Purification, kinetics and inhibition by antimonials of recombinant phosphofructokinase from Schistosoma mansoni.
We reported before on the cloning of a cDNA encoding S. mansoni PFK. In the present investigation we established optimal conditions for expression of the enzyme in insect cells with high yield. The recombinant PFK was purified to homogeneity. Kinetic properties of the pure enzyme were studied with respect to its two substrates, Fru-6-P and ATP, and were compared with properties of mammalian PFK. ATP inhibited the parasite enzyme only at concentrations higher than those which inhibited mammalian muscle PFK. Saturation curves for Fru-6-P showed typical cooperative kinetics. AMP, cAMP and Fru-2,6-bisP activated the enzyme causing reduced apparent Km for Fru-6-P and an increase in maximal activity. Both ATP inhibition and cooperative kinetics for Fru-6-P occur at both pH 6.9 and 8.2. This is a distinct difference from the mammalian enzyme which shows these kinetic properties only at neutral or slightly acidic pH, but not at an alkaline pH. Recombinant PFK is more sensitive to inhibition by the trivalent antimonials, antimony potassium tartrate and Stibophen, than is the mammalian heart muscle enzyme. The inhibition is at least partially antagonized by the sulfhydryl protective reagent, dithiothreitol.
Department of Molecular Pharmacology, Stanford University School of Medicine, CA 94305-5332, USA.
J G
JG
Su
J M
JM
Mansour
T E
TE
Mansour
D000818:Animals; D000966:Antimony Potassium Tartrate / Q000494:pharmacology; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001557:Benzenesulfonates / Q000494:pharmacology; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology; D009942:Organometallic Compounds / Q000494:pharmacology; D010732:Phosphofructokinase-1 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012550:Schistosoma mansoni / Q000201:enzymology* / Q000235:genetics; D012556:Schistosomicides / Q000494:pharmacology; D012756:Sheep; D018411:Spodoptera
D001557:Benzenesulfonates; D017931:DNA Primers; D004791:Enzyme Inhibitors; D009942:Organometallic Compounds; D011994:Recombinant Proteins; D012556:Schistosomicides; C077725:Stibophen; D000966:Antimony Potassium Tartrate; D010732:Phosphofructokinase-1