max]) of the purified GSTs from the three developmental stages were determined for GSH or CDNB
by recording the activity towards a range of GSH (2 to 24 mM) or CDNB
For the catalytic activity of GSTs toward GSH and CDNB
as expressed by the [V.
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.
Change in absorbance per min was converted into micromoles of CDNB
conjugated per min per mg of protein using the extinction coefficient (9.
1% triton X-100) after adding 20 [micro]l reduced glutathione, the reaction was initiated by adding 10 [micro]l CDNB
, and then the plate was shake for few seconds and the optical density was measured at 340 nm once every min to obtained at least 5 time points and its activity was measured as (nmol/min/ml).
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
The reaction solution, which contained 100 mL each of the supernatant, CDNB
in ethanol (0.