Many hormones, neurotransmitters and enzymes are released from cells by exocytosis. The properties of cytosolic proteins that may regulate this process will be investigated. Proteins that bind to secretory vesicle (chromaffin granule) membranes in the presence of Ca2+ will be isolated and the relationship of these proteins to specific intracellular events that occur during exocytosis will be assessed. The following issue will be addressed. 1) Ca2+- dependent, secretory vesicle binding proteins will be isolated from bovine adrenal medullary cytosol. 2) The proteins will be tested for specific activities relevant to the process of exocytosis using model systems constructed from components of the chromaffin cell. This will include tests of chromaffin granule aggregating activity, membrane fusion activity, binding to the cytoplasmic face of plasma membranes, interactions with cytoskeletal elements, modulation of chromaffin granule ATPase or chemiosmotic mechanisms, promotion of clathrin binding, and proteolytic, lipolytic or phosphatase activities. 3) The structural and functional properties of the APT- and Ca- dependent membrane-binding protein, chromobindin A, will be further examined to determined the nature of the ATP requirement and the detailed morphology of this complex protein. 4) the mechanism of action of synexin, calelectrin, and p36, which promote membrane contact and fusion, will be determined. 5) Antisera and monoclonal antibodies to several key vesicle-binding proteins will be prepared and used to assess the concentration and distribution of these proteins in a number of tissues and in cultured secretory cells. 6) Peptides and protein domains will be synthesized or isolated that may serve as specific inhibitors of synexin, calelectin and P36. These inhibitors, as well as specific antibodies, will be introduce into secretory cells in order to critically evaluate the importance of these proteins in secretion. The long term objective of these studies is to elucidate the molecular basis of exocytosis. This may lead to the development of new approaches in the treatment of disorders of hormone, neurotransmitter and enzyme release such as diabetes, neurogenic hypertension, inflamation and various endocrinopathies.
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