Department of Chemistry, Kent State University, Kent, OH, USA.
Electrophoresis. 2011 Jun;32(13):1742-9. doi: 10.1002/elps.201000495.
The study of sphingosine and sphingosine-1-phosphate is now widespread due to their immense role as intra- and extracellular messenger molecules. The balance and interplay of these ceramide metabolites is dependent on the activities of kinase and phosphatase enzymes. Sphingosine and sphingosine-1-phosphate are found in very minute quantities in cells; thus, they require highly sensitive techniques for quantitative analysis. In this study, we developed a quantitative assay for the determination of sphingosine kinase 2 (SphK2) activity both in vitro and with cell lysates, using CE-LIF. Sphingosine fluorescein was used as the substrate. The K(M) of SphK2 for sphingosine fluorescein was 2.8 ± 0.8 μM with a V(max) of 2490 ± 520 μM/min and a k(cat) of 1920 ± 402/s. The inhibition of SphK2 was also investigated using four different inhibitors for which 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole inhibitor was the most potent for the in vitro inhibition of SphK2 while N,N-dimethylsphingosine (DMS) did not inhibit but rather increased SphK2 activity. The fluorescence-based approach for the determination of the enzymatic activity of SphK2 proves to be useful for the quantitative determination of SphK2 activity in vitro and in cell lysates, and could be extended to single-cell analysis or applied in drug screening.
由于鞘氨醇和鞘氨醇-1-磷酸在细胞内和细胞外信使分子中具有重要作用，因此现在对它们的研究非常广泛。这些神经酰胺代谢物的平衡和相互作用取决于激酶和磷酸酶的活性。鞘氨醇和鞘氨醇-1-磷酸在细胞中的含量非常少；因此，它们需要高度灵敏的定量分析技术。在这项研究中，我们使用 CE-LIF 开发了一种用于定量测定鞘氨醇激酶 2（SphK2）活性的方法，既可以在体外进行，也可以用于细胞裂解物。荧光素鞘氨醇用作底物。SphK2 对荧光素鞘氨醇的 K(m)为 2.8±0.8 μM，V(max)为 2490±520 μM/min，k(cat)为 1920±402/s。还使用四种不同的抑制剂研究了 SphK2 的抑制作用，其中 2-（对羟苯胺基）-4-（对氯苯基）噻唑抑制剂对 SphK2 的体外抑制作用最强，而 N，N-二甲基鞘氨醇（DMS）不仅没有抑制作用，反而增加了 SphK2 的活性。用于测定 SphK2 酶活性的基于荧光的方法被证明可用于体外和细胞裂解物中定量测定 SphK2 活性，并且可以扩展到单细胞分析或应用于药物筛选。
