Aconitase Activity Measurement in Cells

Whole-cell extracts were obtained by homogenization of cultured cells in 50 mM Tris–HCl, pH 8, 10% (v/v) glycerol, 5 mM EDTA, 150 mM KCl, 1 mM phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at 10 000 g at 4 °C for 10 min.^{42 (link)} Aconitase activity was measured using an Aconitase Assay Kit (Cayman Chemical, Ann Arbor, MI, USA). 50 μl whole-cell lysates were added to 200 μl of substrate mix, (50 mM Tris/HCl pH 7.4, 0.4 mM NADP, 5 mM Na citrate, 0.6 mM MgCl₂, 0.1% (v/v) Triton X-100 and 1 U isocitrate dehydrogenase) then the reaction was initiated by adding 50 μl of diluted substrate solution followed by incubation at 37 °C for 15 min. Absorbance was monitored by spectrophotometry every minute for 15 min at 340 nm 37 °C to determine the reaction slope. Aconitase activities of cells were then normalized to citrate synthase activities, which were determined using a citrate synthase assay kit (SIGMA, CS0720), as previously described.^{53 (link)}