Purification of homogeneous rat phosphofructokinase isozymes with high specific activities.

The purification of rat muscle and liver phosphofructokinase (PFK) isozymes has been greatly facilitated by column chromatographic separation on immobilized Cibacron Blue F3GA. The homogeneous liver PFK isozyme exhibited a specific activity of greater than 200 units per mg of protein which is nearly two-fold greater than has been previously reported for this isozyme. The yields for this isozyme exceeded 40% of the original activity and the molecular weight of its subunit was about 85,000 as determined by SDS-polyacrylamide gel electrophoresis. The muscle PFK isozyme's specific activity was approximately 265 units/mg of protein which also is about twice the greatest specific activity previously reported. The overall yield for muscle PFK exceeded 50% of the original activity, and the molecular weight of its subunit was approximately 82,000. Using each homogeneous isozyme, antibodies were produced in rabbits; and the immunoglobin-G (IgG) fraction from the sera of these rabbits was highly specific for the PFK isozyme used as an antigen.