Abstract
Cysteine conjugate β-lyase (β-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 β-lyase was estimated to be a homodimer consisting of a 47,400-Da subunit with absorption maxima at 280 and 420-430 am. The specific activity of the purified β-lyase toward S-(1,2-dichloro-vinyl)-L-cysteine (1,2-DCVC) in the presence of α-keto-γ-methiolbutyric acid (KMB) was 6.4 μmol/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 of 0.33 mM and 8.4 μmol/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 μmol/min/mg. Amino acid analysis of the purified β-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 aminotransferases. This suggests that glutamine transaminase K/cysteine conjugate β-lyase is a typical member of the PLP-containing aminotransferase group.
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