Purification and characterization of cytochrome P450 2E2 from hepatic microsomes of neonatal rabbits.
The alcohol-inducible P450 2E subfamily in the rabbit has two known members that differ in only 16 amino acid residues scattered throughout the polypeptide chain. P450 2E1 has been thoroughly characterized, and is known to have diverse inducers and substrates. Little is known, however, about the properties of P450 2E2, since efforts to isolate this isozyme from adult rabbits have been unsuccessful. In the present study, 2E2 was purified to electrophoretic homogeneity from liver microsomes of neonatal rabbits with the use of 4-methylpyrazole as a stabilizing agent. The purified cytochrome was identified as 2E2 by NH2-terminal amino acid sequence analysis as well as by immunoblot analysis with three different antibodies to 2E1. Purified 2E2, in contrast to 2E1, is predominantly low-spin in the presence of 20% glycerol, but is in a mixed high- and low-spin state as the concentration of glycerol is decreased. The catalytic properties of purified 2E1 and 2E2 were compared in the reconstituted system with a variety of substrates, including alcohols, ethers, nitrosamines, and aromatic compounds. Differences between the two enzymes in catalytic activity and in the interaction with cytochrome b5 were observed with some but not all of the substrates tested. Purified 2E1 and 2E2 both consume molecular oxygen relatively rapidly during NADPH oxidation in the absence of an added substrate, and stoichiometric determinations indicated that only about 20% of the O2 was reduced to H2O2, with the remainder apparently undergoing four-electron reduction to water.
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Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor 48109-0606.
Pernecky
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SJ
Coon
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MJ
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification*; D015786:Cytochromes b5 / Q000378:metabolism; D000077604:Fomepizole; D007527:Isoenzymes / Q000302:isolation & purification*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010101:Oxygen Consumption; D010105:Oxygenases / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D011817:Rabbits; D013053:Spectrophotometry
D007527:Isoenzymes; D011720:Pyrazoles; D000077604:Fomepizole; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases