Purification and kinetic properties of phosphofructokinase from Rana ridibunda erythrocytes

Abstract

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 S 0.5 value for fructose-6-phosphate (F6P) was 5.6 mM and the K m 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 μmol/ml RBC), 20% of PFK activity was inhibited. Significant activations under cellular conditions were exercised by AMP and, to a lesser extent, by P 1. 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) μM 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.