PLACENTAL 3B-HYDROXYSTEROID DEHYDROGENASE ISOMERASE

3 β-羟基-δ 4-5-类固醇脱氢酶（3-HSD）和类固醇 δ 4-5-异构酶（异构酶）复合物是一种限速酶（由于 3-HSD）在所有哺乳动物组织中类固醇生物合成的调节剂。 在人类胎盘中，它将胎儿雄激素代谢为雌激素 前体和母体双烯醇酮转化为孕酮。 以来 雌激素通过前列腺素刺激子宫肌层收缩， 孕酮松弛子宫肌层，3-HSD/异构酶可调节 人类劳动的开始。 3-HSD和异构酶活性 C-21（异戊烯）和C-19（雄甾烯）底物已被 从人胚胎的微粒体和线粒体中共纯化 胎盘作为单体Mr= 19，000的四聚体。 孕烯醇酮和 脱氢表雄酮竞争性地抑制每一种 其他人认为，C-21、C-19 3-HSD活动存在于 相同的活性部位。 使用以下方法进行中试亲和烷基化研究： C-21产物类似物16 α-溴乙酰氧基-孕酮和 微粒体酶表明C19和C-21底物是 在2个催化位点相同地修饰，一个用于3-HSD，一个用于3-HSD。 异构酶 该提案将描述微粒体和 线粒体酶：它们是同一种酶， 不同的细胞器 由于酶是膜结合的， 在体内，溶液中酶与结合到 人造磷脂囊泡 一级结构是什么 通过人胎盘cDNA λ gt 11文库确定，和 通过与Edman分析的氨基酸序列比较进行验证 退化？ 为了了解C-19和C-21的活动， 3-HSD和异构酶共存于一个蛋白质上，即活性位点上， 将用亲合烷基化类固醇和辅因子探测。 雄激素和孕酮底物的溴乙酰氧基类似物， 产品沿着与雌激素抑制剂类似物将是 合成以比较4种酶活性的失活， 放射性烷基化活性位点氨基酸，并鉴定肽区 其靠近催化中心。 这些研究将 证明负责催化位点的数量 活性以及“姐妹C-19和C-21类似物”如何在 活性位点。 使用产生的酶进行补充研究 异构酶（雌炔）和3-HSD（2-亚甲基-5 α- 雄甾烷-3 β-01）亲和性烷化剂辅因子类似物将 确认异构酶和脱氢酶活性是否存在 在一个或多个不同的地点，以及其机制 将探索多功能性。 所有结果都将是关于 分娩开始的生理控制。

3beta-Hydroxy-delta 4-5-steroid dehydrogenase (3-HSD) and steroid delta 4-5-isomerase (isomerase) complex is a rate limiting (due to 3-HSD) regulator of steroid biosynthesis in all mammalian tissues. In human placenta, it metabolizes fetal androgens to estrogen precursors and maternal pregnenolone to progesterone. Since estrogens stimulate myometrial contractions via prostaglandins and progesterone relaxes the myometrium, 3-HSD/isomerase may regulate the initiation of human labor. 3-HSD and isomerase activities for C-21 (pregnene) and C-19 (androstene) substrates have been copurified from the microsomes and mitochondria of human term placenta as a tetramer with monomeric Mr=19,000. Pregnenolone and dehydroepiandrosterone competitively inhibit the oxidation of each other to suggest that the C-21, C-19 3-HSD activities reside at the same active site. A pilot affinity alkylation study using the C-21 product analog 16 alpha-bromoacetoxy-progesterone and microsomal enzyme suggest that the C19 and C-21 substrates are modified identically at 2 catalytic sites, one for 3-HSD and one for isomerase. This proposal will characterize the microsomal and the mitochondrial enzymes: are they the same enzyme localized in different cell organelles? Since the enzyme is membrane associated in vivo, is enzyme in solution identical to enzyme bound to artificial phospholipid vesicles? What is the primary structure determine by a human placental cDNA lambda gt11 library, and validated by comparison with amino acid sequences analyzed by Edman degradation? To understand how the C-19 and C-21 activities of both 3-HSD and isomerase coexist on one protein, the active site(s) will be probed with affinity alkylating steroids and cofactors. Bromoacetoxy analogs of androgen and progestin substrates and products along with analogs of estrogen inhibitors will be synthesized to compared inactivation of the 4 enzyme activities, radioalkylate active site amino acids, and identify peptide regions which proximate the catalytic center(s). These studies will demonstrate the number of catalytic sites responsible for the activities and how "sister C-19 and C-21 analogs" align within the active sites(s). Complementary studies using enzyme generated alkylators of isomerase (estryne) and 3-HSD (2-methylene-5alpha- androstan-3 beta-01) affinity alkylators cofactor analogs will confirm whether the isomerase and dehydrogenase activities reside at one or separate sites, and the mechanism(s) of this multiactivity will be explored. All results will be insights on the physiological control of the initiation of labor.