Expression, purification, and characterization of uracil phosphoribosyltransferase from Toxoplasma gondii

Darrick Carter, Robert G.K. Donald, David Roos, Buddy Ullman

Research output: Contribution to journalArticle

30 Scopus citations

Abstract

The coding region derived from a full-length CDNA spanning the uracil phosphoribosyltransferase (UPRT) gene of Toxoplasma gondii has been ligated into a bacterial expression vector and overexpressed in E. coli. Recombinant UPRT protein migrated with a molecular mass of 27 kDa on SDS polyacrylamide gels and was purified to homogeneity by conventional protein purification techniques. In solution, UPRT behaved as a monomer and exhibited K(m)(app) values of 3.5 μM for uracil and 243 μM for phosphoribosylpyrophosphate, respectively. Other naturally occurring pyrimidine or purine bases were not recognized as substrates. [14C]Uracil phosphoribosylation was inhibited by 5-fluorouracil with a K(i) value of 25 μM and was not activated by GTP. Ample quanitities of recombinant enzyme are now available for biochemical and structural studies, facilitating evaluation of UPRT as a possible therapeutic target.

Original languageEnglish (US)
Pages (from-to)137-144
Number of pages8
JournalMolecular and Biochemical Parasitology
Volume87
Issue number2
DOIs
StatePublished - Jul 18 1997

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Keywords

  • Enzyme kinetics
  • Phosphoribosyltransferase
  • Protein purification
  • Pyrimidine metabolism
  • Toxoplasma gondii
  • Uracil phosphoribosyltransferase

ASJC Scopus subject areas

  • Parasitology
  • Molecular Biology

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