Gc protein (Group-specific component) is known to interact strongly with the membranes of all normal human peripheral blood nucleated cells. This protein is a major carrier for Vitamin D metabolites (D), and particularly 25-(OH)D3, and could therefore play an important role in vitamin D and calcium metabolism. Gc also binds with high affinity to monomeric G-actin, a major component of MNC, and may thereby modulate cytoskeletal function in such cells. We have recently found that Gc also interacts with lipid, and particularly long chain unsaturated fatty acids (UFA). This provides a plausible explanation for the extremely stable interaction of this hydropholic serum protein with the lipid bilayer. Moreover, since binding of either 25-(OH)D3 or actin strongly affects interaction with fatty acid, the ability of Gc to act as a carrier for fatty acids and for D3 may be closely related. This proposal aims to further investigate these phenomena. A major goal will be to further elucidate the interaction of Gc with Vitamin D metabolites, G-actin, and with UFA and phospholipids of the MNC membrane. These experiments will involve further development of methods for removal of Gc from the cell membrane. With the use of purification protocols already developed, this will allow studies of individual and cooperative interactions between Gc and vitamin D, G-actin and UFA; membrane and serum forms of Gc will be directly compared. Lipid associated with Gc from cells and from serum will be analyzed, utilizing thin layer chromatography, high performance liquid chromatography and gas chromatography. Circulating and membrane Gc will also be delipidated to further examine binding of Vitamin D metabolites, G-actin and UFA, and the effects of interaction with both combinations thereof. The interaction of Gc with the lipid bilayer will also be investigated at an ultrastructural level by immune electron microscopy performed without fixation using frozen sections cut in an ultracryomicrotome. Purified Gc preparation will then be loaded with various combinations of 25-(OH)D3 or G-actin or both, and uptake by MNC analyzed. Finally, the effects of these phenomena upon the coordinated activity of monocytes and lymphocytes will be examined in relation to standard parameters of activation, proliferation and differentiation. These studies should provide information relevant to the interactions of Gc with 25-(OH)D3 and fatty acids, and may shed further light upon the biological function of this protein.
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