Purification and characterization of an epoxide hydrolase from the peroxisomal fraction of mouse liver.
Epoxide hydrolase (EH) activity has been reported to occur in most subcellular fractions of mouse liver. The EHs in the microsomal and cytosolic fractions have been purified and characterized; however, the nature of the EH(s) in the peroxisomal fraction is not known. Therefore an EH, pEH, was purified from the solubilized 12,000g fraction, which contain peroxisomes. Previous studies have demonstrated that the EH activity in this crude solubilized 12,000g fraction resides mostly in the peroxisomes. Thus the crude 12,000g pellet from mouse liver, free from cytosolic contamination, was sonicated to obtain a 105,000g soluble fraction containing 80% of the original EH activity in this fraction. The pEH was purified, using trans-stilbene oxide (TSO) as substrate, by a combination of affinity and hydroxyapatite chromatography. The purified pEH had a native molecular weight of 57 kDa, a molecular weight of 59 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 5.7. The purified pEH was observed to be immunologically similar to the cytosolic EH (cEH). The kinetics of hydrolysis of TSO, however, were slightly different. Lineweaver-Burk plots for the inhibition of pEH suggest a probable noncompetitive, mixed-type inhibition. The purified pEH thus appears to be very similar to the cEH. There are minor differences between the purified cEH and pEH, particularly in the kinetic parameters. However, these minor differences are insignificant. These results demonstrate that the cEH and pEH are substantially similar, if not identical.
Chang
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Department of Entomology, University of California, Riverside 92521.
Gill
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SS
D000818:Animals; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004851:Epoxide Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007526:Isoelectric Point; D007700:Kinetics; D008099:Liver / Q000201:enzymology / Q000648:ultrastructure*; D008297:Male; D051379:Mice; D008830:Microbodies / Q000201:enzymology*; D008970:Molecular Weight; D010449:Peptide Mapping
D004851:Epoxide Hydrolases