title: A high-performance liquid chromatography-based assay of glutathione transferase omega 1 supported by glutathione or non-physiological reductants.
abstract: The unusual glutathione S-transferase GSTO1 reduces, rather than conjugates, endo- and xenobiotics, and its role in diverse cellular processes has been proposed. GSTO1 has been assayed spectrophotometrically by measuring the disappearance of its substrate, S-(4-nitrophenacyl)glutathione (4-NPG), in the presence of 2-mercaptoethanol that regenerates GSTO1 from its mixed disulfide. To assay GSTO1 in rat liver cytosol, we have developed a high-performance liquid chromatography (HPLC)-based procedure with two main advantages: (i) it measures the formation of the 4-NPG reduction product 4-nitroacetophenone, thereby offering improved sensitivity and accuracy, and (ii) it can use glutathione, the physiological reductant of GSTO1, which is impossible to do with the spectrophotometric procedure. Using the new assay, we show that (i) the GSTO1-catalyzed reduction of 4-NPG in rat liver cytosol also yields 1-(4-nitrophenyl)ethanol, whose formation from 4-nitroacetophenone requires NAD(P)H; (ii) the two assays measure comparable activities with 2-mercaptoethanol or tris(2-carboxyethyl)phosphine used as reductant; (iii) the cytosolic reduction of 4-NPG is inhibited by GSTO1 inhibitors (KT53, 5-chloromethylfluorescein diacetate, and zinc), although the inhibitory effect is strikingly influenced by the type of reductant in the assay and by the sequence of reductant and inhibitor addition. Characterization of GSTO1 inhibitors with the improved assay provides better understanding of interaction of these chemicals with the enzyme.
authors.affiliation: Department of Pharmacology and Pharmacotherapy, Toxicology Section, University of Pécs, Medical School, H-7624 Pécs, Hungary.
authors.forename: Balázs
authors.initials: B
authors.lastname: Németi
authors.affiliation: Department of Pharmacology and Pharmacotherapy, Toxicology Section, University of Pécs, Medical School, H-7624 Pécs, Hungary.
authors.forename: Miklós
authors.initials: M
authors.lastname: Poór
authors.affiliation: Department of Pharmacology and Pharmacotherapy, Toxicology Section, University of Pécs, Medical School, H-7624 Pécs, Hungary. Electronic address: zoltan.gregus@aok.pte.hu.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Gregus
mesh_terms: D000098:Acetophenones / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D055162:Biocatalysis; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D005452:Fluoresceins / Q000737:chemistry / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D008623:Mercaptoethanol / Q000737:chemistry; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley; D013053:Spectrophotometry
chemical_list: D000098:Acetophenones; D004791:Enzyme Inhibitors; D005452:Fluoresceins; C527914:S-(4-nitrophenacyl)glutathione; D009243:NAD; C001972:4-nitroacetophenone; C069306:5-chloromethylfluorescein; D008623:Mercaptoethanol; D005982:Glutathione Transferase; D005978:Glutathione
keywords: 4-Nitroacetophenone; GSTO1; Glutathione; Reduction; S-(4-Nitrophenacyl)glutathione