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‐β‐ethyl styrene oxide and styrene 7,8‐oxide, the specific activities decreasing in the order trans ‐β‐ethyl styrene oxide > styrene 7,8‐oxide > trans ‐stilbene oxide. The enzyme exerts highest activity at pH 7.4. K m and V max of the pure enzyme for trans ‐stilbene oxide were 1.7 μ and 205 nmol × min −1 × 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 < methanol < acetone < isopropanol = N,N‐dimethyl formamide < acetonitrile < tetrahydrofuran. The native enzyme, with a molecutar mass of 120 kDa, consists of two 61‐kDa subunits. Digestion of rat liver cytosolic and microsonal epoxide hydrolase by. three proteases resulted in markedly different peptide maps. Western‐blot analysis with antiserum agnnst rat liver cystosolic epoxide hydrolase revealed a single band with the purified enzyme, and with liver cytosol from control and clofiberate‐iduced 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.