The purification and properties of pig spleen phosphofructokinase

Abstract

Pig spleen phosphofructokinase was purified 800 fold with a yield of 17%. Two iso enzymes that appear to be kinetically identical can be separated by DEAE cellulose column chromatography. In common with the enzyme from other mammalian sources, the spleen enzyme has a pH optimum of 8.2. At pH 7.0 it displays sigmoidal kinetics with respect to fructose 6 phosphate concentration but its co operative behaviour is very dependent on pH, protein concentration and the concentration of MgATP. MgGTP and MgITP can replace MgATP as phosphate donors but, unlike MgATP, these nucleotides do not cause significant inhibition. Mn2+ and Co2+ (as the metal ion ATP complexes) act as cofactors and in the free form are far more inhibitory than free Mg2+. The spleen enzyme responds to a wide variety of potential effector molecules: ADP, AMP, cyclic AMP, aspartate, NH4+, fructose 6 phosphate, fructose 1,6 diphosphate and P(i) all act as either activators or protectors, whereas Mg ATP, Mg2+, citrate, phosphoenol pyruvate and the phosphoglycerates are inhibitors.