MOLECULAR BIOLOGY OF STEROID RECEPTORS IN XENOPUS OOCYTES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 在两栖动物的成熟卵泡中，促性腺激素诱导卵泡细胞产生一种诱导成熟的类固醇-孕酮，它直接作用于卵母细胞以启动成熟。 成熟是停滞在减数分裂前期I的卵母细胞恢复并完成其第一次减数分裂的过程。 卵母细胞成熟的完成是正常受精所必需的。早期的研究表明，孕酮在卵母细胞成熟中的作用涉及高亲和力受体。 然而，对孕酮受体知之甚少。 本研究的目的是分离和纯化受体。 从非洲爪蟾中收集卵母细胞并处理以获得质膜。 用毛地黄皂苷从质膜上溶解孕酮受体，并将其应用于天然稳态聚丙烯酰胺凝胶管以测定其结合活性。 在此过程之后，在管凝胶中观察到孕酮结合活性的单峰。 在不同丙烯酰胺浓度的凝胶中，峰的迁移距离不同。 然后通过将管凝胶置于十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）平板凝胶上来分离管凝胶中的蛋白质。 在银染色的平板中，鉴定出一个单独的斑点，其对应于管凝胶中孕酮结合活性的峰，并且仅与管凝胶中孕酮结合活性的峰共迁移。 该蛋白质从凝胶中电洗脱，并通过梯度SDS-PAGE显示为均质的。 在还原或非还原条件下，蛋白质的分子量约为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。 总之，爪蟾卵母细胞的膜孕酮受体被溶解和分离，并纯化了分子量约为90 KDa的单体蛋白，其具有膜结合孕酮受体的电泳和免疫学特征。

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. Completion of oocyte maturation is required for normal fertilization. Early studies have shown that the action of progesterone in oocyte maturation involves a high-affinity receptor. However, little is known about the 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 determine its binding activity. Following this procedure, a single peak of progesterone binding activity was observed in the tube gels. The migration distance of the peak varied in gels of different acrylamide concentrations. 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 tube gels. 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 about 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.