Isolation of a Pi class glutathione S-transferase (GST) from catfish intestinal mucosa

Glutathione S-transferases (GSTs) are known to play an important role in the intestinal biotransformation of the epoxide and diol-epoxide metabolites of benzo(a)pyrene formed in catfish intestine (James et al. (1994) The Toxicologist 14, 431). Intestinal mucosal cytosol prepared from the proximal, medial and distal regions had GST activity with 1-chloro-2,4-dinitrobenzene, ethacrynic acid and benzo(a)pyrene-4,5-oxide as substrates. Intestinal GSTs were isolated from dialyzed cytosol by glutathione-agarose affinity chromatography. More than 90% of the intestinal GST activity bound to the glutathione (GSH)-agarose. Elution with GSH gave one major peak of activity. The isolated GST fraction was purified 50 to 70-fold with respect to cytosol for activity with 1-chloro-2,4-dinitrobenzene and had high activity with ethacrynic acid and benzo(a)pyrene-4, 5-oxide. SDS-PAGE of the purified GST showed one major band at molecular mass 27 400. Isoelectric focusing showed one major band with pI 7.9, and several minor bands. N-terminal sequence analysis of the purified GST gave 29 amino acids with marked homology (> 63% identical) to mammalian π form GST isozymes and very strong similarity (80%) to a salmon hepatic GST which was shown by immunochemical cross-reactivity to belong to the π class. An antibody to the isolated GST was prepared and used to demonstrate that 1–2% of the protein in cytosol from the proximal intestine of catfish was this π class GST. Catfish hepatic cytosol also cross-reacted strongly with the antibody.