Purification and kinetic properties of phosphofructokinase from Rana ridibunda erythrocytes.
Phosphofructokinase (PFK) from Rana ridibunda erythrocytes was purified about 570-fold by column chromatography on Cibacron Blue Sepharose. The resulting enzyme preparation had a specific activity of 1.94 U/mg protein and a pH maximum of 7.6. The molecular weight as determined by HPLC chromatography was 330,000 Da. The S0.5 value for fructose-6-phosphate (F6P) was 5.6 mM and the Km for ATP 0.87 mM. The enzyme was sensitive to inhibition by ATP which was increased with lower F6P concentrations. At physiological levels of 2,3-diphosphoglycerate (0.35 mumol/ml RBC), 20% of PFK activity was inhibited. Significant activations under cellular conditions were exercised by AMP and, to a lesser extent, by Pi. Micromolar concentrations of fructose-2,6-bisphosphate and glucose-1,6-bisphosphate were also potent activators of the erythrocyte enzyme. Fructose-1,6-bisphosphate (10-50) microM activated the enzyme to a limited extent. With respect to these effects, it is suggested that PFK is a significant enzyme in regulating the glycolytic flux of Rana ridibunda red blood cells. The existence of a regulatory mechanism controlled by the energy status of the red cell, as well as the state of oxygenation of haemoglobin, is discussed, in which PFK occupies a central role.
Kaloyianni
M
M
Laboratory of Animal Physiology, Science School, Aristotle University of Thessaloniki, Greece.
Kotinis
K
K
Gounaris
E G
EG
D019794:2,3-Diphosphoglycerate; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004163:Diphosphoglyceric Acids / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004912:Erythrocytes / Q000201:enzymology*; D005635:Fructosediphosphates / Q000494:pharmacology; D005636:Fructosephosphates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010710:Phosphates / Q000494:pharmacology; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors / Q000097:blood*; D011895:Rana ridibunda / Q000097:blood*
D004163:Diphosphoglyceric Acids; D005635:Fructosediphosphates; D005636:Fructosephosphates; D010710:Phosphates; D019794:2,3-Diphosphoglycerate; D000249:Adenosine Monophosphate; C027618:fructose-6-phosphate; C027652:fructose 2,6-diphosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1