PURIFICATION AND CHARACTERIZATION OF A NEWLY IDENTIFIED ISOFORM OF CYTOCHROME-P450 RESPONSIBLE FOR 3-HYDROXYLATION OF 2,5,2',5'-TETRACHLOROBIPHENYL IN HAMSTER LIVER

被引:27
作者
KOGA, N
KIKUICHINISHIMURA, N
HARA, T
HARADA, N
ISHII, Y
YAMADA, H
OGURI, K
YOSHIMURA, H
机构
[1] FUJITA GAKUEN HLTH UNIV,SCH MED,INST COMPREHENS MED SCI,TOYOAKE,AICHI 47011,JAPAN
[2] KYUSHU UNIV,FAC PHARMACEUT SCI,HIGASHI KU,FUKUOKA 812,JAPAN
关键词
HAMSTER; CYTOCHROME P450; 2,5,2',5'-TETRACHLOROBIPHENYL; PHENOBARBITAL;
D O I
10.1006/abbi.1995.1189
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 aminoterminal 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 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 18 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. (C) 1995 Academic Press, Inc.
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页码:464 / 470
页数:7
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