PURIFICATION AND CHARACTERIZATION OF A CYTOCHROME-P450 FROM LIVER-MICROSOMES OF XENOPUS-LAEVIS

A new cytochrome P450 (P450) has been purified to near homogeneity fro m Xenopus laevis liver microsomes. Two steps of column chromatographie s (n-octylamino Sepharose 4B and Mono Q) and fast protein liquid chrom atofocusing were performed consecutively, and the final preparation co ntaining 19 nmol P450/mg protein gave a single band of 52 kDa on SDS-P AGE at an isoelectric point of 6.7. This enzyme had a common feature o f microsomal P450s in NH2-terminal region, and some of the internal se quences were similar to the corresponding sequences of reported P450s. The purified Xenopus P450 cross-reacted with antibodies against CYP2B 1, rat CYP2E1, and CYP2C13, but not with rat CYP1A1, CYP3A2, or CYP4A1 . Upon reconstitution with rat NADPH-cytochrome P450 reductase and pho spholipid, the Xenopus P450 catalyzed aniline hydroxylation and N-nitr osodimethylamine N-demethylation. Cytochrome b(5) enhanced these react ions. This P450 did not catalyze the hydroxylation of either hexobarbi tal 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 highes t in kidney. Tissue specificity of expression was the same in both mal e and female frogs. (C) 1997 Academic Press.