Glutathione transferases from Anguilla anguilla liver: Identification, cloning and functional characterization

• Published in: Aquatic toxicology 2008-10, Vol.90 (1), p.48-57
• Main Authors: Carletti, Erminia, Sulpizio, Marilisa, Bucciarelli, Tonino, Del Boccio, Piero, Federici, Luca, Di Ilio, Carmine
• Format: Article
• Language: English
• Subjects: Agnatha. Pisces; Amino Acid Sequence; Anguilla - genetics; Anguilla - metabolism; Anguilla anguilla; Animal, plant and microbial ecology; Animals; Applied ecology; Atrazine; Atrazine - pharmacology; Base Sequence; Biological and medical sciences; Chromatography, Affinity - methods; Cloning, Molecular; detection; DNA, Complementary - genetics; Ecotoxicology, biological effects of pollution; eel; enzyme activity; enzyme inhibition; Escherichia coli - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; gene expression; General aspects; Glutathione - chemistry; Glutathione - metabolism; Glutathione S-transferase; glutathione transferase; Glutathione Transferase - antagonists & inhibitors; Glutathione Transferase - genetics; Glutathione Transferase - isolation & purification; Glutathione Transferase - metabolism; high performance liquid chromatography; Isoelectric Focusing; Isoenzymes; Kinetics; Liver; Liver - enzymology; Marine; metabolic detoxification; molecular cloning; Molecular Sequence Data; pollutants; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; Rho class GST; sequence analysis; Substrate Specificity; Vertebrates: general zoology, morphology, phylogeny, systematics, cytogenetics, geographical distribution; water pollution
• ISSN: 0166-445X; 1879-1514
• DOI: 10.1016/j.aquatox.2008.07.015

Glutathione transferases (GSTs) constitute a class of detoxifying enzymes involved in Phase II metabolism. Using GSH-affinity chromatografy followed by HPLC analysis, two GST isoforms were isolated from the Anguilla anguilla liver cytosol. The major GST belongs to the piscine-specific rho class and accounted for about 59% of total GST affinity eluted fraction, while the remaining 41% was represented by a Pi class GST. Both isoforms were cloned, heterologously expressed in Escherichia coli and their enzyme activities were characterized with respect to a broad spectrum of well-known GST substrates. Our data indicate that only a fraction of prototypical GST substrates are conjugated by these enzymes and that Pi class GST has higher specific activity than rho class GST against 1-chloro-2,4-dinitrobenzene (CDNB), ethracrynic acid, 4-nitroquinoline-1-oxide and p-nitrophenyl acetate while trans-2-nonenal is detoxified more efficiently by rho class GST. Analysis of the kinetics parameters of the conjugation against CDNB indicated that the utilization ratio K cat/ K m is slightly higher for rho class GST with respect to pi class GSTs. Finally, to determine the potential for environmental inhibition of the GST isoforms, we examined the effect of the widely used herbicide atrazine as an inhibitor of catalytic activity. The inhibition studies revealed that atrazine was an effective inhibitor of GST–CDNB catalytic activities of both isoforms at micromolar concentrations, suggesting the sensitivity of these isoforms to pesticide inhibition at environmentally relevant concentrations.