Clinical biochemistry · 1996
Use of fructose-2,6-diphosphate to assay for phosphofructokinase activity in human muscle.
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Schliselfeld L H, Danon M J
Аннотация
OBJECTIVE: Use fructose-2,6-diphosphate (fru-2,6-P2) for measuring phosphofructokinase (PFK) activity in muscles.
DESIGN AND METHODS: PFK activity was measured at 2 mmol/L MgCl2 and 5 mmol/L adenosine triphosphate (ATP) (mol/L MgCl2:mol/L ATP 0.4) without and with fru-2,6-P2.
RESULTS: 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-P2; 1.83 +/- 0.91 units/g muscle. Addition of fru-2,6-P2 produced an immediate 20- to 57-fold increase in activity; 52.8 +/- 12.5 units/g muscle. Raising the mol/L ratio of MgCl2 to ATP to 0.87 and higher without fru-2,6-P2 produced 34%-76% of the PFK activity seen with fru-2,6-P2. A PFK deficiency patient had a trace of activity, which was independent of mol/L MgCl2:mol/L ATP and not activated by fru-2,6-P2.
CONCLUSION: The almost complete absence of activity without fru-2,6-P2 at 0.40 mol/L MgCl2:mol/L ATP, and the restoration of maximum activity by fru-2,6-P2 provides an assay for verified PFK activity that could lead to a more accurate diagnosis in patients with PFK deficiency.
DESIGN AND METHODS: PFK activity was measured at 2 mmol/L MgCl2 and 5 mmol/L adenosine triphosphate (ATP) (mol/L MgCl2:mol/L ATP 0.4) without and with fru-2,6-P2.
RESULTS: 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-P2; 1.83 +/- 0.91 units/g muscle. Addition of fru-2,6-P2 produced an immediate 20- to 57-fold increase in activity; 52.8 +/- 12.5 units/g muscle. Raising the mol/L ratio of MgCl2 to ATP to 0.87 and higher without fru-2,6-P2 produced 34%-76% of the PFK activity seen with fru-2,6-P2. A PFK deficiency patient had a trace of activity, which was independent of mol/L MgCl2:mol/L ATP and not activated by fru-2,6-P2.
CONCLUSION: The almost complete absence of activity without fru-2,6-P2 at 0.40 mol/L MgCl2:mol/L ATP, and the restoration of maximum activity by fru-2,6-P2 provides an assay for verified PFK activity that could lead to a more accurate diagnosis in patients with PFK deficiency.