Pyrophosphate-dependent phosphofructokinase from Giardia lamblia: purification and characterization.
The pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) from Giardia lamblia has been purified to homogeneity using two methods. The purified enzyme is shown to be homogenous by its migration as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and a single peak on reverse-phase HPLC. The purified enzyme is a monomeric protein of Mr 64 kDa as determined by SDS-PAGE and native PAGE. The enzyme fractionated as a 67-kDa protein by HPLC gel filtration. The pH optima in either the glycolytic or the gluconeogenic direction is near neutral, which is unlike any of the protozoan or bacterial enzymes. The enzyme displayed typical Michaelis-Menten kinetics. The Km values for pyrophosphate and fructose 6-phosphate were 0.039 +/- 0.005 and 0.25 +/- 0.0196 mM, respectively. The initial velocity for the reverse reaction was also found to be hyperbolic. Different nucleoside triphosphates, including ATP, could not substitute for pyrophosphate as the phosphoryl donor. Similar to the pyrophosphate-dependent enzyme from other anaerobic protozoans and bacteria, the Giardial enzyme was not activated by fructose 2,6-bisphosphate, although this metabolite is a competitive inhibitor with respect to fructose 1,6-bisphosphate in the reverse reaction. The pH optima and the molecular weight properties of the Giardial enzyme are different from those of the two classes of the pyrophosphate-dependent phosphofructokinases.
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Department of Medicine and Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106-4983, USA.
Phillips
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D000818:Animals; D002853:Chromatography, Liquid; D011756:Diphosphates / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D016829:Giardia lamblia / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008970:Molecular Weight; D010732:Phosphofructokinase-1 / Q000145:classification / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D011756:Diphosphates; D010732:Phosphofructokinase-1