title: Molecular cloning and characterization of a glutathione S-transferase from largemouth bass (Micropterus salmoides) liver that is involved in the detoxification of 4-hydroxynonenal.
abstract: We are currently investigating the role of detoxification pathways in protecting against the sublethal effects of chemicals in largemouth bass (Micropterus salmoides). To this end, previous work in our laboratory indicated a remarkable ability of bass liver glutathione S-transferases (GSTs) to detoxify 4-hydroxynonenal (4HNE), a common mutagenic and cytotoxic alpha,beta-unsaturated aldehyde produced during the peroxidation of lipids. In the current study, we observed that GST-mediated 4HNE conjugation in bass liver follows high efficiency single-enzyme Michaelis-Menten kinetics, suggesting that an individual GST isoform is involved in 4HNE detoxification. Using 5' and 3' rapid amplification of cDNA ends (RACE), a full-length GST cDNA of 957 base pairs (bp) in length, containing an open reading frame of 678 bp and encoding a polypeptide of 225 amino acids, has been cloned. Interestingly, a search of the BLAST protein database revealed the presence of homologous GST proteins in the plaice (Pleuronectes platessa), European flounder (Platichthys flesus) and fathead minnow (Pimephales promelas), but not in other fish species. Furthermore, the bass GST protein exhibited little homology with the mammalian GSTA4 subclass of proteins which rapidly metabolize 4HNE. The recombinant 6 x His-tagged expressed GST protein showed high catalytic activity towards 4HNE, while showing moderate or low activity toward other class specific GST substrates. HPLC-GST subunit analysis, followed by sequencing, demonstrated that the isolated bass liver GST subunit constitutes the major GST protein in bass liver, with a molecular mass of 26.4 kDa. In summary, the presence of a highly expressed GST isozyme in bass and several evolutionarily divergent fish species indicates the conservation of an important and distinct detoxification protein that protects against oxidative damage in certain aquatic organisms.
authors.affiliation: Department of Physiological Sciences and Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL, USA.
authors.forename: Adriana M
authors.initials: AM
authors.lastname: Doi
authors.forename: Robert T
authors.initials: RT
authors.lastname: Pham
authors.forename: Erin M
authors.initials: EM
authors.lastname: Hughes
authors.forename: David S
authors.initials: DS
authors.lastname: Barber
authors.forename: Evan P
authors.initials: EP
authors.lastname: Gallagher
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001492:Bass; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009928:Organ Specificity; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D005982:Glutathione Transferase