|
type |
Journal Article |
authors |
Yamauchi, A.; Stijntjes, G. J.; Commandeur, J. N.; Vermeulen, N. P. |
title |
Purification of glutamine transaminase K/cysteine conjugate beta-lyase from rat renal cytosol based on hydrophobic interaction HPLC and gel permeation FPLC |
journal |
Protein Expr Purif |
Activity |
4.4.1.13 |
ui |
94115184 |
year |
(1993) |
volume |
4 |
number |
6 |
pages |
552-62. |
| |
keywords |
Amino Acids/analysis |
abstract |
Cysteine conjugate beta-lyase (beta-lyase, EC 4.4.1.13) was purified to homogeneity from rat renal cytosol using a new and highly efficient method, based on C3-hydrophobic interaction (HI) high-performance liquid chromatography (HPLC) in combination with gel permeation fast protein liquid chromatography. The purity of the enzyme was judged from SDS-PAGE and C18-reversed-phase HPLC. The beta-lyase was estimated to be a homodimer consisting of a 47,400-Da subunit with absorption maxima at 280 and 420-430 nm. The specific activity of the purified beta-lyase toward S-(1,2-dichlorovinyl)-L-cysteine (1,2-DCVC) in the presence of alpha-keto-gamma-methiolbutyric acid (KMB) was 6.4 mumol/min/mg protein, which is by far the highest value so far reported. Kinetic analysis of 1,2-DCVC metabolism by the enzyme in the presence of KMB gave Km and Vmax values of 0.33 mM and 8.4 mumol/min/mg protein, respectively. No significant activity of the purified enzyme was detectable with S-2-benzothiazolyl-L-cysteine up to 2 mM. The purified enzyme also had glutamine transaminase K activity (EC 2.6.1.64) as assayed with phenylalanine and KMB as substrates. This specific activity was 16.0 mumol/min/mg. Amino acid analysis of the purified beta-lyase was carried out and was found to be closely similar to the amino acid composition of five other pyridoxal phosphate (PLP)- containing amino acid amino-transferases. This suggests that glutamine transaminase K/cysteine conjugate beta-lyase is a typical member of the PLP-containing aminotransferase group. |
last changed |
2002/11/12 16:17 |
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