Purification and characterization of a newly identified isoform of cytochrome P450 responsible for 3-hydroxylation of 2,5,2',5'-tetrachlorobiphenyl in hamster liver.
In the hamster liver, 2,5,2',5'-tetrachlorobiphenyl (TCB) is metabolized to 3-hydroxy- and 4-hydroxy-2,5,2',5'-TCB to a similar extent, and formation of the former metabolite is stimulated by phenobarbital pretreatment of the animals, while that of the latter metabolite is stimulated by 3-methylcholanthrene pretreatment. In the present study, we identified a new isoform (designated P450HPB-1) of cytochrome P450 which proved to be phenobarbital-inducible and responsible for 3-hydroxylation of this TCB isomer. This isoform was purified from liver microsomes of phenobarbital-treated hamsters and characterized. P450HPB-1 has a molecular mass of 50 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the absorption maxima of the oxidized form at 417 nm and of the reduced CO-complex form at 450 nm. The sequence of 28 amino acids of P450HPB-1 at the amino-terminal has a 68% similarity with those of rat P450 2B1 and mouse P450 2b10, 57% similarity with that of guinea pig P450GP-1, and 54% similarity with that of guinea pig P450GP-1, and 54% similarity with those of rabbit P450 2B4 and dog P450 2B11. P450HPB-1 in the reconstituted system catalyzed the 3- but not 4-hydroxylation of 2,5,2',5'-TCB, at a rate of 19.0 pmol/min/nmol P450. The isoform also has high catalytic activity for 17-oxidation of testosterone but low activity for the N-demethylation of benzphetamine and 16 alpha- and 16 beta-hydroxylations of testosterone. In microsomal metabolism of 2,5,2',5'-TCB, rabbit antiserum against P450HPB-1 almost completely inhibited 3- but not 4-hydroxylation. Immunoblot analysis of hamster liver microsomes revealed that P450HPB-1 was constitutive and phenobarbital-inducible but was decreased by pretreatment with 3-methylcholanthrene or 3,4,5,3',4'-pentachlorobiphenyl. These results suggest that P450HPB-1 belongs in the P450 2B subfamily and apparently plays a major role in the 3-hydroxylation of 2,5,2',5'-TCB, in hamster liver.
Koga
N
N
Department of Food and Nutrition, Nakamura Gakuen University, Fukuoka, Japan.
Kikuichi-Nishimura
N
N
Hara
T
T
Harada
N
N
Ishii
Y
Y
Yamada
H
H
Oguri
K
K
Yoshimura
H
H
D000595:Amino Acid Sequence; D000818:Animals; D002248:Carbon Monoxide / Q000378:metabolism; D002384:Catalysis; D006224:Cricetinae; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006900:Hydroxylation; D007106:Immune Sera / Q000494:pharmacology; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D008647:Mesocricetus; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010634:Phenobarbital / Q000494:pharmacology; D011078:Polychlorinated Biphenyls / Q000378:metabolism*
D007106:Immune Sera; D007527:Isoenzymes; C009407:2,5,2',5'-tetrachlorobiphenyl; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D011078:Polychlorinated Biphenyls; D010634:Phenobarbital