Thyroid plasma membrane-associated protein kinases: properties and substrates of solubilized and insoluble enzymes.
The protein kinase activities of thyroid plasma membranes were characterized after treatment by the nonionic detergent, Triton X-100. With endogenous substrate the protein kinase activity of intact plasma membranes appeared to be cAMP independent, whereas the solubilized plasma membranes contained a cAMP-dependent protein kinase. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of intact plasma membranes demonstrated approximately 30 protein bands, of which several were substrates for endogenous protein kinase, cAMP had a slight, but reproducible, stimulatory effect on some of these. In solubilized plasma membranes cAMP significantly augmented phosphorylation of at least seven of these proteins. Solubilized plasma membranes bound significantly more cAMP per mg protein than intact plasma membranes. The inability to unequivocally detect cAMP-dependent protein kinase in intact membranes using endogenous substrate probably reflects the much greater activity of the cAMP-independent enzyme activity. The protein kinase activity of intact plasma membranes which was stimulated by cAMP when histone was the substrate was primarily recovered in the solubilized plasma membranes. Most of the protein kinase activity of the intact plasma membranes was insoluble and was not augmented by cAMP. The solubilized protein kinase demonstrated the same Km values for ATP, cAMP, and MgCl2 as did the cytosolic protein kinase of the thyroid. Cytosolic and solubilized protein kinase activities were more sensitive to cAMP and cGMP stimulation when histone and protamine were used as substrates. Both enzyme activities were depressed by protein kinase modulator when histone, but not protamine and casein, were used as substrates. The protein kinase activity of insoluble plasma membranes was not inhibited by the protein kinase modulator.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology*; D000242:Cyclic AMP / Q000494:pharmacology; D003570:Cytidine Triphosphate / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D008565:Membrane Proteins / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012995:Solubility; D013379:Substrate Specificity; D013961:Thyroid Gland / Q000201:enzymology*
D008565:Membrane Proteins; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D000262:Adenylyl Cyclases