Unusual, Metabolite-dependent Solubility Properties of Phosphofructokinase

Abstract

Abstract In the presence of 5 mm MgCl2, fructose 6-phosphate kinases from porcine liver and kidney and a variety of other sources: (a) precipitate at low ammonium sulfate concentrations (0 to 30 mm); (b) are completely soluble at slightly higher concentrations (g100 mm); and (c) do not precipitate at low (0 to 30 mm) ammonium sulfate concentrations if fructose-6-P is present or if Mg2+ is absent and 1 mm ATP is present. Although substrate effects on enzymes are well known, the precipitation-solubilization effects just described are, in many respects, novel. These properties also form the basis for a new, rapid and highly specific method for complete purification of porcine liver and kidney phosphofructokinases, as well as substantial purification of other phosphofructokinases. Porcine liver phosphofructokinase was purified 16,000-fold to homogeneity (specific activity = 100) with a 25% yield in only 8 hours. No conventional salt fractionation or column chromatography is required. The key step is repeated selective precipitation and dissolution of the enzyme by simply changing the concentration of ammonium sulfate in the solution. In contrast to conventional ammonium sulfate fractionation, the enzyme is precipitated and dissolved at lower and higher ionic strengths, respectively. The enzyme has a low isoelectric point (pI = 5). Metaboliteinduced structural transitions are also detectable by polyacrylamide gel electrophoresis. In the absence of metabolites, the enzyme does not penetrate 4.2% polyacrylamide gels but in the presence of fructose-6-P or ATP or ADP, it does.