Physical and Kinetic Properties of Human Phosphofructokinase from Skeletal Muscle and Erythrocytes

Abstract

Human phosphofructokinase was partially purified from skeletal muscle and erythrocytes. Erythrocyte phosphofructokinase migrated faster on electrophoresis, showed greater affinity for DEAE-cellulose in Tris-phosphate buffer at pH 8, was more inhibited by ATP, and was less inhibited by citrate than muscle phosphofructokinase. Antibody to muscle phosphofructokinase inhibited the activity of erythrocyte phosphofructokinase less than that of the muscle enzyme, but the enzymes could not be distinguished by immunodiffusion methods. By sucrose density gradient centrifugation, the molecular weight of muscle phosphofructokinase was estimated to be between 3.8 × 105 and 4.3 × 105 for the smallest active form. When larger amounts of enzyme were centrifuged, a broad peak resulted, suggesting polymerization of the enzyme. The sedimentation coefficient of erythrocyte phosphofructokinase was also concentration-dependent, but a heterogeneous peak was obtained when even small amounts of enzyme were centrifuged so that the molecular weight could not be estimated accurately. In kinetic studies of both enzymes, Lineweaver-Burk plots yielded parallel lines when activity was measured at different fixed concentrations of the second substrate. Both Km and Vmax for either substrate were increased by raising the concentration of the other substrate. The pH maximum for both enzymes was about 8.5 and inhibition at low pH was greater with high ATP. NH4+ and Pi increased Vmax and decreased Km for both substrates.