Aconitase activity assay

RKO cells were plated at 2 million cells in 6 cm plates and treated with 0.1% DMSO, 1 μM PIK-III or 30 μM DFX for 24 hours. Cells were harvested, washed twice with cold PBS and resuspended in assay buffer. Cells were then sonicated and centrifuged to remove insoluble material. Cell lysates were normalized using the DC protein assay (Bio-Rad) to a final total protein concentration of 3.6 mg/ml. The aconitase activity assay (Sigma Aldrich; MAK051) was run according to the manufacturer’s instructions except that the assay incubation was for 120 minutes at 25°C before addition of the developer. A standard curve derived from known concentrations of isocitrate was used to calculate the isocitrate generated from each unknown sample and presented in nmole units.

Yeast cells were washed off plates as described in the EPR experiments. Aconitase activity was determined using a commercially available aconitase activity assay (Sigma-Aldrich, St. Louis, MO, USA). Cells were resuspended in aconitase assay buffer and disrupted. Mechanical disruption of 950 μl of cell suspension was achieved by adding 900 mg of 0.25–0.50 mm glass beads (Carl Roth, Karlsruhe, Germany) and subsequent shaking at 4 °C in 4 pulses of 2 min at 30 Hz in a MM400 bead mill (Retsch, Haan, Germany). Cell debris was removed by centrifugation (13300 rpm, 4 °C, 10 min) and yeast lysate was further treated according to the protocol. Isocitrate was processed in the assay to yield a colorimetrically detectable product at 450 nm and compared to a measured isocitrate standard curve. The aconitase activity was determined using the equation: $$\mathit{\text{Aconitase activity}}\left[\frac{mU}{mL}\right]=\frac{B\times SDF}{T\times V}$$
B [nmol], amount of isocitrate generated; SDF, sample dilution factor; T [min], time reaction incubated in minutes; V [ml], sample volume. The measured aconitase activities were normalized to the protein concentration determined by Bradford assay.