Assay for Amoeba Glutathione S-Transferase

Amoebae (5x 10⁴ cells/mL) were washed in PBS and sonicated on ice using three 10-second bursts at high intensity (Heat Systems Ultrasonic Cell Disruptor) and cooling for 30 seconds on ice between each burst. Protein concentrations were estimated by the method of Bradford using bovine serum albumin as the standard [24 (link)]. DdGSTs activity was determined at 25°C with reduced glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB) as substrates, measuring the increase in spectrophotometric absorbance at 340nm for 5 minutes [25 (link)]. CDNB activity assays of purified, recombinant GSTs (rDdGSTA2, rDdGSTA3) were conducted in accordance with protocols previously established in our laboratory [26 (link)]. Data presented as mean enzyme activity (% control) ± SEM. Statistical significance was determined using the one-sample t-test (mean, 100; two-tailed) vs. control, *p < 0.05.