Regulation of phosphofructokinase in perfused rat heart

Phosphofructokinase from rat heart perfused with epinephrine was purified to homogeneity and various allosteric properties were determined under conditions which approximate physiological concentrations of the substrates, effectors and pH. The molecular weights of the protomer of the enzyme isolated from the hormone-stimulated and the control hearts are both approximately 83,000. The epinephrine-stimulated and the control enzymes contain 1.1 and 0.66 mol of phosphate/mol of protomer, respectively. Both enzymes can be fully phosphorylated by cAMP-dependent protein kinase indicating that the phosphorylation site is new and distinct from the known phosphorylation site of skeletal muscle phosphofructokinase. Pure phosphofructokinase isolated from the epinephrine-stimulated heart is significantly less sensitive to inhibition by ATP and citrate, and the K(0.5) values for Fru-6-P (0.18 mM) and Fru-2,6-P₂ (3 μM) are one-half those for the enzyme from control hearts. In the presence of in vivo concentrations of ATP, citrate, and Fru-6-P at pH 7.1, both enzymes are inactive in the absence of Fru-2,6-P₂. Moreover, the K(0.5) values for Fru-2,6-P₂ of the hormone-stimulated and untreated enzymes are 3 and 6 μM, respectively. These differences in the allosteric properties of phosphofructokinases from the hormone-treated and the control hearts dissapear when the enzymes are dephosphorylated by alkaline phosphatase. Determination of the glycolytic intermediates showed a 2-fold increase in Fru-6-P, Fru-2,6-P₂, and AMP and 13-fold increase in Fru-1,6-P₂. Partially purified Fru-6-P,2-kinase from epinephrine-stimulated and control hearts show K(0.5)(Fru-6-P) = 4 and 15 μM, respectively. These results indicate that rat heart phosphofructokinase in vivo requires Fru-2,6-P₂ for its activity. Epinephrine stimulates phosphorylation of phosphofructokinase which results in a more active form. The hormone also increases Fru-2,6-P₂ which appear to be the result of an activation of Fru-6-P,2-kinase by a covalent modification.