Glutamate dehydrogenase activity colorimetric assay kit

GDH activity was measured with a glutamate dehydrogenase activity colorimetric assay kit (BioVision, Milpitas, Calif., USA) according to the manufacturer’s protocol. Briefly, transfected cells (1 × 10⁶) were homogenized. Glutamate was added to the cells, and the NADH produced by GDH was measured with a Model 680XR Microplate Reader at 450 nm.

The enzyme activity of GLUD1 and GOT were determined using a Glutamate Dehydrogenase Activity Colorimetric Assay Kit (catalog #K729-100, BIoVision, Milpitas, CA, USA) and a Glutamate Oxaloacetate Transaminase Activity Assay Kit (catalog #K753-100, BIoVision), respectively. Briefly, 2 × 10⁶ cells or 50 mg of tissues were homogenized, mixed with the assay buffer, and incubated for 2 min at 37 °C in the dark. The change in absorbance at 450 nm was measured every 5 s for 10 min at 37 °C with a microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. GLS enzyme activity was measured by a Glutaminase Assay Kit (catalog #E-133, Biomedical Research Service Center, Buffalo, NY, USA) according to the manufacturer’s instructions. Cells (2 × 10⁶) were homogenized, mixed with the glutamine solution, and incubated for 2 h at 37 °C. The assay buffer was then added to the samples, followed by incubating for another 1 h at 37 °C. The optical density (OD) at 492 nm was then measured using a microplate spectrophotometer. Each sample was analyzed in triplicate, and the enzyme activity was calculated following the manufacturer’s instruction. Experiments were repeated at least three times.