Purification and characterization of a cytochrome P450 from liver microsomes of Xenopus laevis.
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.
Saito
H
H
School of Pharmaceutical Sciences, Showa University, Tokyo, 142, Japan.
Ohi
H
H
Sugata
E
E
Murayama
N
N
Fujita
Y
Y
Higuchi
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002845:Chromatography; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007150:Immunohistochemistry; D007526:Isoelectric Point; D007668:Kidney / Q000201:enzymology; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D051381:Rats; D016415:Sequence Alignment; D017421:Sequence Analysis; D013379:Substrate Specificity; D014982:Xenopus laevis
D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase