用于可溶性环氧化物水解酶的改良放射性标记底物。

Improved radiolabeled substrates for soluble epoxide hydrolase.

作者信息

Borhan B, Mebrahtu T, Nazarian S, Kurth M J, Hammock B D

机构信息

Department of Chemistry, University of California, Davis 95616, USA.

出版信息

Anal Biochem. 1995 Oct 10;231(1):188-200. doi: 10.1006/abio.1995.1520.

DOI:10.1006/abio.1995.1520

PMID:8678300

Abstract

Two rapid assays for the soluble epoxide hydrolase (sEH) are described. First, a sensitive radiometric assay based on thin-layer chromatography of [(14)C]-cis-9,10-epoxystearic acid and its corresponding diol ((14)C]-9,10-dihydroxystearic acid) is described. The cis fatty acid oxide exhibits higher specific activity of hydration with sEH from mouse, rat, human, and potato compared to trans-stilbene oxide (TSO). The K(m) and V(max) obtained for [(14)C]-cis-9,10-epoxystearic acid with mouse sEH are 11.0 microM and 3460 nmol/min/mg protein, respectively. [(14)C]-cis-9,10- Epoxystearic acid might more closely mimic the structures of natural substrates for sEH. Second, [2-(3)H]-trans-1,3-diphenyl-propene oxide ([(3)H]-tDPPO) and [2-(3)H]-cis-1,3-diphenylpropene oxide ([(3)H]-cDPPO) were synthesized and rapid radiometric assays for epoxide hydrolases (EHs) were developed by differential partitioning of the epoxide into iso-octane and its corresponding diol into aqueous phase containing methanol. It was shown that sEHs from mouse, rat, human, and potato rapidly hydrolyze [(3)H]-tDPPO and in comparison to TSO have 20-,49-,28-, and 7-fold higher rates, respectively. Mouse sEH hydrates [(3)H]-tDPPO at 26,200 nmol/min/mg protein, and a K(m)p4 of 2.80 microM is observed.

摘要

介绍了两种用于可溶性环氧化物水解酶（sEH）的快速检测方法。首先，描述了一种基于[(14)C]-顺式-9,10-环氧硬脂酸及其相应二醇（[(14)C]-9,10-二羟基硬脂酸）薄层层析的灵敏放射性检测方法。与反式芪氧化物（TSO）相比，顺式脂肪酸氧化物与小鼠、大鼠、人及马铃薯的sEH表现出更高的水合比活性。用小鼠sEH测得的[(14)C]-顺式-9,10-环氧硬脂酸的K(m)和V(max)分别为11.0微摩尔和3460纳摩尔/分钟/毫克蛋白。[(14)C]-顺式-9,10-环氧硬脂酸可能更接近sEH天然底物的结构。其次，合成了[2-(3)H]-反式-1,3-二苯基环氧丙烷（[(3)H]-tDPPO）和[2-(3)H]-顺式-1,3-二苯基环氧丙烷（[(3)H]-cDPPO），并通过将环氧化物分配到异辛烷中，将其相应二醇分配到含甲醇的水相中，开发了一种用于环氧化物水解酶（EHs）的快速放射性检测方法。结果表明，小鼠、大鼠、人及马铃薯的sEH能快速水解[(3)H]-tDPPO，与TSO相比，水解速率分别高20、49、28和7倍。小鼠sEH以26200纳摩尔/分钟/毫克蛋白的速率水合[(3)H]-tDPPO，观察到K(m)p4为2.80微摩尔。