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. 1999 Jun 1;340(Pt 2):453–458.

Relationship between cytochrome P450 catalytic cycling and stability: fast degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) in hepatoma cells is abolished by inactivation of its electron donor NADPH-cytochrome P450 reductase.

A Zhukov 1, M Ingelman-Sundberg 1
PMCID: PMC1220271  PMID: 10333489

Abstract

Ethanol-inducible cytochrome P450 2E1 (CYP2E1) involved in the metabolism of gluconeogenetic precursors and some cytotoxins is distinguished from other cytochrome P450 enzymes by its rapid turnover (in vivo half-life of 4-7 h), with ligands to the haem iron, both substrates and inhibitors, stabilizing the protein. CYP2E1 is also known to have a high oxidase activity in the absence of substrate, resulting in the production of reactive oxygen radicals. We suggested that the rapid intracellular turnover of the enzyme may be partly due to covalent modifications by such radicals or to other changes during catalytic cycling, in which case the inhibition of electron supply from NADPH-cytochrome P450 reductase would be expected to stabilize the protein. Fao hepatoma cells, where CYP2E1 showed a half-life of 4 h upon serum withdrawal, were treated for 1 h with 0.3 microM diphenylene iodonium (DPI), a suicide inhibitor of flavoenzymes, which resulted in approximately 90% inhibition of the microsomal NADPH-cytochrome P450 reductase and CYP2E1-dependent chlorzoxazone hydroxylase activities. Subsequent cycloheximide chase revealed that the CYP2E1 half-life increased to 26 h. Neither the degradation rates of total protein, CYP2B1 and NADPH-cytochrome P450 reductase nor the cellular ATP level were affected by DPI under the conditions employed. These results demonstrate for the first time that the short half-life of CYP2E1 in vivo may be largely due to the rapid destabilization of the enzyme during catalytic cycling rather than to the intrinsic instability of the protein molecule.

