Measurement of Dehydrogenase Enzyme Activities

For measurement of pyruvate dehydrogenase (PDH), alpha-ketoglutarate dehydrogenase (KGDH), succinate dehydrogenase (SDH), and malate dehydrogenase (MDH) activities, bacterial cells were suspended in 1× phosphate-buffered saline (PBS; pH 7.4) and adjusted to an OD₆₀₀ of 1.0. An aliquot of 50 ml of cells was collected and transferred to a 1.5-ml centrifuge tube. The cells were resuspended with 1 ml of 1× PBS and disrupted by sonic oscillation for 6 min (200-W total power with 35% output, 2-s pulse, and 3-s pause over ice). Following centrifugation at 12,000 × g for 10 min at 4°C, supernatants were collected. The protein concentration of the supernatants was quantified with a BCA protein concentration determination kit (Beyotime; P0009). Then, 300 μg of proteins was used for determination of enzyme activity. For PDH and KGDH measurement, the reaction mixture contained 0.15 mM 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 2.5 mM MgCl₂, 6.5 mM phenazine methosulfate (PMS), 0.2 mM thiamine PP_i (TPP), and 80 mM sodium pyruvate/alpha-ketoglutaric acid potassium salt, with water added to 200 μl. For SDH and MDH measurement, the reaction mixture contained 0.15 mM MTT, 2.5 mM MgCl₂, 13 mM PMS, and 80 mM sodium succinate/sodium malate, with water added to 200 μl. All of the reaction mixtures were incubated at 37°C, for 5 min for MDH/PDH/KGDH and for SDH for 10 min. Finally, they were measured at 562 nm for colorimetric readings. Light was avoided in all reactions. Experiments were repeated in at least three independent biological replicates.