Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1985 Nov;4(11):2793–2800. doi: 10.1002/j.1460-2075.1985.tb04005.x

Epoxide hydrolase is a marker for the smooth endoplasmic reticulum in rat liver.

M M Galteau, B Antoine, H Reggio
PMCID: PMC554580  PMID: 4065094

Abstract

Epoxide hydrolase (EH, EC 3.3.2.3) was chosen as a potential marker for smooth endoplasmic reticulum, because this enzyme is inducible by drugs such as phenobarbital. The hypothesis was verified in rat liver using immunochemical and immunocytochemical techniques. Antibodies were raised to the purified protein. These antibodies were affinity purified using the enzyme immobilized on Sepharose Ultrogel. The specificity of the antibodies was assayed by immunoelectrotransfer (Western blot). The labelling of rat liver thin frozen sections with protein A-gold particles demonstrated that the antibodies specifically recognised smooth endoplasmic reticulum membranes. Rough endoplasmic reticulum, other intracellular organelles and plasma membrane were unlabelled.

Full text

PDF
2798

Images in this article

Selected References

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

  1. Amar-Costesec A., Wibo M., Thinès-Sempoux D., Beaufay H., Berthet J. Analytical study of microsomes and isolated subcellular membranes from rat liver. IV. Biochemical, physical, and morphological modifications of microsomal components induced by digitonin, EDTA, and pyrophosphate. J Cell Biol. 1974 Sep;62(3):717–745. doi: 10.1083/jcb.62.3.717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Batt A. M., Siest G., Oesch F. Differential regulation of two microsomal epoxide hydrolases in hyperplastic nodules from rat liver. Carcinogenesis. 1984 Sep;5(9):1205–1206. doi: 10.1093/carcin/5.9.1205. [DOI] [PubMed] [Google Scholar]
  3. Beaufay H., Amar-Costesec A., Thinès-Sempoux D., Wibo M., Robbi M., Berthet J. Analytical study of microsomes and isolated subcellular membranes from rat liver. 3. Subfractionation of the microsomal fraction by isopycnic and differential centrifugation in density gradients. J Cell Biol. 1974 Apr;61(1):213–231. doi: 10.1083/jcb.61.1.213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bolender R. P., Weibel E. R. A morphometric study of the removal of phenobarbital-induced membranes from hepatocytes after cessation of threatment. J Cell Biol. 1973 Mar;56(3):746–761. doi: 10.1083/jcb.56.3.746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bornstein W. A., Levin W., Thomas P. E., Ryan D. E., Bresnick E. Comparison of nuclear and microsomal epoxide hydrase from rat liver. Arch Biochem Biophys. 1979 Oct 15;197(2):436–446. doi: 10.1016/0003-9861(79)90265-0. [DOI] [PubMed] [Google Scholar]
  6. Brandtzaeg P. Conjugates of immunoglobulin G with different fluorochromes. I. Characterization by anionic-exchange chromatography. Scand J Immunol. 1973;2(3):273–290. doi: 10.1111/j.1365-3083.1973.tb02037.x. [DOI] [PubMed] [Google Scholar]
  7. Brown W. J., Farquhar M. G. The mannose-6-phosphate receptor for lysosomal enzymes is concentrated in cis Golgi cisternae. Cell. 1984 Feb;36(2):295–307. doi: 10.1016/0092-8674(84)90223-x. [DOI] [PubMed] [Google Scholar]
  8. Burke B., Griffiths G., Reggio H., Louvard D., Warren G. A monoclonal antibody against a 135-K Golgi membrane protein. EMBO J. 1982;1(12):1621–1628. doi: 10.1002/j.1460-2075.1982.tb01364.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  10. Colin-Neiger A., Kauffman I., Boutin J. A., Fournel S., Siest G., Batt A. M., Magdalou J. Assessment of the Mulder and Van Doorn kinetic procedure and rapid centrifugal analysis of UDP-glucuronosyltransferase activities. J Biochem Biophys Methods. 1984 Mar;9(1):69–79. doi: 10.1016/0165-022x(84)90067-8. [DOI] [PubMed] [Google Scholar]
  11. Dansette P. M., DuBois G. C., Jerina D. M. Continous fluorometric assay of epoxide hydrase activity. Anal Biochem. 1979 Sep 1;97(2):340–345. doi: 10.1016/0003-2697(79)90083-6. [DOI] [PubMed] [Google Scholar]
