Additions | Incubation time (min) | Activity (pmol HFC/min/mg protein) (%) | ||
---|---|---|---|---|
9EPh | NADPH | Catalase | ||
− | + | − | 0 | 145 ± 13 (100) |
− | + | − | 5 | 127 ± 4 (88) |
+ | − | − | 5 | 123 ± 7 (83) |
+ | + | − | 0 | 136 ± 7 (94) |
+ | + | − | 5 | 33 ± 3 (23) |
+ | + | + | 5 | 29 ± 4 (20) |
Reaction mixtures (1.0 ml) containing 0.1 mg of microsomal protein from human B lymphoblastoid cells expressing human 2B6, 0.75 μM 9EPh, 120 units of catalase (when included), and 50 mM potassium phosphate buffer, pH 7.4, were preincubated for 3 min at 30°C before the addition of NADPH to a final concentration of 0.2 mM or water to the mixtures without NADPH. The deethylase activity was measured spectrofluorometrically as described under Materials and Methods after the addition of 100 μM EFC and NADPH in the reaction mixtures where NADPH was not previously included. The activities presented are the average of duplicate determinations, and the values in parentheses are the percent activities remaining as compared with the incubation with no 9EPh at time 0.