7 efc

Amlodipine besylate, β-NADPH, ribonuclease A (RNase), deoxyribonuclease I (DNase), resorufin, and 7-BR were purchased from Sigma-Aldrich (St. Louis, MO). 7- Hydroxy-4-(trifluoromethyl) coumarin (7-HFC), and 7-EFC were purchased from Invitrogen (Carlsbad, CA). Nickel-nitrilotriacetic acid affinity resin was from Qiagen (Valencia, CA), and Macroprep CM cation exchange resin was obtained from Bio-Rad Laboratories (Hercules, CA). The QuikChange XL site-directed mutagenesis kit and TOPP3 and JM109 cells were obtained from Stratagene (La Jolla, CA). The molecular chaperone plasmid pGro7, which expresses GroES/EL, was obtained from TAKARA BIO (Shiba, Japan). Recombinant NADPH cytochrome P450 reductase (CPR) and cytochrome b₅ from rat liver were prepared as described previously [32 (link)]. All other chemicals and supplies used were from standard sources.

Cytochrome P450 enzyme activities were measured in liver or kidney homogenates using a fluorogenic substrate, 7-ethoxy-4-trifluoromethyl coumarin (7-EFC) (Invitrogen, Carlsbad, CA), which is a substrate for Cyp2E1 and Cyp1A2, as described (Buters et al., 1993 (link); Chang et al., 2006 (link)). Briefly, a 10% homogenate of liver or kidney tissue was prepared in homogenizing buffer (25 mM 4–2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES), pH 7.5, 5 mM ethylenediaminetetraacetic acid (EDTA), 2 mM dithiothreitol, 0.1 % CHAPS, 1 μl/ml pepstatin, 0.1 μl/ml leupeptin and 0.1 μl/ml apoproteinin). Samples were spun at 16,000×g for 5 min at 4°C and the resulting supernatant diluted 1:5 and incubated in 0.1 M phosphate buffer with 0.4 mg/ml BSA and 1 mM NADPH at 37°C for 5 min. Samples were transferred to a 96 well plate and the reaction was started by addition of 7-EFC with or without 4MP to a final concentration of 50 μM each, and the increase in fluorescence monitored overtime (Excitation: 410 nm, Emission: 510 nm).