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. 1987 Nov 15;248(1):301–304. doi: 10.1042/bj2480301

Monoclonal antibodies against human cytochrome P-450 recognizing different pregnenolone 16 alpha-carbonitrile-inducible rat cytochromes P-450.

T S Barnes 1, P M Shaw 1, M D Burke 1, W T Melvin 1
PMCID: PMC1148537  PMID: 3435446

Abstract

Six murine monoclonal antibodies against human hepatic cytochrome P-450 have been raised, using human liver microsomes (microsomal fractions) or semi-purified human cytochrome P-450 as immunogen. All six antibodies recognized the same highly purified of human liver cytochrome P-450 of molecular mass 53 kDa and gave rise to a single band at 53 kDa on immunoblots of human liver microsomes from 11 individuals. The antibodies also recognized proteins at 52 kDa and 54 kDa on immunoblots of control and induced male-rat liver microsomes, showing four different banding patterns. Antibodies HL4 and HP16 recognized a 52 kDa protein that was only weakly expressed in untreated rats and which was strongly induced by pregnenolone 16 alpha-carbonitrile (PCN) but not by phenobarbitone (PB), 3-methylcholanthrene (3MC), isosafrole (ISF), Aroclor 1254 (ARO), clofibrate or imidazole. HP10 and HL5 recognized a constitutive 52 kDa protein that was weakly induced by PCN but not by the other agents and was suppressed by 3MC and ARO. HP3 recognized a 54 kDa protein that was undetectable in control rats but was strongly induced by PB, PCN, ISF and ARO. HL3 appeared to recognize a combination of the proteins recognized by the other antibodies plus a 54 kDa protein that was weakly expressed in control rats. The constitutive proteins recognized were male-specific.

