Hormone‐Induced Changes in Pyruvate Kinase

The kinetic properties of pyruvate kinase from isolated hepatocytes of fed rat liver incubated with and without glucagon (1 μM) are compared. Glucagon incubation of hepatocytes decreases the apparent affinities of the enzyme for the substrate phosphoenolpyruvate and the allosteric activator fructose 1,6‐bisphosphate while the apparent Ki values for alanine and ATP are lowered. The Km for the second substrate ADP is not influenced by glucagon incubation. The kinetic curves show intermediate plateaus indicative of the presence of two enzyme forms, an active form (called La) and a less active form (called Lb). It is concluded that the effect of glucagon on the kinetic parameters of pyruvate kinase is caused by a shift of the equilibrium between both enzyme forms to the less active Lb side. Comparison of the kinetic parameters of pyruvate kinase of a total homogenate from a fed and a 48‐h‐starved rat indicates that starvation causes a decrease of the apparent affinities of the enzyme for phosphoenolpyruvate and fructose 1,6‐bisphosphate while the apparent Ki values for alanine and ATP are lowered. The Km for ADP is not influenced by starvation. This indicates that a change in kinetic properties of pyruvate kinase also occurs in the intact animal stressing its physiological importance. The change in kinetic parameters introduced by glucagon incubation of hepatocytes and starvation of the intact animal are similar and identical to the changes observed on in vitro phosphorylation of the enzyme purified from rat liver [Ekman et al. (1976) Biochim. Biophys. Acta, 429, 374–382] or from pig liver [Ljungström et al. (1976) Eur. J. Biochem. 68, 497–506]. This indicates that the La and Lb form of pyruvate kinase might represent the dephosphorylated and phosphorylated forms of the enzyme respectively. The glucagon inhibited enzyme is partially reactivated in vitro by incubation at 37°C. This reactivation is promoted by the presence of 10mM Mg2+. The reactivation in vitro can be blocked completely by 20 mM KF. These properties of reactivation are most consistent with a protein‐phosphatase‐mediated process. The reactivation is inhibited by a high Ca2+ concentration (2.6 mM). Incubation in vitro of a liver homogenate of a fed and starved rat with 10 mM Mg2++ 0.5 mM ATP in the presence of F− shows that starvation increases the pyruvate kinase inactivation reaction presumably due to protein kinase activity. It is concluded that hormonal regulation of pyruvate kinase activity is exerted both by changing the degree of (de)phosphorylation of the enzyme and by changing the concentration of the allosteric activator fructose 1,6‐bisphosphate which depends, at least in part, on the hormonal control of the phosphofructokinase–fructose‐1,6‐bisphosphatase cycle.