Evidence in favour of an oxidative N-demethylation of nicotine to nornicotine in tobacco cell cultures

Abstract

Summary Nicotine N -demethylation to nornicotine has been studied in cell-free preparations from cell suspension cultures of Nicotiana tabacum , in attempt to explore the mechanism of this enzymatic reaction. Subcellular fractionation of the homogenate using isopycnic differential centrifugation, together with TEM, showed that most of the enzyme activity (2.8 × 10 −5 U) was present in the intermediate pellet whilst the maximum specific activity of 8.8 × 10 −5 U per mg protein appeared to be associated with the microsomal fraction. The addition of selected putative methyl group acceptors, i.e. putrescine, glycine and ethanolamine, to either dialysed or undialysed enzyme preparations, did not promote enzyme activity. In addition, studies involving Sephadex G-25 gel filtration showed that molecules smaller than 5,000 MW are unlikely to be involved in this reaction. The current findings appear to support an oxidative nicotine N -demethylation and, together with the results from previous studies, provide some tentative evidence for the involvement of cytochrome P-450 in nicotine N -demethylation. The possible mechanism is also discussed.