Billich Andreas, Ettmayer Peter
Novartis Research Institute Vienna, Brunner Strasse 59, A-1235 Vienna, Austria.
Anal Biochem. 2004 Mar 1;326(1):114-9. doi: 10.1016/j.ab.2003.11.018.
Sphingosine kinase enzymatic activity is commonly measured using radiolabeled substrates, with thin-layer chromatography and/or solvent extraction needed to detect the reaction product sphingosine-1-phosphate. We developed a fluorescence-based assay, using a sphingosine derivative labeled with a 7-nitrobenz-2-oxa-1,3-diazole moiety (15-NBD-Sph). Separation of substrate (15-NBD-Sph) from product (the corresponding phosphate) is achieved by extraction with chloroform/methanol at pH 8.5. The phosphate derivative is recovered by >98% in the aqueous phase and is directly detected and quantified by its fluorescence. 15-NBD-Sph is readily phosphorylated by human and murine sphingosine kinases 1 and 2. The suitability of the assay for measuring the activity of the kinases, both in the purified state and when contained in lysates of mammalian cells, was demonstrated. The present method is a convenient alternative to the radiometric assays and is particularly suited to the search for inhibitors of sphingosine kinases.
鞘氨醇激酶的酶活性通常使用放射性标记的底物来测量，需要通过薄层色谱法和/或溶剂萃取来检测反应产物鞘氨醇-1-磷酸。我们开发了一种基于荧光的检测方法，使用一种带有7-硝基苯并-2-恶唑-1,3-二唑部分（15-NBD-Sph）标记的鞘氨醇衍生物。通过在pH 8.5下用氯仿/甲醇萃取，可将底物（15-NBD-Sph）与产物（相应的磷酸盐）分离。磷酸盐衍生物在水相中的回收率>98%，并通过其荧光直接进行检测和定量。15-NBD-Sph很容易被人和小鼠的鞘氨醇激酶1和2磷酸化。该检测方法在测量纯化状态以及哺乳动物细胞裂解物中激酶活性方面的适用性得到了证明。本方法是放射性检测方法的一种便捷替代方法，特别适用于寻找鞘氨醇激酶抑制剂。
