Expression and characterization of dog cytochrome P450 2A13 and 2A25 in baculovirus-infected insect cells.
Dog CYP2A13 and CYP2A25 were coexpressed with dog NADPH-cytochrome P450 reductase (OR) in baculovirus-infected Sf9 insect cells. CYP2A13 effectively catalyzed 7-ethoxycoumarin (7EC) deethylation and coumarin hydroxylation with apparent K(m) values of 4.8 and 2.1 microM, respectively, similar to those observed using dog liver microsomes (7.5 and 0.75 microM, respectively). CYP2A25 exhibited much lower affinity toward 7EC, with an apparent K(m) value of 150 microM, which indicates that CYP2A13 plays a more significant role in the metabolism of these CYP2A substrates. Similar to the dog CYP1A2 enzyme, CYP2A13 efficiently catalyzed phenacetin deethylation with a K(m) value of 3.9 microM, which suggests that phenacetin is not a selective probe for dog CYP1A2 activity. Both dog CYP2A13 and CYP2A25 exhibited little or no catalytic activity toward other common cytochrome P450 probe substrates, including bupropion, amodiaquine, diclofenac, S-mephenytoin, bufuralol, dextromethorphan, midazolam, and testosterone. These results provided additional information about the selectivity of these commonly used probe substrates.
Zhou
Diansong
D
Clinical Pharmacology & DMPK, AstraZeneca Pharmaceuticals, Wilmington, Delaware 19803, USA. Diansong.zhou@AstraZeneca.com
Linnenbach
Alban J
AJ
Liu
Ruifeng
R
Luzietti
Rick A
RA
Harris
Jennifer J
JJ
Booth-Genthe
Catherine L
CL
Grimm
Scott W
SW
D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D002478:Cells, Cultured; D003374:Coumarins / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D004285:Dogs; D015971:Gene Expression Regulation, Enzymologic; D008862:Microsomes, Liver / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis; D010615:Phenacetin / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics / Q000821:virology; D013379:Substrate Specificity
D003374:Coumarins; C017299:7-ethoxycoumarin; D003577:Cytochrome P-450 Enzyme System; C030123:coumarin; D009251:NADPH-Ferrihemoprotein Reductase; D010615:Phenacetin