USE OF FRUCTOSE-2,6-DIPHOSPHATE TO ASSAY FOR PHOSPHOFRUCTOKINASE ACTIVITY IN HUMAN MUSCLE

Objective: Use fructose-2,6-diphosphate (fru-2,6-P-2) for measuring ph osphofructokinase (PFK) activity in muscles. Design and Methods: PFK a ctivity was measured at 2 mmol/L MgCl2 and 5 mmol/L adenosine triphosp hate (ATP) (mol/L MgCl2:mol/L ATP 0.4) without and with fru-2,6-P-2. R esults: Human muscle extracts had little PFK activity when assayed at mol/L MgCl2:mol/L ATP of 0.4 to 0.78 without fru-2,6-P-2; 1.83 +/- 0.9 1 units/g muscle. Addition of fru-2,6-P-2 produced an immediate 20- to 57-fold increase in activity; 52.8 +/- 12.5 units/g muscle. Raising t he mol/L ratio of MgCl2 to ATP to 0.87 and higher without fru-2,6-P-2 produced 34%-76% of the PFK activity seen with fru-2,6-P-2. A PFK defi ciency patient had a trace of activity, which was independent of mol/L MgCl2:mol/L ATP and not activated by fru-2,6-P-2. Conclusion: The alm ost complete absence of activity without fru-2,6-P-2 at 0.40 mol/L MgC l2:mol/L ATP, and the restoration of maximum activity by fru-2,6-P-2 p rovides an assay for verified PFK activity that could lead to a more a ccurate diagnosis in patients with PFK deficiency.