Purification and properties of a new beta-naphthoflavone inducible cytochrome P-450, aryl hydrocarbon hydroxylase from rat kidney.
In rat kidney, beta-naphthoflavone induced 53 kDa and 55 kDa proteins, which were both recognized by the antibodies against rat liver cytochrome P-450 1A1 (55kDa). The major inducible 53 kDa protein was purified from the beta naphthoflavone-treated rat kidney and shown to be a new cytochrome P-450 having a high aryl hydrocarbon hydroxylase activity. Purified cytochrome P-450, named P-450KAh, was homogeneous on SDS-polyacrylamide gel electrophoresis, and the apparent molecular weight was estimated to be 53 kDa. The absorption spectra of the oxidized form of P-450KAh showed a Soret peak at 416 nm, a characteristic of low-spin hemoprotein, and the Soret peak of the reduced cytochrome P-450-CO complex was at 446 nm. In the reconstituted system, purified P-450KAh showed high catalytic activity for benzo[a]pyrene hydroxylation and 7-ethoxycoumarin O-deethylation. P-450KAh could activate genotoxicities of not only B[a]P, but also 2-acetylaminofluorene and aflatoxin B1 on the umu test. These catalytic properties of P-450KAh were almost the same as those of P-4501A1, a major P-450 form having arylhydrocarbon hydroxylase in liver microsomes of 3-methylcholanthrene-treated rats, and P-450KAh could not be distinguished from P-4501A1 even by immunochemical analysis. However, the electrophoretic peptide patterns after alpha-chymotrypsin or trypsin treatment of P-450KAh were different from those of P-4501A1, and the NH2-terminal 11 amino acid sequence of the P-450 was also different from that of P-4501A1 and any other P-450s of rat.
Ohgiya
N
N
Department of Veterinary Biochemistry, School of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan.
Yokota
H
H
Mitsuru
Takahashi
Komoro
S
S
Yuasa
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification*; D001571:Benzoflavones / Q000494:pharmacology*; D002273:Carcinogens / Q000378:metabolism; D004790:Enzyme Induction; D007668:Kidney / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D051381:Rats; D017208:Rats, Wistar; D019324:beta-Naphthoflavone
D001571:Benzoflavones; D002273:Carcinogens; D019324:beta-Naphthoflavone; D001189:Aryl Hydrocarbon Hydroxylases