Sensitive high-performance liquid chromatographic assay for D-amino-acid oxidase activity in mammalian tissues using a fluorescent non-natural substrate, 5-fluoro-D-tryptophan
Sensitive high-performance liquid chromatographic assay for D-amino-acid oxidase activity in mammalian tissues using a fluorescent non-natural substrate, 5-fluoro-D-tryptophan
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DOI：
10.1016/j.chroma.2005.08.043
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发表时间：
2006-02-17
影响因子：
4.1
通讯作者：
Zaitsu, K
中科院分区：
文献类型：
--
作者：
Hamase, K;Nagayasu, R;Zaitsu, K
建立了一种灵敏的哺乳动物组织中D-氨基酸氧化酶（DAO）活性测定方法。以D-色氨酸（D-Trp）类似物作为DAO的底物，发现5-氟-D-色氨酸（D-FrP）为最佳底物。通过酶促反应，将D-FrP转化为高荧光产物5-氟吲哚-3-乙酸（FlAA），并通过具有荧光检测器的RP-HPLC系统测定产物。猪肾DAO的检测限为0.25 μ U，日内和日间精密度分别为4.6%（RSD，n = 5）和13.8%（RSD，5d）。通过本方法，DAO活性在小鼠脑中的详细分布首次使用个体动物，并观察到显着的活动在小脑，延髓和中脑。由于在小组织或有限的组织区域中经常需要灵敏的DAO测定，因此本方法可用于关于DAO和相关D-氨基酸的各种研究。(c)2005 Elsevier B. V.保留所有权利。
A sensitive assay for D-amino-acid oxidase (DAO) activity in mammalian tissues has been established. D-Tryptophan (D-Trp) analogs were tested as substrates for DAO, and 5-fluoro-D-tryptophan (D-FrP) was found to be the best substrate. By the enzymatic reaction, D-FrP was converted to 5-fluoroindole-3-acetic acid (FlAA), a highly fluorescent product, and the product was determined by an RP-HPLC system with a fluorescence detector. The detection limit for purified DAO (from hog kidney) was 0.25 mu U, and the within-day and day-to-day precisions of the assays were 4.6% (RSD, n = 5), and 13.8% (RSD, 5 days), respectively. By the present method, the detailed distribution of DAO activity in the mouse brain was determined using individual animals for the first time, and significant activities were observed in the cerebellum, medulla oblongata and midbrain. Because sensitive DAO assay is frequently required in small tissues or in limited-tissue regions, the present method is useful for various research studies concerning DAO and the related D-amino acids. (c) 2005 Elsevier B.V. All rights reserved.