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Título:
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Isolation and Characterization of a Mutant Dihydrofolate Reductase-Thymidylate Synthase from Methotrexate-Resistant Leishmania Cells
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Autores:
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Arrebola, R ;
Olmo, A ;
Reche, Pedro A ;
Garvey, Edward P. ;
Santi, D V ;
Ruiz Pérez, Luis Miguel ;
González-Pacanowska, D.
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Tipo de documento:
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texto impreso
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Editorial:
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American Society for Biochemistry and Molecular Biology, 1994-04-08
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Dimensiones:
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application/pdf
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Nota general:
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info:eu-repo/semantics/openAccess
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Idiomas:
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Palabras clave:
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Estado = Publicado
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Materia = Ciencias Biomédicas: Biología: Bioquímica
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Materia = Ciencias Biomédicas: Biología: Biología molecular
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Tipo = Artículo
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Resumen:
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The MTX-resistant Leishmania major promastigote cell line D7BR1000 displays extrachromosomal amplified R-region DNA, which contains the gene for dihydrofolate reductase-thymidylate synthase (DHFR-TS) (Garvey, E. P., and Santi, D. V. (1986) Science 233, 535-540). Now we report that these methotrexate (MTX)-resistant cells also possessed a structurally altered DHFR-TS. We have performed the cloning, expression, and characterization of the altered DHFR-TS gene. The DNA sequence of the altered DHFR-TS gene revealed a single base change in position 158 which resulted in the substitution of a methionine in position 53 of DHFR for an arginine. Steady-state measurements of the purified recombinant enzyme indicated that the mutation did not cause significant modifications in the Km for DHFR or TS substrates but lowered the kcat by 4-fold. Of greater interest, there was a modification in the effect on MTX inhibition of DHFR. The initial inhibition complex appeared to have been unaffected by the alteration, but the subsequent slow-binding step of inhibition in the wild-type enzyme is absent in the altered enzyme. Consequently, the overall Ki for MTX was 30-fold greater for the mutant than for the wild-type enzyme. Transfection of L. major with the mutant DHFR-TS gene gives parasites that are capable of growing in medium containing 10 mM methotrexate, showing that the altered DHFR gene is in itself capable of conferring MTX resistance in Leishmania.
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En línea:
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https://eprints.ucm.es/id/eprint/9322/1/01.Arrebola_Reche_JBC_1994.pdf
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