Purification and kinetic properties of phosphofructokinase from dental pulps of rat incisors.

Phosphofructokinase (EC 2.7.1.11) was partially purified 19-fold from dental pulps of rat incisors, by ammonium-sulphate fractionation and phenyl-Sepharose chromatography with a recovery of about 95 per cent. With a 0.05 M tris-HCl buffer, the pH optimum of the enzyme was determined to be 8.4. At this pH, substrate inhibition of the enzyme by either fructose-6-phosphate (F6P) or ATP was not observed, and the relationship between reaction velocity and each substrate concentration was well explained by the Michaelis-Menten equation. The Km values were determined to be 5 X 10(-5) and 1.8 X 10(-4) M for ATP and F6P, respectively. The enzyme, however, showed different catalytic properties at pH 7.6, i.e. the kinetic behaviour was sigmoidal with respect to F6P and it was inhibited by high concentrations of ATP. The Hill coefficient for F6P was determined graphically to be 1.4. At pH 7.6, the enzyme activity was inhibited by citrate and phosphoenolpyruvate (PEP), neither of which showed any inhibitory effect on the enzyme at pH 8.4.