Properties of cytosolic epoxide hydrolase purified from the liver of untreated and clofibrate-treated mice. Characterization of optimal assay conditions, substrate specificity and effects of modulators on the catalytic activity.
We have characterized certain catalytic properties of cytosolic epoxide hydrolases purified from untreated and clofibrate-treated mouse liver. The enzyme activity was found to be sensitive to oxygen, but nitrogen-saturated buffers containing dithiothreitol maintained high activity for at least 12 h at 0 degrees C. Linearity of the hydration of trans-stilbene oxide with time and protein was established, the pH optimum was broad (6.5 to 7.4) and the temperature optimum was close to 50 degrees C for both forms. The activity was independent of ionic strength, with the exception of the control form in the absence of dithiothreitol, where a lower activity was observed at low ionic strength. The activity decreased when ethanol was replaced by acetone or acetonitrile as solvent for the substrate. Tetrahydrofuran was found to be highly inhibitory, while dimethylsulfoxide had less pronounced effects. The apparent Km values were 4.9 microM, 73 microM and 1980 microM for the control form with trans-stilbene oxide, cis-stilbene oxide and styrene oxide as substrates, respectively. The Km values for the enzyme from clofibrate-treated mice were in the same range, although the V values were higher for all three substrates with this form. The highest turnover was found for trans-beta-propylstyrene oxide as substrate, followed by trans-beta-ethylstyrene oxide. Little or no activity was observed with benzo[a]pyrene 4,5-oxide or cholesterol 5,6 alpha-oxide. The enzymes were found to be sensitive to 5,5'-dithiobis(2-nitrobenzoic acid) and a phenylmercuric salt. alpha-Naphthoflavone, beta-naphthoflavone and chalcone derivatives also inhibited the activity, while none of the compounds known to activate microsomal epoxide hydrolase activated the cytosolic forms.
D000818:Animals; D002384:Catalysis; D002994:Clofibrate / Q000494:pharmacology*; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D004851:Epoxide Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D012997:Solvents / Q000494:pharmacology; D013267:Stilbenes / Q000378:metabolism; D013379:Substrate Specificity
D012997:Solvents; D013267:Stilbenes; D004851:Epoxide Hydrolases; D002994:Clofibrate; C025906:stilbene oxide