Fish Muscle Phosphofructokinase: Influences of Protein Concentration on Enzyme Kinetic Behaviour

Phosphofructokinase (PFK) is regulated by multiple mechanisms, one of these being changes in the polymerization state of the enzyme which is influenced by enzyme concentration. To better understand the regulation of PFK in vivo, the present study monitored the effects of changing enzyme concentration and of protein crowding on enzyme activity and properties. Purified PFK from white muscle of rainbow trout (Oncorhynchus mykiss) was characterized under assay conditions that simulated the physiological state of resting muscle [pH 7.2, 7.5 mM MgATP, 0.2 mM fructose-6-phosphate (F6P)], and with experimental manipulation of enzyme concentration in two ways: (a) increasing the amount of PFK (0.1 or 0.6/~g]ml) added, and (b) crowding the enzyme by the addition of polyethylene glycol (PEG) (or other agents) to the assay mixture. A marked decrease in ATP inhibition of the enzyme was observed when PFK was studied in buffer containing PEG, as well as when assays were carried out with the purified enzyme at a concentration of 0.6/~ g/mi, as compared with a 6-fold diluted enzyme. Enzyme concentration also altered the F6P saturation curve, S0.s values for F6P decreasing when PFK concentration was increased or crowding reagents were added. The inclusion of PEG in the assay mixture shifted the pH profile of PFK leftward with much higher enzyme activity, observed as compared to that in the absence of PEG, especially at low pH values. These results provide evidence that the allosteric behaviour of fish muscle PFK is affected by enzyme concentration and support the proposal that the more aggregated forms of the enzyme have higher affinity for fructose 6-phosphate and are less inhibited by Mg.ATP.