Inactivation Assay | Relative Percent Activity3-a |
---|---|
NADPH-GS | 100 |
8-MOP (2.5 μM) | 112 |
8-MOP (2.5 μM), NADPH-GS | 9 |
8-MOP (2.5 μM), NADPH-GS, 1 μM pilocarpine | 35 |
8-MOP (2.5 μM), NADPH-GS, 2 mM sodium cyanide | 13 |
8-MOP (2.5 μM), NADPH-GS, 70 μM K3Fe(CN)6 | 10 |
8-MOP (2.5 μM), NADPH-GS, 60 mM semicarbazide | 9 |
8-MOP (2.5 μM), NADPH-GS, 5 mM methoxylamine | 5 |
8-MOP (2.5 μM), NADPH-GS, 5 mMN-acetylcysteine | 9 |
8-MOP (2.5 μM), NADPH-GS, 5 mM glutathione | 10 |
8-MOP (2.5 μM), NADPH-GS, 5 mM deferoxamine | 10 |
8-MOP (2.5 μM), NADPH-GS, 750 units superoxide dismutase | 10 |
8-MOP (2.5 μM), NADPH-GS, 2000 units catalase | 10 |
8-MOP (2.5 μM), NADPH-GS, overnight dialysis3-b | 10 |
↵3-a Human liver microsomes (HL109, 50 pmol) were exposed to the indicated components for 3 min at 30°C. The remaining P450 2A6 activity was determined by the amount of 7-hydroxycoumarin formed relative to the control (+ NADPH-GS) using an HPLC fluorescence assay. The rate of turnover for the uninhibited reaction in microsomes (HL109) was 5.21 nmol/nmol P450/min.
↵3-b Dialysis was carried out overnight against 1 liter (1000 × incubation volume) of 25 mM potassium phosphate buffer (pH 7.4) after exposure to 8-MOP in the absence (control) and presence of the NADPH-GS.