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MECHANISM OF SEX STEROID BINDING TO MACROMOLECULES

性类固醇与大分子的结合机制

基本信息

批准号：
3311919
负责人：
FREDERICK L SWEET
金额：
$ 20.54万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
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

项目摘要

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.

该项目将继续研究分子因素，确定类固醇激素与特定的大分子上的结合位点。新的亲和标记（定点不可逆抑制剂）类固醇激素的类似物将被合成，在这些研究中用作试剂。包括一个新的雌二醇系列具有14 C-溴乙酸酯侧链的衍生物。他们将习惯于亲和标记17 β-雌二醇脱氢酶的活性位点，分离从人胎盘中提取的新的孕酮和胆固醇类似物， 14 C-溴乙酸酯侧链将被合成，然后用于亲和标记细胞色素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。来自绵羊胎儿的20 α-羟基类固醇氧化还原酶（20 α-HSD）红细胞将被分离，并通过本领域中开发的方法表征。 PI的实验室那么20 α-HSD的活性位点将是亲和的，用底物类似物标记（如上所述）。来自以下每一种的肽 14 C标记的活性位点将在蛋白水解后分离，消化亲和标记的酶。这些肽将被氨基酸序列分析。的氨基酸构型。酶的活性位点将进行比较，以获得以下关系的线索：构型、类固醇结合特性和酶催化。的将分离的羊胎儿20 α-HSD注射到兔中以产生抗体的抗体将用于放射免疫测定子宫内羔羊胎儿血液中的20 α-HSD。这将给出一个侧写 20 α-HSD的生产在整个怀孕的胎儿血液。的 20 AlphaHSD抗体也将用于比较结构绵羊和牛胎儿20 α-HSD之间的关系。亲和标记将输注在体外快速抑制绵羊20 α-HSD的类固醇通过一个插管进入子宫内的羊胎儿，体内酶活性。这可能揭示了20 α-HSD在胎儿发育中的作用。发展体外亲和标记实验的继续与酶（上述）有望进一步我们的理解，促进类固醇结合和酶催化的分子因子活性位点。更深入地了解这些控制雌二醇的因素- 和胆固醇特异性酶反应将推进分子概念类固醇的生物合成。这些信息将提供开发性类固醇的基础，可能有助于控制生殖和胎儿发育。