Glutathione transferases from Anguilla anguilla liver: identification, cloning and functional characterization.
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.
Carletti
Erminia
E
Dipartimento di Scienze Biomediche, Università G. D'Annunzio, via dei Vestini 31, I-66013 Chieti, Italy. e.carletti@dsb.unich.it
Sulpizio
Marilisa
M
Bucciarelli
Tonino
T
Del Boccio
Piero
P
Federici
Luca
L
Di Ilio
Carmine
C
D000595:Amino Acid Sequence; D000811:Anguilla / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001280:Atrazine / Q000494:pharmacology; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007525:Isoelectric Focusing; D007527:Isoenzymes; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity
D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D005978:Glutathione; D001280:Atrazine