The chromaffin vesicles of the adrenal medulla participate in the synthesis, storage and secretion of catecholamines and are analogous to synaptic vesicles in catecholaminergic neurons in the central and peripheral nervous system. Knowledge of the synthesis and factors regulating the synthesis of major functional or constituitive proteins of the vesicles, namely dopamine-beta-hydroxylase and chromogranin A, and their incorporation into chromaffin vesicles will enhance our understanding of the secretory cycle in the adrenal madulla and sympathetic nerves. The objectives of the proposed research are to characterize the synthesis of dopamine-beta-hydroxylase and chromogranin A and their subsequent incorporation into chromaffin vesicles, to determine whether certain factors affect these processes and to determine whether chromaffin vesicle membranes are recycled using primary cultures of adrenal medullary cells. The synthesis of the proteins and their incorporation into chromaffin vesicles will be determined by labeling cell cultures with ?35S!methionine followed by quantitative immunoprecipitation of the labeled proteins with monospecific anti-sera. The radiochemical purity of the immunoprecipitates will be verified by gel electrophresis and autoradiography. Preliminary studies show that adrenal medullary cells labeled with ?35S!methionine secrete a highly labeled protein into the culture medium. This protein is the major labeled protein which appears in the medium and represents about 5% of the ?35S!methionine incorporated into total protein. Dr. Kirshner proposes to: 1) further characterize the synthesis and secretion of the protein; 2) to purify the protein and determine its molecular weight and subunit structure; 3) to establish its identity either as a known protein or as a new protein.
