PLACENTAL 3B-HYDROXYSTEROID DEHYDROGENASE ISOMERASE

3β-羟基-三角洲4-5-类固醇脱氢酶和类固醇 Delta 4-5-异构酶(异构酶)复合体是一种限速(由于 3-羟色胺)是所有哺乳动物组织中类固醇生物合成的调节因子。 在人类胎盘中，它将胎儿雄激素代谢成雌激素 前体和母体孕烯醇酮转孕酮。自.以来 雌激素通过前列腺素和雌激素刺激子宫肌层收缩 孕酮松弛子宫肌层，3-羟色胺/异构酶可能调节 人类劳动的开始。3-羟色胺和异构酶活性 C-21(孕烯)和C-19(雄烯)底物已被 由人体的微生物体和线粒体交配而成 胎盘为四聚体，单体MR=19,000。孕烯醇酮和 脱氢表雄酮竞争性抑制两者的氧化 其他建议C-21，C-19 3-HSD活动驻留在 相同的活动站点。一项中试亲和烷基化研究 C-21产物类似物16α-溴乙酰氧基孕酮和 微粒体酶表明C19和C-21底物是 在两个催化位置进行相同的修饰，一个用于3-HSD，另一个用于3-HSD 异构酶。这项提案将描述微生物体和 线粒体酶：它们是定位于 不同的细胞器？由于该酶是膜相关的 在体内，溶液中的酶是否与酶结合在一起 人造磷脂小泡？它的主要结构是什么 由人胎盘cDNAlambda gt11文库确定，以及 通过与Edman分析的氨基酸序列进行比较来验证 退化？为了了解C-19和C-21的活动是如何 3-羟色胺脱氢酶和异构酶共存于一个蛋白质的活性部位(S) 将用亲和力、烷基化类固醇和辅因子来探索。 雄激素和孕激素底物的溴乙酰氧基类似物和 与雌激素抑制剂类似物一起的产品将是 合成比较4种酶的失活情况， 放射性烷基化活性部位氨基酸，鉴定多肽区 它靠近催化中心(S)。这些研究将 展示了催化部位的数量对 活动以及“姊妹C-19和C-21类似物”如何在 活跃站点(S)。利用酶产生的补充性研究 异构酶的烷化剂(雌酮)和3-HSD(2-亚甲基-5α- 雄激素-3β-01)亲和烷基化辅因子类似物将 确认异构酶和脱氢酶活性是否存在 在一个或多个地点，以及这一过程的机制(S) 将探索多项活动。所有结果都将是对 分娩启动的生理控制。

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.