Please use this identifier to cite or link to this item: http://repository.li.mahidol.ac.th/dspace/handle/123456789/22061
Title: Purification and characterization of dihydrofolate reductase from wild-type and trimethoprim-resistant Mycobacterium smegmatis
Authors: Worachart Sirawaraporn
Rachada Sirawaraporn
Atid Chanpongsri
William R. Jacobs
Daniel V. Santi
Mahidol University
University of California, San Francisco
Albert Einstein College of Medicine of Yeshiva University
Keywords: Immunology and Microbiology;Medicine
Issue Date: 1-Jan-1991
Citation: Experimental Parasitology. Vol.72, No.2 (1991), 184-190
Abstract: Dihydrofolate reductase (DHFR) from extracts of Mycobacterium smegmatis strain mc26 and trimethoprim-resistant mutant mc226 was purified to homogeneity. In crude extracts, the specific activity of the enzyme from the trimethoprim resistant strain was comparable to that from the sensitive strain. The DHFR from both sources was purified using affinity chromatography on MTX-Sepharose followed by Mono Q FPLC. The enzyme has an apparent molecular mass of 23 kDa from gel filtration on Sephadex G-100 and from SDS-PAGE. Amino terminal sequence analysis showed homology with DHFRs from a subset of other gram-positive organisms. The purified enzyme from the trimethoprim-sensitive organism exhibited Kmvalues for H2folate and NADPH of 0.68 ± 0.2 μM and 21 ± 4 μM, respectively. The Kmvalues for H2folate and NADPH for the enzyme from the drug-resistant organism were 1.8 ± 0.4 μM and 5.3 ± 1.5 μM, respectively. A Kcatof 4.5 sec-1was determined for the DHFR from both sources. The enzyme from both sources was competitively inhibited by pyrimethamine and trimethoprim. The Kivalue of trimethoprim, for the enzyme from the drug-resistant organism was about six-fold higher than for the enzyme from drug-sensitive strain. Our data suggest that mutation of DHFR contributes to trimethoprim resistance in the mc226 strain of M. smegmatis. © 1991.
URI: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=0026113224&origin=inward
http://repository.li.mahidol.ac.th/dspace/handle/123456789/22061
ISSN: 10902449
00144894
Appears in Collections:Scopus 1991-2000

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