Expression and Characterization of Recombinant Human-Derived Pneumocystis carinii Dihydrofolate Reductase
作者： Liang Ma , Joseph A. Kovacs
DOI: 10.1128/AAC.44.11.3092-3096.2000
关键词:
摘要: Dihydrofolate reductase (DHFR) is the target of trimethoprim (TMP), which has been widely used in combination with sulfa drugs for treatment and prophylaxis Pneumocystis carinii pneumonia. While rat-derived P. DHFR well characterized, kinetic studies human-derived DHFR, differs from by 38% amino acid sequence, have not reported to date. Here we report on expression characterization recombinant DHFR. The 618-bp coding sequence gene was expressed Escherichia coli. As determined sodium dodecyl sulfate-polyacrylamide gel eletrophoresis, purified enzyme had a molecular mass 25 kDa, consistent that predicted DNA sequence. Kinetic analysis showed K(m) values dihydrofolate NADPH were 2.7 +/- 0.3 14.0 4.3 microM, respectively, are similar those Inhibition revealed both TMP pyrimethamine poor inhibitors K(i) 0.28 0.08 0.065 0.005 while trimetrexate methotrexate potent inhibitors, 0.23 0.03 0.016 0.004 nM, respectively. availability large quantities should facilitate identification antifolate greater potency higher selectivity
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sci-hub.se 参考文章(26)
Peter J. F. Henderson, Steady-state enzyme kinetics with high-affinity substrates or inhibitors. A statistical treatment of dose-response curves. Biochemical Journal. ,vol. 135, pp. 101- 107 ,(1973) , 10.1042/BJ1350101
K. O'Driscoll, C. Borner, S. M. Kahn, I. D. Weinstein, W. Jiang, Construction of defined deletion mutants by thermal cycled fusion: applications to protein kinase C. Technique, a Journal of Methods in Cell and Molecular Biology. ,vol. 2, pp. 27- 30 ,(1990)
Raymond L. Blakley, Eukaryotic Dihydrofolate Reductase Advances in Enzymology - and Related Areas of Molecular Biology. ,vol. 70, pp. 23- 102 ,(2006) , 10.1002/9780470123164.CH2
Anna W.M Zomer, Paul A.M Michels, Fred R Opperdoes, Molecular characterisation of Trypanosoma brucei alkyl dihydroxyacetone-phosphate synthase Molecular and Biochemical Parasitology. ,vol. 104, pp. 55- 66 ,(1999) , 10.1016/S0166-6851(99)00141-3
J. C. Edman, U. Edman, M. Cao, B. Lundgren, J. A. Kovacs, D. V. Santi, Isolation and expression of the Pneumocystis carinii dihydrofolate reductase gene. Proceedings of the National Academy of Sciences of the United States of America. ,vol. 86, pp. 8625- 8629 ,(1989) , 10.1073/PNAS.86.22.8625
David P. Baccanari, Suzanne S. Joyner, Dihydrofolate reductase hysteresis and its effect of inhibitor binding analyses. Biochemistry. ,vol. 20, pp. 1710- 1716 ,(1981) , 10.1021/BI00510A002
Liang Ma, Luciana Borio, Henry Masur, Joseph A. Kovacs, Pneumocystis carinii dihydropteroate synthase but not dihydrofolate reductase gene mutations correlate with prior trimethoprim-sulfamethoxazole or dapsone use. The Journal of Infectious Diseases. ,vol. 180, pp. 1969- 1978 ,(1999) , 10.1086/315148
Joseph A. Kovacs, Carmen J. Allegra, Henry Masur, Characterization of dihydrofolate reductase of Pneumocystis carinii and Toxoplasma gondii Experimental Parasitology. ,vol. 71, pp. 60- 68 ,(1990) , 10.1016/0014-4894(90)90008-Z
Susan L. Rancourt, Virginia K. Walker, Kinetic characterization of dihydrofolate reductase from Drosophila melanogaster. Biochemistry and Cell Biology. ,vol. 68, pp. 1075- 1082 ,(1990) , 10.1139/O90-161
B.L. Hillcoat, P.F. Nixon, R.L. Blakley, Effect of substrate decomposition on the spectrophotometric assay of dihydrofolate reductase Analytical Biochemistry. ,vol. 21, pp. 178- 189 ,(1967) , 10.1016/0003-2697(67)90179-0