Activity: 7-methoxy-4-(aminomethyl)-coumarin (MAMC) O-demethylase
General description
Incubations are set up in a black 96-well plate, and consist of substrate (MAMC) and CYP2D6 Bactosomes in phosphate buffer containing magnesium chloride. Reactions are initiated by adding 40 µl of 5x NADPH generating system [this is added to all wells, including blanks and standards]. Metabolite (HAMC) formation is measured fluorometrically, using detection wavelengths chosen to minimise interference from NADPH and MAMC.
The substrate, MAMC, and metabolite, HAMC, are both available from Cypex.
Incubation conditions
Plate type: black, 96-well, flat-bottomed
Assay buffer: 50 mM potassium phosphate pH 7.4, 5 mM MgCl2
Solvent used to dissolve MAMC: methanol
Final MAMC concentration: 8 µM (for CYP2D6R and CYP2D6LR Bactosomes)
Final CYP2D6 concentration: 4 pmol/200 µl (CYP2D6R); 20 pmol/200 µl (CYP2D6LR)
Incubation time: 5 min
Incubation volume: 200 µl
Incubation temperature: 37°C
Metabolite standard: HAMC, 50 ng/200 µl (final concentration)
Stop reagent: none (continuous monitoring)
This test is also suitable for CYP2D6*2R Bactosomes (use 4 µM MAMC and 4 pmol CYP/200 µl) and CYP2D6*10R Bactosomes (use 40 µM MAMC and 10 pmol CYP/200 µl).
Plate reader parameters
Detection mode: fluorescence, top reading
Excitation wavelength: 429 nm (bandwidth 9 nm)
Emission wavelength: 470 nm (bandwidth 20 nm)
Gain: calculated from HAMC standard
Sampling frequency: 15 s (time linearity) or 20 s (P450 linearity or kinetics)
Time linearity test on CYP2D6R Bactosomes with MAMC as substrate:
This test was run at 8 µM MAMC at a final CYP concentration of 4 pmol/200 µl.
The fluorescence was measured every 15 s.
The reaction is linear for approximately 15 min with CYP2D6R Bactosomes.
Time linearity test on CYP2D6LR Bactosomes with MAMC as substrate:
This test was run at 8 µM MAMC at a final CYP concentration of 20 pmol/200 µl.
The fluorescence was measured every 15 s.
The reaction is linear for approximately 30 min with CYP2D6LR Bactosomes.
P450 linearity test on CYP2D6R Bactosomes with MAMC as substrate:
This test was run at 8 µM MAMC using an incubation time of 5 min.
The fluorescence was measured every 20 s.
The reaction is linear up to 16 pmol/200 µl with CYP2D6R Bactosomes.
P450 linearity test on CYP2D6LR Bactosomes with MAMC as substrate:
This test was run at 8 µM MAMC using an incubation time of 5 min.
The fluorescence was measured every 20 s.
The reaction is linear up to 40 pmol/200 µl with CYP2D6LR Bactosomes.
Kinetics test on CYP2D6LR Bactosomes with MAMC as substrate:
This test was run at 20 pmol CYP/200 µl using an incubation time of 5 min.
The fluorescence was measured every 20 s.
The reaction is characterised by a Vmax of 1.0 pmol/min/pmol CYP and a Km of 10 µM in CYP2D6LR Bactosomes. MAMC concentrations ≥100 µM give rise to substrate activation.
Typical kinetic parameters for different enzyme preparations:
Kinetic parameters were estimated by fitting the experimental data directly to the Michaelis-Menten equation using Microsoft Excel.
