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-2and designated CYP2A14, had in the reconstituted system high activities of 7-ethoxycoumarinO-deethylase (43 nmol/min/nmol P450) and aflatoxin B1activation and a moderate activity of testosterone 15α-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 ofCYP2A1as 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.