ASS1 activity assay

The procedure was carried out essentially as described (64). Briefly, cells treated with or without ADR and x-ray irradiation were washed with PBS and stored frozen at −80°C until use. Frozen cells were resuspended into buffer A [50 mM tris-HCl (pH 8), 10% glycerol, protease inhibitor cocktail (Calbiochem)] and then left on ice for 10 min. After centrifugation for 20 min at 15,000 rpm at 4°C, 10 μg of proteins was added to the reaction buffer [20 mM tris-HCl (pH 7.8), 2 mM adenosine 5′-triphosphate, 2 mM citrulline, 2 mM aspartate, 6 mM MgCl₂, 20 mM KCl, and 0.1 U pyrophosphatase] to a final volume of 0.1 ml. Samples were incubated for 60 min at 37°C. The reactions were stopped by the addition of an equal volume of molybdate buffer (10 mM ascorbic acid, 2.5 mM ammonium molybdate, and 2% sulfuric acid). Accumulation of pyrophosphate, a by-product of argininosuccinate synthesis, was determined spectrophotometrically at 660 nm.