A RAPID RADIOMETRIC ASSAY FOR MAMMALIAN CYTOSOLIC EPOXIDE HYDROLASE
A RAPID RADIOMETRIC ASSAY FOR MAMMALIAN CYTOSOLIC EPOXIDE HYDROLASE
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DOI：
10.1016/0003-2697(80)90551-5
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发表时间：
1980-01-01
影响因子：
2.9
通讯作者：
HAMMOCK, BD
中科院分区：
文献类型：
--
作者：
MULLIN, CA;HAMMOCK, BD
描述了一种快速、灵敏的细胞溶质环氧化物水解酶放射性测定法。该测定基于未反应的1-苯基-1，2-环氧丁烷（β-环丁烷）的高效分配。乙基苯乙烯氧化物）转化为异辛烷，产物二醇保留在水相中。反式-环氧化物是小鼠肝胞液中谷胱甘肽转移酶和环氧化物水解酶的优良底物，在对测定进行适当修改后，可以同时监测这两种酶。反式-β-在3种不同哺乳动物的肝胞质溶胶中，氧化乙基苯乙烯的水合速度比顺式异构体快得多，两种异构体都是微粒体环氧化物水解酶的不良底物。小鼠肝胞质溶胶以69 nmol/min-mg蛋白质水合反式-环氧化物，Km为7.2 × 10 - 6。10-5 M被观察到。用硼三氢化钠还原1-苯基-1-溴-2-丁酮，随后用碱使溴代醇混合物闭环，是放射性标记β-溴代丁酮的高效途径。氧化乙基苯乙烯。所得的顺式和反式环氧化物可通过半制备高效液相色谱法分离。
A rapid and sensitive radiometric assay for cytosolic epoxide hydrolase is described. The assay is based on the highly efficient partitioning of unreacted 1-phenyl-1,2-epoxybutane (.beta.-ethylstyrene oxide) from an aqueous mix into isooctane, with the product diol being retained in the aqueous phase. The trans-epoxide is an excellent substrate for both the glutathione transferase and epoxide hydrolase present in mouse liver cytosol, and both enzymes may be monitored simultaneously after appropriate modifications of the assay are made. Trans-.beta.-ethylstyrene oxide is hydrated much faster than the cis-isomer in the hepatic cytosol of 3 different mammalian species, and both isomers are poor substrates for the microsomal epoxide hydrolase. Mouse liver cytosol hydrates the trans-epoxide at 69 nmol/min-mg protein, and a Km of 7.2 .times. 10-5 M is observed. Reduction of 1-phenyl-1-bromo-2-butanone with sodium borotritide and subsequent ring closure of the bromohydrin mix with base was a highly efficient route for radiolabeling .beta.-ethylstyrene oxide. The resulting cis- and trans-epoxides were separable by semipreparative high-performance liquid chromatography.