STRUCTURE-FUNCTION OF PLASMA SEX STEROID-BINDING PROTEIN

在下一个预算期间，我们打算继续我们的长期 旨在了解类固醇蛋白质现象的研究 分子水平上的相互作用及其意义 存在于血浆中的类固醇激素的作用机制。 我们计划开展实验，研究 性类固醇结合蛋白SBP在肝脏中的生物合成， 影响其表达的因素。 为了实现这一目标，我们 将采取以下两个主要做法： 首先，我们将描述两种特定类固醇的生物化学- 结合蛋白，即性类固醇结合蛋白（SBP） 人和兔血浆，通过测定和比较， 它们的结构和类固醇结合位点。 的氨基酸 通过cDNA克隆确定rSBP的序列并进行比较 与本实验室近两年来测序的hSBP的同源性进行了比较。 的 类固醇结合位点将通过亲和标记来表征 和基团特异性修饰以揭示结构参数 负责结合特异性。 SBP的X射线衍射 晶体将揭示三维排列 类固醇和蛋白质的区别 第二，我们会进行分子生物学研究， 人SBP基因，并确定其调控的分子基础 通过分析它的基因组结构。 我们将使用部分hSBP 我们最近测序了一个cDNA克隆， 全长cDNA并在哺乳动物细胞系中表达。 研究中心- 将进行定向诱变以鉴定所述突变体中的氨基酸。 类固醇结合位点。 我们还将建立HepG2细胞系， 研究各种激素对SBP生物合成的影响。 的 这些信息将有助于临床医生对控制 与SBP水平异常相关的疾病。分子 生物学研究将帮助我们确定 其生物合成。最后，我们要分离并测序一个完整的- 编码人ABP、睾丸雄激素结合的长cDNA 蛋白质，以建立其与人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.