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.
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