CONTROL OF LUTEINIZING HORMONE BIOSYNTHESIS

总体目标是确定涉及的监管步骤 促黄体生成素的糖基化及其如何释放促性腺激素 激素(GnRH)和性腺类固醇调节糖基化、储存和 释放黄体生成素。有四个具体目标：(1)确定促黄体生成素是否 糖基化和释放是通过不同的分化来调节的 细胞内通路。这将通过确定相对角色来完成 钙离子和cAMP信号转导通路的相互作用 调节黄体生成素的糖基化和释放。对黄体生成素糖基化的影响 以及释放已知可调制参与钙离子和钙离子的信号的药物 将研究营地的小路。(2)确定是否不同 促性腺激素释放激素受体相互作用调节黄体生成素的糖基化和释放。这 将通过确定受体交联剂的效果来实现， GnRH相关肽及GnRH拮抗剂对GnRH诱导的黄体生成素的影响 糖基化VS释放。(3)检验共同和共同的假设 黄体生成素的翻译后修饰(即核心和末端 糖基化和硫酸化)受GnRH和性腺类固醇的调节。 这将通过确定(A)促性腺激素释放激素对添加 富含甘露糖的脂质载体核心碳水化合物单位到脱脂蛋白 以及(B)GnRH对硫酸盐和末端加成的影响 除氨基葡萄糖以外的碳水化合物。(4)确定GnRH和 性腺类固醇调节细胞内黄体生成素的降解。这件事会做到的 通过监测荷尔蒙对黄体生成素亚单位的降解。 酶法制备分散的脑垂体前叶细胞 去卵巢的大鼠将被培养在培养瓶或生物支持珠上。 性腺激素释放激素、性腺类固醇或药物的存在或不存在。黄体生成素合成 将通过测量放射性标记前体的并入来进行监测 转化为黄体生成素的蛋白质和寡糖部分。放射性标记的黄体生成素遗嘱 通过免疫沉淀法与特定抗体分离。无线电标记的 免疫沉淀物中的LH亚单位将用十二烷基硫酸钠凝胶进行评估 电泳法。总免疫反应性黄体生成素将通过 放射免疫分析。特定药物对黄体生成素合成和释放的影响 通过测量摄取和测量来区分非特定的毒性影响 将放射性标记的前体掺入总蛋白中并通过 孵育结束时用活性染料检测细胞。

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.