ISOLATION AND CHARACTERIZATION OF A NEW RAT P450 (CYP3A18) CDNA-ENCODING P450(6-BETA-2) CATALYZING TESTOSTERONE 6-BETA-HYDROXYLATIONS AND 16-ALPHA-HYDROXYLATIONS

A cytochrome P450 cDNA, encoding a new form of CYP3A protein, was isol ated from a liver cDNA library of a male rat using anti-P450(6 beta-1) and anti-P450(6 beta-2) antibodies and the CYP3A2 cDNA. The cDNA (CYP 3A18 cDNA) consisted of 1987 nucleotides, in which were contained an o pen reading frame of 1491 bp (corresponding to 497 amino acids), 5'-(5 9 bp) and 3'-noncoding regions (437 bp). The deduced amino acid sequen ce of CYP3A18 cDNA was completely identical in the first 27 N-terminal residues of P450(6 beta-2) previously isolated by us (Nagata et al. J Biochem 1990: 107, 718-725) from livers of rats treated with dexameth asone, and also shared higher extents of similarity with hamster CYP3A 10 (78.5%) than with rat CYP3As previously sequenced (66.3-69.3%). Nor thern blot analyses indicated a male-dominant expression of this new C YP3A mRNA and enhanced expression in dexamethasone- or pregnenolone-16 alpha-carbonitrile (PCN)-treated, but not phenobarbital- or 3-methylc holanthrene-treated rats. Expressed CYP3A18 protein in COS-1 cells mig rated at a position identical to that of purified P450(6 beta-2) on so dium dodecyl sulfate-acrylamide gel electrophoresis and catalyzed 16 b eta- and 6 alpha-hydroxylations of testosterone. In contrast to CYP3A1 and CYP3A2, cytochrome b(5) was not essential for maximal catalytic a ctivities of recombinant CYP3A18 protein. These results, together with ontogenic profiles of CYP3A18 mRNA and P450(6 beta-2) protein, indica te that the newly isolated CYP3A18 cDNA encodes P450(6 beta-2) in rat P450 liver.