The major rationale for this research is to investigate cellular and subcellular aspects of exocytosis. We have been examining secretory granule ATPase and calcium binding. To continue our studies isolated secretory granules will be analyzed for ATPase activity and calcium association. The ATPase of the granule will be investigated in isolated secretory granule membranes. The bicarbonate ion effect on granule ATPase will be tested and compared to the effect of other anions. The effect of calcium and other cations will also be investigated. The role of some specific chemical groups and phosphorylations will be assessed. The effect of fusogens and characterization of specific granule membrane protein will be investigated. The purpose will be to better characterized those factors which influence the granule membrane ATPase. Calcium binding uptake and efflux will also be studied in whole secretory granules. The influence of ATPase activity in the granule will be studied in light of the need for ATP hydrolysis for calcium association with the granules. Several agents or events and their possible role in calcium binding will be investigated. These are: protein kinase phosphorylations, coincubation with plasma membranes, inositol (1,4,5) triphosphate, arachidonic acid and phospholipase A2, chemical group specific agents, and synexin. Synexin binding to secretory granules will also be pursued. Whole parotid cells will be permeabilized with digitonin and in separate experiments, staphlococcal alpha toxin. The benefit provided by the permeabilized cell is the ability to directly alter the intracellular environment. After appropriate parameters have been determined for alpha toxin permeabilization and incubations, the effects and possible mechanism of calcium in promoting amylase release from the cells will be pursued. ATP-dependent phosphorylations, as well as the requirement for ATP in amylase secretion will e evaluated. The effect of cyclic nucleotides and anions will be assessed. Several inhibitors of intracellular processes as well as protein modulators will also be investigated. The purpose is to define more precisely the events in exocytosis, as provided by the permeabilized cell and to draw correlations with the results of the secretory granule data.
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