|
type |
Journal Article |
authors |
Walsh, H. A.; Botting, N. P. |
title |
Purification and biochemical characterization of some of the properties of recombinant human kynureninase |
journal |
Eur J Biochem |
Activity |
3.7.1.3 |
Family |
3.7.1.3 |
sel |
selected |
ui |
11985583 |
year |
(2002) |
volume |
269 |
number |
8 |
pages |
2069-74 |
| |
keywords |
Cloning, Molecular |
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 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 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 nm. 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-hydroxykynurenine (Km = 3.0 microm +/- 0.10) and was inhibited by L-kynurenine (Ki = 20 microm), d-kynurenine (Ki = 12 microm) and a synthetic substrate analogue D,L-3,7-dihydroxydesaminokynurenine (Ki = 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. |
last changed |
2009/05/20 16:59 |
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