TAILIEUCHUNG - Báo cáo Y học: Purification and biochemical characterization of some of the properties of recombinant human kynureninase

Recombinant human kynureninase (L-kynurenine hydrolase, EC ) was purified to homogeneity (60-fold) from Spodoptera frugiperda (Sf9) cells infected with baculovirus containing the kynureninase gene. The purification protocol comprised ammonium sulfate precipitation and several chromatographic steps, including DEAE–Sepharose CL-6B, hydroxyapatite, strong anionic and cationic separations. The purity of the enzyme was determined by SDS/ PAGE, and the molecular mass verified by MALDI-TOF MS. The monomeric molecular mass of kDa determined was of the predicted molecular mass. A UV absorption spectrum of the holoenzyme resulted in a peak at 432 nm. . | Eur. J. Biochem. 269 2069-2074 2002 FEBS 2002 doi Purification and biochemical characterization of some of the properties of recombinant human kynureninase Harold A. Walsh and Nigel P. Botting School of Chemistry University of St Andrews St Andrews Fife Scotland UK Recombinant human kynureninase L-kynurenine hydrolase EC was purified to homogeneity 60-fold from Spodoptera frugiperda Sf9 cells infected with baculovirus containing the kynureninase gene. The purification protocol comprised ammonium sulfate precipitation and several chromatographic steps including DEAE-Sepharose CL-6B hydroxyapatite strong anionic and cationic separations. The purity of the enzyme was determined by SDS PAGE and the molecular mass verified by MALDI-TOF MS. The monomeric molecular mass of kDa determined was of the predicted molecular mass. A UV absorption spectrum of the holoenzyme resulted in a peak at 432 nm. The optimum pH was and the enzyme displayed a strong dependence on the ionic strength of the buffer for optimum activity. This cloned enzyme was highly specific for 3-hydroxykynurenine Km M and was inhibited by L-kynurenine Ki 20 M D-kynurenine Ki 12 M and a synthetic substrate analogue D L-3 7-dihydroxydesaminokynurenine Ki 100 dm . The activity concentration profile for kynureninase from this source was sigmoidal in all instances. There appeared to be partial inhibition by substrate and excess pyridoxal 5 -phosphate was found to be inhibitory. Keywords kynureninase kynurenine neuroprotection quinolinic acid tryptophan metabolism. Rapid progress in the pathophysiology of human diseases has always been hampered by the availability of human tissue aesthetics and ethical considerations. The principle aim of this study was to express a clone of human kynureninase in an appropriate host that would yield sufficient quantities of protein to permit identification and biochemical characterization of the enzyme and .

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