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Selected References

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  1. Adams D. J., Seilman S., Amelizad Z., Oesch F., Wolf C. R. Identification of human cytochromes P-450 analogous to forms induced by phenobarbital and 3-methylcholanthrene in the rat. Biochem J. 1985 Dec 15;232(3):869–876. doi: 10.1042/bj2320869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adesnik M., Atchison M. Genes for cytochrome P-450 and their regulation. CRC Crit Rev Biochem. 1986;19(3):247–305. doi: 10.3109/10409238609084657. [DOI] [PubMed] [Google Scholar]
  3. Beaune P. H., Umbenhauer D. R., Bork R. W., Lloyd R. S., Guengerich F. P. Isolation and sequence determination of a cDNA clone related to human cytochrome P-450 nifedipine oxidase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8064–8068. doi: 10.1073/pnas.83.21.8064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beaune P., Kremers P., Letawe-Goujon F., Gielen J. E. Monoclonal antibodies against human liver cytochrome P-450. Biochem Pharmacol. 1985 Oct 1;34(19):3547–3552. doi: 10.1016/0006-2952(85)90732-4. [DOI] [PubMed] [Google Scholar]
  5. Burke M. D., Falzon M., Milton A. S. Decreased hepatic microsomal cytochrome P450 due to indomethacin: protective roles of 16,16-dimethylprostaglandin F2 alpha and inducing agents. Biochem Pharmacol. 1983 Feb 1;32(3):389–397. doi: 10.1016/0006-2952(83)90516-6. [DOI] [PubMed] [Google Scholar]
  6. Dannan G. A., Guengerich F. P., Kaminsky L. S., Aust S. D. Regulation of cytochrome P-450. Immunochemical quantitation of eight isozymes in liver microsomes of rats treated with polybrominated biphenyl congeners. J Biol Chem. 1983 Jan 25;258(2):1282–1288. [PubMed] [Google Scholar]
  7. Distlerath L. M., Reilly P. E., Martin M. V., Davis G. G., Wilkinson G. R., Guengerich F. P. Purification and characterization of the human liver cytochromes P-450 involved in debrisoquine 4-hydroxylation and phenacetin O-deethylation, two prototypes for genetic polymorphism in oxidative drug metabolism. J Biol Chem. 1985 Jul 25;260(15):9057–9067. [PubMed] [Google Scholar]
  8. Elshourbagy N. A., Guzelian P. S. Separation, purification, and characterization of a novel form of hepatic cytochrome P-450 from rats treated with pregnenolone-16 alpha-carbonitrile. J Biol Chem. 1980 Feb 25;255(4):1279–1285. [PubMed] [Google Scholar]
  9. Gelboin H. V., Friedman F. K. Monoclonal antibodies for studies on xenobiotic and endobiotic metabolism. Cytochromes P-450 as paradigm. Biochem Pharmacol. 1985 Jul 1;34(13):2225–2234. doi: 10.1016/0006-2952(85)90776-2. [DOI] [PubMed] [Google Scholar]
  10. Gonzalez F. J., Song B. J., Hardwick J. P. Pregnenolone 16 alpha-carbonitrile-inducible P-450 gene family: gene conversion and differential regulation. Mol Cell Biol. 1986 Aug;6(8):2969–2976. doi: 10.1128/mcb.6.8.2969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Guengerich F. P., Martin M. V., Beaune P. H., Kremers P., Wolff T., Waxman D. J. Characterization of rat and human liver microsomal cytochrome P-450 forms involved in nifedipine oxidation, a prototype for genetic polymorphism in oxidative drug metabolism. J Biol Chem. 1986 Apr 15;261(11):5051–5060. [PubMed] [Google Scholar]
  12. Kalow W. Genetic variation in the human hepatic cytochrome P-450 system. Eur J Clin Pharmacol. 1987;31(6):633–641. doi: 10.1007/BF00541288. [DOI] [PubMed] [Google Scholar]
  13. Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. doi: 10.1038/256495a0. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Mendelson C. R., Wright E. E., Evans C. T., Porter J. C., Simpson E. R. Preparation and characterization of polyclonal and monoclonal antibodies against human aromatase cytochrome P-450 (P-450AROM), and their use in its purification. Arch Biochem Biophys. 1985 Dec;243(2):480–491. doi: 10.1016/0003-9861(85)90525-9. [DOI] [PubMed] [Google Scholar]
  16. Molowa D. T., Schuetz E. G., Wrighton S. A., Watkins P. B., Kremers P., Mendez-Picon G., Parker G. A., Guzelian P. S. Complete cDNA sequence of a cytochrome P-450 inducible by glucocorticoids in human liver. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5311–5315. doi: 10.1073/pnas.83.14.5311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nebert D. W., Adesnik M., Coon M. J., Estabrook R. W., Gonzalez F. J., Guengerich F. P., Gunsalus I. C., Johnson E. F., Kemper B., Levin W. The P450 gene superfamily: recommended nomenclature. DNA. 1987 Feb;6(1):1–11. doi: 10.1089/dna.1987.6.1. [DOI] [PubMed] [Google Scholar]
  18. Park S. S., Waxman D. J., Miller H., Robinson R., Attisano C., Guengerich F. P., Gelboin H. V. Preparation and characterization of monoclonal antibodies to pregnenolone 16-alpha-carbonitrile inducible rat liver cytochrome P-450. Biochem Pharmacol. 1986 Sep 1;35(17):2859–2867. doi: 10.1016/0006-2952(86)90477-6. [DOI] [PubMed] [Google Scholar]
