Purification and properties of recombinant Pneumocystis carinii dihydrofolate reductase
作者： Worachart Sirawaraporn , Jeffrey C. Edman , Daniel V. Santi
DOI: 10.1016/1046-5928(91)90088-Z
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摘要: Abstract Pneumocystis carinii dihydrofolate reductase (DHFR) expressed in Escherichia coli was purified to homogeneity a single step using methotrexate-Sepharose affinity chromatography. The enzyme migrated as 24-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. sequence of the first 26 amino acids from N-terminus accord with that predicted DNA sequence. showed broad pH optimum maximum activity over range 6 7. activated by salts, maximal twofold activation at 50 150 mM KCl and 200 NaCl. Urea 2.5 M also increased twofold. Kinetic analysis revealed K m values for NADPH were 1.8 1.4 μM, respectively, k cat 70 s −1 . Inhibition studies verified trimethoprim pyrimethamine poor inhibitors P. DHFR little selectivity human DHFR. Trimetrexate piritrexim much more potent enzyme, but these are
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sci-hub.se 参考文章(24)
Sidney Futterman, ENZYMATIC REDUCTION OF FOLIC ACID AND DIHYDROFOLIC ACID TO TETRAHYDROFOLIC ACID Journal of Biological Chemistry. ,vol. 228, pp. 1031- 1038 ,(1957) , 10.1016/S0021-9258(18)70678-9
S Singer, R Ferone, L Walton, L Elwell, Isolation of a dihydrofolate reductase-deficient mutant of Escherichia coli. Journal of Bacteriology. ,vol. 164, pp. 470- 472 ,(1985) , 10.1128/JB.164.1.470-472.1985
D P Baccanari, R L Tansik, S S Joyner, M E Fling, P L Smith, J H Freisheim, Characterization of Candida albicans dihydrofolate reductase. Journal of Biological Chemistry. ,vol. 264, pp. 1100- 1107 ,(1989) , 10.1016/S0021-9258(19)85059-7
J E Parrillo, J Moss, J C Swan, J A Kovacs, J L Halpern, H Masur, Identification of antigens and antibodies specific for Pneumocystis carinii. Journal of Immunology. ,vol. 140, pp. 2023- 2031 ,(1988)
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
Walter T. Hughes, Sandor Feldman, Subhash C. Chaudhary, Michael J. Ossi, Frederick Cox, S.K. Sanyal, Comparison of pentamidine isethionate andtrimethoprim-sulfamethoxazole in the treatment of Pneumocystis carinii pneumonia The Journal of Pediatrics. ,vol. 92, pp. 285- 291 ,(1978) , 10.1016/S0022-3476(78)80028-6
S.M. Read, D.H. Northcote, Minimization of variation in the response to different proteins of the Coomassie blue G dye-binding assay for protein. Analytical Biochemistry. ,vol. 116, pp. 53- 64 ,(1981) , 10.1016/0003-2697(81)90321-3
DREW J WINSTON, WILLIAM K LAU, ROBERT PETER GALE, LOWELL S YOUNG, Trimethoprim-Sulfamethoxazole for the Treatment of Pneumocystis carinii Pneumonia Annals of Internal Medicine. ,vol. 92, pp. 762- 769 ,(1980) , 10.7326/0003-4819-92-6-762
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
Jeffrey M. Blaney, Corwin Hansch, Carlo Silipo, Antonio Vittoria, Structure-activity relationships of dihydrofolated reductase inhibitors Chemical Reviews. ,vol. 84, pp. 333- 407 ,(1984) , 10.1021/CR00062A002