CHARACTERIZATION AND PURIFICATION OF LH/HCG RECEPTORS AND ADENYLATE CYCLASE

LH/HCG 受体和腺苷酸环化酶的表征和纯化

• 批准号: 4693724
• 负责人: M L DUFAU
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/4693724
• 关键词: Leydig cells; adenylate cyclase; affinity chromatography; calcium metabolism; cell membrane; chemical structure; chorionic gonadotropin; crosslink; density gradient ultracentrifugation; enzyme complex; gel electrophoresis; gel filtration chromatography; guanine nucleoside; hormone regulation /control mechanism; luteinizing hormone; ovary; phosphorylation; pituitary gonadal axis; prolactin; radioimmunoassay; radiotracer; silver impregnation; testis

This aspect of the program is concerned with a) characterization of gonadotropin and prolactin receptors of the testis and ovary; b) the physical and functional relationships of the LH receptor site and adenylate cyclase; and c) cAMP-dependent and independent phosphorylation during gonadotropin action. In studies on the properties of testicular lactogern receptors, detergent-solubilized preparations had higher binding capacity than the membrane fraction, due to unmasking of a receptor pool. The crosslinked hormone-receptor complexes were distributed between two subunit sets with Mr of 113 & 103K or 59 & 53K, equivalent to 91 & 81K and 37 & 31K (calculated) for the free receptor. The existence of bands with close Mrs could be attributed to differences in glycosylation or phosphorylation. Parallel crosslinking experiments with other tissues (i.e. ovary, mammary gland, liver and kidney) showed similar resolution. As in the ovary, the 37K species appears to be contained within the 80K species through disulfide linkage. Variants of low ahd high affinity subunits were usually indistinguishable in cross-linking studies with ovarian soluble preparations. In contrast, silver staining profiles of purified ovarian receptors (20.4 nmol/mg protein) showed 88K and 95K variants of the high Mr subunits, while the low Mr subunit was observed as a single band of 41K. The purified receptor subunits were biologically active as demonstrated by hormone binding to free receptors transblotted to nitrocellulose. We have devised a procedure for purification of microgram quantities of active lactogen receptors from rat ovaries. Also, we have shown that extensive internalization and degradation of bound hCG is not required for the early phase of gonadotropic desensitization in the Leydig cell. Studies of the hormone and G-nucleotide effect on membrane phosphorylation have shown that in the presence of submaximal levels of GTP, oLH induces significant increase in phosphorylation of a 44K protein. These findings and our previous observations, suggest that hormone binding to the LH receptor induces a G-nucleotide/Ca2+ dependent phosphorylation of the 44K protein.

该计划的这一方面涉及a)特征 睾丸和卵巢的促性腺激素和催乳素受体；b) 黄体生成素受体位置和腺苷的物理和功能关系 C)cAMP依赖和独立的磷酸化。 促性腺激素的作用。睾丸催乳素的性质研究 受体，洗涤剂增溶制剂有较高的结合能力 而不是膜部分，这是因为受体池被揭开了面纱。这个 交联型激素受体复合体分布在两个亚基之间 集合的MR为113&103K或59&53K，相当于91&81K和37&31K (计算)用于自由受体。具有接近MRS的带的存在性 可能归因于糖基化或磷酸化的差异。 与其他组织(如卵巢、乳腺)的平行交联实验 腺体、肝脏和肾脏)显示相似的分辨率。正如在卵巢中一样， 37K个物种似乎包含在80K个物种中，通过 二硫键。低亲和力亚基和高亲和力亚基的变体通常是 在卵巢可溶性的交联性研究中无法区分 准备工作。相比之下，纯化卵巢的银染图谱 受体(20.4nmol/mg蛋白)显示高MR的88K和95K变异体 低MR亚基为一条41K的单一条带。 纯化的受体亚基具有生物活性。 与游离受体结合的激素转印到硝化纤维。我们有 设计了一种提纯微克量活性物质的方法 大鼠卵巢中的催乳素受体。此外，我们已经展示了广泛的 早期不需要结合的hCG内化和降解 间质细胞中促性腺激素脱敏的时相。对中国传统文化的研究 激素和G-核苷酸对膜磷酸化的影响已表明 在GTP水平低于最大值的情况下，OLH诱导显著 44K蛋白的磷酸化增加。这些发现和我们的 先前的观察表明，荷尔蒙与黄体生成素受体结合 诱导44K蛋白的G-核苷酸/钙离子依赖的磷酸化。