[Establishment of a method for determining the sphingosine kinase activity and its initial application].

AIM:To establish the methods for determining the activity of sphingosine kinase(SPK) and the content of sphingosine 1-phosphate (S1P) in biological samples. METHODS:The ECV304 cells were transfected with pcDNA3 vector encoding Flag-labeled SPK gene. The expression of SPK was measured by Western blot assay and the activity of SPK was determined by enzymatic reaction, isotope incorporation and thin-layer chromatography methods. The S1P in biological samples was extracted, digested by alkaline phosphatase and then catalyzed by SPK. The S1P contents were determined according to the amounts of products. RESULTS:SPK gene transfection could enhance the expression and activity of SPK in cells markedly, and the cellular S1P was also increased obviously. HGF stimulation could increase the activity of SPK and cellular S1P in ECV304 cells. CONCLUSION:Methods for determining the activity of SPK and the content of SPK in biological samples were established.