Summary
The microsomal epoxide hydrolase (mEH) catalyzes the hydrolysis of reactive epoxides which are formed by the action of cytochromes P450 from xenobiotics. In addition the mEH has been found to mediate the transport of bile acids. For the mEH it has been shown that it is cotranslationally inserted into the endoplasmic reticulum. Here we demonstrate that the amino-terminal twenty amino acid residues of this protein serve as its single membrane anchor signal sequence and that the function of this sequence can be also supplied by a cytochrome P450 (CYP2B1) anchor signal sequence.
In addition we present data showing that the membrane anchor signal sequence of the mEH is dispensable for the catalytic activity of this protein. Our results indicate that it might be feasable to invert the topology of the mEH in the membrane of the endoplasmic reticulum without affecting the catalytic activity of this protein. With this strategy it will be possible to investigate whether the membrane topology of xenobiotic metabolizing enzymes is important for their role in chemical carcinogenesis.
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 302).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
Abbreviations
- ER:
-
endoplasmic reticulum
- mEH:
-
microsomal epoxide hydrolase
- δmEH:
-
truncated epoxide hydrolase
- CYP:
-
EH
- cytochrome P450:
-
mEH fusionprotein
- PAGE:
-
polyacrylamide gel electrophoresis
- PMSF:
-
phenylmehylsulfonylfluoride
References
Davies, R.L., Crespi, C.L., Rudo, K., Turner, T.R. and Langenbach, R. (1989) Development of a human cell line by selection and drug-metabolizing gene transfection with increased capacity to activate promutagens. Careinogenesis, 10, 885–891.
Gesch, F. (1973) Mammalian epoxide hydrases. Inducible enzymes catalysing the inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and olefinic compounds. Xenobiotica, 3, 305–340.
Grover, P.L. (1986) Pathways involved in the metabolism and activation of polycyclic aromatic hydrocarbons. Xenobiotica, 16, 915–931.
Wood, A.W., Levin W., Lu A.Y.H., Yagi H., Hernandez O., Jerina D.M. and Conney A.H. (1976) Metabolism of benzo (a) pyrene and benzo (a) pyrene derivatives to mutagenic products by highly purified hepatic microsomal enzymes. J. Biol. Chem., 251, 4882–4890.
Bar-Nun, S., Kreibich G., Adesnik M., Alterman L., Negishi M. and Sabatini D.D. (1980) Synthesis and insertion of cytochrome P-450 into endoplasmic reticulum membranes. Proc. Natl. Acad. Sci. USA., 77, 965–969.
De Lemos-Chiarandini, C, Frey A.B., Sabatini D.D. and Kreibich G. (1987) Determination of the membrane topology of the phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4 using site specific antibodies. J. Cell. Biol., 104, 209–219.
Szczesna-Skorupa, E., Browne N., Mead D. and Kemper B. (1988) Positive charges at the NH2 terminus convert the membrane anchor signal peptide of cytochrome P-450 to a secretory signal peptide. Proc. Natl. Acad. Sci. USA., 85, 738–742.
Okada, Y., Frey A.B., Guenthner T.M., Oesch F., Sabatini D.D. and Kreibich G. (1982) Studies on the biosynthesis of microsomal membrane proteins. Eur. J. Biochem., 122, 393–402.
Gonzales, F.J. and Kasper C.B. (1980) Biochem. Biophys. Res.Commun., 93, 1254–1258.
Craft, J.A., Baird S., Lamont M. and Burchell B. (1990) Membrane topology of epoxide hydrolase. Biochem. Biophys. Acta, 1046, 32–39.
Waechter, F., Bentley P., German M., Oesch F. and Stäubli W. (1982) Immuno-electron-microscopic studies on the subcellular distribution of rat liver epoxide hydrolase and the effect of phenobarbitone and 2-acetamidofluorene treatment. Biochem. J., 202, 677–686.
Alves, C, Vondippe P., Amoui M. and Levy D. (1993) Bile acid transport into hepatocyte smooth endoplasmic reticulum vesicles is mediated by microsomal epoxide hydrolase, a membrane protein exhibiting two distinct topological orientations. J Biol Chem, 268, 20148–20155.
Porter, T.D., Beck T.W. and Kasper C.B. (1986) Complementary DNA and amino acid sequence of rat liver microsomal, xenobiotic epoxide hydrolase. Arch. Biochem. Biophys., 248, 121–129.
Yabusaki, Y., Murakami H., Sakaki T., Shibata M. and Ohkawa H. (1988) Genetically engineered modification of P450 monooxygenases: Functional analysis of the amino-terminal hydrophobic region and hinge region of the P450/reductase fused enzyme. DNA, 7, 701–711.
Sagara, Y., Barnes H.J. and Waterman M.R. (1993) Expression in Escherichia coli of functional P450c17 lacking its hydrophobic amino-terminal signal anchor. Arch. Biochem. Biophys., 304, 272–278.
Larson, J.R., Coon M.J. and Porter T.D. (1993) Alcohol inducible cytochrome P450IIEl lacking the hydrophobic NH2-terminal segment retains catalytic activity and is membrane bound when expressed in E. coli. J. Biol. Chem., 266, 7321–7324.
Friedberg, T., Lollmann B., Becker R., Holler R. and Oesch F. (1994) The microsomal epoxide hydrolase has a single membrane signal anchor sequence which is dispensable for the catalytic activity of this protein. Biochem J, 303, 967–972.
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.
Glatt, H.R., Gemperlein I., Setiabudi F., Platt K.L. and Oesch F. (1990) Expression of xenobiotic metabolising enzymes in propagatable cell cultures and induction of micronuclei by 13 compounds. Mutagenesis, 5, 241–249.
Clark, B.J. and Waterman M.R. (1991) Heterologous expression of mammalian P450 in COS cells. In Waterman, M.R. and Johnson, E.F. (eds), Methods in Enzymology. Vol. 206. Academic Press, San Diego, pp. 100–108.
Clark, B.J. and Waterman M.R. (1991) The hydrophobic amino-terminal sequence of bovine 17a hydroxylase is required for the expression of a functional hemoprotein in COS 1 cells. J. Biol. Chem., 266, 5898–5904.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1996 Springer Science+Business Media New York
About this chapter
Cite this chapter
Friedberg, T., Löllmann, B., Becker, R., Holler, R., Arand, M., Oesch, F. (1996). Investigating the Role of the Microsomal Epoxide Hydrolase Membrane Topology and Its Implication for Drug Metabolism Pathways. In: Snyder, R., et al. Biological Reactive Intermediates V. Advances in Experimental Medicine and Biology, vol 387. Springer, Boston, MA. https://doi.org/10.1007/978-1-4757-9480-9_3
Download citation
DOI: https://doi.org/10.1007/978-1-4757-9480-9_3
Publisher Name: Springer, Boston, MA
Print ISBN: 978-1-4757-9482-3
Online ISBN: 978-1-4757-9480-9
eBook Packages: Springer Book Archive