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. 1986 May 1;235(3):859–868. doi: 10.1042/bj2350859

Purification and characterization of a previously unreported form of cytochrome P-448 from the liver of 3-methylcholanthrene-pretreated rats.

S L Seidel, T K Shires
PMCID: PMC1146766  PMID: 3753450

Abstract

At least four hepatic isoenzymes of cytochrome P-450 were purified and characterized from rats treated with 3-methylcholanthrene. A monoclonal antibody developed against one of the forms (designated cytochrome P-450 MC-B) and polyclonal antibodies against others were used to demonstrate that form MC-B is immunologically distinct from other methylcholanthrene-inducible forms. Limited N-terminal amino acid sequencing showed that cytochrome P-450 MC-B has a primary structure that differs from the N-terminal sequences of other established rat isoenzymes. Cytochrome P-450 MC-B has a minimum Mr of 53,000, a CO-reduced spectral maximum at 448 nm, a Soret maximum of 417 nm in the absolute oxidized spectrum and a pattern of substrate preferences that differs from those of the other methylcholanthrene-induced forms. The other forms (MC-A, MC-C and MC-D) share characteristics with isoenzymes previously reported by other investigators.

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Selected References

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  1. Botelho L. H., Ryan D. E., Levin W. Amino acid compositions and partial amino acid sequences of three highly purified forms of liver microsomal cytochrome P-450 from rats treated with polychlorinated biphenyls, phenobarbital, or 3-methylcholanthrene. J Biol Chem. 1979 Jul 10;254(13):5635–5640. [PubMed] [Google Scholar]
  2. Botelho L. H., Ryan D. E., Yuan P. M., Kutny R., Shively J. E., Levin W. Amino-terminal and carboxy-terminal sequence of hepatic microsomal cytochrome P-450d, a unique hemoprotein from rats treated with isosafrole. Biochemistry. 1982 Mar 16;21(6):1152–1155. doi: 10.1021/bi00535a007. [DOI] [PubMed] [Google Scholar]
  3. COCHIN J., AXELROD J. Biochemical and pharmacological changes in the rat following chronic administration of morphine nalorphine and normorphine. J Pharmacol Exp Ther. 1959 Feb;125(2):105–110. [PubMed] [Google Scholar]
  4. Cheng K. C., Schenkman J. B. Testosterone metabolism by cytochrome P-450 isozymes RLM3 and RLM5 and by microsomes. Metabolite identification. J Biol Chem. 1983 Oct 10;258(19):11738–11744. [PubMed] [Google Scholar]
  5. Conney A. H. Pharmacological implications of microsomal enzyme induction. Pharmacol Rev. 1967 Sep;19(3):317–366. [PubMed] [Google Scholar]
  6. Gard A. L., Pigott R., Dutton G. R. A solid-phase beta-galactosidase ELISA for detecting and quantifying monoclonal antibody binding to dissociated cell cultures of postnatal rodent cerebellum. J Neurosci Methods. 1983 May;8(1):51–60. doi: 10.1016/0165-0270(83)90051-1. [DOI] [PubMed] [Google Scholar]
  7. Goldstein J. A., Linko P., Luster M. I., Sundheimer D. W. Purification and characterization of a second form of hepatic cytochrome P-448 from rats treated with a pure polychlorinated biphenyl isomer. J Biol Chem. 1982 Mar 10;257(5):2702–2707. [PubMed] [Google Scholar]
  8. Greenlee W. F., Poland A. An improved assay of 7-ethoxycoumarin O-deethylase activity: induction of hepatic enzyme activity in C57BL/6J and DBA/2J mice by phenobarbital, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin. J Pharmacol Exp Ther. 1978 Jun;205(3):596–605. [PubMed] [Google Scholar]
  9. Guengerich F. P., Dannan G. A., Wright S. T., Martin M. V., Kaminsky L. S. Purification and characterization of liver microsomal cytochromes p-450: electrophoretic, spectral, catalytic, and immunochemical properties and inducibility of eight isozymes isolated from rats treated with phenobarbital or beta-naphthoflavone. Biochemistry. 1982 Nov 9;21(23):6019–6030. doi: 10.1021/bi00266a045. [DOI] [PubMed] [Google Scholar]
  10. Guengerich F. P., Dannan G. A., Wright S. T., Martin M. V., Kaminsky L. S. Purification and characterization of microsomal cytochrome P-450s. Xenobiotica. 1982 Nov;12(11):701–716. doi: 10.3109/00498258209038945. [DOI] [PubMed] [Google Scholar]
  11. Guengerich F. P. Separation and purification of multiple forms of microsomal cytochrome P-450. Activities of different forms of cytochrome P-450 towards several compounds of environmental interest. J Biol Chem. 1977 Jun 10;252(11):3970–3979. [PubMed] [Google Scholar]
  12. Haniu M., Ryan D. E., Iida S., Lieber C. S., Levin W., Shively J. E. NH2-terminal sequence analyses of four rat hepatic microsomal cytochromes P-450. Arch Biochem Biophys. 1984 Dec;235(2):304–311. doi: 10.1016/0003-9861(84)90202-9. [DOI] [PubMed] [Google Scholar]
