Rapid purification of cytosolic epoxide hydrolase from normal and clofibrate-treated animals by affinity chromatography

G. D. Prestwich, B. D. Hammock

Research output: Contribution to journalArticle

27 Scopus citations

Abstract

Epoxide hydrolase from liver cytosol (cEH) of both normal and clofibrate-treated mice can be bioselectively absorbed onto an affinity column prepared from epoxy-activated Sepharose and 7-methoxycitronellyl thiol. The free ligand is a modest inhibitor of cEH (I50, ≃ 3 x 10-4 M) and lacks the epoxide function necessary for it to be turned over as a substrate. This study demonstrates that a methoxy group can be used to mimic an oxirane in a vertebrate system. Bioselective elution of cEH can be accomplished with several chalcone oxides, which are selective potent inhibitors (I50, 1-50 x 10-7 M), and activity can be recovered by dialysis. This procedure thus enhances the purification by offering independent opportunities for selective binding and selective elution. Conservatively, a 40%-80% recovery of partially inhibited enzyme activity can be achieved in 4-48 hr with a 30- to 90-fold purification. The purified cEH from clofibrate-induced animals was essentially homogeneous by NaDodSO4/PAGE and had an apparent subunit molecular weight of 58,000. The cEHs from normal and clofibrate-induced animals appeared identical by NaDodSO4/PAGE. Since the cEH activity in normal and clofibrate-treated animals is due to the same enzyme, the increase in cEH activity caused by selected hypolipidemic agents appears to be true induction.

Original languageEnglish (US)
Pages (from-to)1663-1667
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume82
Issue number6
StatePublished - 1985
Externally publishedYes

ASJC Scopus subject areas

  • General
  • Genetics

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