TY - JOUR
T1 - Expression, purification, and characterization of uracil phosphoribosyltransferase from Toxoplasma gondii
AU - Carter, Darrick
AU - Donald, Robert G.K.
AU - Roos, David
AU - Ullman, Buddy
N1 - Funding Information:
This work was supported by Grant AI-31808 (D.S.R. and B.U.) from the National Institute of Allergy and Infectious Disease. D.C. was supported in part by an N.L. Tartar Trust Fellowship from the Medical Research Foundation of Oregon. D.S.R. and B.U. are both Burroughs Wellcome Fund Scholars in Molecular Parasitology, and this work was supported in part by grants from the Burroughs Wellcome Fund.
PY - 1997
Y1 - 1997
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 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.
AB - 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.
KW - Enzyme kinetics
KW - Phosphoribosyltransferase
KW - Protein purification
KW - Pyrimidine metabolism
KW - Toxoplasma gondii
KW - Uracil phosphoribosyltransferase
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U2 - 10.1016/S0166-6851(97)00058-3
DO - 10.1016/S0166-6851(97)00058-3
M3 - Article
C2 - 9247925
AN - SCOPUS:0030738303
SN - 0166-6851
VL - 87
SP - 137
EP - 144
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
IS - 2
ER -