Estimation of phosphofructokinase activity in homogenates of rat heart using a new isotopic assay at low substrate concentration

A new method is described for the determination of phosphofructokinase activity in crude extracts of heart. The assay does not require the prior removal of NADH oxidase from extracts and allows application of hexose-6-phosphate substrate concentrations that approach physiological. The method uses [5- 3H]Hexose-6-phosphate as substrate and the rate of the reaction is determined by the rate of formation of [ 3H 2O]. Inhibition by citrate and dependence on ATP as well as responses to other known inhibitors and activators suggest that the assay is specific for phosphofructokinase. Application of the new assay indicated that perfusion of the isolated rat heart with 10 μ m epinephrine markedly activated phosphofructokinase. Thus, the present assay method for phosphofructokinase in heart extracts is considered applicable to assessing acute changes in the activity of this key glycolytic enzyme.