An assay for sphingosine kinase activity using biotinylated sphingosine and streptavidin-coated membranes

Analytical Biochemistry
Jane L RobertsS M Pitson

Abstract

Sphingosine kinase catalyses the phosphorylation of sphingosine to generate sphingosine 1-phosphate, a lipid signaling molecule implicated in roles in a diverse range of mammalian cell processes through its action as both a ligand for G-protein-coupled cell-surface receptors and an apparent intracellular second messenger. This paper describes a rapid, sensitive, and reproducible assay for sphingosine kinase activity using biotinylated sphingosine (biotinyl-Sph) as a substrate and capturing the phosphorylated product with streptavidin-coated membranes. We have shown that both human sphingosine kinase 1 and 2 (hSK1 and hSK2) can efficiently phosphorylate biotinyl-Sph, with K(m) values similar to those of sphingosine. The assay utilizing this substrate has high sensitivity for hSK1 and hSK2, with detection limits in the low-femtomole range for both purified recombinant enzymes. Importantly, we have also demonstrated the capacity of this assay to measure endogenous sphingosine kinase activity in crude cell extracts and to follow changes in this activity following sphingosine kinase activation. Together, these results demonstrate the potential utility of this assay in both cell-based analysis of sphingosine kinase signaling pathways...Continue Reading

References

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Citations

Jan 17, 2008·Analytical Chemistry·Katherine J LeeNancy L Allbritton
Feb 12, 2008·The Journal of Biological Chemistry·Tamara M LeclercqStuart M Pitson
May 6, 2014·Journal of Lipid Research·Santiago LimaSarah Spiegel

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