小鼠肾上腺C19-类固醇5β-还原酶的亚细胞定位及特性
Subcellular localization and properties of mouse adrenal C19-steroid 5beta-reductase.
作者信息
Collins W, Cameron E H
出版信息
Biochem J. 1975 Apr;147(1):165-74. doi: 10.1042/bj1470165.
The localization and some characteristics of mouse adrenal C19-steroid 5 beta-reductase were determined by the incubation of subcellular fractions of mouse adrenal tissue with [7 alpha-3H]androst-4-ene-3,17-dione. This enzyme was present only in the soluble fraction and was NADPH-dependent, although a small activity in the presence of NADH was also detected. The soluble fraction also contained 3alpha-, 3beta- and a small amount of 17 beta-hydroxy steroid dehydrogenase. These and other steroid-metabolizing enzymes present in the remaining subcelluar fractions are also described briefly. To measure 5 beta-androstane-3,17-dione production by the mouse adrenal soluble fraction, all 5 beta products first had to be oxidized to 5 beta-androstane-3,17-dione, and the recovery of radio-activity between the substrate androst-4-ene-3,17-dione and product 5 beta-androstane-3,17-dione of 96.1 +/-3.2% validated this technique. C19-steroid 5 beta-reductase has a pH optimum of 6.5 and at low substrate concentrations the Km and Vmax. for 5 beta reduction of [7 alpha-3H]androst-4-ene-ene-3,17-dione was 2.22 times 10(-6) "/- 0.48 times 10(-6) M and 450+/- 53 pmol/min per mg of protein respectively. At high substrate concentration, inhibition of the reaction occurred, which was shown to be due to increasing product concentration.
通过将小鼠肾上腺组织的亚细胞组分与[7α-³H]雄甾-4-烯-3,17-二酮一起孵育，确定了小鼠肾上腺C19-类固醇5β-还原酶的定位和一些特性。这种酶仅存在于可溶性组分中，并且依赖于NADPH，不过在存在NADH时也检测到了少量活性。可溶性组分中还含有3α-、3β-和少量的17β-羟基类固醇脱氢酶。还简要描述了存在于其余亚细胞组分中的这些和其他类固醇代谢酶。为了测量小鼠肾上腺可溶性组分产生5β-雄甾烷-3,17-二酮的情况，所有5β产物首先必须被氧化为5β-雄甾烷-3,17-二酮，并且底物雄甾-4-烯-3,17-二酮与产物5β-雄甾烷-3,17-二酮之间放射性的回收率为96.1±3.2%，这验证了该技术。C19-类固醇5β-还原酶的最适pH为6.5，在低底物浓度下，[7α-³H]雄甾-4-烯-3,17-二酮5β还原的Km和Vmax分别为2.22×10⁻⁶±0.48×10⁻⁶M和450±53pmol/分钟每毫克蛋白质。在高底物浓度下，反应受到抑制，这表明是由于产物浓度增加所致。
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