BIOCHEMICAL STUDIES ON THE LH-HCG RECEPTOR

LH-HCG 受体的生化研究

• 批准号: 3318767
• 负责人: Brij Saxena
• 金额: $ 10.6万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-02-01 至 1991-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3318767
• 关键词: Primates; affinity chromatography; chorionic gonadotropin; corpus luteum; disulfide bond; gel electrophoresis; genetic manipulation; hormone receptor; laboratory mouse; luteinizing hormone; membrane reconstitution /synthesis; molecular cloning; monoclonal antibody; phospholipids; protein reconstitution; protein sequence; protein structure; ultracentrifugation

A receptor preparation specific to luteinizing hormone and to human chorionic gonadotropin has been isolated from bovine corpora lutea and is available in our laboratory in sufficient quantities to permit biochemical, structural and immunological studies proposed here. Subunits of the receptor of 85,000 and 38,000 molecular weight are also available in the purified form. The isolated receptor and its subunits are largely protein in nature with minor moieties of carbohydrates and lipids. We propose to perform recombination experiments to confirm that the 85K and 38K fragments are disulfide-linked subunits of the receptor molecule. Reconstitution experiments, where receptors are taken out of the biological membrane and put back into an artificial membrane environment will also be performed for the LH-hCG receptor using phospholipids. We will characterize the protein moieties by SDS-gel electrophoresis, ultracentrifugal analyses, peptide mapping, end group analyses and partial amino acid sequence determination. A library of monoclonal antibodies to the LH-hCG receptor and its subunits will be produced. The antibodies will be purified and mapped for their specificities. The monoclonal antibodies will be utilized in structural analyses of the receptor; in the immunocytochemical localization of the receptor; to follow the biochemical fate of the hormone-receptor complex in the cell. The monoclonal antibodies will be immobolized on a solid phase and used to characterize receptor fractions by affinity chromatography. The monoclonal antibodies will also be used in the cloning of genetic material. The cloned DNA sequences will be utilized to alternatively confirm the sequence of the receptor and subunits. The monoclonal antibodies can be used for the development of more specific assays of the receptor. Monoclonal antibodies, because of their selectivity, specificity and unlimited productibility, can lend themselves for clinical use when coupled to a drug. These studies are vital to understand the mechanism of hormone action at the molecular level during normal and abnormal gonadal function.

一种对促黄体生成激素具有特异性的受体制剂， 人绒毛膜促性腺激素已从牛 在我们的实验室中， 允许生物化学、结构和免疫学 在此提出的研究。 85，000的受体亚单位， 38，000分子量也可以纯化形式获得。 分离的受体及其亚基在本质上主要是蛋白质 含有少量的碳水化合物和脂质。 我们建议 进行复合实验，以确认85 K和 38 K片段是受体的二硫键连接亚基 分子。 重组实验，其中受体被 从生物膜中分离出来， 膜环境也将用于LH-hCG 受体使用磷脂。 我们将描述这种蛋白质 通过SDS-凝胶电泳，超电泳分析， 肽图谱、末端基团分析和部分氨基酸 序列测定 单克隆抗体库， 将产生LH-hCG受体及其亚单位。 的 抗体将被纯化并针对其特异性作图。 的 单克隆抗体将用于结构分析， 受体;在受体的免疫细胞化学定位; 来追踪受体复合物的生化命运 在细胞中。 将单克隆抗体固定在 固相并用于通过亲和力表征受体级分 层析 单克隆抗体也将用于 遗传物质的克隆。 克隆的DNA序列将 可用于替代地确认受体的序列 和子单元。 所述单克隆抗体可用于 开发更特异性的受体测定法。 单克隆 抗体，因为它们的选择性，特异性和无限 生产性，可以借给自己的临床使用时，耦合 一种药物。 这些研究对于理解 激素在分子水平上的作用， 性腺功能异常