DIVALENT-CATIONS BUT NOT OTHER ACTIVATORS ENHANCE PHOSPHORYLASE KINASES AFFINITY FOR GLYCOGEN-PHOSPHORYLASE

TO better understand the physical interaction between glycogen phospho rylase-b (P-b) and its only known kinase, phosphorylase kinase (PbK), and the relationship of this interaction to the activation of PbK, dir ect binding studies are necessary. By utilizing an enzyme-linked immun osorbent assay, a method was developed for measuring the binding of Pb K to immobilized P-b under a variety of experimental conditions. A mon oclonal antibody specific for the alpha subunit of PbK that had no eff ect on the phosphorylation of P-b by PbK or on the interaction of PbK with its known effecters was used to detect PbK bound to plated P-b. H yperbolic binding curves were obtained regardless of whether the conce ntration of PbK or P-b was varied, and the assay detected changes in r elative affinity caused by certain effectors of the kinase. The allost eric effector ADP, alkaline pH, and phosphorylation by cAMP-dependent protein kinase, all activators of PbK, did not cause significant chang es in its relative affinity for P-b; however, Ca2+ and Mg2+ ions, whic h also stimulate PbK, increased its affinity for P-b, with Mg2+ being more effective. Mn2+, which inhibits the P-b conversion activity of Pb K, was found to be the most potent enhancer of its affinity for P-b. T hese results indicate that activation of PbK is not necessarily caused by an increase in its affinity for P-b, although divalent cations may enhance binding. Inclusion of ATP analogs in the binding assay with C a2+ and Mg2+ to simulate catalytic assay conditions did not further af fect the apparent affinity for P-b, which is consistent with the previ ously reported rapid equilibrium random bi-bi kinetic mechanism for P- b conversion.