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

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Abstract
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 MW range of 250,000-30,000 were observed by means of the sodium dodecyl sulfate electrophoresis and autoradiography. On the basis of the criteria of MW, selective stimulation of the phosphorylation by cAMP (as compared with cGMP) and immunoprecipitation by specific antibodies, band 5 (MW 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 cAMP in presence of Mg2+ and Zn2+, respectively, and immunoreactivity toward antibodies showed band 6 to contain 2 forms of the regulatory subunits of the type II cAMP-dependent protein kinase, distinguishable by their MW (56,000 and 52,000, respectively). Phosphorylation of band 7 (MW 29,800) was stimulated 2-3 times by Ca2+ and calmodulin in the concentration range of both agents believed to occur in the secretory tissues under physiological conditions.

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