Identification and partial purification of hamster microsomal cytochrome P-450 isoenzymes

Abstract

We have identified and partially purified three forms of cytochrome P-450 from hamster liver microsomes. Phenobarbital (PB) treatment induced three major polypeptides with relative mobilities ( M r ) of 47,000, 50,000 and 51,500. The 47,000 polypeptide was assigned as epoxide hydrolase, since it was also enhanced by trans-stilbene oxide (TSO) treatment. Two polypeptides ( M r = 48,500 and 53,500) were induced by both 3-methylcholanthrene (3-MC) and β-naphthoflavone (BNF) treatments. Treatment with Aroclor 1254 induced three polypeptides ( M r = 48,500, 50,000 and 53,500), indicating the induction of both drug- and carcinogen-inducible cytochrome P-450s. Liver microsomal benzo[ a]pyrene hydroxylase activity was not affected significantly by any of these inducers. In contrast, it was induced 2- to 3-fold in lung microsomes by 3-MC, BNF or Aroclor 1254 treatment. Benzphetamine N-demethylase and 7-ethoxycoumarin O-deethylase activities, expressed as nmoles of product formed per min per mg of liver microsomal protein, were increased 3- to 4-fold by either PB or Aroclor treatment. The activity of 7-ethoxycoumarin O-deethylase was the only one enhanced significantly by 3-methylcholanthrene or β-naphthoflavone treatment in liver microsomes. Pregnenolone-16-α-carbonitrile (PCN) and TSO did not alter any of these activities. The major polypeptides induced by PB ( M r = 50,000) and 3-MC ( M r = 48,500 and 53,500 respectively) were partially purified, to a specific content of 6–10 nmoles P-450/mg of protein and were active in catalyzing N-demethylation of benzphetamine, hydroxylation of benzo[ a]pyrene, and O-deethylation of 7-ethoxycoumarin with different substrate specificity. None of these isoenzymes immuno-cross-reacted with antibodies prepared against rabbit cytochrome P-450 LM2 or P-450 LM4.