Insulin-mediated regulation of heart atrial and ventricular 6-phosphofructo-1-kinase.

Atrial 6-phosphofructo-1-kinase activity from the hearts of diabetic rats was decreased by 50%, but ventricular 6-phosphofructo-1-kinase activity was found not to be insulin-sensitive. This decrease in atrial 6-phosphofructo-1-kinase activity during diabetes was characterized by diminished levels of all three types of 6-phosphofructo-1-kinase subunits. As shown by immunological titration and column chromatography, the population of native 6-phosphofructo-1-kinase isozymes in the ventricles was not measurably affected during insulin deprivation. However, the atrial isozyme population in diabetic rat heart appeared to contain, on a relative basis, higher levels of the isozymic forms containing the L-type subunit. Measurement of the levels of this subunit indicated that in diabetic atria it was less affected than the other subunits. In the ventricles, insulin deficiency did not promote significant losses of fructose-2,6-P2; but, in diabetic rats, the atrial levels of this activator were decreased by 80% and subsequently restored by insulin treatment. These data suggest that any insulin-mediated effects on ventricular 6-phosphofructo-1-kinase activity and resultant effects on ventricular glycolysis do not appear to be exerted through changes in enzyme concentration, but probably through changes in modulators other than fructose-2,6-P2. In contrast to the ventricles, it appears that insulin exerts its effects on atrial 6-phosphofructo-1-kinase activity and, in part, influences atrial glycolysis through alteration of fructose-2,6-P2 levels, enzyme concentration, and isozymic content.

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.