  12. Fleck A., Begg D. The estimation of ribonucleic acid using ultraviolet absorption measurements. Biochim Biophys Acta. 1965 Nov 8;108(3):333–339. doi: 10.1016/0005-2787(65)90025-0. [DOI] [PubMed] [Google Scholar]
  13. Fowler S., Remacle J., Trouet A., Beaufay H., Berthet J., Wibo M., Hauser P. Analytical study of microsomes and isolated subcellular membranes from rat liver. V. Immunological localization of cytochrome b5 by electron microscopy: methodology and application to various subcellular fractions. J Cell Biol. 1976 Nov;71(2):535–550. doi: 10.1083/jcb.71.2.535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gill S. S., Hammock B. D. Epoxide hydrolase activity in the mitochondrial fraction of mouse liver. Nature. 1981 May 14;291(5811):167–168. doi: 10.1038/291167a0. [DOI] [PubMed] [Google Scholar]
  15. Gonzalez F. J., Kasper C. B. Differential inducibility of nuclear envelope epoxide hydratase by trans-stilbene oxide and phenobarbital. Mol Pharmacol. 1982 Mar;21(2):511–516. [PubMed] [Google Scholar]
  16. Gram T. E. Separation of hepatic smooth and rough microsomes associated with drug-metabolizing enzymes. Methods Enzymol. 1974;31:225–237. doi: 10.1016/0076-6879(74)31024-5. [DOI] [PubMed] [Google Scholar]
  17. Green J., Griffiths G., Louvard D., Quinn P., Warren G. Passage of viral membrane proteins through the Golgi complex. J Mol Biol. 1981 Nov 15;152(4):663–698. doi: 10.1016/0022-2836(81)90122-4. [DOI] [PubMed] [Google Scholar]
  18. Griffiths G., Simons K., Warren G., Tokuyasu K. T. Immunoelectron microscopy using thin, frozen sections: application to studies of the intracellular transport of Semliki Forest virus spike glycoproteins. Methods Enzymol. 1983;96:466–485. doi: 10.1016/s0076-6879(83)96041-x. [DOI] [PubMed] [Google Scholar]
  19. Guenthner T. M., Hammock B. D., Vogel U., Oesch F. Cytosolic and microsomal epoxide hydrolases are immunologically distinguishable from each other in the rat and mouse. J Biol Chem. 1981 Apr 10;256(7):3163–3166. [PubMed] [Google Scholar]
  20. Hammock B. D., Gill S. S., Stamoudis V., Gilbert L. I. Soluble mammalian epoxide hydratase: action on juvenile hormone and other terpenoid epoxides. Comp Biochem Physiol B. 1976;53(2):263–265. doi: 10.1016/0305-0491(76)90045-6. [DOI] [PubMed] [Google Scholar]
  21. Heinemann F. S., Ozols J. The covalent structure of hepatic microsomal epoxide hydrolase. II. The complete amino acid sequence. J Biol Chem. 1984 Jan 25;259(2):797–804. [PubMed] [Google Scholar]
  22. Hiller G., Weber K. Golgi detection in mitotic and interphase cells by antibodies to secreted galactosyltransferase. Exp Cell Res. 1982 Nov;142(1):85–94. doi: 10.1016/0014-4827(82)90412-8. [DOI] [PubMed] [Google Scholar]
  23. Howell K. E., Ito A., Palade G. E. Endoplasmic reticulum marker enzymes in Golgi fractions--what does this mean? J Cell Biol. 1978 Nov;79(2 Pt 1):581–589. doi: 10.1083/jcb.79.2.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jerina D. M., Daly J. W. Arene oxides: a new aspect of drug metabolism. Science. 1974 Aug 16;185(4151):573–582. doi: 10.1126/science.185.4151.573. [DOI] [PubMed] [Google Scholar]
  25. Jones A. L., Fawcett D. W. Hypertrophy of the agranular endoplasmic reticulum in hamster liver induced by phenobarbital (with a review on the functions of this organelle in liver). J Histochem Cytochem. 1966 Mar;14(3):215–232. doi: 10.1177/14.3.215. [DOI] [PubMed] [Google Scholar]
  26. Kamdem L., Magdalou J., Siest G. Effects of aflatoxin B1 on the activity of drug-metabolizing enzymes in rat liver. Toxicol Appl Pharmacol. 1981 Sep 30;60(3):570–578. doi: 10.1016/0041-008x(81)90344-6. [DOI] [PubMed] [Google Scholar]
  27. Kamdem L., Siest G., Magdalou J. Differential toxicity of aflatoxin B1 in male and female rats: relationship with hepatic drug-metabolizing enzymes. Biochem Pharmacol. 1982 Oct 1;31(19):3057–3062. doi: 10.1016/0006-2952(82)90080-6. [DOI] [PubMed] [Google Scholar]
