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 (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.
Institute of Toxicology, University of Mainz, Germany.
T
T
Friedberg
B
B
Löllmann
R
R
Becker
R
R
Holler
F
F
Oesch
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology; D002474:Cell-Free System; D018076:DNA, Complementary; D004285:Dogs; D004851:Epoxide Hydrolases / Q000235:genetics / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals / Q000235:genetics*; D051381:Rats; D013698:Templates, Genetic
D018076:DNA, Complementary; D021382:Protein Sorting Signals; D004851:Epoxide Hydrolases