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 cytoch rome P-450 1A1(55 kDa). The major inducible 53 kDa protein was purifie d from the beta-naphthoflavone-treated rat kidney and shown to be a ne w cytochrome P-450 having a high aryl hydrocarbon hydroxylase activity . Purified cytochrome P-450, named P-450K(Ah), was homogeneous on SDS- polyacrylamide gel electrophoresis, and the apparent molecular weight was estimated to be 53 kDa. The absorption spectra of the oxidized for m of P-450K(Ah) 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-450K (Ah) showed high catalytic activity for benzo[a]pyrene hydroxylation a nd 7-ethoxycoumarin O-deethylation. P-450K(Ah) could activate genotoxi cities of not only B[a]P, but also 2-acetylaminofluorene and aflatoxin B-1 on the umu test. These catalytic properties of P-450K, were almos t same as that of P-4501A1, a major P-450 form having arylhydrocarbon hydroxylase in liver microsomes of 3-methylcholanthrene-treated rats, and P-450K(Ah) could not be distinguished from P-4501A1 even by immuno chemical analysis. However, the electrophoretic peptide patterns after alpha-chymotrypsin or trypsin treatment of P-450K(Ah) were different from those of P-4501A1, and the NH2-terminal 11 amino acid sequence of the P-450 was also different from those of P-4501A1 and any other P-4 50s of rat.