α ketoglutarate dehydrogenase activity colorimetric assay kit

After drug treatment, 1 mL of mixture was centrifuged at 8000 rpm for 5 min and the cell pellet was washed once with fresh broth medium. After collection, the cells were immediately transferred to ice. The activities of MDH and α-KGDH in cells were detected using Malate Dehydrogenase Assay Kit and α-Ketoglutarate Dehydrogenase Activity Colorimetric Assay Kit (Sigma, St. Louis, MO, USA), respectively, according to manufacturer’s instructions. The activities of MDH and α-KGDH in cells were determined based on the formation of NADH which was detected with absorbance at 450 nm. One unit of both MDH and α-KGDH is the amount of enzyme that generates 1.0 μmol of NADH per minute at 37 °C and pH 9.5. Oxaloacetate concentration in the sample was measured using the Oxaloacetate Assay Kit (Sigma, St. Louis, MO, USA) according to manufacturer’s instructions and determined based on fluorometric (Excitation/Emission = 535 nm/587 nm) product that is proportional to the oxaloacetate present.

Enzymatic activities of the predicted citrate synthase, aconitase and α-ketoglutarate dehydrogenase were tested using the Citrate Synthase Assay kit, Aconitase Activity Assay kit and α-ketoglutarate Dehydrogenase Activity Colorimetric Assay kit, respectively (Sigma-Aldrich, St. Louis, MS, USA). IDH activity assays were performed using the isocitrate dehydrogenase activity assay kit (Sigma-Aldrich, St. Louis, MS, USA) and monitored with a Synergy HT microplate reader (BioTek, Winooski, VT, USA). Reactions were carried out in duplicate at 37 °C in a 96-well plate containing a final volume of 100 µL in each well. Conversion of the isocitrate to α-ketoglutarate was monitored for 30 min following changes in absorbance at 450 nm, corresponding to the production of NADH. A NADH standard curve was constructed, allowing enzyme quantification and calculation of specific activity. In addition, IDH activity was assessed on mature viral particles purified as described above, and on human IDH (Sigma-Aldrich; St. Louis, MS, USA). Initial velocities were calculated using Gen5.1 software (BioTek), and the obtained mean values were fitted using the Michaelis–Menten equation in Prism 6 (GraphPad Software, San Diego, CA, USA).

The Mitochondria Isolation Kit for Cultured Cells (Thermo Fisher Scientific, Germany) was used to extract mitochondria of ISKNV-infected or uninfected cells. An Isocitrate Dehydrogenase Activity Assay Kit (Sigma-Aldrich, Germany) and an α-Ketoglutarate Dehydrogenase Activity Colorimetric Assay Kit (Sigma-Aldrich, Germany) were used to detect the enzyme activity in cells. Briefly, mitochondria from 5 × 10⁶ cells were homogenized in 300 μL of assay buffer to prepare sample solution. Twenty μL of sample solution was pipetted into the wells of a 96-well plate. Then, a reaction mixture (8 μL developer, and 2 μL α-KGDH substrate or 2 μL IDH substrate) was added to each well. For IDH2 activity analysis, 2 μL of NADP+ was added additionally to the reaction mixture. After 3 min, the absorbance at 450 nm was measured every minute in a microplate reader (Infinite M200 Pro, Tecan, Switzerland) until the value of the most active sample is greater than the value of the highest standard. IDH2 activity was defined as the production of 1.0 μM NADPH per minute. α-KGDH activity was defined as the production of 1.0 μM NADH per minute. Three replicates of each sample were performed. For samples exhibiting significant background, we included a sample blank by omitting the substrate.