Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1975 Sep;150(3):561–564. doi: 10.1042/bj1500561

Ring-andN-hydroxylation of 2-acetamidofluorene by rat liver reconstituted cytochrome P-450 enzyme system.

P D Lotlikar, K Zaleski
PMCID: PMC1165772  PMID: 813632

Abstract

The N- and ring-hydroxylation of 2-acetamidofluorene were studied with a reconstituted cytochrome P-450 enzyme from microsomal fractions of liver from both control and 3-methylcholanthrene-pretreated rats. Proteinase treatment and Triton X-100 solubilization were two important steps for partial purification of the cytochrome P-450 fraction. Both cytochrome P-450 and NADPH-cytochrome c reductase fractions were required for optimum N- and ring-hydroxylation activity. Hydroxylation activity was determined by the source of cytochrome P-450 fraction; cytochrome P-450 fraction from pretreated animals was severalfold more active than the fraction from controls. Formation of N-hydroxylated metabolites with reconstituted systems from both control and pretreated animals was greater than that with their respective whole microsomal fractions.

Full text

PDF
562

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alvares A. P., Schilling G., Levin W., Kuntzman R. Studies on the induction of CO-binding pigments in liver microsomes by phenobarbital and 3-methylcholanthrene. Biochem Biophys Res Commun. 1967 Nov 30;29(4):521–526. doi: 10.1016/0006-291x(67)90515-3. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Lotlikar P. D. Effects of 3-methylcholanthrene pretreatment on microsomal hydroxylation of 2-acetamidofluorene by various rat hepatomas. Biochem J. 1970 Jul;118(3):513–518. doi: 10.1042/bj1180513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Lotlikar P. D., Enomoto M., Miller J. A., Miller E. C. Species variations in the N- and ring-hydroxylation of 2-acetylaminofluorene and effects of 3-methylcholanthrene pretreatment. Proc Soc Exp Biol Med. 1967 Jun;125(2):341–346. doi: 10.3181/00379727-125-32086. [DOI] [PubMed] [Google Scholar]
  5. Lotlikar P. D., Luha L., Zaleski K. Reconstituted hamster liver microsomal enzyme system for N-hydroxylation of the carcinogen 2-acetylaminofluorene. Biochem Biophys Res Commun. 1974 Aug 19;59(4):1349–1355. doi: 10.1016/0006-291x(74)90462-8. [DOI] [PubMed] [Google Scholar]
  6. Lotlikar P. D., Zaleski K. Inhibitory effect of carbon monoxide on the N- and ring-hydroxylation of 2-acetamidofluorene by hamster hepatic microsomal preparations. Biochem J. 1974 Nov;144(2):427–430. doi: 10.1042/bj1440427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Lu A. Y., Junk K. W., Coon M. J. Resolution of the cytochrome P-450-containing omega-hydroxylation system of liver microsomes into three components. J Biol Chem. 1969 Jul 10;244(13):3714–3721. [PubMed] [Google Scholar]
  8. Lu A. Y., Levin W. The resolution and reconstitution of the liver microsomal hydroxylation system. Biochim Biophys Acta. 1974 Sep 16;344(2):205–240. doi: 10.1016/0304-4157(74)90004-5. [DOI] [PubMed] [Google Scholar]
  9. Lu A. Y., Levin W., West S., Jacobson M., Ryan D., Kuntzman R., Conney A. H. The role of cytochrome P-450 and P-448 in drug and steroid hydroxylations. Ann N Y Acad Sci. 1973;212:156–174. doi: 10.1111/j.1749-6632.1973.tb47594.x. [DOI] [PubMed] [Google Scholar]
  10. Lu A. Y., West S. B. Reconstituted liver microsomal enzyme system that hydroxylates drugs, other foreign compounds, and endogenous substrates. 3. Properties of the reconstituted 3,4-benzypyrene hydroxylase system. Mol Pharmacol. 1972 Sep;8(5):490–500. [PubMed] [Google Scholar]
  11. Lu A. Y., West S. B., Vore M., Ryan D., Levin W. Role of cytochrome b5 in hydroxylation by a reconstituted cytochrome P-450-containing system. J Biol Chem. 1974 Nov 10;249(21):6701–6709. [PubMed] [Google Scholar]
  12. MILLER E. C., MILLER J. A., BROWN R. R., MACDONALD J. C. On the protective action of certain polycyclic aromatic hydrocarbons against carcinogenesis by aminoazo dyes and 2-acetylaminofluorene. Cancer Res. 1958 May;18(4):469–477. [PubMed] [Google Scholar]
  13. MILLER J. A., CRAMER J. W., MILLER E. C. The N- and ringhydroxylation of 2-acetylaminofluorene during carcinogenesis in the rat. Cancer Res. 1960 Jul;20:950–962. [PubMed] [Google Scholar]
  14. Masters B. S., Baron J., Taylor W. E., Isaacson E. L., LoSpalluto J. Immunochemical studies on electron transport chains involving cytochrome P-450. I. Effects of antibodies to pig liver microsomal reduced triphosphopyridine nucleotide-cytochrome c reductase and the non-heme iron protein from bovine adrenocortical mitochondria. J Biol Chem. 1971 Jul 10;246(13):4143–4150. [PubMed] [Google Scholar]
  15. Miller J. A. Carcinogenesis by chemicals: an overview--G. H. A. Clowes memorial lecture. Cancer Res. 1970 Mar;30(3):559–576. [PubMed] [Google Scholar]
  16. 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]
  17. Strobel H. W., Lu A. Y., Heidema J., Coon M. J. Phosphatidylcholine requirement in the enzymatic reduction of hemoprotein P-450 and in fatty acid, hydrocarbon, and drug hydroxylation. J Biol Chem. 1970 Sep 25;245(18):4851–4854. [PubMed] [Google Scholar]
  18. Thorgeirsson S. S., Jollow D. J., Sasame H. A., Green I., Mitchell J. R. The role of cytochrome P-450 in N-hydroxylation of 2-acetylaminofluorene. Mol Pharmacol. 1973 May;9(3):398–404. [PubMed] [Google Scholar]
  19. Weisburger J. H., Weisburger E. K. Biochemical formation and pharmacological, toxicological, and pathological properties of hydroxylamines and hydroxamic acids. Pharmacol Rev. 1973 Mar;25(1):1–66. [PubMed] [Google Scholar]
  20. van der Hoeven T. A., Coon M. J. Preparation and properties of partially purified cytochrome P-450 and reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase from rabbit liver microsomes. J Biol Chem. 1974 Oct 10;249(19):6302–6310. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES