The Mg2+ requirements of nonactivated and activated rat liver phosphorylase kinase

Abstract

Incubation of rat liver phosphorylase kinase in the presence of MgATP results in a time‐dependent increase in activity, i.e., activation. Determination of the magnitude of activation depends, in large part, on the relative concentrations of Mg²⁺ and ATP used in the phosphorylase kinase activity assay, such that as the Mg²⁺ to ATP ratio increases less activation is detectable. Prior to activation, maximal activity of nonactivated phosphorylase kinase requires a 2–3 fold molar excess of Mg²⁺ (i.e., free Mg²⁺) over ATP. MgATP‐dependent activation of the enzyme results in an alteration in the free Mg²⁺ requirement such that the activity of the activated enzyme is sharply inhibited by the free cation. Inhibition by free Mg²⁺ of the activated enzyme is rapidly reversed by removal of free Mg²⁺ but is not affected by addition of Ca²⁺. Both nonactivated and activated forms of enzyme appear to be inhibited by free ATP^4–. The results show that the use of high concentrations of free Mg²⁺ in the phosphorylase kinase activity assay can blunt or completely obscure changes in enzyme activity following activation of the enzyme.