A broad research program is proposed to continue and develop strategies to investigate the multiple functions of the plasma vitamin D binding protein (DBP, Gc globulin). The vitamin D- sterol and actin-binding domains of the protein will be characterized by isolating and analyzing proteolytic and chemical cleavage products for the presence of chemically cross-linked, radioactive sterol analogues and the high-affinity binding to monomeric actin. Physicochemical, affinity and immunoaffinity chromatography will be employed to isolate DBP from homozygotes having the Gc-1f and Gc-2 phenotype, as well as earlier examples (fish) of the protein, thereby allowing further definition of the functional differences of genetic and evolutionary forms. Selective immunoprobes, developed with hybridoma technology, will allow characterization of the orientation of cell associated DBP by light and electron microscopy. The source of cell-associated DBP will be defined, and receptor sites sought. The function of cell-surface DBP will be sought in leukocytes and cell lines. Studies are designed to test the putative roles of DBP in: Fc receptor activity; association with mIgG; co-chemoattractant with C5a; endotoxin or LPS-binder and inactivator; protection from human immunodeficiency virus infection. The role of plasma DBP in the vivo disposition of actin moieties will be analyzed. The influences of DBP and other carriers in the egress of vitamin D3 from cutaneous synthetic sites will be studied in isolated, perfusion systems, and the influences of plasma carriers on cellular access of sterols in vitro will be defined. These studies should lead to an improved understanding of the roles of DBP in sterol transport and cellular access, in protection of the organism from endotoxins and actin forms liberated into the circulation, and in cell surface- cytoskeleton relationships, cell structure and motility.
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