Characterization and cloning of avian-hepatic glutathione S-transferases

Cytosolic glutathione S-transferases (GSTs) were isolated from 1-day-old Le ghorn chick livers by glutathione (GSH)-affinity chromatography. After samp le loading and extensive washing with 0.2 M NaCl, the column was sequential ly eluted with 5 mM GSH and 1 mM S-hexylglutathione. The isolated GSTs were subjected to reverse-phase HPLC, electrospray ionization-MS, N-terminal an d internal peptide sequencing analyses. The proteins recovered from the 5 m M GSH eluant were predominantly cGSTM1. A protein (cGSTM1') with an N-termi nal amino acid sequence identical to that of cGSTM1 but with the initiator methionine retained and a novel class-mu isozyme (cGSTM2*) were also recove red from this fraction. Nine class-alpha isozymes with distinctive molecula r 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 h ave had their complete (cGSTA1 and cGSTA2) or partial (cGSTA3) cDNA sequenc es 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 we re obtained. A clone encoding the C-terminal 187 residues of cGSTA5 was als o isolated.