HUMAN PLACENTAL 3-BETA-HYDROXY-5-ENE-STEROID DEHYDROGENASE AND STEROID 5-]4-ENE-ISOMERASE - PURIFICATION FROM MITOCHONDRIA AND KINETIC PROFILES, BIOPHYSICAL CHARACTERIZATION OF THE PURIFIED MITOCHONDRIAL AND MICROSOMAL-ENZYMES
HUMAN PLACENTAL 3-BETA-HYDROXY-5-ENE-STEROID DEHYDROGENASE AND STEROID 5-]4-ENE-ISOMERASE - PURIFICATION FROM MITOCHONDRIA AND KINETIC PROFILES, BIOPHYSICAL CHARACTERIZATION OF THE PURIFIED MITOCHONDRIAL AND MICROSOMAL-ENZYMES
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DOI：
10.1016/0022-4731(89)90296-3
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发表时间：
1989-08-01
影响因子：
4.1
通讯作者：
STRICKLER, RC
中科院分区：
文献类型：
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作者：
THOMAS, JL;MYERS, RP;STRICKLER, RC
在人胎盘中，3 β-羟基-5-烯-类固醇脱氢酶和类固醇5 →4-烯异构酶是一种存在于微粒体和线粒体中的酶复合物，可从双烯醇酮合成孕酮，从胎儿脱氢表雄酮硫酸盐合成雄烯二酮。线粒体酶的脱氢酶和异构酶活性使用连续的胆酸盐溶解，离子交换色谱法（DEAE-Toyopolysin 650 S），和羟基磷灰石色谱法（Bio-Gel HT）共纯化（733倍）。 通过SDS-聚丙烯酰胺凝胶电泳（单体Mr = 41，000）中的单一蛋白条带、恒定比酶活性（Mr = 77，000）下的凝胶过滤和单一NH 2-末端序列证明了酶的均一性。测定了双烯醇酮的氧化动力学常数（Km = 1.6 μ M，Vmax = 48.6 nmol/min/mg）和脱氢表雄酮（Km = 2.4 μ M，Vmax = 48.5 nmol/min/mg）和用于5-异戊烯-3，20-二酮的异构化（Km = 9.3 μ M，Vmax = 914.2 nmol/min/mg）和5-雄甾烯-3，17-二酮（Km = 27.6 μ M，Vmax = 888.4 nmol/min/mg）。混合底物的研究表明，脱氢酶和异构酶的活动利用各自的藜芦烯和雄甾烯底物竞争。狄克逊分析表明，产物类固醇，孕酮和雄烯二酮，是C-21和C-19脱氢酶活性的竞争性抑制剂。从线粒体和微粒体纯化的酶在底物利用、产物抑制和辅因子（NAD+）还原方面具有相似的动力学曲线（平均Km . ±.使用C-19和C-21脱氢酶底物的SD = 26.4。0.8μ M，平均Vmax = 73.2 ±。1.3 nmol/min/mg）。来自两种细胞器的纯酶在分子量和亚基组成、最适pH（pH 9.8，脱氢酶; pH 7.5，异构酶）、最适温度（37 ℃）、储存和溶液中的稳定性、二价阳离子的作用和27个氨基酸的单一NH 2-末端序列方面表现出相同的生物物理性质。这些结果表明，线粒体和微粒体酶是定位于不同细胞器的相同蛋白质。
In human placenta, 3.beta.-hydroxy-5-ene-steroid dehydrogenase and steroid 5 .fwdarw. 4-ene-isomerase, an enzyme complex found in microsomes and mitochondria, synthesizes progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate. The dehydrogenase and isomerase activities of the mitochondrial enzyme were copurified (733-fold) using sequential cholate solubilization, ion exchange chromatography (DEAE-Toyopearl 650S), and hydroxylapatite chromatography (Bio-Gel HT). Enzyme homogeneity was demonstrated by a single protein band in SDS-polyacrylamide gel electrophoresis (monomeric Mr = 41,000), gel filtration at constant specific enzyme activity (Mr = 77,000), and a single NH2-terminal sequence. Kinetic constants were determined for the oxidation of pregnenolone (Km = 1.6 .mu.M, Vmax = 48.6 nmol/min/mg) and dehydroepiandrosterone (Km = 2.4 .mu.M, Vmax = 48.5 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione (Km = 9.3 .mu.M, Vmax = 914.2 nmol/min/mg) and 5-androstene-3,17-dione (Km = 27.6 .mu.M, Vmax = 888.4 nmol/min/mg. Mixed substrate studies showed that the dehydrogenase and isomerase activities utilize their respective pregnene and androstene substrates competitively. Dixon analysis demonstrated that the product steroids, progesterone and androstenedione, are competitive inhibitors of the C-21 and C-19 dehydrogenase activities. Enzyme purified from mitochondria and microsomes had similar kinetic profiles with respect to substrate utilization, product inhibition, and cofactor (NAD+) reduction (mean Km .+-. SD using C-19 and C-21 dehydrogenase substrates = 26.4 .+-. 0.8 .mu.M, mean Vmax = 73.2 .+-. 1.3 nmol/min/mg). Pure enzyme from both organelles exhibited identical biophysical properties in terms of molecular weight and subunit composition, pH optima (pH 9.8, dehydrogenase; pH 7.5, isomerase), temperature optimum (37.degree.C), stability in storage and solution, effects of divalent cations, and the single NH2-terminal sequence of 27 amino acids. These results suggest that the mitochondrial and microsomal enzymes are the same protein localized in different organelles.