Mouse liver phenobarbital-inducible P450 system: purification, characterization, and differential inducibility of four cytochrome P450 isozymes from D2 mouse.
Three novel cytochrome P450 isozymes were purified from phenobarbital (PB)-treated D2 mouse liver microsomes and compared to the previously characterized coumarin 7-hydroxylase, P450Coh. The molecular masses were 56.5, 55, 51, and 49.5 kDa, and the peaks of the reduced CO complexes were at 450, 447.5, 451.5, and 449 nm for P450PBI, P450PBII, P450PBIII, and P450Coh, respectively. The NH2-terminal sequences suggest that these isozymes belong to the P450 gene subfamilies 2B, 1A, 2C, and 2A, respectively. On the basis of reconstituted activities and microsomal immunoinhibition studies, P450Coh was the sole catalyst of coumarin 7-hydroxylation. P450PBI was the major isozyme catalyzing the high Km 7-pentoxyresorufin O-dealkylation. This reaction was also mediated at a slower rate by the low Km isozyme, P450PBII. P450PBIII contributed significantly to the microsomal O-deethylation of 7-ethoxyresorufin and N-demethylation of benzphetamine. Western blotting and dot immunobinding analyse of microsomes showed that the induction patterns of the isozymes were different. PB and TCPO-BOP induced all isozymes variably: P450PBI (19- and 31-fold), P450PBII (2- and 3-fold), P450PBIII (9- and 4-fold), and P450Coh (about 2-fold). Pyrazole induced only P450Coh, while all other isozymes were decreased by 30 to 60%. The changes in the microsomal amounts of these isozymes correlated generally well with the variation in the immunoinhibitable enzyme activities. On the basis of the structural and catalytic properties, immunochemical characteristics, and induction profiles, all three isozymes were different from each other and from the previously characterized P450Coh. This mouse PB-inducible P450 model may be valuable in further studies on the induction mechanisms of PB and TCPOBOP.
Honkakoski
P
P
Department of Pharmacology and Toxicology, University of Kuopio, Finland.
Lang
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D003429:Cross Reactions; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D015151:Immunoblotting; D005779:Immunodiffusion; D007527:Isoenzymes / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D007700:Kinetics; D051379:Mice; D008811:Mice, Inbred DBA; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000032:analysis; D008969:Molecular Sequence Data; D010634:Phenobarbital / Q000494:pharmacology*; D051381:Rats; D013379:Substrate Specificity
D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6; D010634:Phenobarbital