title: Liquid chromatography-electrospray mass spectrometry study of cysteine-10 S-glutathiolation in recombinant glutathione S-transferase of Ochrobactrum anthropi.
abstract: Glutathione S-transferase of Ochrobactrum anthropi (OaGST), a bacterium isolated from soils contaminated by xenobiotic pollutants, was recently purified, cloned and characterised in our laboratories. The recombinant OaGST (rOaGST), highly expressed in Escherichia coli, when purified by glutathione-affinity chromatography and then analysed by electrospray ionisation mass spectrometry (ESI-MS), has evidenced a disulphide bond with glutathione (S-glutathiolation), which was removable by reduction with 2-mercaptoethanol. Enzymatic digestion of rOaGST with endoproteinase Glu-C, followed by liquid chromatography (LC)-ESI-MS analyses of the peptide mixtures under both reducing and not reducing conditions, have shown that glutathione was covalently bound to the Cys10 residue of rOaGST. Furthermore, LC-ESI-MS analyses of overexpressed rOaGST in Escherichia coli crude extracts, with and without incubation with glutathione, have not shown any S-glutathiolation of the recombinant enzyme.
authors.affiliation: G. Paone Environmental Health Center, Mario Negri Institute for Pharmacological Research, Consorzio Mario Negri Sud, Via Nazionale, 66030 Santa Maria Imbaro, Chieti, Italy. celli@negrisud.it
authors.forename: Nicola
authors.initials: N
authors.lastname: Celli
authors.forename: Agata
authors.initials: A
authors.lastname: Motos-Gallardo
authors.forename: Antonio
authors.initials: A
authors.lastname: Tamburro
authors.forename: Bartolo
authors.initials: B
authors.lastname: Favaloro
authors.forename: Domenico
authors.initials: D
authors.lastname: Rotilio
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D003545:Cysteine / Q000378:metabolism*; D004269:DNA, Bacterial; D005982:Glutathione Transferase / Q000378:metabolism*; D008969:Molecular Sequence Data; D020621:Ochrobactrum anthropi / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D004269:DNA, Bacterial; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D003545:Cysteine