PURIFICATION AND KINETIC-PROPERTIES OF PHOSPHOFRUCTOKINASE FROM RANA-RIDIBUNDA ERYTHROCYTES

Phosphofructokinase (PFK) from Rana ridibunda erythrocytes was purifie d 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 S-0.5 value for fructose-6-ph osphate (F6P) was 5.6 mM and the K-m for ATP 0.87 mM. The enzyme was s ensitive to inhibition by ATP which was increased with lower F6P conce ntrations. At physiological levels of 2,3-diphosphoglycerate (0.35 mu mol/ml RBC), 20% of PFK activity was inhibited. Significant activation s under cellular conditions were exercised by AMP and, to a lesser ext ent, by P-i. Micromolar concentrations of fructose-2,6-bisphosphate an d glucose-1,6-bisphosphate were also potent activators of the erythroc yte enzyme. Fructose-1,6-bisphosphate (10-50) mu M activated the enzym e 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 mechanis m controlled by the energy status of the red cell, as well as the stat e of oxygenation of haemoglobin, is discussed, in which PFK occupies a central role.