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 and cyclic AMP binding components was very similar in both preparations. Fractionation and characterization of membrane phosphoproteins have shown that protein III and comigrating minor protein are substrates for protein kinase but membrane sialoglycoproteins are not phosphorylated.