Glutathione s-transferase activity per mg weight and its specific activity were examined using CDNB
as GST substrate for different life stages of different insecticideresistant strains and susceptible control.
, 1-chloro-2,4-dinitrobenzene; CMNA, cyano(6-methoxy-2-naphthyl)methyl actetate; CMNPC, cyano(6-methoxy-naphthalen-2-yl)muthyl trans-[3-phenylaxiran-2-yl) methyl] carbonate; EROD, ethoxyresurufin; Octanoyl-MP, N-(6-methoxypyridin-3-yl) octanamide.
Although the presence of activity towards CDNB
or other substrates is suggestive of the presence of enzymes from certain GST classes, it can by no means be regarded as proof of the presence of that specific class of GSTs.
For the CDNB
endpoint method, only acidified clear supernatants were used: sample aliquots (0.
The GST activity was expressed in amount of conjugate constituted by GSH and CDNB
catalyzed by GST per unit time per mg of enzyme (mol min-1 mg-1 protein).
After centrifugation, the CDNB
conjugate was measured spectrophotometrically at 340 [eta]m.
GSH and CDNB
in GST assay buffer were added to the supernatant and mixed.
Kinetic, electrophoretic, and immunoblotting data confirmed the presence of GST-[pi] in the purified enzyme preparations; specific activity was measured using CDNB
Change in absorbance per min was converted into micromoles of CDNB
conjugated per min per mg of protein using the extinction coefficient (9.
It is based on the conjugation reaction between GST and a substrate, CDNB
(1-chloro 2, 4 dinitrobenzene) in the presence of a cofactor: glutathione (GSH).
Working solution (phosphate buffer 100mM, GSH 1mM, EDTA 60mM), CDNB
(10mM) and sample were placed in a cuvette and mixed.
The final reaction mixture contained 1 mM CDNB
and 1 mM GSH in 0.