Isolation and characterization of muscle phosphofructokinases with varying degrees of phosphorylation.

Phosphofructokinase has been purified from the skeletal muscle of a rat (200 g) and a rabbit (3 kg) 20 h after intraperitoneal injection of [32P]phosphate (40 mCi). The specific radioactivities of the labeled phosphofructokinases were 3000 and 140 cpm/mg, respectively. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate and peptide mapping of the tryptic digest of the rat enzyme showed that one specific peptide is phosphorylated and that the phosphate is covalently linked to a serine residue. Rabbit muscle phosphofructokinase has been separated by DEAE-cellulose chromatography into two fractions with lower and higher phosphate contents, which contain an average of 0.3 and 0.8 mol of phosphate/mol of enzyme (320,000 daltons), respectively. The lower phosphate form differs from the phosphofructokinase fractions reported by other workers not only in the phosphate content but also in its aggregation state. Sedimentation velocity patterns of the lower phosphate fraction showed two major peaks (12 and 18 S) and also a minor peak (24 S), but no 30 S material. The higher phosphate fraction, however, consisted mainly of 30 S material (40%) and also 24, 18, and 12 S polymers. Upon decreasing the protein concentration from 7 to 0.4 mg/ml, the 24 and 18 S polymers in the lower and the higher phosphate fractions dissociated to 12 S material in a stepwise manner. The relative amount of 30 S material in the latter fraction remained constant, indicating that it is a stable polymer. Additional information concerning the dissociation process came from examination of three subfractions from molecular sieve chromatography of the lower phosphate and higher phosphate fractions. Within each set of three subfractions, the phosphate content was the same. However, all subfractions contained several polymeric species, with smaller polymers appearing in the trailing subfractions. The polymers appearing at comparable positions in the elution profiles were always smaller in the lower phosphate than the higher phosphate fraction. Thus, there is heterogeneity among the polymers of a given size, in phosphate content and perhaps conformation. We conclude that the association-dissociation system is too complex to be described in terms of any simple model. The specific activities of over 20 samples of phosphofructokinase with different phosphate content (0.2 to