Properties of cytosolic epoxide hydrolase purified from the liver of untreated and clofibrate-treated mice. Purification procedure and physicochemical characterization of the pure enzymes

Abstract

Cytosolic epoxide hydrolase was purified from the liver of untreated and clofibrate-treated male C57B1/6 mice. The purification procedure involves chromatography on DEAE-cellulose, phenyl-Sepharose and hydroxyapatite, takes 2 days to perform and results in a 120-fold purification and .apprx. 35% yield of the enzyme from untreated mice. The purified enzyme is a dimer with a molecular mass of 120 kDa [kilodaltons], at Stokes'' radius of 4.2 nm, a frictional ratio of 1.0 and an isoelectric point of 5.5. The subunits behave identically upon isoelectric focusing in 8 M urea and only 1 band with a molecular mass of 60 kDa is seen after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The form purified from clofibrate-treated mice had very similar properties and was apparently identical to the control form as judged by amino acid analysis and peptide mapping as well. The cytosolic enzyme is clearly different from microsomal epoxide hydrolase isolated from rat liver. Ouchterlony immunodiffusion using antibodies raised in rabbits towards the control form of cytosolic epoxide hydrolase revealed identity between the 2 forms of cytosolic epoxide hydrolase, but no reaction with the microsomal epoxide hydrolase was observed. Large structural differences between the cytosolic and microsomal forms of epoxide hydrolase in the liver are indicated.