Expression and characterization of recombinant pyruvate kinase from Toxoplasma gondii tachyzoites

Takuya Maeda, Tomoya Saito, Yoshiyo Oguchi, Miki Nakazawa, Tsutomu Takeuchi, Takashi Asai

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17 Citations (Scopus)

Abstract

We have cloned a cDNA encoding Toxoplasma gondii pyruvate kinase and obtained the full-length recombinant enzyme with a calculated molecular mass of 57.5 kDa. The predicted amino acid sequence of T. gondii pyruvate kinase exhibited a highest identity (63%) to that of Eimeria tenella pyruvate kinase and a lower identity of less than 25% to the pyruvate kinases from other organisms. Southern blot analysis indicated that the pyruvate kinase gene existed as a single copy in the T. gondii tachyzoite. The active recombinant enzyme contained four subunits and produced a strongly sigmoid saturation curve with phosphoenolpyruvate as the variable substrate. Fructose 1,6-diphosphate, a general activating factor of pyruvate kinase in most species, did not affect the enzyme activity. However, glucose 6-phosphate radically activated the enzyme. Fructose 2,6-diphosphate suppressed the reaction velocity at a higher concentration of phosphoenolpyruvate. These properties indicate that pyruvate kinase activity in T. gondii is regulated by unusual phosphorylated sugars.

Original languageEnglish
Pages (from-to)259-265
Number of pages7
JournalParasitology Research
Volume89
Issue number4
Publication statusPublished - 2003 Mar 1

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ASJC Scopus subject areas

  • Parasitology
  • veterinary(all)
  • Insect Science
  • Infectious Diseases

Cite this

Maeda, T., Saito, T., Oguchi, Y., Nakazawa, M., Takeuchi, T., & Asai, T. (2003). Expression and characterization of recombinant pyruvate kinase from Toxoplasma gondii tachyzoites. Parasitology Research, 89(4), 259-265.