Properties of locust muscle 6-phosphofructokinase and their importance in the regulation of glycolytic flux during prolonged flight

Abstract

6-Phosphofructokinase (PFK, EC 2.7.1.11) from the flight muscle of the locust (Locusta migratoria) was purified to a specific activity of 80 μmol min−1 (mg protein)−1 (at 25°C). 1. The enzyme is made up from subunits ofMr-81600, and the smallest catalytically active form is likely to be a tetramer. 2. PFK activity is markedly affected by the pH of the assay; the optimum pH was at about 8. 3. Physiological concentrations of ATP strongly inhibit locust PFK by shifting the S0.5 for fructose 6-phosphate (concentration required for 50% of maximum activity) out of the physiological concentration range. At pH 7.4 and about physiological concentrations of ATP, the curve of PFK activity against the concentration of fructose 6-phosphate is highly sigmoidal with S0.5 several hundred-fold higher than the concentration of fructose 6-phosphate in vivo. 4. The sigmoidicity and the S0.5 values for fructose 6-phosphate are decreased by addition of activators such as NH 4 + , inorganic phosphate, AMP and fructose 2,6-bisphosphate. The sensitivity of the response to these activators is increased by synergistic effects; marked synergistic effects were observed between inorganic phosphate and NH 4 + , fructose 2,6-bisphosphate or AMP as well as between AMP and fructose 2,6-bisphosphate. 5. Fructose 1,6-bisphosphate and fructose 2,6-bisphosphate are both activators of locust muscle PFK but the latter is much more potent than the former. Either of these compounds modifies the effect of the other; when phosphofructokinase is activated by fructose 2,6-bisphosphate, addition of fructose 1,6-bisphosphate results in inhibition of the enzyme activity; and when fructose 1,6-bisphosphate is present in the assay, higher concentrations of fructose 2,6-bisphosphate are required to increase the activity of the enzyme. 6. At low AMP concentrations, fructose 2,6-bisphosphate is not very effective in activating PFK, but AMP is effective at low concentrations of fructose 2,6-bisphosphate. 7. The properties of locust PFK suggest that the marked decrease in the concentration of fructose 2,6-bisphosphate that occurs in the locust flight muscle in the early stages of flight is, in part, responsible for the decrease in PFK activity and thus conservation of carbohydrate during prolonged flight, when lipid is the major fuel. At any stage of flight, however, an increase in energy demand could bring about an increase in PFK activity via increased concentrations of AMP and phosphate.