A major phenobarbital-inducible P450 isozyme, CYP2A14, in the Chinese hamster liver: purification, characterization, and cDNA cloning.
Phenobarbital, a potent inducer of CYP2B isozyme of cytochrome P450, induces mainly CYP2A but not CYP2B in the Chinese hamster liver. A major isozyme inducible by phenobarbital was purified by column chromatography from Chinese hamster livers. This isozyme, named P450CH2A-2 and designated CYP2A14, had in the reconstituted system high activities of 7-ethoxycoumarin O-deethylase (43 nmol/min/nmol P450) and aflatoxin B1 activation and a moderate activity of testosterone 15alpha-hydroxylase and coumarin 7-hydroxylase. The N-terminal amino acid sequence of the purified protein had a high homology with those of CYP2A proteins. cDNA of this isozyme was analyzed by screening a Chinese hamster liver cDNA library with cDNA of CYP2A1 as a probe, and the obtained clone encoded a protein of 494 amino acids with a calculated molecular mass of 56.4 kDa. The N-terminal amino acid sequence of 20 residues was identical to that derived from the purified protein. The deduced amino acid sequence of the clone had a high identity with most of CYP2A proteins (>65%) and thus was designated CYP2A14. Immunoblot and Northern blot analyses demonstrated that this isozyme was induced markedly by phenobarbital but not with 3-methylcholanthrene and constitutes one of the major components in livers of phenobarbital-treated Chinese hamsters.
Fukuhara
M
M
Department of Pharmaceutical Sciences, National Institute of Public Health, Tokyo, 108-8638, Japan. fukuhara@iph.go.jp
Kurose
K
K
Aiba
N
N
Matsunaga
N
N
Omata
W
W
Kato
K
K
Kimura
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D018076:DNA, Complementary / Q000302:isolation & purification*; D004790:Enzyme Induction / Q000187:drug effects; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D008862:Microsomes, Liver / Q000737:chemistry / Q000201:enzymology*; D008969:Molecular Sequence Data; D010634:Phenobarbital / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D007527:Isoenzymes; C116010:cytochrome P-450 CYP2A14 (hamster); D003577:Cytochrome P-450 Enzyme System; D010634:Phenobarbital