Further analysis of the mode of inhibition and activation of Novikoff ascites tumor phosphofructokinase.

The Novikoff ascites tumor phosphofructokinase showed a lag in the onset of inhibition by adenosine triphosphate or citrate. Under identical assay conditions, Ehrlich ascites tumor P-fructokinase and the rabbit muscle enzyme were immediately inhibited by ATP. Advantage was taken of this lag period by first incubating the enzyme with ATP for 2 min before the assay of enzyme activity. After the first incubation, the ATP inhibition of tumor P-fructokinase became immediate. This inhibition can be reversed by the addition of orthophosphate, AMP, or fructose-6-P prior to the addition of all the necessary components for assaying enzyme activity. Both the inhibition and the reactivation of the enzyme were found to be dependent on enzyme concentrations. The lower the enzyme concentration, the stronger is the inhibition and the slower the reactivation.