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Expression, purification, and characterization of uracil phosphoribosyltransferase from Toxoplasma gondii.
Mol Biochem Parasitol. 1997 Aug; 87(2):137-44.MB

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 microM for uracil and 243 microM 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 Ki value of 25 microM and was not activated by GTP. Ample quantities of recombinant enzyme are now available for biochemical and structural studies, facilitating evaluation of UPRT as a possible therapeutic target.

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Authors+Show Affiliations

Carter D
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland 97201-3098, USA.
Donald RG
No affiliation info available
Roos D
No affiliation info available
Ullman B
No affiliation info available

MeSH

AnimalsBase SequenceDNA PrimersEscherichia coliGene ExpressionGenes, ProtozoanKineticsMolecular WeightMutagenesis, Site-DirectedPentosyltransferasesRecombinant ProteinsSubstrate SpecificityToxoplasma

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

9247925

Citation

Carter, D, et al. "Expression, Purification, and Characterization of Uracil Phosphoribosyltransferase From Toxoplasma Gondii." Molecular and Biochemical Parasitology, vol. 87, no. 2, 1997, pp. 137-44.
Carter D, Donald RG, Roos D, et al. Expression, purification, and characterization of uracil phosphoribosyltransferase from Toxoplasma gondii. Mol Biochem Parasitol. 1997;87(2):137-44.
Carter, D., Donald, R. G., Roos, D., & Ullman, B. (1997). Expression, purification, and characterization of uracil phosphoribosyltransferase from Toxoplasma gondii. Molecular and Biochemical Parasitology, 87(2), 137-44.
Carter D, et al. Expression, Purification, and Characterization of Uracil Phosphoribosyltransferase From Toxoplasma Gondii. Mol Biochem Parasitol. 1997;87(2):137-44. PubMed PMID: 9247925.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Expression, purification, and characterization of uracil phosphoribosyltransferase from Toxoplasma gondii. AU - Carter,D, AU - Donald,R G, AU - Roos,D, AU - Ullman,B, PY - 1997/8/1/pubmed PY - 1997/8/1/medline PY - 1997/8/1/entrez SP - 137 EP - 44 JF - Molecular and biochemical parasitology JO - Mol Biochem Parasitol VL - 87 IS - 2 N2 - 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 microM for uracil and 243 microM 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 Ki value of 25 microM and was not activated by GTP. Ample quantities of recombinant enzyme are now available for biochemical and structural studies, facilitating evaluation of UPRT as a possible therapeutic target. SN - 0166-6851 UR - https://www.unboundmedicine.com/medline/citation/9247925/Expression_purification_and_characterization_of_uracil_phosphoribosyltransferase_from_Toxoplasma_gondii_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0166-6851(97)00058-3 DB - PRIME DP - Unbound Medicine ER -