PLACENTAL 3B-HYDROXYSTEROD DEHYDDROGENASE INSOMERASE

3β-羟基类固醇脱氢酶/β4-5异构酶(3-HSD/异构酶)，a 限速酶复合体存在于大多数类固醇代谢组织中， 不是从人胎盘中提纯出来的，在那里它是均匀分布的 在线粒体和微生物体之间。这些酶的活性是 使用脱氧胆酸钠从线粒体共溶解(Edwards等人， 1976年)。在这份提案中提供了初步数据，Brij58 胎盘微粒体的治疗共增溶3-羟色胺和异构酶。这个 申请人建议从两者中提纯溶解的3-HSD/异构酶 细胞器通过硫酸铵沉淀、亲和层析和 凝胶过滤层析。3-羟色胺和异构酶活性是否应该 接下来将进行单独的3-HSD活动。纯酶均一性 将演示：在十二烷基硫酸钠和非变性聚丙烯酰胺上的单一条带 电泳法.单氨基末端.非特异性抗体的形成 兔的免疫。 从微粒体和线粒体中纯化的3-HSD/异构酶将被 分别表征：相对分子质量；亚基组成；氨基 酸组成；氨基末端序列；溶液稳定性；pH和 最佳温度；底物和辅因子动力学分布；以及产品 抑制动力学研究。这些观察将证明 线粒体和微粒体酶之间的相似性。 为了解决孕烯和雄烯底物是否在 以及3-HSD和异构酶活性是否位于 相同或分离的中心在纯化的蛋白质上，亲和烷基化 并提出了放射性烷基化的研究方向。亲和烷基化类似物 底物(孕烯醇酮、脱氢表雄酮)、产物(孕酮、 雄烯二酮)和抑制物(雌酮、马齿豆素)类固醇； 酶促亲和烷化剂(雌酮)和烷化辅因子 将使用类似物(例如：氟磺酰苯甲酰腺苷)。配置文件 从酶失活、底物和辅因子保护 失活和放射性烷基化的氨基酸应该回答这些问题 关于3-HSD/异构酶的多重活性。 如果胎儿来源的脱氢表雄酮和母体孕烯醇酮 在同一部位氧化，胎儿可引起“黄体酮撤退” 通过3-HSD允许前列腺素介导的子宫肌层收缩。 3-HSD/异构酶的纯化和研究将阐明两者的重要性 这种酶在启动分娩和类固醇的一连串事件中的作用 大分子结合部位的新陈代谢。

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.