TABLE 2
Metabolism of NNK by heterologously expressed CYP2A13.1 and CYP2A13.2
Incubation mixtures contained 100 mM sodium phosphate buffer, pH 7.4, 1.0 mM EDTA, 3.0 mM MgCl2, an NADPH-generating system (5.0 mM glucose 6-phosphate, 1.0 mM NADPH, and 1.5 units of glucose-6-phosphate dehydrogenase), 2 to 50 μM NNK (containing 1 μCi of [5-3H]NNK), 5.0 mM sodium bisulfite, 20 or 40 pmol of purified rat CPR, and 5 or 10 pmol of CYP2A13, in a total volume of 0.4 ml. Reactions were carried out at 37°C for 5 to 10 min. NNK metabolites were determined using radiometric HPLC, as described under Materials and Methods. The Km and Vmax values represent means ± S.D. from three separate determinations.