Dept. of Biomedicinal Chemistry, Institute for Advanced Chemistry of Catalonia (IQAC), Spanish National Research Council (CSIC), Barcelona, Spain.
Chem Phys Lipids. 2012 Feb;165(2):225-31. doi: 10.1016/j.chemphyslip.2012.01.001. Epub 2012 Jan 12.
Sphingosine-1-phosphate lyase (SGPL1) is the last enzyme in the catabolism of sphingolipids. It catalyzes the retroaldolic cleavage of long chain base phosphates into phosphoethanolamine and a fatty aldehyde. In this article we report on an easy and sensitive procedure to determine SPL activity. The assays uses C17-sphinganine-1-phosphate as substrate and the aldehyde product, pentadecanal, is quantified as its pentafluorobenzyloxime derivative by GC/MS. Derivatization of pentadecanal is performed as a one-step reaction, and the oxime product is directly injected for GC/MS analysis without any further purification. Acquisition in selected ion monitoring mode allows very high sensitivity, with a limit of detection of 281fmol. The assay is linear with both protein concentration and incubation time up to 20μg and 40min, respectively. The K(m) value obtained (6μM) is similar to that for the natural substrate sphingosine-1-phosphate. Using this method, FTY720 and deoxypyridoxine phosphate inhibited SPL with similar potencies to those reported.
鞘氨醇-1-磷酸裂解酶（SGPL1）是鞘脂类物质代谢的最后一种酶。它催化长链碱基磷酸的反醛裂解，生成磷酸乙醇胺和脂肪酸醛。本文报道了一种简单、灵敏的 SPL 活性测定方法。该测定法以 C17-鞘氨醇-1-磷酸为底物，通过 GC/MS 定量测定醛产物十五烷醛作为其五氟苯甲肟衍生物。十五烷醛的衍生化是一步反应，肟产物无需进一步纯化即可直接进样进行 GC/MS 分析。选择离子监测模式采集可获得非常高的灵敏度，检测限为 281fmol。该测定法的线性范围分别为 20μg 蛋白和 40min 孵育时间。获得的 K(m)值（6μM）与天然底物鞘氨醇-1-磷酸相似。使用该方法，FTY720 和去氧吡啶酮磷酸对 SPL 的抑制作用与报道的相似。
