Identification and partial purification of hamster microsomal cytochrome P-450 isoenzymes.
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 (Mr) 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 (Mr = 48,500 and 53,500) were induced by both 3-methylcholanthrene (3-MC) and beta-naphthoflavone (BNF) treatments. Treatment with Aroclor 1254 induced three polypeptides (Mr = 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 beta-naphthoflavone treatment in liver microsomes. Pregnenolone-16-alpha-carbonitrile (PCN) and TSO did not alter any of these activities. The major polypeptides induced by PB (Mr = 50,000) and 3-MC (Mr = 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-450LM2 or P-450LM4.
D000818:Animals; D006224:Cricetinae; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D007527:Isoenzymes / Q000302:isolation & purification*; D008168:Lung / Q000201:enzymology; D008297:Male; D008647:Mesocricetus; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D010105:Oxygenases / Q000302:isolation & purification
D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases