PLACENTAL 3B-HYDROXYSTEROD DEHYDDROGENASE INSOMERASE

3 β-羟基类固醇脱氢酶/δ 4-5异构酶（3-HSD/异构酶），a 在大多数类固醇代谢组织中发现的限速酶复合物， 还没有从人体胎盘中纯化出来， 在线粒体和微粒体之间。 酶活性为 使用脱氧胆酸钠从线粒体共溶解（Edwards等， 1976年）。 初步数据显示，Brij 58 胎盘微粒体的处理共溶解3-HSD和异构酶。 的 申请人提出从这两者中纯化溶解的3-HSD/异构酶 硫酸铵沉淀，亲和层析，和 凝胶过滤色谱法。 3-HSD和异构酶活性 将遵循单独的3-HSD活性。 纯化酶的均一性 将证明：SDS和非变性聚丙烯酰胺上的单一条带 电泳;单NH 2-末端;非特异性抗体形成 兔子的免疫 从微粒体和线粒体中纯化的3-HSD/异构酶将被 分别表征：分子量;亚基组成;氨基 酸组成; NH 2-末端序列;溶液稳定性; pH和 最佳温度;底物和辅因子动力学曲线;和产物 抑制动力学研究 这些观察将证明 线粒体和微粒体酶的相似性 为了解决双烯和雄甾烯底物是否被氧化， 相同的活性位点以及3-HSD和异构酶活性是否位于 相同或不同的中心对纯化的蛋白质，亲和烷基化 和放射性烷基化研究提出。 的亲和烷基化类似物 底物（双烯醇酮，脱氢表雄酮），产物（孕酮， 雄烯二酮）和抑制剂（雌酮、马烯雌酮）类固醇; 酶产生的亲和性烷基化剂（雌炔）;和烷基化辅因子 将使用类似物（例如：氟磺酰基苯甲酰基腺苷）。 简档 从酶失活，底物和辅因子的保护， 灭活和氨基酸的放射性烷基化应该回答这些问题 关于3-HSD/异构酶的多种活性。 如果胎儿来源的脱氢表雄酮和母体去甲烯醇酮是 氧化在同一地点，胎儿可以诱导“孕酮撤退” 通过3-HSD，使胰高血糖素介导的子宫肌层收缩。 3-HSD/异构酶的纯化和研究将阐明其重要性 这种酶在引发分娩和类固醇的级联反应中的作用 在大分子结合位点的代谢。

3Beta-Hydroxysteroid dehydrogenase/delta 4-5 isomerase (3-HSD/isomerase), a rate-limiting enzyme complex found in most steroid-metabolizing tissues, has not been purified from human placenta where it is equally distributed between the mitochondria and microsomes. The enzyme activities were co-solubilized from mitochondria using sodium deoxycholate (Edwards et al., 1976). Preliminary data is presented in this proposal that Brij 58 treatment of placental microsomes co-solubilizes 3-HSD and isomerase. The applicant proposes to purify solubilized 3-HSD/isomerase from both organelles by ammonium sulfate precipitation, affinity chromatography, and gel filtration chromatography. Should the 3-HSD and isomerase activities separate, 3-HSD activity will be followed. Purified enzyme homogeneity will be demonstrated: single band on SDS and non-denaturing polyacrylamide electrophoresis; single NH2-terminus; non-specific antibody formation by immunization of rabbits. 3-HSD/isomerase purified from microsomes and mitochondria will be separately characterized: molecular weight; subunit composition; amino acid composition; NH2-terminus sequence; stability in solution; pH and temperature optimums; substrate and cofactor kinetic profile; and product inhibition kinetic studies. These observations will demonstrate the degree of similarity between the mitochondrial and microsomal enzymes. To address whether pregnene and androstene substrates are oxidized at the same active site and whether the 3-HSD and isomerase activities reside at the same or separate centers on the purified protein, affinity alkylation and radioalkylation studies are proposed. Affinity alkylating analogs of substrate (pregnenolone, dehydroepiandrosterone), product (progesterone, androstenedione), and inhibitor (estrone, equilenin) steroids; an enzyme-generated affinity alkylator (estryne); and alkylating cofactor analogs (eg: fluorosulfonylbenzoyladenosine) will be used. The profiles from enzyme inactivation, substrate and cofactor protection from inactivation, and amino acids radioalkylated should answer these questions regarding the multiple activities of 3-HSD/isomerase. If dehydroepiandrosterone of fetal origin and maternal pregnenolone are oxidized at the same site, the fetus can induce "progesterone withdrawal" via 3-HSD allowing prostaglandin-mediated myometrial contractions. Purification and study of 3-HSD/isomerase will clarify both the importance of this enzyme in the cascade of events which initiate labor and steroid metabolism at a macromolecular binding site.