Hepatic pyruvate kinase. Quantitative measurements of phosphorylation in vitro and in the isolated rat hepatocyte.

The phosphorylation of rat liver pyruvate kinase was studied in isolated rat hepatocytes and with the crystalline enzyme. Hepatocytes were incubated with ["PI orthophosphate and isotopically labeled pyruvate kinase was quantitatively isolated using a resin-coupled antibody. The molar extent of phosphorylation of the enzyme was calculated from the amount of radioactivity in the enzyme and the specific radioactivity of [y32P]ATp determined simultaneously. A low steady state level of phosphorylation of pyruvate kinase was observed in hepatocytes incubated for 50 min in the absence of hormonal stimuli (0.4 P/tetramer). The activity ratio (enzyme activity at 1.8 m~ P-enolpyruvate, -fructose-1,6-Pz/+fructose-1,6-Pz) for the low phosphatecontaining form of the enzyme was 0.68. Exposure of the hepatocyte to 1 p~ glucagon for 5-10 min increased the phosphorylation state to 1.7 P/tetramer and decreased the activity ratio by 60%. An inverse, linear relationship between the activity ratio and the phosphorylation state of the enzyme up to 2 P/tetramer was observed both in studies with the hepatocyte and with the crystalline nzyme phosphorylated using CAMP-dependent protein kinase from beef heart. In no situation was greater than 2P/tetramer observed in the present investigation. With the crystallized enzyme, a single protein band migrating with an apparent M, = 62,000 was seen after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing in the presence of 8 M urea, however, revealed heterogeneity in the subunit composition. Five bands, three of which were not influenced by phosphorylation, could be resolved by isoelectric focusing. Despite the heterogeneity in the subunits with respect to phosphorylation, the purified enzyme was capable of binding 3.7 molecules of fructose-1,6-bisphosphate/tetramer. The results of this investigation differ from previous reports and suggest that some of the subunits of hepatic pyruvate kinase are modified such that they lose the capacity to be phosphorylated. Furthermore, nonphosphorylatable forms of the enzyme may occur in the intact cell and are not an artifact of purification.