Nature of N-Nitrosodimethylamine Demethylase and Its Inhibitors¹

The present study was undertaken to examine the nature of the low K_m (K_mI) form of rat liver microsomal N-nitrosodimethylamine demethylase (NDMAd) and its inhibition by organic compounds which are commonly present in the assay mixture. Using radiometric and colorimetric assay methods with an NADPH-generating system consisting of 0.4 mm NADP, 10 mm glucose-6-phosphate, and glucose-6-phosphate dehydrogenase (0.4 units/ml), K_m values of 40–50 µm were obtained. These K_m values were lower than the values of 60–80 µm reported previously. This decrease was due to the elimination of inhibitors such as glycerol in the assay mixture. Glycerol was a competitive inhibitor, and this observation explained in part why purified P-450_ac (acetone-inducible form of P-450), displayed a higher K_m value in a reconstituted NDMAd system, which contained glycerol, than in microsomes. Semicarbazide which had been used in many previous assays of NDMAd was also found to be a competitive inhibitor of this enzyme. Other inhibitors studied include the commonly used solvents dimethylsulfoxide, acetone, ethylene glycol, dimethylformamide, ethyl acetate, benzene, and hexane as well as thiol compounds dithiothreitol and mercaptoethanol. Although very low K_m values (10–20 µm) for N-nitrosodimethylamine metabolism were reported in studies with perfused liver, liver slices, and isolated liver cells, we believe that the K_mI form of liver NDMAd is responsible for the metabolism and activation of N-nitrosodimethylamine in the rat liver.