Binding of adenosine 3',5'-monophosphate dependent protein kinase regulatory subunit to immobilized cyclic nucleotide derivatives

Abstract

Several cyclic nucleotide derivatives with aminoalkyl side chains attached to the purine ring were synthesized and their interactions with adenosine 3'',5''-monophosphate cyclic AMP [cAMP] dependent protein kinase were studied before and after immobilization to CNBr-activated Sepharose 4B. The soluble N6-substituted derivatives were as effective as cAMP itself in activating protein kinase and were more effective than 8-substituted cAMP derivatives, the 2-substituted cAMP derivatives and the cGMP derivatives were the least effective. All of the synthetic derivatives tested were poor substrates for beef heart phosphodiesterase being hydrolyzed at rates less than 2% for that of cAMP itself. All of the immobilized cyclic nucleotides interacted with protein kinase in a biospecific manner as judged by the following criteria: the immobilized cyclic nucleotides competed with cAMP for the binding sites on protein kinase; the analogous spacer-arm did not compete; and the effects of enzyme concentration, MgATP and cleavage of the cyclic phosphate ring on the interactions of protein kinase with the immobilized cyclic nucleotides were the same as previously shown for free cAMP. The immobilized ligands were bound with the same order of effectiveness as the analogous soluble ligand. The observed Ka for the activation of 0.005 .mu.M protein kinase by N6-H2N(CH2)2-cAMP was increased from 0.23 to 3 .mu.M by the process of immobilization. This increase was unaffected by the coupling density and spacer-arm length. The observed Kb for 0.10 .mu.M protein kinase binding to immobilized N6-H2N(CH2)2-cAMP was increased as the molecular sieving exclusion limit of the matrix used was decreased indicating that at least part of this decrease in apparent affinity upon immobilization is due to exclusion of the enzyme from a portion of the matrix and therefore from a fraction of the immobilized ligand molecules.