PURIFICATION AND PROPERTIES OF PHOSPHOFRUCTOKINASE FROM DICTYOSTELIUM-DISCOIDEUM

Phosphofructokinase (PFruK) from the slime mold Dictyostelium discoide um has been purified to homogeneity over 15000-fold with a 29% yield. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the final preparation revealed a single band of 95 kDa. The native molecular ma ss was determined by gel filtration to be 382 kDa, indicating that the enzyme is a homotetramer. An antibody raised in rabbits against the 9 5-kDa band immunoprecipitated PFruK activity while it did not react wi th the enzyme from yeast and mammalian cells. The apparent pI was 6.8 and the pH optimum was 7.6. The enzyme had an activation energy (E(a)) of 29.1 kJ/mol. The amino acid composition was distinctive in having high Ser, Gly and Glx and low Ala, Val and Tyr compared with other euk aryotic PFruKs. Enzyme activity did not have a sigmoidal saturation cu rve for fructose 6-phosphate, was only mildly inhibited by MgATP at ac idic pH values, was not affected by enzyme concentration and was insen sitive to any of the typical allosteric effecters of PFruKs from other sources. However, the enzyme binds fructose 2,6-bisphosphate as indic ated by protection against thermal denaturation. Treatment with cAMP-d ependent protein kinase led to phosphorylation of the enzyme without c hange in activity. The metabolic significance of these properties and their relationship to structure/function are discussed.