Purification and Properties of D-Fructose 1,6-Diphosphatase from Swine Kidney

Abstract d-Fructose 1,6-diphosphatase was extensively purified from swine kidney by a simplified procedure which involved chromatography on cellulose phosphate, ammonium sulfate fractionation, and separation on Sephadex G-200. The final specific activity of the enzyme was 25.2 µmoles per min per mg and 34% of the activity present in the crude extract was recovered. The stability of the purified enzyme was greatly increased by the addition of 0.01 m β-mercaptoethylamine and 0.0005 m 8-hydroxyquinoline. The purified enzyme was sensitive to inhibition by adenosine monophosphate and high concentrations of d-fructose 1,6-diphosphate. The preparation was essentially homogeneous after electrophoresis, ultracentrifugation, and elution from cellulose phosphate and Sephadex G-200 columns. Ultracentrifugal sedimentation analysis revealed the presence of a major symmetrical peak with s20, ω = 7.5 and a minor symmetrical peak with s20, ω = 3.6. The proportion of the minor peak was increased by dialysis of the enzyme preparation. The molecular weight of swine kidney d-fructose 1,6-diphosphatase calculated from data obtained by ultracentrifugation, chromatography on Sephadex G-200, and sucrose density gradient centrifugation was 129,500 and that of the minor component was about 65,000. The turnover number of the enzyme was 3250 moles of d-fructose 1,6-diphosphate converted to fructose 6-phosphate and inorganic phosphate per min per mole of enzyme. Analysis of the amino acid composition indicated the presence of a comparatively large amount of basic amino acids, particularly lysine.