Publications

• Differences in soluble P-450 hemoproteins from livers of rats treated with phenobarbital and 3-methylcholanthrene.

  Fujita T., Mannering G.J.

  Chem Biol Interact 3:264-265(1971) [PubMed] [EuropePMC]

• The biochemistry of aromatic amines. III. Enzymic hydroxylation by rat-liver microsomes.

  BOOTH J., BOYLAND E.

  Biochem J 66:73-78(1957) [PubMed] [EuropePMC]

• Characterization of three forms of rabbit microsomal cytochrome P-450 by peptide mapping utilizing limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis.

  Johnson E.F., Zounes M.C., Muller-Eberhard U.

  Arch Biochem Biophys 192:282-289(1979) [PubMed] [EuropePMC]

• Properties of electrophoretically homogeneous phenobarbital-inducible and beta-naphthoflavone-inducible forms of liver microsomal cytochrome P-450.

  Haugen D.A., Coon M.J.

  Procedures are described for the isolation of two forms of rabbit liver microsomal liver microsomal cytochrome P-450 (P-450LM) in homogeneous state. They are designated by their relative electrophoretic mobilities on polyacrylamide gel in the presence of sodium dodecyl sulfate as P-450LM2 and P-45 ... >> More

  Procedures are described for the isolation of two forms of rabbit liver microsomal liver microsomal cytochrome P-450 (P-450LM) in homogeneous state. They are designated by their relative electrophoretic mobilities on polyacrylamide gel in the presence of sodium dodecyl sulfate as P-450LM2 and P-450LM4. P-450LM2, which was isolated from phenobarbital-induced animals, has a subunit molecular weight of 48,700. The best preparations contain 20 nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. P-450LM4, which is induced by beta-naphthoflavone but is also present in phenobarbital-induced and untreated animals, was isolated from all three sources and found to have a subunit molecular weight of 55,300. The best preparations contain 17nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. Some of the purified preparations of the cytochromes, although electrophoretically homogeneous, contain apoenzyme due to heme loss during purification. The purified proteins contain no detectable NADPH-cytochrome P-450 reductase, cytochrome b5, or NADH-cytochrome b5 reductase, and only low levels of phospholipid (about 1 molecule per subunit). Amino acid analysis indicated that P-450LM2 and P-450LM4 are similar in composition, but the latter protein has about 60 additional residues. The COOH-terminal amino acid of P-450LM2 is arginine, as shown by carboxypeptidase treatment, whereas that of P-450LM4 is lysine. NH2-terminal amino acid residues could not be detected. Carbohydrate analysis indicated that both cytochromes contain 1 residue of glucosamine and 2 of mannose per polypeptide subunit. The optical spectra of the oxidized and reduced cytochromes and carbon monoxide complexes were determined. Oxidized P-450LM2 has maxima at 568, 535, and 418 nm characteristic of a low spin hemeprotein, and P450LM4 from beta-naphthoflavone-induced, phenobarbital-induced, or control microsomes has maxima at 645 and 394 nm, characteristic of the high spin state. The spectrum of -450lm4 becomes similar to that of P-450LM2 at high protein concentrations or upon the addition of detergent (Renex), whereas the spectrum of P-450LM2 is unaffected by the protein concentration or the presence of detergent. Electron paramagnetic resonance spectrometry of the purified cytochromes indicated that oxidized -450lm2 is in the low spin state, whereas P-450LM4 is largely, but not entirely, in the high spin state. << Less

  J Biol Chem 251:7929-7939(1976) [PubMed] [EuropePMC]

• Aminopyrine metabolism by multiple forms of cytochrome P-450 from rat liver microsomes: simultaneous quantitation of four aminopyrine metabolites by high-performance liquid chromatography.

  Imaoka S., Inoue K., Funae Y.

  Four aminopyrine metabolites generated by hepatic microsomes were simultaneously assayed by high-performance liquid chromatography. The metabolites were 4-monomethylaminoantipyrine (MAA), 4-aminoantipyrine (AA), 3-hydroxymethyl-2-methyl-4-dimethylamino-1-phenyl-3-pyrazoline-5-one (AM-OH), and one ... >> More

  Four aminopyrine metabolites generated by hepatic microsomes were simultaneously assayed by high-performance liquid chromatography. The metabolites were 4-monomethylaminoantipyrine (MAA), 4-aminoantipyrine (AA), 3-hydroxymethyl-2-methyl-4-dimethylamino-1-phenyl-3-pyrazoline-5-one (AM-OH), and one unidentified metabolite. MAA was the major metabolite generated by the microsomes; its formation was induced by phenobarbital but not by 3-methylcholanthrene. Female rats had lower N-demethylation activity of aminopyrine than male rats. The production of AA by microsomes was low. The formation of AM-OH was strongly induced by phenobarbital, but treatment with 3-methylcholanthrene reduced its formation. These differences in the microsomal aminopyrine monooxygenase activity are dependent on the relative amounts of the individual cytochrome P-450 isozymes. Therefore, we examined aminopyrine metabolism in a reconstituted system with purified cytochrome P-450s. P-450 UT-2 (P-450h) had high aminopyrine N-demethylation and hydroxylation activities, but P-450 F-2 (P-450i) had low N-demethylation activity and no hydroxylation activities, but P-450 F-2 (P-450i) had low N-demethylation activity and no hydroxylation activity. P-450 PB-4 (P-450b) and P-450 PB-5 (P-450e) had high aminopyrine hydroxylation activity and their N-demethylation activity also was high. The 3-methylcholanthrene-inducible forms P-450 MC-1 (P-450d) and MC-5 (P-450c) had aminopyrine N-demethylation activity but no hydroxylation activity. P-450 UT-4 (RLM2) is a unique form that produced a large amount of the unknown metabolite. P-450 UT-7 had the highest N-demethylation activity. Addition of cytochrome b5 to the reconstituted system enhanced the aminopyrine hydroxylation activities of P-450s UT-1, UT-2, PB-2, and PB-5. Also, the N-demethylation activities of P-450s UT-1, PB-1, PB-2, and MC-1 were increased by cytochrome b5. Metyrapone inhibited the catalytic activities of P-450s PB-4, PB-5, MC-1, and MC-5, and especially those of P-450s UT-4, and UT-7. The kinetics of the four major cytochrome P-450s (P-450 UT-2, UT-4, PB-4, and MC-5) for aminopyrine N-demethylation and hydroxylation activities were studied. P-450s PB-4 and UT-2 had similar Km values (0.50 and 0.62 mM, respectively) in aminopyrine N-demethylation activity.(ABSTRACT TRUNCATED AT 400 WORDS) << Less

  Arch Biochem Biophys 265:159-170(1988) [PubMed] [EuropePMC]