Purification of glutamine transaminase K/cysteine conjugate beta-lyase from rat renal cytosol based on hydrophobic interaction HPLC and gel permeation FPLC.
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.
Yamauchi
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A
Department of Pharmacochemistry, Free University, Amsterdam, The Netherlands.
Stijntjes
G J
GJ
Commandeur
J N
JN
Vermeulen
N P
NP
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D013437:Carbon-Sulfur Lyases; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D007668:Kidney / Q000201:enzymology*; D008190:Lyases / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D017421:Sequence Analysis; D000637:Transaminases / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D000637:Transaminases; C427605:glutamine - phenylpyruvate transaminase; C021481:kynurenine-oxoglutarate transaminase; D008190:Lyases; D013437:Carbon-Sulfur Lyases; C019969:S-alkylcysteine lyase