ABSTRACT
We previously purified a cytochrome P450 (P450) from liver microsomes of adult female Xenopus laevis. In this study, we screened a cDNA library of Xenopus liver to isolate the cDNA clone coding for this P450. The 5'-end of the resultant cDNA was truncated at the N-terminal region and extended by method of rapid amplification of cDNA end to give the complete coding sequence. Amino acid sequence showed this clone to be 36% to 55% identical to members of the CYP2 family and less than 31% identical to members of other gene families, and to belong to the CYP2Q subfamily. This gene is expressed constitutively in the livers of adult male and female frogs, and is significantly induced by dexamethasone administration.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Dexamethasone/pharmacology , Liver/enzymology , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/metabolism , DNA, Complementary , Enzyme Induction , Female , Male , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Messenger/metabolism , Xenopus laevisABSTRACT
A new cytochrome P450 (P450) has been purified to near homogeneity from Xenopus laevis liver microsomes. Two steps of column chromatographies (n-octylamino Sepharose 4B and Mono Q) and fast protein liquid chromatofocusing were performed consecutively, and the final preparation containing 19 nmol P450/mg protein gave a single band of 52 kDa on SDS-PAGE at an isoelectric point of 6.7. This enzyme had a common feature of microsomal P450s in NH2-terminal region, and some of the internal sequences were similar to the corresponding sequences of reported P450s. The purified Xenopus P450 cross-reacted with antibodies against CYP2B1, rat CYP2E1, and CYP2C13, but not with rat CYP1A1, CYP3A2, or CYP4A1. Upon reconstitution with rat NADPH-cytochrome P450 reductase and phospholipid, the Xenopus P450 catalyzed aniline hydroxylation and N-nitrosodimethylamine N-demethylation. Cytochrome b5 enhanced these reactions. This P450 did not catalyze the hydroxylation of either hexobarbital or testosterone. Thus, the catalytic activities of this P450 were comparable with those of mammalian CYP2E1. Expression of this P450 was observed in liver, kidney, lung, and testis, and the level was highest in kidney. Tissue specificity of expression was the same in both male and female frogs.
