MECHANISM OF SEX STEROID BINDING TO MACROMOLECULES

该项目将继续研究分子因素 确定类固醇激素与特异性结合的机制 大分子上的结合部位。新的亲和力标记(站点定向 不可逆抑制剂)类固醇激素类似物将被合成用于 在这些研究中用作试剂。其中包括一系列新的雌二醇 具有14C-溴乙酸酯侧链的衍生物。他们将习惯于 亲和标记17β-雌二醇脱氢酶的活性部位 来自人类胎盘。新的黄体酮和胆固醇类似物 将合成14C-溴乙酸酯侧链，并用于亲和 细胞色素P-450(类固醇特异性)侧链活性部位的标记 裂解酶，最近从猪的睾丸和肾上腺中分离出来。 绵羊胎儿20Alpha-羟基类固醇氧化还原酶(20Alpha-HSD) 红血球将被分离出来，并用在 派的实验室。那么20Alpha-HSD的活性部位将是亲和的 用底物类似物标记(如上所述)。来自每一个的多肽 14C标记的活性部位将在蛋白分解后被分离 亲和标记酶的消化。这些多肽将受到 进行氨基酸序列分析。其氨基酸构型的研究 将比较酶活性部位，以寻找它们之间关系的线索 构型、类固醇结合特性和酶催化。这个 分离的绵羊胎儿20Alpha-HSD将注射到兔体内产生 抗体。抗体将通过放射免疫测定法进行检测。 20宫内羔羊胎儿血液中的α-HSD。这将提供一份个人资料 在整个妊娠过程中，胎儿血液中有20%的α-HSD产生。这个 20AlphaHSD抗体也将用于比较结构 绵羊与牛胎20Alpha-HSD的关系亲和标记 体外快速抑制绵羊20Alpha-HSD的类固醇将被注入 通过插管进入子宫中的羔羊胚胎，试图阻止这种 体内的酶活性。这可能揭示了20Alpha-HSD在胎儿发育中的作用 发展。体外亲和标记实验的继续 这些酶(如上)有望进一步加深我们对 促进类固醇结合和酶催化作用的分子因素 活动站点。对这些控制雌二醇的因素有了更深入的了解- 黄体酮特异性的酶反应将推进分子概念 类固醇的生物合成。这一信息预计将提供 为开发可能对控制疾病有用的性类固醇的基础 生殖和胎儿发育。

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.