  13. Hawkes R., Niday E., Gordon J. A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem. 1982 Jan 1;119(1):142–147. doi: 10.1016/0003-2697(82)90677-7. [DOI] [PubMed] [Google Scholar]
  14. Imai Y., Ito A., Sato R. Evidence for biochemically different types of vesicles in the hepatic microsomal fraction. J Biochem. 1966 Oct;60(4):417–428. doi: 10.1093/oxfordjournals.jbchem.a128453. [DOI] [PubMed] [Google Scholar]
  15. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Lu A. Y., West S. B. Multiplicity of mammalian microsomal cytochromes P-45. Pharmacol Rev. 1979 Dec;31(4):277–295. [PubMed] [Google Scholar]
  18. NETTER K. J., SEIDEL G. AN ADAPTIVELY STIMULATED O-DEMETHYLATING SYSTEM IN RAT LIVER MICROSOMES AND ITS KINETIC PROPERTIES. J Pharmacol Exp Ther. 1964 Oct;146:61–65. [PubMed] [Google Scholar]
  19. Nebert D. W., Gelboin H. V. Substrate-inducible microsomal aryl hydroxylase in mammalian cell culture. I. Assay and properties of induced enzyme. J Biol Chem. 1968 Dec 10;243(23):6242–6249. [PubMed] [Google Scholar]
  20. OMURA T., SATO R. THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. I. EVIDENCE FOR ITS HEMOPROTEIN NATURE. J Biol Chem. 1964 Jul;239:2370–2378. [PubMed] [Google Scholar]
  21. Reik L. M., Levin W., Ryan D. E., Thomas P. E. Immunochemical relatedness of rat hepatic microsomal cytochromes P-450c and P-450d. J Biol Chem. 1982 Apr 10;257(7):3950–3957. [PubMed] [Google Scholar]
  22. Ryan D. E., Iida S., Wood A. W., Thomas P. E., Lieber C. S., Levin W. Characterization of three highly purified cytochromes P-450 from hepatic microsomes of adult male rats. J Biol Chem. 1984 Jan 25;259(2):1239–1250. [PubMed] [Google Scholar]
  23. Ryan D. E., Thomas P. E., Levin W. Hepatic microsomal cytochrome P-450 from rats treated with isosafrole. Purification and characterization of four enzymic forms. J Biol Chem. 1980 Aug 25;255(16):7941–7955. [PubMed] [Google Scholar]
  24. Ryan D. E., Thomas P. E., Reik L. M., Levin W. Purification, characterization and regulation of five rat hepatic microsomal cytochrome P-450 isozymes. Xenobiotica. 1982 Nov;12(11):727–744. doi: 10.3109/00498258209038947. [DOI] [PubMed] [Google Scholar]
  25. Scearce R. M., Eisenbarth G. S. Production of monoclonal antibodies reacting with the cytoplasm and surface of differentiated cells. Methods Enzymol. 1983;103:459–469. doi: 10.1016/s0076-6879(83)03032-3. [DOI] [PubMed] [Google Scholar]
  26. Seidel S. L., Shawver L. K., Shires T. K. Detection of phenobarbital-induced cytochrome P-450 in rat hepatic microsomes using an enzyme-linked immunosorbent assay. Arch Biochem Biophys. 1984 Mar;229(2):519–531. doi: 10.1016/0003-9861(84)90183-8. [DOI] [PubMed] [Google Scholar]
  27. Tarr G. E. A general procedure for the manual sequencing of small quantities of peptides. Anal Biochem. 1975 Feb;63(2):361–370. doi: 10.1016/0003-2697(75)90358-9. [DOI] [PubMed] [Google Scholar]
  28. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Waxman D. J., Ko A., Walsh C. Regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by cytochrome P-450 isozymes purified from phenobarbital-induced rat liver. J Biol Chem. 1983 Oct 10;258(19):11937–11947. [PubMed] [Google Scholar]
  30. Waxman D. J., Walsh C. Cytochrome P-450 isozyme 1 from phenobarbital-induced rat liver: purification, characterization, and interactions with metyrapone and cytochrome b5. Biochemistry. 1983 Sep 27;22(20):4846–4855. doi: 10.1021/bi00289a035. [DOI] [PubMed] [Google Scholar]
  31. Waxman D. J., Walsh C. Phenobarbital-induced rat liver cytochrome P-450. Purification and characterization of two closely related isozymic forms. J Biol Chem. 1982 Sep 10;257(17):10446–10457. [PubMed] [Google Scholar]
  32. Wolf C. R., Oesch F. Isolation of a high spin form of cytochrome P-450 induced in rat liver by 3-methylcholanthrene. Biochem Biophys Res Commun. 1983 Mar 16;111(2):504–511. doi: 10.1016/0006-291x(83)90335-2. [DOI] [PubMed] [Google Scholar]
  33. Yasukochi Y., Masters B. S. Some properties of a detergent-solubilized NADPH-cytochrome c(cytochrome P-450) reductase purified by biospecific affinity chromatography. J Biol Chem. 1976 Sep 10;251(17):5337–5344. [PubMed] [Google Scholar]
  34. Yuan P. M., Ryan D. E., Levin W., Shively J. E. Identification and localization of amino acid substitutions between two phenobarbital-inducible rat hepatic microsomal cytochromes P-450 by micro sequence analyses. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1169–1173. doi: 10.1073/pnas.80.5.1169. [DOI] [PMC free article] [PubMed] [Google Scholar]

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