Cytosolic glutathione S-transferases (GSTs) were isolated from 1-day-old Leghorn chick livers by glutathione (GSH)-affinity chromatography. After sample loading and extensive washing with 0.2 M NaCl, the column was sequentially eluted with 5 mM GSH and 1 mM S-hexylglutathione. The isolated GSTs were subjected to reverse-phase HPLC, electrospray ionization-MS, N-terminal and internal peptide sequencing analyses. The proteins recovered from the 5 mM GSH eluant were predominantly cGSTM1. A protein (cGSTM1′) with an N-terminal amino acid sequence identical to that of cGSTM1 but with the initiator methionine retained and a novel class-mu isozyme (cGSTM2*) were also recovered from this fraction. Nine class-alpha isozymes with distinctive molecular masses were identified from the 1 mM S-hexylglutathione eluant. Three of these proteins are probably variants with minor amino acid substitutions of other isozymes. Of the six remaining class-alpha isozymes, three of them have had their complete (cGSTA1 and cGSTA2) or partial (cGSTA3) cDNA sequences reported previously in the literature. A chicken liver cDNA library was screened with oligonucleotides generated from the cGSTA2 sequence as probes. Clones that encompass the complete coding regions of cGSTA3 and cGSTA4 were obtained. A clone encoding the C-terminal 187 residues of cGSTA5 was also isolated.

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