Divalent cations but not other activators enhance phosphorylase kinase's affinity for glycogen phosphorylase.
To better understand the physical interaction between glycogen phosphorylase-b (P-b) and its only known kinase, phosphorylase kinase (PbK) and the relationship of this interaction to the activation of PbK, direct binding studies are necessary. By utilizing an enzyme-linked immunosorbent assay, a method was developed for measuring the binding of PbK to immobilized P-b under a variety of experimental conditions. A monoclonal antibody specific for the alpha subunit of PbK that had no effect on the phosphorylation of P-b by PbK or on the interaction of PbK with known effectors was used to detect PbK bound to plated P-b. Hyperbolic binding curves were obtained regardless of whether the concentration of Pbk or P-b was varied, and the assay detected changes in relative affinity caused by certain effectors of the kinase. The allosteric effector ADP, alkaline pH, and phosphorylation by cAMP-dependent protein kinase, all activators of PbK, did not cause significant changes in its relative affinity for P-b; however, Ca2+ and Mg2+ ions, which also stimulate PbK, increased its affinity for P-b, with Mg2+ being more effective. Mn2+, which inhibits the P-b conversion activity of PbK, was found to be the most potent enhancer of its affinity for P-b, although divalent cations may enhance binding. Inclusion of ATP analogs in the binding assay with Ca2+ and Mg2+ to stimulate catalytic assay conditions did not further affect the apparent affinity for P-b, which is consistent with the previously reported rapid equilibrium random bi-bi kinetic mechanism for P-b conversion.
Xu
Y H
YH
Department of Biochemistry, College of Medicine, University of Tennessee, Memphis, 38163, USA.
Wilkinson
D A
DA
Carlson
G M
GM
D000818:Animals; D001667:Binding, Competitive; D002413:Cations, Divalent / Q000494:pharmacology*; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D004800:Enzymes, Immobilized / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology; D010764:Phosphorylase Kinase / Q000378:metabolism*; D006005:Phosphorylases / Q000378:metabolism*; D011817:Rabbits
D002413:Cations, Divalent; D004800:Enzymes, Immobilized; D006005:Phosphorylases; D010764:Phosphorylase Kinase