Nicotine N-demethylase in cell-free preparations from tobacco cell cultures

• Published in: Phytochemistry (Oxford) Vol. 42; no. 2; pp. 325 - 329
• Main Authors: Hao, Dong-yun, Yeoman, Michael M.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Ltd 01.05.1996; Elsevier
• Subjects: Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Metabolism; Metabolism. Physicochemical requirements; Plant physiology and development; N-demethylation; nicotine N-demethylase; Solanaceae; tobacco cell culture; Nicotiana tabacum; NADPH; Cell culture; Enzyme; Metabolism; Demethylation; Enzymatic activity; Dicotyledones; Angiospermae; Spermatophyta; Kinetics; Cell free system; Nicotine
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/0031-9422(95)00868-3

The activity of the enzyme(s) catalysing the bioconversion of nicotine to nornicotine has been demonstrated, for the first time, in cell-free preparations from tobacco cell cultures. Using 14C-assay, it has been shown that a maximal specific activity of ca 0.2 pkat mg protein −1 is present exclusively in the supernatant fraction after centrifugation at 8 800 g. The enzyme has a pH optimum between 9.0 and 9.5, and a temperature optimum between 25 and 30°, while the V max and the apparent K m are 7.6 × 10 −2 pkat and 7.4 μM of 14C-nicotine, respectively. The decline in enzyme activity, following the removal of some endogenous co-factors and co-enzymes by dialysis of the crude enzyme preparation, can be restored and indeed enhanced by the addition of NADPH, suggesting that this enzyme may be NADPH dependent.