CHARACTERIZATION OF RAT LUNG EPOXIDE (STYRENE OXIDE) HYDRASE WITH A MODIFIED RADIOACTIVE ASSAY OF IMPROVED SENSITIVITY

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  • 1 January 1977
    • journal article
    • research article
    • Vol. 37  (4) , 1075-1082
Abstract
The epoxide hydrase assay developed by Oesch et al. using [3H]styrene oxide as substrate was modified in 3 ways for use with rat lung microsomes. The substrates was purified before use, the volume of the incubation mixture was scaled down 4-fold and the incubation time was extended to 45 min (activity was linear for at least 60 min). These modifications increased the sensitivity of the assay procedure 75- to 150-fold. The procedure was linear with lung microsomal protein up to at least 1.8 mg protein per incubation mixture. This modified assay for epoxide hydrase was used to characterize the enzyme in rat lung. It apparent Vmax is 0.5 nmol of styrene glycol formed per min mg microsomal protein, and its apparent Km was 0.11-0.25 mM. The pH optimum is around 9.7. On subcellular fractionation of lung tissue, expoxide hydrase distributes in the same manner as a marker for the endoplasmic reticulum (NADPH-cytochrome c reductase) and in a different way from markers for the nuclei, mitochondria, concentric lamellar organelles, lysosomes, Golgi membranes, plasma membrane and soluble cytoplasm. The specific activity of epoxide hydrase in rough and smooth lung microsomes is about the same. Treatment of rats i.p. with the carcinogen methylcholanthrene (3 injections of 20 mg/kg), phenobarbital (5 daily injections of 80 mg/kg) or styrene oxide (5 daily injections of 40 mg/kg) did not induce lung microsomal epoxide hydrase activity. 1,1,1-Trichloropropene-2,3-oxide was an uncompetitive inhibitor, and cyclohexene oxide was a noncompetitive inhibitor of this enzyme. Ethanol and butanol activate the epoxide hydrase of lung microsomes at low concentrations and inhibit it at higher concentrations.