3',5'-cyclic adenosine monophosphate- and Ca2+-calmodulin-dependent endogenous protein phosphorylation activity in membranes of the bovine chromaffin secretory vesicles: identification of two phosphorylated components as tyrosine hydroxylase and protein kinase regulatory subunit type II.
Membranes of the secretory vesicles from bovine adrenal medulla were investigated for the presence of the endogenous protein phosphorylation activity. Seven phosphoprotein bands in the molecular weight range of 250,000 to 30,000 were observed by means of the sodium dodecyl sulphate electrophoresis and autoradiography. On the basis of the criteria of molecular weight, selective stimulation of the phosphorylation by cyclic AMP (as compared with cyclic GMP) and immunoprecipitation by specific antibodies, band 5 (molecular weight 60,300) was found to represent the phosphorylated form of the secretory vesicle-bound tyrosine hydroxylase. The electrophoretic mobility, the stimulatory and inhibitory effects of cyclic AMP in presence of Mg2+ and Zn,2+ respectively, and immunoreactivity toward antibodies showed band 6 to contain two forms of the regulatory subunits of the type II cyclic AMP-dependent protein kinase, distinguishable by their molecular weights (56,000 and 52,000, respectively). Phosphorylation of band 7 (molecular weight 29,800) was stimulated about 2 to 3 times by Ca2+ and calmodulin in the concentration range of both agents believed to occur in the secretory tissues under physiological conditions.
D000313:Adrenal Medulla / Q000378:metabolism / Q000648:ultrastructure; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D000242:Cyclic AMP / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D046911:Macromolecular Substances; D008970:Molecular Weight; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D046911:Macromolecular Substances; D000242:Cyclic AMP; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases; D002118:Calcium