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Selected References

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  1. Barmada S., Kienle E., Koop D. R. Rabbit P450 2E1 expressed in CHO-K1 cells has a short half-life. Biochem Biophys Res Commun. 1995 Jan 17;206(2):601–607. doi: 10.1006/bbrc.1995.1085. [DOI] [PubMed] [Google Scholar]
  2. Bell L. C., Guengerich F. P. Oxidation kinetics of ethanol by human cytochrome P450 2E1. Rate-limiting product release accounts for effects of isotopic hydrogen substitution and cytochrome b5 on steady-state kinetics. J Biol Chem. 1997 Nov 21;272(47):29643–29651. doi: 10.1074/jbc.272.47.29643. [DOI] [PubMed] [Google Scholar]
  3. Berlett B. S., Stadtman E. R. Protein oxidation in aging, disease, and oxidative stress. J Biol Chem. 1997 Aug 15;272(33):20313–20316. doi: 10.1074/jbc.272.33.20313. [DOI] [PubMed] [Google Scholar]
  4. Blanck J., Ristau O., Zhukov A. A., Archakov A. I., Rein H., Ruckpaul K. Cytochrome P-450 spin state and leakiness of the monooxygenase pathway. Xenobiotica. 1991 Jan;21(1):121–135. doi: 10.3109/00498259109039456. [DOI] [PubMed] [Google Scholar]
  5. Correia M. A., Davoll S. H., Wrighton S. A., Thomas P. E. Degradation of rat liver cytochromes P450 3A after their inactivation by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine: characterization of the proteolytic system. Arch Biochem Biophys. 1992 Sep;297(2):228–238. doi: 10.1016/0003-9861(92)90666-k. [DOI] [PubMed] [Google Scholar]
  6. Dai Y., Rashba-Step J., Cederbaum A. I. Stable expression of human cytochrome P4502E1 in HepG2 cells: characterization of catalytic activities and production of reactive oxygen intermediates. Biochemistry. 1993 Jul 13;32(27):6928–6937. doi: 10.1021/bi00078a017. [DOI] [PubMed] [Google Scholar]
  7. Davies K. J. Protein damage and degradation by oxygen radicals. I. general aspects. J Biol Chem. 1987 Jul 15;262(20):9895–9901. [PubMed] [Google Scholar]
  8. Doussière J., Vignais P. V. Diphenylene iodonium as an inhibitor of the NADPH oxidase complex of bovine neutrophils. Factors controlling the inhibitory potency of diphenylene iodonium in a cell-free system of oxidase activation. Eur J Biochem. 1992 Aug 15;208(1):61–71. doi: 10.1111/j.1432-1033.1992.tb17159.x. [DOI] [PubMed] [Google Scholar]
  9. Ekström G., Ingelman-Sundberg M. Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 (P-450IIE1). Biochem Pharmacol. 1989 Apr 15;38(8):1313–1319. doi: 10.1016/0006-2952(89)90338-9. [DOI] [PubMed] [Google Scholar]
  10. Eliasson E., Johansson I., Ingelman-Sundberg M. Ligand-dependent maintenance of ethanol-inducible cytochrome P-450 in primary rat hepatocyte cell cultures. Biochem Biophys Res Commun. 1988 Jan 15;150(1):436–443. doi: 10.1016/0006-291x(88)90539-6. [DOI] [PubMed] [Google Scholar]
  11. Eliasson E., Johansson I., Ingelman-Sundberg M. Substrate-, hormone-, and cAMP-regulated cytochrome P450 degradation. Proc Natl Acad Sci U S A. 1990 Apr;87(8):3225–3229. doi: 10.1073/pnas.87.8.3225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Eliasson E., Mkrtchian S., Ingelman-Sundberg M. Hormone- and substrate-regulated intracellular degradation of cytochrome P450 (2E1) involving MgATP-activated rapid proteolysis in the endoplasmic reticulum membranes. J Biol Chem. 1992 Aug 5;267(22):15765–15769. [PubMed] [Google Scholar]
  13. Gatley S. J., Sherratt S. A. The effects of diphenyleneiodonium on mitochondrial reactions. Relation of binding of diphenylene[125I]iodonium to mitochondria to the extent of inhibition of oxygen uptake. Biochem J. 1976 Aug 15;158(2):307–315. doi: 10.1042/bj1580307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gorsky L. D., Koop D. R., Coon M. J. On the stoichiometry of the oxidase and monooxygenase reactions catalyzed by liver microsomal cytochrome P-450. Products of oxygen reduction. J Biol Chem. 1984 Jun 10;259(11):6812–6817. [PubMed] [Google Scholar]
  15. Guengerich F. P. Oxidation-reduction properties of rat liver cytochromes P-450 and NADPH-cytochrome p-450 reductase related to catalysis in reconstituted systems. Biochemistry. 1983 Jun 7;22(12):2811–2820. doi: 10.1021/bi00281a007. [DOI] [PubMed] [Google Scholar]
