Adenosine 3':5'-monophosphate-regulated phosphorylation of erythrocyte membrane proteins. Separation of membrane-associated cyclic adenosine 3':5'-monophosphate-dependent protein kinase from its endogenous substrates.
An adenosine 3':5'-monophosphate (cyclic AMP)-binding protein in the human erythrocyte plasma membrane was isotopically labeled using a photoaffinity analog of cyclic AMP, N6-(ethyl 2-diazomalonyl) cyclic [3H]AMP. The cyclic AMP-binding site is located in a polypeptide chain having a molecular weight of 48,000. Cyclic AMP-binding protein and cyclic AMP-dependent protein kinase were solubilized with 0.5% Triton X-100 in 56 mM sodium borate, pH 8, but 32P-labeled membrane phosphoproteins were retained in the Triton-insoluble fraction, suggesting that the membrane-associated binding protein is not a primary substrate for protein kinase. Triton-solubilized and membrane-associated protein kinase activities were stimulated 15- and 17-fold by cyclic AMP, suggesting that the degree of association between the catalytic anc cyclic AMP-binding components was very similar in both preparations. Fractionation and characterization of membrane phosphoproteins have shown that protein III and a co-migrating minor protein are substrates for protein kinase but membrane sialoglycoproteins are not phosphorylated.
D000818:Animals; D001665:Binding Sites; D001798:Blood Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology*; D004912:Erythrocytes / Q000187:drug effects / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D008970:Molecular Weight; D010750:Phosphoproteins / Q000096:biosynthesis*; D011485:Protein Binding; D011494:Protein Kinases / Q000097:blood*; D011955:Receptors, Drug
D001798:Blood Proteins; D010750:Phosphoproteins; D011955:Receptors, Drug; D000242:Cyclic AMP; D011494:Protein Kinases; D005033:Ethylmaleimide