. 5 alpha-Dihydrotestosterone competitively inhibits 20 alpha-reductase activity with a Ki value of 102 microM.