Recombinant SaGAPDH activity was determined by measuring the absorbance change at 340 nm and 25°C resulting from the production of NADH. The reaction was carried out in a 150 µl assay mixture containing 20 mM Tris–HCl (pH 8.7), 0.36 µM SaGAPDH, 1.25 mM NAD+, 1.25 mM ethylenediaminetetraacetic acid and 15 mM sodium arsenate. The reaction was started by the addition of 0.25 mM glyceraldehyde 3-phosphate. Initial reaction rates were calculated as described recently [25 (link)], by determining the slope in the linear part of the curve during the first 80 s of the reaction (GraphPad, linear regression function). The percentage of SaGAPDH activity was calculated as: Rate of inactivated/Rate of untreated × 100%. The results are presented as mean ± SEM from at least three separate experiments.
For the inactivation experiments, SaGAPDH was preincubated with 1 µM, 10 µM, 100 µM and 10 mM H2O2 for 10 min or with 10 mM CoASSCoA for 30 min. About 2 µl of the mixture was then added to the assay mixture and the remaining activity was measured as described. To reduce it, the enzyme was incubated with 10 mM DTT for 15 min. After treatments, excess H2O2, CoASSCoA and DTT were removed using Micro Biospin 6 columns (Bio-Rad).
For the inactivation experiments, SaGAPDH was preincubated with 1 µM, 10 µM, 100 µM and 10 mM H2O2 for 10 min or with 10 mM CoASSCoA for 30 min. About 2 µl of the mixture was then added to the assay mixture and the remaining activity was measured as described. To reduce it, the enzyme was incubated with 10 mM DTT for 15 min. After treatments, excess H2O2, CoASSCoA and DTT were removed using Micro Biospin 6 columns (Bio-Rad).
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