Purification and characterization of rat-liver cytosolic epoxide hydrolase.
Rat liver cytosolic epoxide hydrolase has been purified and characterized. The enzyme was purified from tiadenol-induced rat liver 540-fold with respect to trans-stilbene oxide as a substrate. Similar purification was obtained with the substrates trans-beta-ethyl styrene oxide and styrene 7,8-oxide, the specific activities decreasing in the order trans-beta-ethyl styrene oxide greater than styrene 7,8-oxide greater than trans-stilbene oxide. The enzyme exerts highest activity at pH 7.4 Km and Vmax of the pure enzyme for trans-stilbene oxide were 1.7 microM and 205 nmol x min-1 x mg protein-1 respectively. With trans-stilbene oxide as a substrate, the inhibition by organic solvents (2.5% by vol.) increased in the order ethanol less than methanol less than acetone less than isopropanol = N,N-dimethyl formamide less than acetonitrile less than tetrahydrofuran. The native enzyme, with a molecular mass of 120 kDa, consists of two 61-kDa subunits. Digestion of rat liver cytosolic and microsomal epoxide hydrolase by three proteases resulted in markedly different peptide maps. Western-blot analysis with antiserum against rat liver cytosolic epoxide hydrolase revealed a single band with the purified enzyme, and with liver cytosol from control and clofibrate-induced rats. No cross-reactivity was observed with purified rat microsomal epoxide hydrolase or microsomes. A positive reaction at the same molecular mass was obtained with liver cytosol of mouse, guinea pig, Syrian hamster and New Zealand white rabbit but not with that of green monkey.
D000818:Animals; D003600:Cytosol / Q000201:enzymology*; D004851:Epoxide Hydrolases / Q000302:isolation & purification*; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007120:Immunochemistry; D007526:Isoelectric Point; D008099:Liver / Q000201:enzymology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008862:Microsomes, Liver / Q000201:enzymology; D008970:Molecular Weight; D010447:Peptide Hydrolases; D010449:Peptide Mapping; D051381:Rats; D011916:Rats, Inbred F344; D011919:Rats, Inbred Strains; D012997:Solvents; D013379:Substrate Specificity
D012997:Solvents; D004851:Epoxide Hydrolases; D010447:Peptide Hydrolases