CONTROL OF LUTEINIZING HORMONE BIOSYNTHESIS

黄体生成素生物合成的控制

• 批准号: 3311140
• 负责人: TSUEI-CHU LIU
• 金额: $ 12.73万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-05-01 至 1990-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3311140
• 关键词: calcium metabolism; cyclic nucleoside monophosphate; estrogens; glycosyltransferase; gonadotropin releasing factor; hormone analog; hormone regulation /control mechanism; luteinizing hormone; ovariectomy; peptide hormone biosynthesis; pituitary gland; radioimmunoassay; radionuclide double label; scintillation counter; secretion

The overall aims are to determine the regulatory steps involved in glycosylation of luteinizing hormone (LH) and how gonadotropin-releasing hormone (GnRH) and gonadal steroids regulate glycosylation, storage, and release of LH. There are four specific aims: (1) To determine if LH glycosylation and release are regulated via different divergent intracellular pathways. This will be done by determining the relative role of, and interaction between, the putative Ca2+ and cAMP pathways in regulating LH glycosylation and release. The effects on LH glycosylation and release of drugs known to modulate signals involved in the Ca2+ and cAMP pathways will be studied. (2) To determine if different GnRH-receptor interactions regulate glycosylation and release of LH. This will be done by determining the effects of receptor cross-linkers, GnRH-associated peptide, and GnRH antagonists on GnRH-induced LH glycosylation vs. release. (3) To test the hypothesis that both co- and post-translational modification of LH (i.e., core- and terminal glycosylation and sulfation) are regulated by GnRH and gonadal steroids. This will be done by determining (a) the effect of GnRH on addition of mannose-rich lipid-carrier core carbohydrate units to the apoprotein moiety, and (b) the effect of GnRH on addition of sulfate and terminal carbohydrates other than glucosamine to LH. (4) To determine if GnRH and gonadal steroids modulate intracellular LH degradation. This will be done by monitoring LH subunit degradation in response to hormones. Enzymatically dispersed anterior pituitary cells prepared from ovariectomized rats will be cultured in flasks or on biosupport beads in the presence or absence of GnRH, gonadal steroids, or drugs. LH synthesis will be monitored by measuring incorporation of radiolabeled precursors into the protein and oligasaccharide moieties of LH. Radiolabeled LH will be isolated by immunoprecipitation with specific antibodies. Radiolabeled LH subunits in the immunoprecipitates will be assessed by SDS gel electrophoresis. Total immunoreactive LH will be measured by radioimmunoassay. Specific drugs effects on LH synthesis and release will be distinguished from non-specific toxic effects by measuring uptake and incorporation of radiolabeled precursors into total protein and by examining the cells with vital dye at the end of incubation.

总体目标是确定涉及的监管步骤， 促黄体生成激素（LH）的糖基化和促性腺激素释放激素如何 激素（GnRH）和性腺类固醇调节糖基化，储存， 释放LH。 具体目标有四个：（1）确定LH是否 糖基化和释放通过不同的趋异因子调节， 细胞内途径 这将通过确定相对作用来完成 以及它们之间的相互作用，假定的Ca 2+和cAMP途径， 调节LH糖基化和释放。 对LH糖基化的影响 和释放已知调节Ca 2+和Ca 2+信号的药物， 将研究cAMP途径。 (2)以确定是否不同 GnRH-受体相互作用调节LH的糖基化和释放。 这 将通过确定受体交联剂的作用来完成， GnRH相关肽和GnRH拮抗剂对GnRH诱导的LH的影响 糖基化与释放。 (3)为了验证这一假设， LH的翻译后修饰（即，核心和终端 糖基化和硫酸化）由GnRH和性腺类固醇调节。 这将通过确定（a）GnRH对添加 富含甘露糖的脂质载体核心碳水化合物单元到脱辅基蛋白 （B）GnRH对硫酸根和末端的加成的影响 除了葡萄糖胺以外的碳水化合物。 (4)为了确定GnRH和 性腺类固醇调节细胞内LH降解。 为此将 通过监测LH亚单位降解对激素的反应。 酶促分散的垂体前叶细胞， 将卵巢切除的大鼠培养在烧瓶中或生物支持珠上， 是否存在GnRH、性腺类固醇或药物。 LH合成 将通过测量放射性标记前体的掺入来监测 进入LH的蛋白质和寡糖部分。 放射性标记的LH将 用特异性抗体通过免疫沉淀分离。 放射标记 免疫沉淀物中的LH亚单位将通过SDS凝胶进行评估 电泳 将通过以下方法测量总免疫反应性LH： 放射免疫法 特定药物对LH合成和释放的影响将 通过测量摄入量，将其与非特异性毒性效应区分开来， 将放射性标记的前体掺入总蛋白中， 在孵育结束时用活体染料检查细胞。