Acinar cell clumps from the rabbit parotid gland will be isolated by published procedures involving enzymatic and mechanical means. The effect of pharmacological agents will be examined on cyclic AMP-dependent phosphorylation, cyclic nucleotide concentrations and amylase secretion. Phosphorylation of two plasma membrane proteins (34,000, 30,000 M.W.) will be examined as a function of amylase release and cyclic nucleotide concentrations in the cells. These proteins, located in the plasma membrane, increase in phosphorylation when the cells are exposed to isoproterenol. Phosphorylation is specific to Beta-receptor stimulation. Protocols will include the following drugs: isoproterenol, carbachol, phenylephrine, propranolol, colchicine, A-23187 and substance P. The intent is to clarify the effects of the various drugs on cyclic nucleotide concentrations, phosphorylation and secretion, and thereby better establish the role of cyclic AMP-dependent phosphorylation in the secretory process. Emphasis will be on the interaction of the various agents with isoproterenol-induced secretion and the course of phosphorylation and cyclic nucleotide levels as stimulated secretion is altered by the various agents. The second part of the research is concerned with the isolation of the proteins which are subject to increased phosphorylation with isoproterenol. These proteins, the major one being the 34,000 M.W. protein, will be isolated by gel chromatography and gel electrophoresis after solubilization. Protein from the microsomal fraction of the cell whic binds to secretory granules will be isolated using a secretory granule affinity column. Any binding protein will be compared to the phosphorylated protein and the role of phosphorylation on binding, if any, will be assessed. Isolated proteins will be tested as to their affinity to secretory granules and the ability to promote membrane-membrane interactions between granules and between granules and plama membranes. These membrane-membrane interactions will be assessed by sucrose gradient centrifugation, amylase release from granules, light scattering and secretory granule affinity columns.
Showing the most recent 10 out of 14 publications
