An assay system for measuring the acute production of sphingosine 1-phosphate in intact monolayers
An assay system for measuring the acute production of sphingosine 1-phosphate in intact monolayers
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DOI：
10.1016/j.ab.2007.08.002
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发表时间：
2007-12-15
影响因子：
2.9
通讯作者：
Wattenberg, Binks W.
中科院分区：
文献类型：
--
作者：
Siow, Deanna L.;Wattenberg, Binks W.
鞘氨醇激酶 (SK) 是一种信号酶，可磷酸化鞘氨醇以产生 1-磷酸鞘氨醇。鞘氨醇和 1-磷酸鞘氨醇 (SIP) 属于一类生物活性鞘脂代谢物，它们在许多细胞过程中至关重要，但通常具有相反的生物学功能。多项研究已证明鞘氨醇激酶的细胞内定位是其信号传导功能的关键方面。迄今为止，已开发出鞘氨醇激酶活性测定法来测量裂解物中的活性。本地化效果消失的地方。在这里，我们概述了一个系统，其中可以使用外源添加的放射性标记 ATP 而不是氚化鞘氨醇来测量完整细胞中 SIP 的生成速率。 ATP 进入非渗透性纳米层的令人惊讶的能力是使该测定简单、高效且廉价的一个方面，但又足够灵敏以测量内源酶活性。该测定在动力学和底物依赖性方面表现良好。总体而言，该测定对于未来研究鉴定完整细胞中 SIP 生产中的 chanlyes 是理想的，例如由鞘氨醇激酶的差异细胞内靶向产生的细胞。 (C) 2007 Elsevier Inc. 保留所有权利。
Sphingosine kinase (SK) is a signaling enzyme that phosphorylates sphingosine to produce sphingosine 1-phosphate. Sphingosine and sphingosine 1-phosphate (S I P) belong to a class of bioactive sphingolipid metabolites that are critical in a number of cellular processes, yet often have opposing biological functions. The intracellular localization of sphingosine kinase has been demonstrated in multiple studies to be a critical aspect of its signaling function. To date, assays of sphingosine kinase activity have been developed for measuring activity in lysates. where the effects of localization are lost. Here we outline a system in which the rate of production of S I P can be measured in intact cells using exogenously added radiolabeled ATP instead of tritiated sphingosine. The surprising ability of ATP to enter unpermeabilized nionolayers is one aspect that makes this assay simple, efficient, and inexpensive, yet sensitive enough to measure endogenous enzyme activity. The assay is well behaved in terms of kinetics and substrate dependence. Overall, this assay is ideal for future studies to identify chanlyes in SIP production in intact cells such as those that result from the differential intracellular targeting of sphingosine kinase. (C) 2007 Elsevier Inc. All rights reserved.