Determination of sphingosine kinase activity for cellular signaling studies
Determination of sphingosine kinase activity for cellular signaling studies
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DOI：
10.1021/ac702305q
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发表时间：
2008-03-01
影响因子：
7.4
通讯作者：
Allbritton, Nancy L.
中科院分区：
文献类型：
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作者：
Lee, Katherine J.;Mwongela, Simon M.;Allbritton, Nancy L.
由于鞘氨醇和1-磷酸鞘氨醇在细胞信号转导中的重要性，对它们浓度的调节越来越受到人们的关注。此外，调节鞘氨醇代谢物细胞内和细胞外水平的新药物在临床前和临床试验中显示出良好的前景。在本工作中，建立了一种基于毛细管电泳联用激光诱导荧光检测的定量方法来检测鞘氨醇和鞘氨醇-1-磷酸的相互转化。该方法可以用纯化的酶在体外同时测定磷酸酶和磷酸酶的活性。其荧光底物的Km值为38+/-18µM，V-max为0.4+/-0.2µM/min，k(CAT)为3900 S(-1)。神经鞘氨酸激酶的药理抑制作用也呈浓度依赖关系。此外，荧光底物很容易被哺乳动物细胞吸收，这使得研究活细胞中鞘氨醇激酶的内源性活性成为可能。该方法适用于大量细胞裂解物或极少量完整细胞的使用。这一新的方法在体外和基于细胞的分析中在灵敏度、定量和人力方面都优于标准方法。
Regulation of sphingosine and sphingosine-1-phosphate concentrations is of growing interest due to their importance in cellular signal transduction. Furthermore, new pharmaceutical agents moderating the intracellular and extracellular levels of sphingosine metabolites are showing promise in preclinical and clinical trials. In the present work, a quantitative assay relying on capillary electrophoresis with laser-induced fluorescence detection was developed to measure the interconversion of sphingosine and sphingosine-1-phosphate. The assay was demonstrated to be capable of determining the in vitro activity of both kinase and phosphatase using purified enzymes. The Km of sphingosine kinase for its fluorescently labeled substrate was 38 +/- 18 mu M with a V-max of 0.4 +/- 0.2 mu M/ min and a k(cat) of 3900 s(-1). Pharmacologic inhibition of sphingosine kinase in a concentration-dependent manner was also demonstrated. Moreover, the fluorescent substrate was shown to be readily taken up by mammalian cells making it possible to study the endogenous activity of sphingosine kinase activity in living cells. The method was readily adaptable to the use of either bulk cell lysates or very small numbers of intact cells. This new methodology provides enhancements over standard methods in sensitivity, quantification, and manpower for both in vitro and cell-based assays.