BINDING, BIOSYNTHESIS AND ACTION OF STEROIDS

该项目将继续研究结构和结构的共性 类固醇结合蛋白的机制。重点抓好三个方面 哺乳动物类固醇转换酶：1)人胎盘雌二醇 17β-脱氢酶，已被认为与生理作用有关 维持和调节适当的雌二醇浓度 随着分娩的临近，母体/胎儿单位中；2)牛睾丸 多功能酶--20α-羟基类固醇脱氢酶 有一种被认为是精子必需的醛糖还原酶 维持；3)马胎盘雌二醇17α/17β-脱氢酶 它们提供了研究结构/功能的机会 利用表观底物的两种酶之间的关系。这个 利用亲和力研究这些酶的天然形式 标记类固醇底物(定点不可逆抑制物)将 继续鉴定烷基化残基和多肽 其中包含了它们。此外，从cDNA中获得的基因产物 将通过亲和标记来检查每个酶的编码 技术和以前应用于 天然蛋白质。两种基因编码区的点突变研究 酶将允许对结构/功能关系进行具体分析 对于改变的基因产物中的特定氨基酸残基。那里 是每种原生酶的持续、充足的来源。这个 人雌二醇17β-脱氢酶和牛的表达蛋白 20α-羟基类固醇脱氢酶/醛糖还原酶目前 由于之前在我们的合作和合作中所做的努力 实验室。目前正在进行获得编码该基因的c DNA的工作 马脱氢酶。醛糖还原酶的晶体结构为 现在已经知道了。最终目标是验证为 点突变与晶体结构研究的结果 表达的蛋白质，这将明确地定义空间 活动期氨基酸残基与类固醇、辅因子的关系 这些酶中每一种酶的位置。

This project will continue the study of commonality in the structure and mechanism of steroid-binding proteins. Efforts will focus on three mammalian steroid-interconverting enzymes: 1) human placental estradiol 17beta-dehydrogenase which has been implicated in the physiologic role of maintaining and regulating the appropriate estradiol concentrations in the maternal/fetal unit as delivery approaches; 2) bovine testicular 20alpha-hydroxysteroid dehydrogenase, a multi-functional enzyme identical with aldose reductase which has been suggested to be necessary for sperm maintenance; 3) equine placental estradiol 17alpha/17beta-dehydrogenases which offer the opportunity to investigate the structure/function relationships between two enzymes which utilize epimeric substrates. The studies on the native form of each of these enzymes utilizing affinity labeling steroid substrates (site-directed irreversible inhibitors) will continue with the identification of alkylated residues and the peptides which contain them. In addition, the gene product obtained from cDNA which encodes each enzyme will be examined by affinity labeling techniques and the rigorous kinetic analyses previously applied to the native proteins. Study of point mutations within the cDNA encoding each enzyme will allow specific analysis of structure/function relationships for particular amino acid residues in the altered gene product. There is a continuing, sufficient source available for each native enzyme. The expressed protein for human estradiol 17beta-dehydrogenase and bovine 20alpha-hydroxysteroid dehydrogenase/aldose reductase are currently available as a result of previous efforts in our and collaborating laboratories. Work is presently underway to obtain the cDNA encoding the equine dehydrogenases. The crystalline structure of aldose reductase is now known. The ultimate goal is to validate the affinity labeling the point mutation results with the crystallographic structural studies of the expressed proteins, which will unequivocally define the spatial relationships of amino acid residues, steroid and cofactor at the active site of each of these enzymes.