Measurement of NAD+/NADH ratio

Intracellular NAD⁺ and NADH levels were measured using the EnzyChrom NAD⁺/NADH Assay Kit (BioAssay Systems). Briefly, cells were homogenized in either 100 μl of NAD⁺ extraction buffer (for NAD⁺ determination) or 100 μl of NADH extraction buffer (for NADH determination). Samples were heated at 60°C for 5 min, and then mixed with 20 μl of assay buffer and 100 μl of the opposite extraction buffer to neutralize the extracts. Next, samples were briefly vortexed, and centrifuged at 14,000 rpm for 5 min. Supernatants were subjected to NAD⁺/NADH assays based on the lactate dehydrogenase cycling reaction, in which the generated NADH reduces a tetrazolium salt to a purple colored formazan product. NAD⁺ and NADH levels were quantified by measuring the increase in formazan at 570 nm using a microplate reader.

To prepare the cell-free enzyme system, 1 μM KL1333 was mixed with 200 μM NADH and 200 μM NAD in 50 mM Tris-HCl (pH 7.5) buffer containing 0.14% BSA (total volume, 200 μl). Reactions were initiated by addition of rhNQO1 (10 mU), and assay mixtures were incubated for 1 h at 37°C. Twenty microliters of reaction mixture were mixed with either 100 μl of NAD⁺ extraction buffer or 100 μl of NADH extraction buffer. Samples were heated at 60°C for 5 min, and then mixed with 20 μl of assay buffer and 100 μl of the opposite extraction buffer to neutralize the extracts. Next, samples were briefly vortexed, and centrifuged at 14,000 rpm for 5 min. Supernatants were subjected to NAD⁺/NADH assays, and NAD⁺ and NADH levels were measured on a microplate reader by monitoring absorbance at 570 nm.