  28. Knowles R. G., Burchell B. A simple method for purification of epoxide hydratase from rat liver. Biochem J. 1977 May 1;163(2):381–383. doi: 10.1042/bj1630381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kreibich G., Ulrich B. L., Sabatini D. D. Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes. J Cell Biol. 1978 May;77(2):464–487. doi: 10.1083/jcb.77.2.464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kupfer A., Dennert G., Singer S. J. Polarization of the Golgi apparatus and the microtubule-organizing center within cloned natural killer cells bound to their targets. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7224–7228. doi: 10.1073/pnas.80.23.7224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kupfer A., Louvard D., Singer S. J. Polarization of the Golgi apparatus and the microtubule-organizing center in cultured fibroblasts at the edge of an experimental wound. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2603–2607. doi: 10.1073/pnas.79.8.2603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Levin W., Lu A. Y., Thomas P. E., Ryan D., Kizer D. E., Griffin M. J. Identification of epoxide hydrase as the preneoplastic antigen in rat liver hyperplastic nodules. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3240–3243. doi: 10.1073/pnas.75.7.3240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Louvard D., Morris C., Warren G., Stanley K., Winkler F., Reggio H. A monoclonal antibody to the heavy chain of clathrin. EMBO J. 1983;2(10):1655–1664. doi: 10.1002/j.1460-2075.1983.tb01640.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Louvard D., Reggio H., Warren G. Antibodies to the Golgi complex and the rough endoplasmic reticulum. J Cell Biol. 1982 Jan;92(1):92–107. doi: 10.1083/jcb.92.1.92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lu A. Y., Thomas P. E., Ryan D., Jerina D. M., Levin W. Purification of human liver microsomal epoxide hydrase. Differences in the properties of the human and rat enzymes. J Biol Chem. 1979 Jul 10;254(13):5878–5881. [PubMed] [Google Scholar]
  38. Meyer D. I., Krause E., Dobberstein B. Secretory protein translocation across membranes-the role of the "docking protein'. Nature. 1982 Jun 24;297(5868):647–650. doi: 10.1038/297647a0. [DOI] [PubMed] [Google Scholar]
  39. Meyer D. I., Louvard D., Dobberstein B. Characterization of molecules involved in protein translocation using a specific antibody. J Cell Biol. 1982 Feb;92(2):579–583. doi: 10.1083/jcb.92.2.579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Morré D. J., Cheetham R. D., Nyquist S. E. A simplified procedure for isolation of golgi apparatus from rat liver. Prep Biochem. 1972;2(1):61–69. doi: 10.1080/00327487208061453. [DOI] [PubMed] [Google Scholar]
  41. Mulder G. J., van Doorn A. B. A rapid NAD+-linked assay for microsomal uridine diphosphate glucuronyltransferase of rat liver and some observations on substrate specificity of the enzyme. Biochem J. 1975 Oct;151(1):131–140. doi: 10.1042/bj1510131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Novikoff A. B., Novikoff P. M., Stockert R. J., Becker F. F., Yam A., Poruchynsky M. S., Levin W., Thomas P. E. Immunocytochemical localization of epoxide hydrase in hyperplastic nodules induced in rat liver by 2-acetylaminofluorene. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5207–5211. doi: 10.1073/pnas.76.10.5207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Oesch F. Drug detoxification: epoxide hydrolase. Prog Clin Biol Res. 1983;135:81–105. [PubMed] [Google Scholar]
  44. Oesch F., Golan M. Specificity of mouse liver cytosolic epoxide hydrolase for K-region epoxides derived from polycyclic aromatic hydrocarbons. Cancer Lett. 1980 May;9(3):169–175. doi: 10.1016/0304-3835(80)90083-x. [DOI] [PubMed] [Google Scholar]
