Protein lysates centrifuged at 35,000 rpm (105,000 g) for 1 h at 4 °C were used for ADH and CYP2E1 activity assays. The high-speed cytosolic fractions (supernatants) were used for ADH activity assay, while the microsomal fractions in phosphate-buffered saline were used for CYP2E1 activity assay. ADH catalytic activity was assayed by formation of reduced nicotinamide adenine dinucleotide (NADH) from NAD oxidation as described [39]. Briefly, 50 μl of cytosolic protein (100 μg protein) was added to a 430-μl reaction mixture containing 0.5 M Tris-HCl, 0.01 M dithiothreitol, and 0.5 M EtOH in 1.5 ml disposable cuvette. A 20 μl of 90 mM NAD was added to prewarmed cuvettes, and changes in optical density were read at 340 nm using the time-drive kinetics for 10 min at 30-s intervals. ADH activity was calculated from nmol of NADH formed/h/ml, and the specific activity of ADH was expressed as nanomoles per hour per milligram cellular protein.
