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Título:
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Expression and characterization of the Trypanosoma cruzi dihydrofolate reductase domain
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Autores:
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Reche, Pedro A ;
Arrebola, R ;
Santi, D V ;
González-Pacanowska, D. ;
Ruiz Pérez, Luis Miguel
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Tipo de documento:
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texto impreso
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Editorial:
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Elsevier, 1995
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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: Microbiología
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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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We have cloned and expressed in Escherichia coli a 702-base pair gene coding for the dihydrofolate reductase (DHFR) domain of the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Trypanosoma cruzi. The DHFR domain was purified to homogeneity by methotrexate-Sepharose chromatography followed by an anion-exchange chromatography step in a mono Q column, and displayed a single 27-kDa band on SDS-PAGE. Gel filtration showed that the catalytic domain was expressed as a monomer. Kinetic parameters were similar to those reported for the wild-type bifunctional enzyme with Km values of 0.75 microM for dihydrofolate and 16 microM for NADPH and a kcat value of 16.5 s-1. T. cruzi DHFR is poorly inhibited by trimethoprim and pyrimethamine and the inhibition constants were always lower for the bifunctional enzyme. The binding of methotrexate was characteristic of a class of inhibitors that form an initial complex which isomerizes slowly to a tighter complex and are referred to as 'slow, tight-binding' inhibitors. While the slow-binding step of inhibition was apparently unaffected in the individually expressed DHFR domain, the overall inhibition constant was two-fold higher as a consequence of the superior inhibition constant value obtained for the initial inhibitory complex.
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En línea:
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https://eprints.ucm.es/id/eprint/9353/1/04.Reche_etal_JMP_1995.pdf
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