MOLECULAR BIOLOGY OF STEROID RECEPTORS IN XENOPUS OOCYTES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在两栖动物成熟的卵泡中，促性腺激素诱导卵泡细胞产生一种成熟诱导类固醇，孕酮，它直接作用于卵母细胞，启动成熟。成熟是处于减数分裂前期I期的卵母细胞恢复并完成第一次减数分裂的过程。这个过程包括染色体凝聚、生发泡破裂和第一极体的挤出。成熟完成后，卵母细胞减数分裂再次停止在中期II。完成卵母细胞成熟是正常排卵和受精所必需的。早期的研究表明，孕酮在卵母细胞成熟中的作用涉及一个高亲和力和低容量的受体。然而，人们对细胞表面孕激素受体的特性知之甚少。本研究的目的是分离纯化该受体。从非洲爪哇采集卵母细胞，处理后得到质膜。用洋地黄素从质膜上增溶孕酮受体，并将其应用于自然稳态的聚丙烯酰胺凝胶管，分离其配体结合活性。在这一过程之后，沿着试管凝胶一致地观察到黄体酮结合活性的单峰，该活性在存在过量的未标记黄体酮的情况下是饱和的。不同浓度的丙烯酰胺凝胶中峰的迁移距离不同。天然相对分子质量分析表明，孕酮结合蛋白约为107 KDa。将试管凝胶放置在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳板上，分离试管凝胶中的蛋白质。在银染切片中，发现了与不同浓度的酰胺的一维管状凝胶中的孕酮结合活性峰值相对应的单一斑点，并且该斑点唯一地与其共同迁移。该蛋白经电洗脱后，经梯度SDS-PAGE鉴定为均一蛋白。在还原或非还原条件下，蛋白质的相对分子质量约为90 KDa。针对该90 KDa蛋白的特异性免疫血清显著抑制卵母细胞膜制剂的孕酮结合活性。综上所述，本文对非洲爪哇卵母细胞的膜孕酮受体进行了溶解和分离，并纯化了一种具有膜结合孕酮受体的电泳学和免疫学特性的单体蛋白，其相对分子质量约为90 KDa。(由NIH Grant P20 RR016480支持)。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In full-grown ovarian follicles of amphibians gonadotropin induces the follicular cells to produce a maturation-inducing steroid, progesterone, that acts directly on the oocyte to initiate maturation. Maturation is the process by which oocytes arrested in prophase I of meiosis resume and complete their first meiotic division. This process involves chromosome condensation, germinal vesicle breakdown, and extrusion of the first polar body. At the completion of maturation, oocyte meiosis once again becomes arrested at metaphase II. Completion of oocyte maturation is required for normal ovulation and fertilization. Early studies have shown that the action of progesterone in oocyte maturation involves a high-affinity and low-capacity receptor. However, little is known about the characteristics of the cell surface progesterone receptor. The objective of this study is to isolate and purify the receptor. Oocytes were collected from Xenopus laevis and processed to obtain plasma membrane. Progesterone receptor was solubilized from plasma membranes with digitonin and applied to native steady state polyacrylamide gel tubes to isolate its ligand binding activity. Following this procedure, a single peak of progesterone binding activity which was saturable in the presence of excess unlabeled progesterone was consistently observed along the tube gels. The migration distance of the peak varied in gels of different acrylamide concentrations. Native molecular weight analysis showed the progesterone binding protein to be about 107 KDa. Proteins in the tube gels were then separated by placing the tube gels on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) slab gels. In silver-stained slabs, a single spot was identified that corresponded to, and exclusively co-migrated with, the peak of the progesterone binding activity in the first-dimension tube gels of different acylamide concenrations. This protein was electro-eluted from the gels and shown to be homogenous by gradient SDS-PAGE. The molecular weight of the protein under reducing or nonreducing conditions was approximately 90 KDa. A specific immune serum directed against this 90 KDa protein significantly inhibited the progesterone binding activity of oocyte membrane preparations. In conclusion, the membrane progesterone receptor of Xenopus oocytes was solubilized and isolated and a monomeric protein at the molecular weight of approximately 90 KDa with electrophoretic and immunologic characteristics of the membrane-bound progesterone receptor was purified. (Supported by NIH Grant P20 RR016480).