We are proposing to continue our long term studies towards understanding the biochemistry and physiology of the plasma sex steroid-binding protein, SBP. The project is divided into two parts: The first will comprise biochemical studies describing the chemical environment of steroid-binding sites to explain how steroids are bound to protein surfaces. We will attempt to identify amino acid residues in the steroid binding sites of both human and rabbit SBP by affinity labeling, and to determine the adjacent amino acid sequence. Since human SBP binds both E2 and T whereas rabbit SBP only binds T, a comparison should reveal the structural elements responsible for binding specificity. We will also undertake an x-ray diffraction analysis on SBP crystals and elucidate the entire amino acid sequence of SBP in an attempt to describe the three-dimensional structure of a steroid-binding protein. The second objective is to understand the role of SBP in the mechanism of action of steroid hormones in relation to reproductive biology. We will test a new hypothesis implicating SBP as a specific carrier of sex steroids across membranes. To this end, we will attempt to identify a SBP receptor on the plasma membrane by incubating 125I-hSBP with MCF-7 and HEC-50 cells in culture, and by in vivo infusion of 125I-rSBP into rabbits followed by autoradiographic examination of reproductive tissues in the electron microscope. We will also induce a long-term SBP withdrawal in the rhesus monkey by infusing F(ab)2 fragments of SBP antibodies and study the effects on the menstrual cycle and pregnancy (parturition).
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