EPOXIDE HYDROLASE ACTIVITIES IN THE MICROSOMES AND THE SOLUBLE FRACTION FROM VICIA-SATIVA SEEDLINGS

Epoxide hydrolases (EC 3.3.2.3) in the microsomal and soluble fraction s of 4 d old Vicia sativa seedlings hydrolyze 9,10-epoxystearic acid t o 9,10-dihydroxystearic acid. No alteration of epoxide hydrolase activ ity was observed after treatment of the seedlings with 8 mM phenobarbi tal or 0.5 mM 2,4-dichlorophenoxyacetic acid compared to control plant s. Treatment with 1 mM clofibrate decreased the activity in the micros omes by 37%, but had no effect on the activity in the soluble fraction . Four different inhibitors of mammalian epoxide hydrolases were teste d. Preincubation of either subcellular fraction with 4-fluorochalcone oxide had no effect. Weak inhibition (25% in microsomes and 16% in the soluble fraction) was observed following preincubation with 1,1,1-tri chloropropene-2,3-oxide. After preincubation with (2S,3S)-(-)-3-(4-nit rophenyl)-glycidol, we measured a 45% decrease in soluble epoxide hydr olase activity whereas no change was observed in the microsomal epoxid e hydrolase activity. The 2R, 3R enantiomer did not affect activity in either fraction. Lowering the pH from 9 to 7.4 stimulated the activit y by 366% in the cytosol but only by 72% in the microsomes. After incu bation of a racemic mixture of 9,10-epoxystearic acid with the microso mal fraction, the chirality of the residual epoxide was 69/31 in favor of the 9S, 10R enantiomer. We determined K-m of 5.2+/-0.5 and 2.5+/-0 .4 mu M and V-max (observed) of 198+/-4.7 and 404+/-10 pmol min(-1) mg (-1) for the 9S, 10R and 9R, 10S enantiomers, respectively.