A study of adenosine 3′–5′ cyclic monophosphate binding sites of human erythrocyte membranes using 8‐azidoadenosine 3′–5′ cyclic monophosphate, a photoaffinity probe

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Abstract
An earlier report (1a) has shown the utility of 8-N3cAMP (8-azidoadenosine-3′, 5′-cyclic monophosphate) as a photoaffinity probe for cAMP binding sites in human erythrocyte membranes. The increased resolution obtained using a linear-gradient SDS polyacrylamide gel system now shows that: (1) both cAMP and 8-N3cAMP stimulate the phosphorylation by [γ-32P]-ATP of the same red cell membrane proteins; (2) the protein of approximately 48,000 molecular weight whose phosphorylation by [γ-32P]-ATP is stimulated by cAMP and 8-N3cAMP migrates at a solwer rate than the protein in the same molecular weight range which is heavily photolabeled with [32P]-8-N3cAMP; (3) other cyclic nucleotide binding sites exist besides those initailly reported; (4) the variation in the ratio of incorporation of 32P-8-N3cAMP into the two highest affinity binding sites appears to be the result of a specific proteolysis of the larger protein.