Glucagon-stimulated phosphorylation of pyruvate kinase in hepatocytes.

A 32P-labeled pyruvate kinase (L-type isozyme) was elicited in response to glucagon and has been isolated from suspensions of hepatocytes incubated with inorganic [32P]phosphate. The pyruvate kinase in crude extracts of hepatocytes was stabilized by ammonium sulfate fractionation, 30% glycerol, and 50 mM KF and the enzyme was isolated by immunoprecipitation with anti-liver pyruvate kinase immunoglobulin. In the absence of glucagon, incorporation of 32P into pyruvate kinase was linear for up to 60 min. However, addition of 1 pM glucagon to hepatocytes incubated in the presence of inorganic [32P]phosphate resulted in a 3-fold increase in the incorporation of 32P into pyruvate kinase within 5 min. Immunoelectrophoresis of the hepatocyte extracts followed by autoradiography demonstrates that 32P is located with the pyruvate kinaseanti-liver pyruvate kinase immunoglobulin precipitation band. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated immune complex indicates that 32P is covalently bound to the dissociated subunits of pyruvate kinase. Incubation of the hepatocytes in 1 pM glucagon resulted in a 60% decrease of pyruvate kinase activity when assayed in subsaturating concentrations of phosphoenolpyruvate and in the absence of the activator fructose 1,6-bisphosphate. Furthermore, the pyruvate kinase in glucagon-treated hepatocytes has a decreased affinity for phosphoenolpyruvate and a decreased affinity for fructose 1,6-bisphosphate. Both the rapid decrease in pyruvate kinase activity and the rapid increase in phosphorylation of the enzyme in response to addition of glucagon are consistent with phosphorylation of pyruvate kinase being a hormone-sensitive biochemical modification to regulate its activity in uivo.