Purification in a single step and kinetic characterization of the pyruvate kinase of Trypanosoma brucei

John P. Barnard, Peter L. Pedersen

Research output: Contribution to journalArticle

Abstract

The pyruvate kinase of Trypanosoma brucei can be purified to homogeneity in one step by affinity elution from a phosphocellulose column with the substrate phosphoenolpyruvate (PEP) and the allosteric activator fructose-2,6-diphosphate (FDP). The purified enzyme has a specific activity of 175 μmol min-1 (mg protein)-1 and a subunit molecular mass of 59 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinetic studies of the pure enzyme show that an increase in the PEP concentration decreases the apparent Km for adenosine diphosphate (ADP) and that an increase in the ADP concentration decreases the half saturation point (S0.5) for PEP. Likewise, the allosteric activator FDP decreases both the apparent Km for ADP and the S0.5 for PEP. ADP concentrations above 0.2 mM inhibit trypanosomal pyruvate kinase.

Original languageEnglish (US)
Pages (from-to)141-147
Number of pages7
JournalMolecular and Biochemical Parasitology
Volume31
Issue number2
DOIs
StatePublished - Nov 1988

Keywords

  • Affinity elution
  • Bioenergetics
  • Enzyme kinetics
  • Enzyme purification
  • Pyruvate kinase
  • Trypanosoma brucei

ASJC Scopus subject areas

  • Parasitology
  • Molecular Biology

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