Liquid chromatography-electrospray mass spectrometry study of cysteine-10 S-glutathiolation in recombinant glutathione S-transferase of Ochrobactrum anthropi.
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.
Celli
Nicola
N
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
Motos-Gallardo
Agata
A
Tamburro
Antonio
A
Favaloro
Bartolo
B
Rotilio
Domenico
D
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*
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D003545:Cysteine