Purification and biochemical characterisation of some of the properties of recombinant human kynureninase

HA Walsh, Nigel Peter Botting

Research output: Contribution to journalArticlepeer-review

31 Citations (Scopus)

Abstract

Recombinant human kynureninase (L-kynurenine hydrolase, EC 3.7.1.3) 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 DEAF-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 52.4 kDa determined was > 99.99% of the predicted molecular mass. A UV absorption spectrum of the holoenzyme resulted in a peak at 432 mn. The optimum pH was 8.25 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-hydroxykynureiune (K-m = 3.0 muM +/- 0.10) and was inhibited by L-kynurenine (K-i = 20 muM), D-kynurenine (K-i = 12 muM) and a synthetic substrate analogue D,L-3,7-dihydroxydesaminokynurenine (K-i = 100 nM). 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.

Original languageEnglish
Pages (from-to)2069-2074
Number of pages6
JournalEuropean Journal of Biochemistry
Volume269
DOIs
Publication statusPublished - Apr 2002

Keywords

  • kynureninase
  • kynurenine
  • neuroprotection
  • quinolinic acid
  • tryptophan metabolism
  • EXPRESSION
  • CLONING
  • BRAIN
  • ACID
  • RAT

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