REGULATION OF RABBIT MUSCLE PHOSPHOFRUCTOKINASE BY PHOSPHORYLATION

Muscle phosphofructokinase is one of the glycolytic enzymes whose part itioning between the particulate and soluble fractions in skeletal mus cle is linked to the biological activity of the muscle. The formation of the enzyme-actin complex is apparently regulated by phosphorylation of the enzyme, In order to understand the role of phosphorylation on the regulatory mechanism of phosphofructokinase, the self-association of the phosphorylated and dephosphorylated forms of phosphofructokinas e was studied by investigating the sedimentation velocity at pH 7.0 an d 23 degrees C in different solvent constituents. The results show tha t both the phosphorylated and dephosphorylated forms of the enzyme exh ibit the same mechanism of assembly, The effects of allosteric effecte rs are dependent on the phosphorylation state of the enzyme, The prese nce of 0.2 mM fructose-6-phosphate, one of the two substrates, leads t o a significant enhancement in the formation of octomers without alter ing the equilibrium constant for tetramerization for either phosphoryl ated or dephosphorylated enzyme. The presence of 10 mM citrate, an all osteric inhibitor, leads to the formation of a significant amount of d imer, an inactive form of the enzyme. Citrate decreases the propensiti es of the dephosphorylated and phosphorylated forms of the enzyme to t etramerize 3000 times and 100 times, respectively. Based on the mode o f subunit assembly, bimodal sedimentation velocity profiles can be obt ained by simulation. Furthermore, simulation showed that the seemingly very different profiles reported in the literature can be accounted f or by various combinations of equilibrium constants. In summary, this study showed that the propensity of subunit assembly is affected diffe rentially by specific metabolites and the phosphorylation state of pho sphofructokinase.