  16. Johansson I., Ekström G., Scholte B., Puzycki D., Jörnvall H., Ingelman-Sundberg M. Ethanol-, fasting-, and acetone-inducible cytochromes P-450 in rat liver: regulation and characteristics of enzymes belonging to the IIB and IIE gene subfamilies. Biochemistry. 1988 Mar 22;27(6):1925–1934. doi: 10.1021/bi00406a019. [DOI] [PubMed] [Google Scholar]
  17. Johansson I., Ingelman-Sundberg M. Benzene metabolism by ethanol-, acetone-, and benzene-inducible cytochrome P-450 (IIE1) in rat and rabbit liver microsomes. Cancer Res. 1988 Oct 1;48(19):5387–5390. [PubMed] [Google Scholar]
  18. Koop D. R., Morgan E. T., Tarr G. E., Coon M. J. Purification and characterization of a unique isozyme of cytochrome P-450 from liver microsomes of ethanol-treated rabbits. J Biol Chem. 1982 Jul 25;257(14):8472–8480. [PubMed] [Google Scholar]
  19. Kukielka E., Cederbaum A. I. DNA strand cleavage as a sensitive assay for the production of hydroxyl radicals by microsomes: role of cytochrome P4502E1 in the increased activity after ethanol treatment. Biochem J. 1994 Sep 15;302(Pt 3):773–779. doi: 10.1042/bj3020773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Morgan E. T., Koop D. R., Coon M. J. Catalytic activity of cytochrome P-450 isozyme 3a isolated from liver microsomes of ethanol-treated rabbits. Oxidation of alcohols. J Biol Chem. 1982 Dec 10;257(23):13951–13957. [PubMed] [Google Scholar]
  21. Murray M., Zaluzny L., Farrell G. C. Selective reactivation of steroid hydroxylases following dissociation of the isosafrole metabolite complex with rat hepatic cytochrome P-450. Arch Biochem Biophys. 1986 Dec;251(2):471–478. doi: 10.1016/0003-9861(86)90354-1. [DOI] [PubMed] [Google Scholar]
  22. Nelson D. R., Koymans L., Kamataki T., Stegeman J. J., Feyereisen R., Waxman D. J., Waterman M. R., Gotoh O., Coon M. J., Estabrook R. W. P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics. 1996 Feb;6(1):1–42. doi: 10.1097/00008571-199602000-00002. [DOI] [PubMed] [Google Scholar]
  23. Neve E. P., Eliasson E., Pronzato M. A., Albano E., Marinari U., Ingelman-Sundberg M. Enzyme-specific transport of rat liver cytochrome P450 to the Golgi apparatus. Arch Biochem Biophys. 1996 Sep 15;333(2):459–465. doi: 10.1006/abbi.1996.0415. [DOI] [PubMed] [Google Scholar]
  24. O'Donnell B. V., Tew D. G., Jones O. T., England P. J. Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase. Biochem J. 1993 Feb 15;290(Pt 1):41–49. doi: 10.1042/bj2900041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rashba-Step J., Turro N. J., Cederbaum A. I. Increased NADPH- and NADH-dependent production of superoxide and hydroxyl radical by microsomes after chronic ethanol treatment. Arch Biochem Biophys. 1993 Jan;300(1):401–408. doi: 10.1006/abbi.1993.1054. [DOI] [PubMed] [Google Scholar]
  26. Roberts B. J. Evidence of proteasome-mediated cytochrome P-450 degradation. J Biol Chem. 1997 Apr 11;272(15):9771–9778. doi: 10.1074/jbc.272.15.9771. [DOI] [PubMed] [Google Scholar]
  27. Roberts B. J., Shoaf S. E., Jeong K. S., Song B. J. Induction of CYP2E1 in liver, kidney, brain and intestine during chronic ethanol administration and withdrawal: evidence that CYP2E1 possesses a rapid phase half-life of 6 hours or less. Biochem Biophys Res Commun. 1994 Dec 15;205(2):1064–1071. doi: 10.1006/bbrc.1994.2774. [DOI] [PubMed] [Google Scholar]
  28. Ronis M. J., Johansson I., Hultenby K., Lagercrantz J., Glaumann H., Ingelman-Sundberg M. Acetone-regulated synthesis and degradation of cytochrome P450E1 and cytochrome P4502B1 in rat liver [corrected]. Eur J Biochem. 1991 Jun 1;198(2):383–389. doi: 10.1111/j.1432-1033.1991.tb16026.x. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Sadano H., Omura T. Turnover of two drug-inducible forms of microsomal cytochrome P-450 in rat liver. J Biochem. 1983 May;93(5):1375–1383. doi: 10.1093/oxfordjournals.jbchem.a134272. [DOI] [PubMed] [Google Scholar]
