Reassessment of the cold-labile nature of phosphofructokinase from a hibernating ground squirrel.
This study reassesses the proposal that cellular conditions of low temperature and relative acidosis during hibernation contribute to a suppression of phosphofructokinase (PFK) activity which, in turn, contributes to glycolytic rate suppression during torpor. To test the proposal that a dilution effect during in vitro assay of PFK was the main reason for activity loss (tetramer dissociation) at lower pH values, the influence of the macromolecular crowding agent, polyethylene glycol 8000 (PEG), on purified skeletal muscle PFK from Spermophilus lateralis was evaluated at different pH values (6.5, 7.2 and 7.5) and assay temperatures (5, 25 and 37degrees C). A 78 +/- 2.5% loss of PFK activity during 1 h incubation at 5 degrees C and pH 6.5 was virtually eliminated when 10% PEG was present (only 7.0 +/- 1.5% activity lost). The presence of PEG also largely reversed PFK inactivation at pH 6.5 at warmer assay temperatures and reversed inhibitory effects by high urea (50 or 400 mM). Analysis of pH curves at 5 degrees C also indicated that approximately 70% of activity would remain at intracellular pH values in hibernator muscle. The data suggest that under high protein concentrations in intact cells that the conditions of relative acidosis, low temperature or elevated urea during hibernation would not have substantial regulatory effects on PFK.
MacDonald
J A
JA
Institute of Biochemistry, College of Natural Sciences, Carleton University, Ottawa, ON, Canada.
Storey
K B
KB
D000818:Animals; D003080:Cold Temperature; D004789:Enzyme Activation; D004795:Enzyme Stability; D006019:Glycolysis; D006605:Hibernation; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology; D010732:Phosphofructokinase-1 / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D012589:Sciuridae / Q000378:metabolism* / Q000502:physiology; D012997:Solvents / Q000494:pharmacology
D012997:Solvents; D011092:Polyethylene Glycols; D010732:Phosphofructokinase-1