Characterization and cloning of avian-hepatic glutathione S-transferases.
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.
Hsieh
C H
CH
Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, Republic of China.
Liu
L F
LF
Tsai
S P
SP
Tam
M F
MF
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D005982:Glutathione Transferase