Use of fructose-2,6-diphosphate to assay for phosphofructokinase activity in human muscle.
OBJECTIVE
Use fructose-2,6-diphosphate (fru-2,6-P2) for measuring phosphofructokinase (PFK) activity in muscles.


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.


CONCLUSIONS
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.
Schliselfeld
L H
LH
Department of Pathology, New York Medical College, Valhalla, NY 10595, USA.
Danon
M J
MJ
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001706:Biopsy; D004789:Enzyme Activation; D005635:Fructosediphosphates / Q000494:pharmacology*; D005636:Fructosephosphates / Q000494:pharmacology; D006003:Glycogen / Q000032:analysis; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000201:enzymology*; D010732:Phosphofructokinase-1 / Q000172:deficiency / Q000378:metabolism*; D006005:Phosphorylases / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism
D005635:Fructosediphosphates; D005636:Fructosephosphates; C027618:fructose-6-phosphate; C027652:fructose 2,6-diphosphate; D000255:Adenosine Triphosphate; D006003:Glycogen; D007770:L-Lactate Dehydrogenase; D006005:Phosphorylases; D010770:Phosphotransferases; D010732:Phosphofructokinase-1; D008274:Magnesium