Studies on Heart Phosphofructokinase

Sheep heart phosphofructokinase was extracted from a sedimentable fraction of homogenates after incubation with ATP and MgSO4. The extracted enzyme was fully active and soluble. The enzyme was purified by a procedure which involved ethanol fractionation, extraction from the ethanol fraction, DEAE-cellulose chromatography, and ammonium sulfate fractionation. The final specific activity of the enzyme was 120 to 157. Enzyme recovery was about 55% of the enzyme activity in the original extract. The purified enzyme was crystallized. The crystals were hexagonal in shape and had approximately the same specific activity as the purified enzyme. Examination on starch gel electrophoresis showed that the enzyme moved as a diffuse patch with enzyme activity through the entire patch. Ultracentrifugal sedimentation analysis revealed the presence of either a schlieren pattern with a single asymmetrical peak or a pattern with a minor light peak and a major heavy peak. The value for s20,w varied from 22.5 to 50, according to the degree of poylmerization of the enzyme. The sedimentation coefficient as determined on the sucrose gradient was found to be concentration-dependent. The lowest value was s20,w 15.2. Sodium chloride (2 m) caused dissociation of the enzyme, accompanied by a decrease in its catalytic activity.