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Thyroid Plasma Membrane-Associated Protein Kinases:Properties and Substrates of Solubilized and Insoluble Enzymes^*

Clinical Research Unit and the Department of Medicine, University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania 15261

Address reprint requests to: James B. Field, M.D.; Director, Clinical Research Unit; 3304 Presbyterian-University Hospital; Pittsburgh, PA 15261.

Abstract

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

Footnotes

^* This work was supported by USPHS Grant AM 06865 from the NIH.

Received February 6, 1978.