  45. Oesch F. Mammalian epoxide hydrases: inducible enzymes catalysing the inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and olefinic compounds. Xenobiotica. 1973 May;3(5):305–340. doi: 10.3109/00498257309151525. [DOI] [PubMed] [Google Scholar]
  46. Olmsted J. B. Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. J Biol Chem. 1981 Dec 10;256(23):11955–11957. [PubMed] [Google Scholar]
  47. REMMER H., MERKER H. J. EFFECT OF DRUGS ON THE FORMATION OF SMOOTH ENDOPLASMIC RETICULUM AND DRUG-METABOLIZING ENZYMES. Ann N Y Acad Sci. 1965 Mar 12;123:79–97. doi: 10.1111/j.1749-6632.1965.tb12247.x. [DOI] [PubMed] [Google Scholar]
  48. Reggio H., Bainton D., Harms E., Coudrier E., Louvard D. Antibodies against lysosomal membranes reveal a 100,000-mol-wt protein that cross-reacts with purified H+,K+ ATPase from gastric mucosa. J Cell Biol. 1984 Oct;99(4 Pt 1):1511–1526. doi: 10.1083/jcb.99.4.1511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Remacle J., Fowler S., Beaufay H., Amarcostesec A., Berthet J. Analytical study of microsomes and isolated subcellular membranes from rat liver. VI. Electron microscope examination of microsomes for cytochrome b5 by means of a ferritin-labeled antibody. J Cell Biol. 1976 Nov;71(2):551–564. doi: 10.1083/jcb.71.2.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Roth J., Berger E. G. Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae. J Cell Biol. 1982 Apr;93(1):223–229. doi: 10.1083/jcb.93.1.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Stasiecki P., Oesch F., Bruder G., Jarasch E. D., Franke W. W. Distribution of enzymes involved in metabolism of polycyclic aromatic hydrocarbons among rat liver endomembranes and plasma membranes. Eur J Cell Biol. 1980 Apr;21(1):79–92. [PubMed] [Google Scholar]
  52. Stäubli W., Hess R., Weibel E. R. Correlated morphometric and biochemical studies on the liver cell. II. Effects of phenobarbital on rat hepatocytes. J Cell Biol. 1969 Jul;42(1):92–112. doi: 10.1083/jcb.42.1.92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Tokuyasu K. T. A technique for ultracryotomy of cell suspensions and tissues. J Cell Biol. 1973 May;57(2):551–565. doi: 10.1083/jcb.57.2.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Vainio H. Drug hydroxylation and glucuronidation in liver microsomes of phenobarbital-treated rats. Xenobiotica. 1973 Nov;3(11):715–725. doi: 10.3109/00498257309151596. [DOI] [PubMed] [Google Scholar]
  55. Vogel-Bindel U., Bentley P., Oesch F. Endogenous role of microsomal epoxide hydrolase. Ontogenesis, induction inhibition, tissue distribution, immunological behaviour and purification of microsomal epoxide hydrolase with 16 alpha, 17 alpha-epoxyandrostene-3-one as substrate. Eur J Biochem. 1982 Aug;126(2):425–431. [PubMed] [Google Scholar]
  56. Waechter F., Bentley P., Bieri F., Stäubli W., Völkl A., Fahimi H. D. Epoxide hydrolase activity in isolated peroxisomes of mouse liver. FEBS Lett. 1983 Jul 25;158(2):225–228. doi: 10.1016/0014-5793(83)80583-3. [DOI] [PubMed] [Google Scholar]
  57. Waechter F., Bentley P., Germann M., Oesch F., Stäubli W. Immuno-electron-microscopic studies on the subcellular distribution of rat liver epoxide hydrolase and the effect of phenobarbitone and 2-acetamidofluorene treatment. Biochem J. 1982 Mar 15;202(3):677–686. doi: 10.1042/bj2020677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wang K., Feramisco J. R., Ash J. F. Fluorescent localization of contractile proteins in tissue culture cells. Methods Enzymol. 1982;85(Pt B):514–562. doi: 10.1016/0076-6879(82)85050-7. [DOI] [PubMed] [Google Scholar]
  59. Wang P., Meijer J., Guengerich F. P. Purification of human liver cytosolic epoxide hydrolase and comparison to the microsomal enzyme. Biochemistry. 1982 Nov 9;21(23):5769–5776. doi: 10.1021/bi00266a007. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES