Recombinant glutathione S-transferase 3-3 expressed in Spodoptera frugiperda (SF9) cells with the use of a baculovirus expression system was modified with 1 mM-iodoacetamide. Amino acid analysis indicated that 0.79 +/- 0.15 cysteine residue was modified per enzyme subunit. The S-carbaminomethylated protein retains the GSH-conjugating activity. Glutathione S-transferase 3-3 modified with iodo[14C]acetamide was digested with Achromobacter proteinase I and the resulting peptides were separated by h.p.l.c. The modified peptides were pooled and further digested with Staphylococcus aureus V8 proteinase. Isotope-labelled peptides were isolated and collected for N-terminal sequence analysis. By this procedure, cysteine-86 was identified as the major S-carbaminomethylated residue. Verification of this findings came from the use of site-directed mutagenesis in which this cysteine was replaced by serine (C86S mutant). The C86S mutant is enzymically active. Therefore cysteine-86 is not needed for the conjugation of GSH with electrophilic compounds on glutathione S-transferase 3-3.
Research Article| August 15 1991
Cysteine-86 is not needed for the enzymic activity of glutathione S-transferase 3-3
J C Hsieh;
S C Huang;
W L Chen;
Y C Lai;
Biochem J (1991) 278 (1): 293–297.
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J C Hsieh, S C Huang, W L Chen, Y C Lai, M F Tam; Cysteine-86 is not needed for the enzymic activity of glutathione S-transferase 3-3. Biochem J 15 August 1991; 278 (1): 293–297. doi: https://doi.org/10.1042/bj2780293
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