Many hormones and neurotransmitters are released from cells by exocytosis. The properties of cytosolic proteins that may regulate this process will be investigated. Soluble proteins that bind to secretory vesicle (chromaffin granule) membranes in the presence of Ca++ will be isolated by Ca++ dependent affinity chromatography of bovine adrenal medullary or liver cytosol fractions on bovine chromaffin granule membranes bound to Sepharose 4B. This group of proteins includes synexin, which mediates membrane interactions, calmodulin, which relays the Ca++ signal to other proteins, protein kinase C, which is a phospholipid and Ca++ activated protein kinase that may regulate enzyme activities through phosphorylation, and an additional 18 proteins the functions of which have not been identified. The following will be determined: which proteins in or binding to the vesicle membrane are substrates for the kinase; which proteins bind calmodulin; the physical and biochemical properties of proteins that require ATP to bind to the membrane; the relationship between peptide maps of the affinity prepared proteins with those of known proteins, in particular coated vesicle proteins that may be involved in secretory vesicle membrane recovery; the immunochemical cross-reaction between synexin and other vesicle-binding proteins; and whether these proteins will modulate those functions of isolated chromaffin granules that are thought to be involved in exocytosis such as ATP dependent chemiosmotic lysis and synexin dependent granule fusion. In addition, secretogogue-dependent changes in the level of phosphorylation of proteins in cultured chromaffin cells will be determined and the phosphorylated proteins will be compared with the substrates for protein kinase C identified in isolated cell fractions. The long-term objective of these studies is to elucidate the molecular basis of exocytosis. The knowledge generated may assist in the treatment of disorders of hormone and neurotransmitter release such as diabetes, neurogenic hypertension and various endocrinopathies.
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