Expression and characterization of recombinant pyruvate kinase from Toxoplasma gondii tachyzoites

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

Research output: Contribution to journalArticle

16 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

Fingerprint

tachyzoites
pyruvate kinase
Pyruvate Kinase
Toxoplasma
Toxoplasma gondii
Phosphoenolpyruvate
Enzymes
fructose
enzymes
Eimeria tenella
Glucose-6-Phosphate
glucose 6-phosphate
Sigmoid Colon
Southern Blotting
Southern blotting
Amino Acid Sequence
amino acid sequences
Complementary DNA
enzyme activity
molecular weight

ASJC Scopus subject areas

  • Parasitology

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.

Expression and characterization of recombinant pyruvate kinase from Toxoplasma gondii tachyzoites. / Maeda, Takuya; Saito, Tomoya; Oguchi, Yoshiyo; Nakazawa, Miki; Takeuchi, Tsutomu; Asai, Takashi.

In: Parasitology Research, Vol. 89, No. 4, 01.03.2003, p. 259-265.

Research output: Contribution to journalArticle

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, vol. 89, no. 4, pp. 259-265.
Maeda T, Saito T, Oguchi Y, Nakazawa M, Takeuchi T, Asai T. Expression and characterization of recombinant pyruvate kinase from Toxoplasma gondii tachyzoites. Parasitology Research. 2003 Mar 1;89(4):259-265.
Maeda, Takuya ; Saito, Tomoya ; Oguchi, Yoshiyo ; Nakazawa, Miki ; Takeuchi, Tsutomu ; Asai, Takashi. / Expression and characterization of recombinant pyruvate kinase from Toxoplasma gondii tachyzoites. In: Parasitology Research. 2003 ; Vol. 89, No. 4. pp. 259-265.
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