Multiple forms of cytochrome P-450 in kidney cortex microsomes of rabbits treated with 3-methylcholanthrene.
Cytochrome P-450 was purified from kidney cortex microsomes of rabbits treated with 3-methylcholanthrene. 6-Amino-n-hexyl-Sepharose 4B column chromatography of the cholate-solubilized microsomes yielded two cytochrome P-450 fractions, one of which was eluted from the column with 20 mM potassium phosphate buffer in the presence of 0.4% cholate and 0.08% Emulgen 913. This fraction was partially purified to a specific content of 4.49 nmol of cytochrome P-450/mg of protein. This P-450 fraction catalyzed myristate omega- and (omega-1)-hydroxylation with a turnover rate of 5.0 nmol/nmol of cytochrome P-450 in a reconstituted system containing NADPH-cytochrome c reductase, cytochrome b5 and phosphatidylethanolamine. It had no benzo(a)pyrene hydroxylation activity. The other cytochrome P-450 fraction, which was eluted from the column with 0.1 M potassium phosphate buffer in the presence of 0.4% cholate and 0.08% Emulgen 913, was purified to a specific content of 12.0 nmol of cytochrome P-450/mg of protein. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the final preparation gave a major polypeptide band with a molecular weight of 58,000. This cytochrome P-450 showed a maximal peak at 448 nm in the carbon monoxide difference spectrum of its reduced form. Its absolute spectrum of the oxidized form had low-spin characteristics. It catalyzed benzo(a)pyrene hydroxylation with a turnover rate of 3.63 nmol/nmol of cytochrome P-450 in a reconstituted system containing NADPH-cytochrome c reductase and phosphatidylcholine, whereas little myristate hydroxylation activity was detected. The results demonstrate the occurrence of multiple forms of cytochrome P-450 in rabbit kidney cortex microsomes.
D000818:Animals; D001579:Benzopyrene Hydroxylase / Q000378:metabolism; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007672:Kidney Cortex / Q000201:enzymology*; D007700:Kinetics; D008748:Methylcholanthrene / Q000494:pharmacology*; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology; D011817:Rabbits
D010743:Phospholipids; D009243:NAD; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System; D001579:Benzopyrene Hydroxylase