Rabbit skeletal muscle phosphorylase kinase. Comparison of glycogen synthase and phosphorylase as substrates.

The Ca”-stimulated phosphorylation of rabbit muscle glycogen synthase (EC 2.4.1.11) catalyzed by phosphorylase kinase (EC 2.7.1.38) was characterized and compared with the reaction involving phosphorylase (EC 2.4.1.1). By comparing reaction rates at equal concentrations (on a mass basis), the activity ratio of phosphorylase/synthase varied from 30 (0.05 mg/ml) to approximately 8 (1 mg/ml). A basically parallel behavior of the synthase and phosphorylase phosphorylation rates was observed when phosphorylase kinase activity was modified as follows: (a) by varying the pH from 6.8 and 8.2, (b) activation of phosphorylase kinase, measured at pH 6.8, by tryptic proteolysis, (c) activation of phosphorylase kinase, measured at pH 6.8, by phosphorylation of the kinase through the action of the catalytic subunit of cyclic AMP-dependent protein kinase, (d) by varying the free Ca2+ concentration, and (e) by following the inactivation of phosphorylase kinase when incubated at 37°C. We infer that the same catalytic center is probably involved in both phosphorylation reactions and that the mechanisms of activation by Ca’+, limited proteolysis, or phosphorylation have certain essential features in common. ADP (2 mu) produced parallel inhibitions (about 75%) of both the synthase and phosphorylase phosphorylation rates. KC1 (0.2 M) and Pi (20 mM) displayed differential inhibition of the synthase reaction (37% and 51%, respectively) compared with the phosphorylase reaction (29% and 8%, respectively). Glycogen (8 mg/ml) caused a 6-fold increase in the rate of phosphorylase phosphorylation but only a 13% increase for the synthase reaction. The phosphorylation of synthase (at 0.28 mg/ml) catalyzed by phosphorylase kinase was unaltered by the simultaneous presence of phosphorylase (at 0.28 mg/ml), whereas the presence of synthase caused at least a 50% decrease in the rate of phosphorylase phosphorylation.