Purification and characterization of N1-methylnicotinamide oxidases I and II separated from rat liver.

N1-Methylnicotinamide (1-CH3Nmd) oxidases I and II of the livers of Wistar strain male rats were separated by DEAE-cellulose column chromatography and purified by AcA 34 gel filtration, Affi-Gel blue column chromatography, and sucrose density gradient centrifugation. These two enzymes were purified 568- and 82-fold with yields of 1.4 and 0.1% of the total activity in the liver homogenate, respectively. They showed the same molecular weight of 300,000 by AcA 34 gel filtration and a single protein band with a molecular weight of 150,000 in sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. The difference in antigenicities of these two enzymes was observed with double immunodiffusion and immunoprecipitation using antibody produced in rabbits against each enzyme. Furthermore, these two enzymes were found to be different from each other in the heat stability, optimum pH values, Km values for 1-CH3Nmd, and response to various inhibitors.