A rapid assay for assessment of sphingosine kinase inhibitors and substrates.
A rapid assay for assessment of sphingosine kinase inhibitors and substrates.
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DOI：
10.1016/j.ab.2011.01.003
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发表时间：
2011-04-15
影响因子：
2.9
通讯作者：
Lynch KR
中科院分区：
文献类型：
--
作者：
Kharel Y;Mathews TP;Kennedy AJ;Houck JD;Macdonald TL;Lynch KR
鞘氨醇激酶催化磷酸盐从ATP转移到鞘氨醇以产生鞘氨醇1-磷酸，鞘氨醇1-磷酸是一种重要的生物活性脂质分子，其介导多种细胞信号传导过程。鞘氨醇激酶酶活性的常规测定使用[γ-32 P]ATP和鞘氨醇作为底物，通过有机萃取回收放射性标记的S1 P产物，通过薄层色谱法显示，并通过液体闪烁计数进行定量。虽然该测定法是灵敏和准确的，但它是缓慢和劳动密集型的，因此排除了同时筛选多于几种抑制剂化合物。在此，我们描述了一种快速且可重复的鞘氨醇激酶的96孔测定法。我们的方法，它利用了有限的溶解度的S1 P，检测放射性S1 P粘附到板的闪烁接近计数。我们的方法避免了萃取到有机溶剂中，反应后转移和色谱法。此外，我们的测定能够评估抑制剂和底物，并可以检测细胞和组织提取物中的内源性鞘氨醇激酶活性。鞘氨醇激酶的动力学参数，Km，和Ki值的抑制剂确定与我们的测定和传统的测定是无法区分的。这些结果证明，我们的测定非常适合于鞘氨醇激酶抑制剂的化学文库的筛选。
Sphingosine kinases catalyze the transfer of phosphate from ATP to sphingosine to generate sphingosine 1-phosphate, an important bioactive lipid molecule that mediates a diverse range of cell signaling processes. The conventional assay of sphingosine kinase enzymatic activity uses [γ-32P]ATP and sphingosine as substrates with the radiolabeled S1P product recovered by organic extraction, displayed by thin-layer chromatography and quantified by liquid scintillation counting. While this assay is sensitive and accurate, it is slow and labor intensive and thus precludes the simultaneous screening of more than a few inhibitor compounds. Herein we describe a 96 well assay for sphingosine kinases that is rapid and reproducible. Our method, which takes advantage of the limited solubility of S1P, detects radioactive S1P adhering to the plate by scintillation proximity counting. Our procedure obviates extraction into organic solvents, post-reaction transfers and chromatography. Further, our assay enables assessment of both inhibitors and substrates, and can detect endogenous sphingosine kinase activity in cell and tissue extracts. The sphingosine kinase kinetic parameter, Km, and the Ki values of inhibitors determined with our assay and the conventional assay were indistinguishable. These results document that our assay is well suited for the screening of chemical libraries of sphingosine kinase inhibitors.
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影响因子：
2.9
作者：
Vessey, DA;Kelley, M;Karliner, JS
通讯作者：
Karliner, JS
影响因子：
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影响因子：
4.8
作者：
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通讯作者：
Lynch, KR