Immuno-electron-microscopic studies on the subcellular distribution of rat liver epoxide hydrolase and the effect of phenobarbitone and 2-acetamidofluorene treatment

Waechter, F.; Bentley, Philip; Germann, Markus W.; Oesch, Franz; Stäubli, W.

doi:10.1042/bj2020677

Cited by 10 publications

(7 citation statements)

References 43 publications

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“…This value would indicate that these proteins have an Neyt/Cexo orientation in the membrane of the ER. Experimental data indicate that the rat mEH has an Nexo/Ceyt orientation in the membrane [10,11]; however, these findings are at variance with a recent report on the membrane topology of mEH [12].…”

Section: Discussioncontrasting

confidence: 75%

“…A co-translational insertion into the ER was also shown for mEH, and the N-terminus of this protein is not proteolytically processed during its insertion into membranes [8,9]. There is evidence that this enzyme has a similar orientation in the membrane to that of microsomal cytochromes P-450 [10,11]. However, a recent report [12] demonstrated that mEH was identical with a bile acid transport protein which could assume two opposite orientations within the membrane.…”

Section: Introductionmentioning

confidence: 99%

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The microsomal epoxide hydrolase has a single membrane signal anchor sequence which is dispensable for the catalytic activity of this protein

Friedberg¹,

Löllmann²,

Becker³

et al. 1994

Biochemical Journal

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The microsomal epoxide hydrolase (mEH) catalyses the hydrolysis of reactive epoxides which are formed by the action of cytochromes P-450 from xenobiotics. In addition it has been suggested that mEH might mediate the transport of bile acids. For the mEH it has been shown that it is co-translationally inserted into the endoplasmic reticulum. Here we demonstrate that the N-terminal 20 amino acid residues of this protein serve as its single membrane anchor signal sequence and that the function of this sequence can also be supplied by a cytochrome P-450 (CYP2B1) anchor signal sequence. The evidence supporting this conclusion is as follows: (i) the rat mEH and a CYP2B1-mEH fusion protein, in which the CYP2B1 membrane anchor signal sequence replaced the N-terminal 20 amino acid residues of mEH, was co-translationally inserted into dog pancreas microsomes in a cell-free translation system, whereas a truncated epoxide hydrolase with a deletion of the 20 N-terminal amino acid residues was not co-translationally inserted. (ii) The mEH and the CYP2B1-mEH fusion protein, but not the truncated epoxide hydrolase, were anchored in microsomes in a cell-free translation system and in membrane fractions derived from fibroblasts which expressed these proteins heterologously. These fibroblasts were also used to evaluate the significance of the mEH membrane anchor for the catalytic activity of mEH. The mEH, the truncated mEH and the CYP-EH fusion protein were found to be enzymically active. This result shows that the membrane anchor signal sequence of mEH is dispensable for the catalytic activity of this protein. However, truncated mEH was only expressed at low levels, which might indicate that this protein is unstable.

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Section: Discussioncontrasting

confidence: 75%

Section: Introductionmentioning

confidence: 99%

The microsomal epoxide hydrolase has a single membrane signal anchor sequence which is dispensable for the catalytic activity of this protein

Friedberg¹,

Löllmann²,

Becker³

et al. 1994

Biochemical Journal

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“…We therefore decided to invert the membrane topology of mEH as an important step in the evaluation of the resulting toxicological consequences. In the course of these studies we have firmly established the mEH membrane topology, which until now has been disputed [13,14,17].…”

Section: Discussionmentioning

confidence: 99%

“…These values suggest that mEH from these species have exclusively a type II orientation in the membrane of the ER [11]. However, on the basis of labelling experiments with membrane-impermeant protein-reactive fluorescent probes, and on immuno-electron microscopy, there is evidence that the mEHlike cytochrome P-450 has a type I membrane orientation [13,14]. If this is correct, the sequence of events catalysed by cytochrome P-450 and by mEH, leading to the formation of the ultimate carcinogenic metabolites of polycyclic aromatic hydrocarbons, would take place exclusively at the cytosolic face of the ER, allowing highly efficent metabolic channelling [15].…”

Section: Introductionmentioning

confidence: 99%

The catalytic activity of the endoplasmic reticulum-resident protein microsomal epoxide hydrolase towards carcinogens is retained on inversion of its membrane topology