  19. Pelkonen O., Pasanen M., Kuha H., Gachalyi B., Kairaluoma M., Sotaniemi E. A., Park S. S., Friedman F. K., Gelboin H. V. The effect of cigarette smoking on 7-ethoxyresorufin O-deethylase and other monooxygenase activities in human liver: analyses with monoclonal antibodies. Br J Clin Pharmacol. 1986 Aug;22(2):125–134. doi: 10.1111/j.1365-2125.1986.tb05239.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Reik L. M., Levin W., Ryan D. E., Maines S. L., Thomas P. E. Monoclonal antibodies distinguish among isozymes of the cytochrome P-450b subfamily. Arch Biochem Biophys. 1985 Nov 1;242(2):365–382. doi: 10.1016/0003-9861(85)90221-8. [DOI] [PubMed] [Google Scholar]
  21. Reubi I., Griffin K. J., Raucy J., Johnson E. F. Use of a monoclonal antibody specific for rabbit microsomal cytochrome P-450 3b to characterize the participation of this cytochrome in the microsomal 6 beta- and 16 alpha-hydroxylation of progesterone. Biochemistry. 1984 Sep 25;23(20):4598–4603. doi: 10.1021/bi00315a013. [DOI] [PubMed] [Google Scholar]
  22. Sesardic D., Boobis A. R., McQuade J., Baker S., Lock E. A., Elcombe C. R., Robson R. T., Hayward C., Davies D. S. Inter-relatedness of some isoenzymes of cytochrome P-450 from rat, rabbit and human, determined with monoclonal antibodies. Biochem J. 1986 Jun 1;236(2):569–577. doi: 10.1042/bj2360569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Shimada T., Misono K. S., Guengerich F. P. Human liver microsomal cytochrome P-450 mephenytoin 4-hydroxylase, a prototype of genetic polymorphism in oxidative drug metabolism. Purification and characterization of two similar forms involved in the reaction. J Biol Chem. 1986 Jan 15;261(2):909–921. [PubMed] [Google Scholar]
  24. Song B. J., Gelboin H. V., Park S. S., Friedman F. K. Epitope-relatedness and phenotyping of hepatic cytochromes P-450 with monoclonal antibodies. Biochem J. 1985 Nov 1;231(3):671–676. doi: 10.1042/bj2310671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Thomas P. E., Reidy J., Reik L. M., Ryan D. E., Koop D. R., Levin W. Use of monoclonal antibody probes against rat hepatic cytochromes P-450c and P-450d to detect immunochemically related isozymes in liver microsomes from different species. Arch Biochem Biophys. 1984 Nov 15;235(1):239–253. doi: 10.1016/0003-9861(84)90273-x. [DOI] [PubMed] [Google Scholar]
  26. Thomas P. E., Reik L. M., Ryan D. E., Levin W. Characterization of nine monoclonal antibodies against rat hepatic cytochrome P-450c. Delineation of at least five spatially distinct epitopes. J Biol Chem. 1984 Mar 25;259(6):3890–3899. [PubMed] [Google Scholar]
  27. Wang P. P., Beaune P., Kaminsky L. S., Dannan G. A., Kadlubar F. F., Larrey D., Guengerich F. P. Purification and characterization of six cytochrome P-450 isozymes from human liver microsomes. Biochemistry. 1983 Nov 8;22(23):5375–5383. doi: 10.1021/bi00292a019. [DOI] [PubMed] [Google Scholar]
  28. Watkins P. B., Wrighton S. A., Maurel P., Schuetz E. G., Mendez-Picon G., Parker G. A., Guzelian P. S. Identification of an inducible form of cytochrome P-450 in human liver. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6310–6314. doi: 10.1073/pnas.82.18.6310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Waxman D. J., Dannan G. A., Guengerich F. P. Regulation of rat hepatic cytochrome P-450: age-dependent expression, hormonal imprinting, and xenobiotic inducibility of sex-specific isoenzymes. Biochemistry. 1985 Jul 30;24(16):4409–4417. doi: 10.1021/bi00337a023. [DOI] [PubMed] [Google Scholar]
  30. Wolf C. R., Seilman S., Oesch F., Mayer R. T., Burke M. D. Multiple forms of cytochrome P-450 related to forms induced marginally by phenobarbital. Differences in structure and in the metabolism of alkoxyresorufins. Biochem J. 1986 Nov 15;240(1):27–33. doi: 10.1042/bj2400027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wong T. K., Domin B. A., Bent P. E., Blanton T. E., Anderson M. W., Philpot R. M. Correlation of placental microsomal activities with protein detected by antibodies to rabbit cytochrome P-450 isozyme 6 in preparations from humans exposed to polychlorinated biphenyls, quaterphenyls, and dibenzofurans. Cancer Res. 1986 Feb;46(2):999–1004. [PubMed] [Google Scholar]
  32. Wrighton S. A., Campanile C., Thomas P. E., Maines S. L., Watkins P. B., Parker G., Mendez-Picon G., Haniu M., Shively J. E., Levin W. Identification of a human liver cytochrome P-450 homologous to the major isosafrole-inducible cytochrome P-450 in the rat. Mol Pharmacol. 1986 Apr;29(4):405–410. [PubMed] [Google Scholar]
  33. Wrighton S. A., Schuetz E. G., Watkins P. B., Maurel P., Barwick J., Bailey B. S., Hartle H. T., Young B., Guzelian P. Demonstration in multiple species of inducible hepatic cytochromes P-450 and their mRNAs related to the glucocorticoid-inducible cytochrome P-450 of the rat. Mol Pharmacol. 1985 Sep;28(3):312–321. [PubMed] [Google Scholar]

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