STRUCTURE-FUNCTION OF PLASMA SEX STEROID-BINDING PROTEIN

在接下来的预算期间，我们打算继续我们的长期计划 旨在了解类固醇蛋白现象的研究 分子水平上的相互作用及其意义 在血浆中存在对类固醇激素作用机制的研究。 我们计划启动实验来研究细胞的调节 性类固醇结合蛋白SBP在肝脏和肝脏的生物合成 影响其表达的因素。为了实现这一目标，我们 我们会采取两项主要措施，分别是： 首先，我们将描述两种特定类固醇的生物化学- 结合蛋白，即性激素结合蛋白(SBP) 人和兔血浆，通过测定和比较 它们的结构和类固醇结合部位。氨基酸 RSBP的序列将通过克隆和比较确定 与我们实验室过去两年测得的hSBP序列一致。这个 类固醇结合位点将通过亲和标记来表征 以及分组特定修改以揭示结构参数 负责绑定的专一性。SBP的X射线衍射 晶体将被用来揭示三维排列 类固醇和蛋白质之间。 其次，我们将进行分子生物学研究，以克隆 人SBP基因及其调控的分子基础 通过对其基因组结构的分析。我们将使用部分hSBP 我们最近进行了测序以分离或构建 获得全长c DNA并在哺乳动物细胞系中表达。站点- 将进行定向突变，以确定氨基酸在 类固醇结合部位。我们还将建立HepG2细胞系，以 研究不同激素对SBP生物合成的影响。这个 信息将帮助有兴趣控制的临床医生 与SBP异常水平相关的疾病。分子 生物学研究将帮助我们确定 它的生物合成。最后，我们将分离并测序一个完整的- 人睾丸雄激素结合蛋白ABP的编码长度 蛋白质，以确定其与人的SBP的关系。

During the coming budget period we intend to continue our long term studies designed at understanding the phenomenon of steroid-protein interaction at the molecular level and the implication of its existence in plasma on the mechanism of action of steroid hormones. We plan to initiate experiments to study the regulation of the biosynthesis of the sex steroid-binding protein, SBP, in liver and factors influencing its expression. To accomplish that goal we will undertake two major approaches which are as follows: Firstly, we will describe the biochemistry of two specific steroid- binding proteins, i.e. the sex-steroid-binding proteins (SBP) of human and rabbit plasma, through determination and comparison of their structures and steroid-binding sites. The amino acid sequence of rSBP will be determined by cDNA cloning and compared to that of hSBP sequenced in our lab in the past two years. The steroid binding sites will be characterized by affinity labeling and group specific modification to reveal the structural parameters responsible for binding specificity. X-ray diffraction of SBP crystals will be done to reveal the three-dimensional arrangement between steroid and protein. Secondly, we will engage in molecular biology studies to clone the human SBP gene and determine the molecular basis of its regulation by analysis of its genomic structure. We will use the partial hSBP cDNA clone we have recently sequenced to isolate or construct a full-length cDNA and express it in mammalian cell lines. Site- directed mutagenesis will be done to identify amino acids in the steroid binding site. We will also establish HepG2 cell lines to study the effect of various hormones on SBP biosynthesis. The information will help clinicians interested in controlling disorders associated with abnormal levels of SBP. The molecular biology studies will help us determine the site and regulation of its biosynthesis. Finally, we will isolate and sequence a full- length cDNA coding for human ABP, the testis androgen binding protein, to establish its relationship to human SBP.