Characterization of rat lung epoxide (styrene oxide) hydrase with a modified radioactive assay of improved sensitivity.

The epoxide hydrase assay developed by Oesch et al. (Biochim. Biophys. Acta, 227: 685-691, 1971) using [3H]styrene oxide as substrate was modified in three ways for use with rat lung microsomes: the substrate 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 found to be linear for at least 60 min). These modifications increased the sensitivity of the assay procedure 75- to 150-fold. The procedure was found to be 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. Its apparent vmax is 0.5 nmole of styrene glycol formed per min per mg microsomal protein, and its apparent Km was 0.11 to 0.25 mM. The pH optimum is around 9.7. Upon subcellular fractionation of lung tissue, expoxide hydrase distributes in the same manner as a marker for the endoplasmic reticulum (reduced nicotinamide adenine dinucleotide phosphate-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 aobut the same. Treatment i.p. of rats with 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 shown to be 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.

Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver.

Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver have been investigated. After perfusing the lung to remove contaminating blood, this organ was found to have an apparent concentration of glutathione (2mM) which is approx. 20% of that found in the liver. Both organs contain very low levels of glutathione disulfide. Neither phenobarbital nor methylcholanthrene had a significant effect on the levels of reduced glutathione in lung and liver. In addition, the activities of some glutathione-metabolizing enzymes--glutathione reductase and glutathione S-transferase activity assayed with four different substrates--were observed to be 5-to 60-fold lower in lung tissue than in the liver.

The topology of epoxide hydratase and benzpyrene monooxygenase in the endoplasmic reticulum of rat liver.

The distributions of benzpyrene monooxygenase and epoxide hydratase in subfractions of liver microsomes from control and from phenobarbital- and methylcholanthrene-treated rats have been investigated. The specific activities of these enzymes in rough and smooth microsomes from control and phenobarbital-treated animals are approximately the same, whereas after methylcholanthrene treatment benzpyrene monooxygenase is four times higher and epoxide hydratase twice as high in the rough vesicles. Further subfractionation of rough and smooth microsomes by rate differential centrifugation revealed the distributions of both enzymes among microsomal vesicles to be highly heterogeneous. Comparison of these distributions leads to the conclusion that the benzpyrene monooxygenase system and epoxide hydratase may form a complex of unique stoichiometry in the membrane of microsomes from control rats, but that such a complex is not consistent with the distributions obtained after methylcholanthrene induction. Studies with proteases and the nonpenetrating chemical reagent diazobenzene sulfonate suggest that epoxide hydratase may be buried deeply in the hydrophobic phase of the membrane of the hepatic endoplasmic reticulum.