A click chemistry mediated in vivo activity probe for dimethylarginine dimethylaminohydrolase.
A click chemistry mediated in vivo activity probe for dimethylarginine dimethylaminohydrolase.
复制标题
点击化学介导的二甲基精氨酸二甲氨基水解酶体内活性探针。
DOI：
10.1021/ja906432e
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发表时间：
2009
影响因子：
15
通讯作者：
Fast,Walter
中科院分区：
文献类型：
--
作者：
Wang,Yun;Hu,Shougang;Fast,Walter
N ω，Nω-二甲基精氨酸（ADMA）是一种内源性的一氧化氮合酶抑制剂，是心血管疾病的生物标志物。ADMA的浓度由其分解代谢酶二甲基精氨酸二甲氨基水解酶（DDAH）的两种亚型控制，DDAH的失调已被研究为内皮功能障碍的介导因素。两部分点击化学介导的基于活性的探针N-丁-3-炔基-2-氯乙脒显示标记HEK 293 T细胞中表达的myc标记的DDAH-1，但不是无活性突变体或抑制的酶。双色蛋白质印迹技术用于测定DDAH-1的可逆抑制剂N5-（1-亚氨基丙基）-L-鸟氨酸的体内IC 50值，表明该化合物的生物利用度及其与活性位点结合的竞争。该探针为分析DDAH-1在正常和病理生理状态下的活性提供了一种新的工具，并应允许对内皮功能障碍的病因学进行更有意义的研究。
AsymmetricNω,Nω-dimethyl-l-arginine (ADMA) is an endogenously produced inhibitor of human nitric oxide synthase and an emerging biomarker for cardiovascular disease. Concentrations of ADMA are controlled by two isoforms of its catabolic enzyme dimethylarginine dimethylaminohydrolase (DDAH), the dysregulation of which has been studied as a mediating factor for endothelial dysfunction. A two-part, click-chemistry mediated activity-based probe,N-but-3-ynyl-2-chloroacetamidine, is shown to label myc-tagged DDAH-1 expressed in HEK 293T cells, but not an inactive mutant or inhibited enzyme. A two-color Western blotting technique is used to determine thein vivoIC50value for a reversible inhibitor of DDAH-1,N5-(1-iminopropyl)-l-ornithine, indicating this compound’s bioavailability and its competition for binding to the active site. This probe provides a novel tool for the analysis of DDAH-1 activity in normal and pathophysiological states and should allow more meaningful studies of the etiology of endothelial dysfunction.