Prestwich G D, Hammock B D
Proc Natl Acad Sci U S A. 1985 Mar;82(6):1663-7. doi: 10.1073/pnas.82.6.1663.
Epoxide hydrolase from liver cytosol (cEH) of both normal and clofibrate-treated mice can be bioselectively adsorbed onto an affinity column prepared from epoxy-activated Sepharose and 7-methoxycitronellyl thiol. The free ligand is a modest inhibitor of cEH (I50, approximately equal to 3 X 10(-4) M) and lacks the epoxide function necessary for it to be turned over as a substrate. This study demonstrates that a methoxy group can be used to mimic an oxirane in a vertebrate system. Bioselective elution of cEH can be accomplished with several chalcone oxides, which are selective potent inhibitors (I50, 1-50 X 10(-7) M), and activity can be recovered by dialysis. This procedure thus enhances the purification by offering independent opportunities for selective binding and selective elution. Conservatively, a 40%-80% recovery of partially inhibited enzyme activity can be achieved in 4-48 hr with a 30- to 90-fold purification. The purified cEH from clofibrate-induced animals was essentially homogeneous by NaDodSO4/PAGE and had an apparent subunit molecular weight of 58,000. The cEHs from normal and clofibrate-induced animals appeared identical by NaDodSO4/PAGE. Since the cEH activity in normal and clofibrate-treated animals is due to the same enzyme, the increase in cEH activity caused by selected hypolipidemic agents appears to be true induction.
正常小鼠和用氯贝丁酯处理过的小鼠肝脏胞液中的环氧化物水解酶（cEH）都能被生物选择性地吸附到由环氧活化的琼脂糖和7-甲氧基香茅基硫醇制备的亲和柱上。游离配体是cEH的一种适度抑制剂（半数抑制浓度I50约为3×10⁻⁴ M），并且缺乏作为底物被转化所需的环氧化物官能团。这项研究表明，在脊椎动物系统中，甲氧基可用于模拟环氧乙烷。cEH的生物选择性洗脱可以用几种查尔酮氧化物来完成，这些查尔酮氧化物是选择性强效抑制剂（I50为1 - 50×10⁻⁷ M），并且活性可以通过透析恢复。因此，该方法通过提供选择性结合和选择性洗脱的独立机会来提高纯化效果。保守估计，在4 - 48小时内可实现部分抑制的酶活性40% - 80%的回收率，纯化倍数为30至90倍。用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（NaDodSO4/PAGE）分析，从用氯贝丁酯诱导的动物中纯化得到的cEH基本均一，其亚基表观分子量为58,000。用NaDodSO4/PAGE分析，正常动物和用氯贝丁酯诱导的动物的cEH看起来是相同的。由于正常动物和用氯贝丁酯处理的动物中的cEH活性是由同一种酶引起的，所以某些降血脂药物引起的cEH活性增加似乎是真正的诱导作用。
