CHEMISTRY AND BIOLOGY OF HUMAN AND OTHER GONADOTROPINS

与结构和功能有关的拟议研究 人绒毛膜促性腺激素(HCG)与其受体的关系 继续我们正在进行的研究。人绒毛膜促性腺激素及其亚基和大鼠 促黄体生成素受体在异源真核细胞中表达 表达系统、杆状病毒-昆虫细胞系统。荷尔蒙及其相关基因 亚基高效表达，可通过 免疫亲和层析。因为重组激素是 类似于天然激素及其亚单位在免疫学和 生物学特性，它们可以取代天然的尿液激素 所有免疫诊断应用程序。更重要的是，它一直是 人绒毛膜促性腺激素中可以用硒蛋氨酸代替蛋氨酸。这个 重组人绒毛膜促性腺激素具有较大的应用潜力 它的三维结构的测定。重组人 在杆状病毒系统中表达的黄体生成素/人绒毛膜促性腺激素受体主要是 一种二聚体。本研究的具体目的是双重的。我。 促性腺激素的结构与功能关系的研究 将包括：(1)重组蛋白的制备和表征 大规模的亚硒硫酰人绒毛膜促性腺激素和尝试获得X射线 用于三维结构分析的衍射级晶体。 (2)重组人绒毛膜促性腺激素及其亚基拆分 糖类在人绒毛膜促性腺激素功能中的作用问题 去糖化的人绒毛膜促性腺激素。(3)多肽和碳水化合物 重组人绒毛膜促性腺激素及其亚基的链。(4)重组人绒毛膜促性腺激素 HCGalpha和hCGbeta的大规模应用，以进一步评估其在 活体生物学特性。(5)hCGalpha和hCGalpha的糖类突变体 HCGbeta在杆状病毒表达系统中的作用 单个N-连接的碳水化合物与hCG的功能有关。二、关于 大鼠黄体生成素/人绒毛膜促性腺激素受体的结构和功能将包括 重组大鼠黄体生成素/人绒毛膜促性腺激素的制备与鉴定 受体。(2)重组1-294个氨基酸残基胞外 多肽及其亚硒基类似物。(3)各种删除 昆虫细胞中黄体生成素/人绒毛膜促性腺激素受体突变体的研究 受体的配基结合域和(4)激素的描绘 促黄体生成素/人绒毛膜促性腺激素受体的Gs结合部位及其可能的参与 配基或Gs结合中的胞外和胞质环 分别进行了分析。(5)黄体生成素受体与GTP的同源重组 结合蛋白(Gs)和Gs和腺苷环化酶或磷脂酶 C从大鼠卵巢膜中提取脂质体研究其作用 碳水化合物在激素和受体信号传递机制中的作用 转导。以上所有的研究都应该提高我们的认识 对人类生育能力的调节，从而促进 更安全的避孕方法。

The proposed studies relating to the structure and function relationships in human choriogonadotropin (hCG) and its receptor are a continuation of our ongoing studies. HCG and its subunits and the rat lutropin receptor have been expressed in an heterologous eukaryotic expression system, baculovirus-insect cell system. The hormone and its subunits were expressed in a high efficiency and could be purified by immunoaffinity chromatography. Since the recombinant hormones are similar to the native hormone and its subunits in immunological and biological properties, they can replace the native urinary hormones in all immunodiagnostic applications. More importantly, it has been possible to substitute selenomethionine for methionine in hCG. The recombinant selenomethionyl hCG has considerable potential in the determination of its three dimensional structure. The recombinant LH/hCG receptor expressed in baculovirus system occurs predominantly as a dimer. The specific aims of the present studies are twofold. I. Studies on the structure and function relationships of gonadotropins will include the preparation and characterization of: (1) Recombinant selenomethionyl hCG on a large scale and to attempt to obtain x-ray diffraction grade crystals for three dimensional structural analysis. (2) Recombinant unglycosylated hCG and its subunits to resolve the question of the carbohydrate role in the function of hCG and selenomethionyl unglycosylated hCG. (3) The polypeptide and carbohydrate chains of recombinant hCG and its subunits. (4) Recombinant hCG, hCGalpha and hCGbeta on a large scale for further evaluation of their in vivo biological properties. (5) Carbohydrate mutants of hCGalpha and hCGbeta in baculovirus expression system to evaluate the contribution of individual N-linked carbohydrates to the function of hCG. II. Studies on the structure and function of rat LH/hCG receptor will include the preparation and characterization of: (1) Recombinant rat LH/hCG receptor. (2)Recombinant 1-294 amino acid residue extracellular polypeptide and its selenomethionyl analog. (3) Various deletion mutants of LH/hCG receptor in insect cells in order to delineate the ligand binding domain of the receptor and (4) Delineation of the hormone and Gs binding sites of the LH/hCG receptor and the possible involvement of extracellular and cytoplasmic loops in the ligand or Gs binding respectively. (5) Homologous reconstitution of LH receptor with GTP binding protein (Gs) and with Gs and adenylate cyclase or phospholipase C from rat ovarian membranes in liposomes to study the role of carbohydrate in the hormone and the receptor in the mechanism of signal transduction. All of the above studies should enhance our understanding of the regulation of human fertility and thus facilitate the development of safer methods of contraception.