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West Indian med. j ; 32(Suppl): 27, 1983.
Artigo em Inglês | MedCarib | ID: med-6142

RESUMO

Despite the use of a variety of anti-folate agent in chemotherapy, detailed understanding of the metabolism of folates is scant due largely to the instability of folate coenzymes in vitro, and the low levels of the enzymes involved in vivo. Commercially available tetrahydrofolate preparations contain at least 25 percent unspecified impurities, and are racemic mixture of biologically active and inactive isomers. The object of this study was to investigate the application of purified substrates in the isolation of folate-metabolising enzymes by affinity chromatography. Methylenetetrahydrofolate, which is both oxygen - and photo-labile, was prepared by reducing folic acid with borohydride and subsequent reaction with formaldehyde. The biologically active isomer, d-methylenetetrahydrofolate, was isolate by ion-exchange chromatgraphy, with a yield of 35 percent from folic acid. Methylenetetrahdrofolate reductase, an allosteric enzyme regulating the biosynthesis of S-adenosylmethionine, was partially purified from rat liver by ion-exchange chromatography and applied to a column of the affinity matrix procion red HE-3B - sepharose. The loaded column was washed with buffer to remove unbound proteins and active enzyme was bio-specifically eluted with a gradient of d-methylenetetrahydrofolate with greater than 90 percent recovery of activity. Hereditary deficiency of this enzyme has been implicated in cases of schizophrenia and homocystinuria. The quantitative determination of enzyme levels in vivo, and kinetic data obtained from purified preparations in vitro are prerequisites for the understanding of metabolic fluxes in the intact animal. The technique investigated in this study may facilitate analysis of other folate-requiring enzymes (AU)


Assuntos
21003 , Ratos , Enzimas/isolamento & purificação , Cromatografia de Afinidade/estatística & dados numéricos , Cromatografia por Troca Iônica/estatística & dados numéricos , Metilenotetra-Hidrofolato Desidrogenase (NADP)/isolamento & purificação
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