A fluorescent plate reader assay for ceramide kinase
A fluorescent plate reader assay for ceramide kinase
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DOI：
10.1016/j.ab.2007.12.024
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发表时间：
2008-04-15
影响因子：
2.9
通讯作者：
Rosen, Hugh
中科院分区：
文献类型：
--
作者：
Don, Anthony S.;Rosen, Hugh
神经酰胺激酶及其产物神经酰胺i -磷酸参与细胞增殖和存活、胞质磷脂酶A的激活(2)、肥大细胞脱颗粒和吞噬作用。目前的神经酰胺激酶活性测定采用[(32)P]ATP，通过有机萃取和薄层色谱将标记产物与多余的ATP分离。我们开发了一种神经酰胺激酶荧光板检测方法，使用市售的C6-NBD神经酰胺（N-{6-[（7-硝基-2-1,3-苯并恶二唑-4-基）氨基]己醇}- d -红-鞘氨醇）。我们的分析是基于单一氯仿/甲醇萃取后底物和产物的差异分配。该产品在生理pH值下进入水相，直接在平板阅读器中定量。底物可以使用无脂肪酸白蛋白或洗涤剂/脂质混合胶束递送，我们发现使用白蛋白而不是洗涤剂胶束可以检测到脂质与酶的相互作用，否则可能会被忽视。我们的方法对体外和培养细胞中测定神经酰胺激酶活性都很有用，与传统的测定方法相比，它有几个优点，包括更快的速度，一次进行大量重复测定的能力，以及消除与使用放射性物质相关的环境和安全问题。(c) 2007爱思唯尔公司版权所有。
Ceramide kinase and its product ceramide I-phosphate have been implicated in cellular proliferation and survival, activation of cytosolic phospholipase A(2), mast cell degranulation, and phagocytosis. Current assays for ceramide kinase activity employ [ (32) P]ATP, with separation of labeled product from excess ATP by organic extraction and thin-layer chromatography. We have developed a fluorescent plate reader assay for ceramide kinase that uses commercially available C6-NBD ceramide (N-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl) amino]hexanoyl}- D-erythro-sphingosine). Our assay is based on the differential partitioning of substrate and product following a single chloroform/methanol extraction. The product, which partitions into the aqueous phase at physiological pH, is quantitated directly in a plate reader. The substrate may be delivered using either fatty acid-free albumin or detergent/lipid mixed micelles, and we have found that the use of albumin rather than detergent micelles allows one to detect lipid interactions with the enzyme that might otherwise go unnoticed. Our method is useful for assaying ceramide kinase activity both in vitro and in cultured cells, and it offers several advantages over the conventional assay, including greater speed, the ability to run a larger number of assay replicates at one time, and the elimination of environmental and safety issues associated with the use of radioactive materials. (c) 2007 Elsevier Inc. All rights reserved.