Phosphofructokinase from a vertebrate facultative anaerobe: effects of temperature and anoxia on the kinetic parameters of the purified enzyme from turtle white muscle.
The effects of low temperature and anoxia were determined on phosphofructokinase (PFK) purified from white skeletal muscle of the freshwater turtle, Pseudemys scripta. These effects were assayed by comparing PFK kinetic constants measured at a high (20 degrees C) and low (6 degrees C) temperature using enzyme obtained from animals held under normoxic and anoxic conditions. When assayed at 20 degrees C, PFK from anoxic animals had a lower Ka for phosphate, a lower Ka for AMP and showed no inhibition with increasing concentrations of ATP (up to 10 mM) when compared to enzyme from normoxic animals. At 6 degrees C, anoxic enzyme had a higher Km for fructose 6-phosphate and a higher I50 value for citrate with respect to normoxic enzyme. Decreasing temperature also had a differential effect on PFK kinetic parameters depending on the source of the enzyme. When normoxic enzymes were compared at 20 and 6 degrees C, the enzyme measured at 6 degrees C showed a lower Km for ATP and a lower Ka for AMP. Comparison of anoxic enzymes at these two temperatures showed that anoxic PFK at 6 degrees C had a higher Ka for phosphate, a higher Ka for AMP, and a larger Hill coefficient. A comparison of maximal velocities at varying temperature showed that normoxic enzyme (Q10 = 2.22) was more temperature sensitive than the anoxic enzyme (Q10 = 1.80). It is possible to interconvert the normoxic and anoxic forms of PFK by incubating normoxic enzyme with the active subunit of protein kinase, suggesting that the kinetic changes observed during anoxia resulted from enzyme phosphorylation. These data are discussed with respect to the mechanisms underlying white muscle function during diving and hibernation in red-eared turtles.
Brooks
S P
SP
Institute of Biochemistry, Carleton University, Ottawa, Ontario, Canada.
Storey
K B
KB
D000255:Adenosine Triphosphate / Q000032:analysis; D000818:Animals; D000860:Hypoxia / Q000201:enzymology; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D010732:Phosphofructokinase-1 / Q000302:isolation & purification*; D010766:Phosphorylation; D013696:Temperature; D014426:Turtles / Q000378:metabolism*
D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1