Multiple activities on phosphorylase kinase. 2. Different specificities toward the protein substrates phosphorylase b, troponin, and phosphorylase kinase

Abstract

Phosphorylase kinase exhibits 3 kinds of enzymatic activities. A partial activity, A0, catalyzes the phosphorylation of phosphorylase b, troponin I, and phosphorylase kinase itself (autophosphorylation); A1 can utilize only phosphorylase b and phosphorylase kinase as the substrate, whereas A2 can utilize only phosphorylase b and troponin T. Stimulation of A1 by Ca2+ coincides with an increase in the number of sites that can undergo self-phosphorylation ranging from .apprx. 35-70 mol of phosphate incorporated/1.28 .times. 106 g of proteins. Inhibition of A0 and A1 by millimolar Ca2+ is accompanied by a decrease in substrate availability during self-phosphorylation. NH4Cl (150 mM) strongly inhibits the availability of troponin as a substrate. In the course of self-phosphorylation, the activities A0 and A1 are both stimulated moderately by an increase in pH; however, only A1 shows some inhibition by 150 mM NH4Cl. Millimolar Ca2+ inhibits A1 and A2 as measured by self-phosphorylation or tropinin phosphorylation, as observed with the phosphorylation of phosphorylase b. The rate of self-phosphorylation varies as a function of substrate concentration (Km = 68 nM at 10 mM Mg2+ and 184 .mu.M Ca2+, pH 9.0). The data indicate that both Ca2+ activation and inhibition seem to be mediated by phosphorylase kinase itself rather than by the substrates.