Abstract
Publisher Summary This chapter describes an assay method, purification, and properties of cAMP-dependent protein kinase—soluble and particulate—from swine kidney. The homogeneous preparations of protein kinase isolated from swine kidney catalyze the phosphorylation of purified homologous glycogen synthase, phosphorylase kinase, phosphofructokinase, pyruvate kinase, and fructose-l,6-bisphosphatase. The regulation of these enzymes through the action of the cyclic AMP-dependent forms of protein kinase may mediate the intracellular effects of hormones on the adenylate cyclase system in kidney. The enzymic assay is based on the ability of protein kinase to catalyze conversion of glycogen synthase to a form that is dependent on the presence of glucose 6-phosphate for activity. The rate of decrease in the activity of glycogen synthase is measured in the absence of glucose 6-phosphate. The purification process of the cytosolic catalytic subunit of protein kinase involves homogenization, chromatography on cellulose phosphate, pH 5.5 treatment, chromatography on diethylaminoethyl (DEAE)-cellulose, chromatography on Blue Dextran–Sepharose 4B, chromatography on cellulose phosphate, and gel filtration on Sephadex G-100.
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