Friedberg¹,

Holler²,

Löllmann³

et al. 1996

Biochemical Journal

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Diol epoxides formed by the sequential action of cytochrome P-450 and the microsomal epoxide hydrolase (mEH) in the endoplasmic reticulum (ER) represent an important class of ultimate carcinogenic metabolites of polycyclic aromatic hydrocarbons. The role of the membrane orientation of cytochrome P-450 and mEH relative to each other in this catalytic cascade is not known. Cytochrome P-450 is known to have a type I topology. According to the algorithm of Hartman, Rapoport and Lodish [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5786-5790], which allows the prediction of the membrane topology of proteins, mEH should adopt a type II membrane topology. Experimentally, mEH membrane topology has been disputed. Here we demonstrate that, in contrast with the theoretical prediction, the rat mEH has exclusively a type I membrane topology. Moreover we show that this topology can be inverted without affecting the catalytic activity of mEH. Our conclusions are supported by the observation that two mEH constructs (mEHg1 and mEHg2), containing engineered potential glycosylation sites at two separate locations after the C-terminal site of the membrane anchor, were not glycosylated in fibroblasts. However, changing the net charge at the N-terminus of these engineered mEH proteins by +3 resulted in proteins (++mEHg1 and ++mEHg2) that became glycosylated and consequently had a type II topology. The sensitivity of these glycosylated proteins to endoglycosidase H indicated that, like the native mEH, they are still retained in the ER. The engineered mEH proteins were integrated into membranes as they were resistant to alkaline extraction. Interestingly, an insect mEH with a charge distribution in its N-terminus similar to ++mEHg1 has recently been isolated. This enzyme might well display a type II topology instead of the type I topology of the rat mEH. Importantly, mEHg1, having the natural cytosolic orientation, as well as ++mEHg1, having an artificial huminal orientation, displayed rather similar substrate turnovers for the mutagenic metabolite benzo[a]pyrene 4,5-oxide. To our knowledge this is the first report demonstrating that topological inversion of a protein within the membrane of the ER has only a moderate effect on its enzymic activity, despite differences in folding pathways and redox environments on each side of the membrane. This observation represents an important step in the evaluation of the influence of mEH membrane orientation in the cascade of events leading to the formation of ultimate carcinogenic metabolites, and for studying the general importance of metabolic channelling on the surface of membranes.

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“…The microsomal epoxide hydrolase which has been studied in detail [reviews 3,4] has been purified from rat, rabbit and human livers [5 -71. This enzyme has immunological properties very similar to those of the epoxide hydrolase found in nuclear membranes [8,9]. On the other hand, the cytoplasmic hydrolase appears to differ markedly from the microsomal hydrolase, and the two enzymes are immunologically distinguishable [lo].…”

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confidence: 89%

Purification and Characterization of a Soluble Epoxide Hydrolase from Rabbit Liver

et al. 1982

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A method for the extensive purification of rabbit liver cytoplasmic epoxide hydrolase is described. The endproduct, which was purified 550-fold with respect to the cytosolic fraction, appeared to be more than 85 ' %, pure. Results indicate that the enzyme is a dimer of molecular weight I10000 and consists of two subunits,which are identical or very similar ( M , 57000) and possess N-terminal serine. Evidence for the existence of aggregates of higher molecular weight was also obtained. The catalytic properties of the cytoplasmic enzyme with styrene oxide as substrate ( K , = 3.4 m M ; V = 3480 nmol mg-' min-') differed markedly from the values published for the microsomal hydrolase.

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Immuno-electron-microscopic studies on the subcellular distribution of rat liver epoxide hydrolase and the effect of phenobarbitone and 2-acetamidofluorene treatment

Cited by 10 publications

References 43 publications

The microsomal epoxide hydrolase has a single membrane signal anchor sequence which is dispensable for the catalytic activity of this protein

The microsomal epoxide hydrolase has a single membrane signal anchor sequence which is dispensable for the catalytic activity of this protein

The catalytic activity of the endoplasmic reticulum-resident protein microsomal epoxide hydrolase towards carcinogens is retained on inversion of its membrane topology

Purification and Characterization of a Soluble Epoxide Hydrolase from Rabbit Liver

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