  31. Sakai H., Park S. S., Kikkawa Y. Differential oxidase activity of hepatic and pulmonary microsomal cytochrome P-450 isozymes after treatment with cytochrome P-450 inducers. Biochem Biophys Res Commun. 1992 Sep 30;187(3):1262–1269. doi: 10.1016/0006-291x(92)90439-r. [DOI] [PubMed] [Google Scholar]
  32. Salo D. C., Pacifici R. E., Lin S. W., Giulivi C., Davies K. J. Superoxide dismutase undergoes proteolysis and fragmentation following oxidative modification and inactivation. J Biol Chem. 1990 Jul 15;265(20):11919–11927. [PubMed] [Google Scholar]
  33. Shiraki H., Guengerich F. P. Turnover of membrane proteins: kinetics of induction and degradation of seven forms of rat liver microsomal cytochrome P-450, NADPH-cytochrome P-450 reductase, and epoxide hydrolase. Arch Biochem Biophys. 1984 Nov 15;235(1):86–96. doi: 10.1016/0003-9861(84)90257-1. [DOI] [PubMed] [Google Scholar]
  34. Sligar S. G., Cinti D. L., Gibson G. G., Schenkman J. B. Spin state control of the hepatic cytochrome P450 redox potential. Biochem Biophys Res Commun. 1979 Oct 12;90(3):925–932. doi: 10.1016/0006-291x(79)91916-8. [DOI] [PubMed] [Google Scholar]
  35. Sligar S. G. Coupling of spin, substrate, and redox equilibria in cytochrome P450. Biochemistry. 1976 Nov 30;15(24):5399–5406. doi: 10.1021/bi00669a029. [DOI] [PubMed] [Google Scholar]
  36. Song B. J., Veech R. L., Park S. S., Gelboin H. V., Gonzalez F. J. Induction of rat hepatic N-nitrosodimethylamine demethylase by acetone is due to protein stabilization. J Biol Chem. 1989 Feb 25;264(6):3568–3572. [PubMed] [Google Scholar]
  37. Steward A. R., Wrighton S. A., Pasco D. S., Fagan J. B., Li D., Guzelian P. S. Synthesis and degradation of 3-methylcholanthrene-inducible cytochromes P-450 and their mRNAs in primary monolayer cultures of adult rat hepatocytes. Arch Biochem Biophys. 1985 Sep;241(2):494–508. doi: 10.1016/0003-9861(85)90575-2. [DOI] [PubMed] [Google Scholar]
  38. Stuehr D. J., Fasehun O. A., Kwon N. S., Gross S. S., Gonzalez J. A., Levi R., Nathan C. F. Inhibition of macrophage and endothelial cell nitric oxide synthase by diphenyleneiodonium and its analogs. FASEB J. 1991 Jan;5(1):98–103. doi: 10.1096/fasebj.5.1.1703974. [DOI] [PubMed] [Google Scholar]
  39. Tew D. G. Inhibition of cytochrome P450 reductase by the diphenyliodonium cation. Kinetic analysis and covalent modifications. Biochemistry. 1993 Sep 28;32(38):10209–10215. doi: 10.1021/bi00089a042. [DOI] [PubMed] [Google Scholar]
  40. Thurman R. G., Ley H. G., Scholz R. Hepatic microsomal ethanol oxidation. Hydrogen peroxide formation and the role of catalase. Eur J Biochem. 1972 Feb;25(3):420–430. doi: 10.1111/j.1432-1033.1972.tb01711.x. [DOI] [PubMed] [Google Scholar]
  41. Tindberg N., Ingelman-Sundberg M. Expression, catalytic activity, and inducibility of cytochrome P450 2E1 (CYP2E1) in the rat central nervous system. J Neurochem. 1996 Nov;67(5):2066–2073. doi: 10.1046/j.1471-4159.1996.67052066.x. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Voorman R., Aust S. D. Inducers of cytochrome P-450d: influence on microsomal catalytic activities and differential regulation by enzyme stabilization. Arch Biochem Biophys. 1988 Apr;262(1):76–84. doi: 10.1016/0003-9861(88)90170-1. [DOI] [PubMed] [Google Scholar]
  44. Watkins P. B., Wrighton S. A., Schuetz E. G., Maurel P., Guzelian P. S. Macrolide antibiotics inhibit the degradation of the glucocorticoid-responsive cytochrome P-450p in rat hepatocytes in vivo and in primary monolayer culture. J Biol Chem. 1986 May 15;261(14):6264–6271. [PubMed] [Google Scholar]
  45. 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]
  46. Yang M. X., Cederbaum A. I. Characterization of cytochrome P4502E1 turnover in transfected HepG2 cells expressing human CYP2E1. Arch Biochem Biophys. 1997 May 1;341(1):25–33. doi: 10.1006/abbi.1997.9907. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Zhukov A., Ingelman-Sundberg M. Selective fast degradation of cytochrome P-450 2E1 in serum-deprived hepatoma cells by a mechanism sensitive to inhibitors of vesicular transport. Eur J Biochem. 1997 Jul 1;247(1):37–43. doi: 10.1111/j.1432-1033.1997.00037.x. [DOI] [PubMed] [Google Scholar]

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