Reassessment of the cold-labile nature of phosphofructokinase from a hibernating ground squirrel

This study reassesses the proposal that cellular conditions of low temperat ure and relative acidosis during hibernation contribute to a suppression of phosphofructokinase (PFK) activity which, in turn, contributes to glycolyt ic rate suppression during torpor. To test the proposal that a dilution eff ect during in vitro assay of PFK was the main reason for activity loss (tet ramer dissociation) at lower pH values, the influence of the macromolecular crowding agent, polyethylene glycol 8000 (PEG), on purified skeletal muscl e PFK from Spermophilus lateralis was evaluated at different pH values (6.5 , 7.2 and 7.5) and assay temperatures (5, 25 and 37 degreesC). A 78 +/- 2.5 % loss of PFK activity during 1 h incubation at 5 degreesC and pH 6.5 was v irtually eliminated when 10% PEG was present (only 7.0 +/- 1.5% activity lo st). The presence of PEG also largely reversed PFK inactivation at pH 6.5 a t warmer assay temperatures and reversed inhibitory effects by high urea (5 0 or 400 mM). Analysis of pH curves at 5 degreesC also indicated that 70% o f activity would remain at intracellular pH values in hibernator muscle. Th e data suggest that under high protein concentrations in intact cells that the conditions of relative acidosis, low temperature or elevated urea durin g hibernation would not have substantial regulatory effects on PFK.