Membrane topology of epoxide hydrolase.
The amino acid sequences of epoxide hydrolase from rat, rabbit and human have been subjected to hydropathy analysis and a novel model for the membrane topology of this enzyme is presented. The enzyme would appear to be retained in microsomal membranes by a single transmembrane segment located at the N-terminus and the majority (96%) of the protein is exposed at the cytosolic membrane surface. This model is significantly different from a scheme suggested by analysis of the rat enzyme alone which proposed six transmembrane domains (Porter et al. (1988) Arch. Biochem. Biophys. 248, 121-129). Experiments with rat microsomal membranes were conducted to distinguish between the two models and used proteolytic enzymes and non-permeant chemical probes. Epoxide hydrolase of intact and permeabilised membranes was resistant to digestion by a number of proteinases. However, this is likely to be related to a compact fold of the protein rather than membrane association since purified, delipidated enzyme preparations were also resistant to proteolysis. While the use of proteinases did not provide useful membrane topological information, experiments with the fluorescent probe, 3-azido-2,7-naphthalenedisulphonate strongly support the view that the majority of the protein is indeed exposed at the cytosolic surface of the membranes. The analysis illustrates the caution which must be employed in the formulation of topological models based on hydropathy plots alone and the value of considering homologous proteins.
Craft
J A
JA
Department of Biological Sciences, Glasgow College, U.K.
Baird
S
S
Lamont
M
M
Burchell
B
B
D000818:Animals; D015153:Blotting, Western; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004851:Epoxide Hydrolases; D004852:Epoxy Compounds / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000648:ultrastructure*; D008862:Microsomes, Liver / Q000201:enzymology* / Q000648:ultrastructure; D008970:Molecular Weight; D010449:Peptide Mapping; D011487:Protein Conformation; D051381:Rats; D012995:Solubility; D014357:Trypsin / Q000494:pharmacology
D004852:Epoxy Compounds; D008565:Membrane Proteins; C013690:styrene oxide; D004851:Epoxide Hydrolases; D014357:Trypsin