Cloning and characterization of the major hepatic glutathione S-transferase from a marine teleost flaffish, the plaice (Pleuronectes platessa), with structural similarities to plant, insect and mammalian Theta class isoenzymes

Abstract

A cDNA clone (PLGSTA) of 896 bp, containing an open reading frame encoding a 225-amino-acid polypeptide of M, 25 723, was isolated from a cDNA library constructed in Agtl 1 from the liver of a marine teleost flatfish, the plaice (Pleuronectes platessa). The identification of this cDNA as that coding for the subunit of the major cytosolic glutathione A cDNA clone (PLGSTA) of 896 bp, containing an open reading frame encoding a 225-amino-acid polypeptide of M(r) 25,723, was isolated from a cDNA library constructed in lambda gt11 from the liver of a marine teleost flatfish, the plaice (Pleuronectes platessa). The identification of this cDNA as that coding for the subunit of the major cytosolic glutathione S-transferase of plaice liver, GST-A, was supported by its heterologous expression in and purification of its protein product from Escherichia coli. The recombinant-derived protein exhibited identical M(r) and immunoreactivity and a similar substrate specificity to GST-A previously isolated from plaice liver. Comparison of the deduced amino acid sequence of the plaice GST-A polypeptide with the primary structures of GSTs from other Phyla revealed that it showed the greatest similarity to plant, insect and mammalian Theta class GSTs. Southern blot analysis of plaice DNA hybridized to the PLGSTA cDNA showed a banding pattern indicative of the presence of a single gene. Northern blot analysis of a variety of plaice tissues showed hybridizing bands of approx. 1100 nucleotides in all tissues tested, with the highest relative amounts in liver and intestinal mucosa. A marked increase in hybridization intensity was observed in hepatic RNA samples from plaice treated with trans-stilbene oxide, suggesting that GST-A is induced by epoxides in this species.