MECHANISM OF SEX STEROID BINDING TO MACROMOLECULES

性类固醇与大分子的结合机制

• 批准号: 3311917
• 负责人: FREDERICK L SWEET
• 金额: $ 22.27万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-01-01 至 1989-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3311917
• 关键词: affinity chromatography; chemical aggregate; chemical synthesis; corpus luteum; embryo /fetus; enzyme structure; erythrocytes; estradiol; hormone analog; hormone regulation /control mechanism; progesterone; radioimmunoassay; radiotracer; sex hormones; steroid hormone metabolism; tritium

This project will continue to investigate the molecular factors that determine the mechanism of the binding of steroid hormones to specific binding sites on macromolecules. New affinity labeling (site-directed irreversible inhibitor) analogs of steroid hormones will be synthesized for use as reagents in these studies. Included is a new series of estradiol derivatives with 14C-bromoacetate side chains. They will be used to affinity label the active site of 17Beta-estradiol dehydrogenase, isolated from human placenta. New progesterone and cholesterol analogs with 14C-bromoacetate side chains will be synthesized and then used to affinity label the active site of cytochrome P-450 (steroid-specific) side chain cleavage enzymes, recently isolated from porcine testis and adrenals. 20Alpha-Hydroxysteroid oxidoreductase (20Alpha-HSD) from ovine fetal erythrocytes will be isolated and characterized by methods developed in the PI's laboratory. Then the active site of 20Alpha-HSD will be affinity labeled with substrate analogs (described above). Peptides from each of the 14C-labeled active sites will be isolated following proteolytic digestion of the affinity labeled enzymes. The peptides will be subjected to amino acid sequence analysis. The amino acid configurations of the enzyme active sites will be compared for clues to the relationships among configurations, steroid binding characteristics, and enzyme catalysis. The isolated ovine fetal 20Alpha-HSD will be injected into rabbits to generate antibodies. The antibodies will be used to measure by radioimmunoassay 20Alpha-HSD in blood from lamb fetuses in utero. This will give a profile of 20Alpha-HSD production in fetal blood throughout pregnancy. The 20AlphaHSD antibody will also be used to compare the structural relationship between ovine and bovine fetal 20Alpha-HSD. Affinity labeling steroids which rapidly inhibit ovine 20Alpha-HSD in vitro will be infused through a cannula into lamb fetuses in utero in an attempt to block this enzyme activity in vivo. This may reveal the role of 20Alpha-HSD in fetal development. Continuation of the in vitro affinity labeling experiments with the enzymes (above) are expected to further our understanding of the molecular factors which promote steroid binding and catalysis at the enzyme active sites. Greater insights into these factors which control estradiol- and progesterone-specific enzyme reactions will advance molecular concepts of steroid biosynthesis. This information is expected to provide foundation for developing sex steroids that may be useful in controlling reproduction and also fetal development.

该项目将继续研究导致这一现象的分子因素。 确定类固醇激素与特定物质结合的机制 大分子上的结合位点。 新的亲和标记（定点 不可逆抑制剂）类固醇激素类似物将被合成 在这些研究中用作试剂。 包括一系列新的雌二醇 具有14C-溴乙酸侧链的衍生物。 他们将习惯于 亲和标记 17β-雌二醇脱氢酶的活性位点，分离 来自人类胎盘。 新的黄体酮和胆固醇类似物 14C-溴乙酸侧链将被合成，然后用于亲和力 标记细胞色素 P-450（类固醇特异性）侧链的活性位点 裂解酶，最近从猪睾丸和肾上腺中分离出来。 来自羊胎儿的 20α-羟基类固醇氧化还原酶 (20α-HSD) 红细胞将通过开发的方法进行分离和表征 PI的实验室。 那么20Alpha-HSD的活性位点将是亲和力 用底物类似物标记（如上所述）。 来自每个的肽 14C 标记的活性位点将在蛋白水解后被分离 亲和标记酶的消化。 肽将受到 至氨基酸序列分析。 的氨基酸构型 将比较酶活性位点以寻找之间关系的线索 配置、类固醇结合特性和酶催化。 这 分离的羊胎20Alpha-HSD将注射到兔子体内产生 抗体。 该抗体将用于通过放射免疫分析进行测量 20 子宫内羔羊胎儿血液中的α-HSD。 这将给出一个配置文件 整个怀孕期间胎儿血液中 20Alpha-HSD 的产生量。 这 20AlphaHSD抗体也将用于比较结构 绵羊和牛胎儿 20Alpha-HSD 之间的关系。 亲和标记 将输注在体外快速抑制绵羊 20Alpha-HSD 的类固醇 通过插管进入子宫内的羔羊胎儿，试图阻止这种情况 体内酶的活性。 这可能揭示了 20Alpha-HSD 在胎儿中的作用 发展。 继续体外亲和标记实验 与酶（上面）的结合有望进一步加深我们对 促进类固醇结合和酶催化的分子因素 活跃站点。 对控制雌二醇的这些因素有更深入的了解 黄体酮特异性酶反应将推进分子概念 类固醇的生物合成。 该信息预计将提供 开发可能有助于控制性类固醇的基础 生殖和胎儿发育。