Fluorometric assay of nicotinamide methyltransferase with a new substrate, 4-methylnicotinamide

A fluorometric method for the assay of nicotinamide methyltransferase has been established using rat liver 9000 x g supernatant fluid as a model enzyme preparation. 1,4-Dimethylnicotinamide formed enzymatically from a new substrate, 4-methylnicotinamide, is quantified by means of its fluorescence reaction with 4-methoxybenzaldehyde in aqueous alkali. The lower limit of determination of 1,4-dimethylnicotinamide is 100 pmol in the enzymatic reaction mixture. The apparent Km values for 4-methylnicotinamide and for nicotinamide, which is a known substrate for this enzyme, were 0.19 and 0.13 mM, respectively, whereas the relative activity of 4-methylnicotinamide as a methyl acceptor was about 1.5 times the value of nicotinamide.