CdGSTP1-1 was heterologously expressed in Eschericia coli BL21(DE3) as a ~24 kDa soluble protein and showed to be catalyticly active towards the model substrate 1-chloro-2,4-dinitrobenzene
4); 25 uL of 30 mmol L-1 1-chloro-2,4-dinitrobenzene
was used to activate the reaction, and changes in absorbance were measured within 2 min at 340 nm.
GST activity was measured using 1-chloro-2,4-dinitrobenzene
(CDNB) as a substrate by the method of Habig and Jacoby .
Louis, Missouri, USA): nicotinamide adenine dinucleotide phosphate (NADP); glycylglycine; glucose-6-phosphate; l-y-glutamyl-3-carboxyl-4nitronilide; epinephrine; hydrogen peroxide; glutathione (GSH); 5,50-dithio-bis-2-nitrobenzoic acid; thiobarbituric acid; and 1-chloro-2,4-dinitrobenzene
The glutathione S-transferase activity was measured using the 1-chloro-2,4-dinitrobenzene
Por exemplo, o protetor benoxacor induz enzimas GST (glutationa S transferase) em Festuca arundinacea, favorecendo a metabolizacao do compostos 1-chloro-2,4-dinitrobenzene
(DEL BUONO et al.
elicited increase in vacuolar glutathione-S-conjugate transport activity.
Reduced glutathione (GSH), 1-chloro-2,4-dinitrobenzene
(CDNB), reduced nicotinamide adenine dinucleotide phosphate (NADPH), amidopyrine, cytochrome C, thiobarbituric acid and all other chemicals were purchased from Sigma Chemical Company (Saint Louis, USA).
Among spectrophotometric determinations, the GSH5,5'-dithio-bis(2-nitrobenzoic acid) "recycling assay" (40) and the glutathione S-transferase 1-chloro-2,4-dinitrobenzene
(CDNB) endpoint method (41) are the most commonly used.
The active sites of the two structures, which were quite different, represented snapshots along the reaction coordinate for the enzyme-catalyzed reaction of glutathione with 1-chloro-2,4-dinitrobenzene
and revealed specific interactions between the enzyme, intermediate, and product that are important for catalysis.
The reagents used for the experiments included [alpha]-naphthylacetate ([alpha]-NA), Eserine, AlbuMin bovine V (BSA), Coomassie Brilliant Blue G250, 1-chloro-2,4-dinitrobenzene
(CDNB),reduced glutathione (GSH), Fast Blue B salt, Sodium dodecyl sulfate (SDS), EDTA, 1,4-dithioerythritol (DTT), phenylmethanesulfonyl fluoride (PMSF) and 4-nitroanisole (PNA),which were all bought from Sigma.
P-Nitroanisol (BODI), Fast blue B salt (Urchem) 1-Naphthol (Kermel), L-Glutathione reduced (Wolsen), 1-Naphthyl acetate (SCR), Sodium darbital (ZIBO) 1-Chloro-2,4-dinitrobenzene
(ABCR), p-NPP (Amresco), NADPH (Wolsen).