An Adenosine 3′:5′‐Monophosphate/Adenosine Binding Protein from Mouse Liver

Abstract

Synthetic and naturally occurring adenosine derivatives were tested for their ability to inhibit the binding of cyclic [3H]AMP and [3H]adenosine to the activated cyclic AMP/adenosine binding protein from mouse liver [Ueland, P. M. and Døskeland, S. O. (1977) J. Biol. Chem. 252, 677–689]. The structural requirements for inhibition of the binding of the two ligands were different, as demonstrated by the existence of adenosine derivatives that inhibited the binding of [3H]adenosine but not the binding of cyclic [3H]AMP and vice versa. The interaction of the binding protein with AMP, ADP and adenine was investigated in more detail. The binding of radioactive AMP and ADP to the protein was determined under equilibrium conditions. Competition studies indicate that AMP binds to the cyclic AMP binding site with an apparent dissociation constant of 2 × 10−6 M and to a low‐affinity site. The double‐reciprocal plot for the binding of ADP deviated slightly from linearity suggesting the possibility of heterogeneity also for the ADP binding sites. Competition studies indicate that this nucleotide binds mainly to the cyclic AMP binding site.Cyclic AMP has previously been shown to activate its own sites under certain conditions (30°C, in the presence of KCl and Mg2+). This phenomenon has been termed homologous activation of binding sites [Ueland, P. M. and Døskeland, S. O. (1978) J. Biol. Chem., in the press]. Similar results were obtained with AMP and ADP.Displacement of [3H]adenosine binding from the sites specific for this adenine derivative was observed with unlabelled adenosine, adenine, AMP and ADP with decreasing efficiency in the order mentioned. This indicates that adenine, AMP and possibly ADP interact with the adenosine binding sites. Adenine and AMP inhibited the activation of the cyclic AMP binding capacity by ATP as previously shown for adenosine [Ueland, P. M. and Døskeland, S. O. (1978) Arch. Biochem. Biophys. 185, 195–203]. The inhibitory power closely parallelled the displacement of [3H]adenosine binding. Adenine was also demonstrated to inhibit the homologous activation of the cyclic AMP binding site.