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

29 Citations (Scopus)

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 - 1997

Fingerprint

uracil phosphoribosyltransferase
Toxoplasma
Uracil
Guanosine Triphosphate
Fluorouracil
Proteins
Escherichia coli
Enzymes
Genes

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Parasitology

Cite this

Expression, purification, and characterization of uracil phosphoribosyltransferase from Toxoplasma gondii. / Carter, Darrick; Donald, Robert G K; Roos, David; Ullman, Buddy.

In: Molecular and Biochemical Parasitology, Vol. 87, No. 2, 1997, p. 137-144.

Research output: Contribution to journalArticle

Carter, Darrick ; Donald, Robert G K ; Roos, David ; Ullman, Buddy. / Expression, purification, and characterization of uracil phosphoribosyltransferase from Toxoplasma gondii. In: Molecular and Biochemical Parasitology. 1997 ; Vol. 87, No. 2. pp. 137-144.
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