Purification and properties of a pyrophosphate-dependent phosphofructokinase from Toxoplasma gondii.
Inorganic pyrophosphate-dependent phosphofructokinase was identified in toxoplasma gondii and purified to near homogeneity from the crude extracts. The purified enzyme displayed one major protein band which coincided with enzyme activity on nondenaturing polyacrylamide gel electrophoresis. This phosphofructokinase had a molecular weight of 100,000 determined by gel filtration and was composed of one type of subunit with the molecular weight of 45,000 estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the enzyme is a homodimer. Some kinetic parameters of the purified enzyme were investigated in the forward and the reverse directions. The substrate saturation curves for fructose 6-phosphate and pyrophosphate were all hyperbolic. The apparent Km values for fructose 6-phosphate and for pyrophosphate were 2.7 x 10(-4) M and 3.3 x 10(-5) M respectively. Kinetics for Fru-1,2-P2 and for Pi in the reverse reaction were also hyperbolic. The activity of this enzyme was magnesium-dependent. Nucleoside triphosphates and polyphosphates did not serve as phosphate donor and the enzyme activity was not altered in the presence of any of these nucleotides. As in the case of pyrophosphate-dependent phosphofructokinases from other anaerobic eukaryotes the Toxoplasma enzyme was not activated by fructose 2,6-biphosphate.
Peng
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Department of Pharmacology, Stanford University School of Medicine, CA 94305.
Mansour
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D000818:Animals; D005635:Fructosediphosphates / Q000378:metabolism; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D014122:Toxoplasma / Q000201:enzymology*
D005635:Fructosediphosphates; D009711:Nucleotides; D010710:Phosphates; D010770:Phosphotransferases; C022502:pyrophosphate-fructose 6-phosphate 1-phosphotransferase; C029063:fructose-1,6-diphosphate