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Enzymatic Measurement of Sphingosine 1-Phosphate

https://doi.org/10.1006/abio.1999.4157Get rights and content
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Abstract

Sphingosine 1-phosphate (SPP) is a sphingolipid metabolite which has novel dual actions acting as both an intracellular second messenger and a ligand for a family of G protein-coupled receptors. This paper describes a rapid enzymatic method to quantify mass levels of SPP in serum, mammalian tissues, and cultured cells. The assay utilizes an alkaline lipid extraction to selectively separate SPP from other phospholipids and sphingolipids, including sphingosine. Extracted SPP is efficiently converted to sphingosine by alkaline phosphatase treatment. Sphingosine thus formed is then quantitatively phosphorylated to [32P]SPP using recombinant sphingosine kinase and [γ-32P]ATP. With this procedure we were able to obtain reproducible measurements of SPP over a broad range from 0.25 pmol to 2.5 nmol. In various rat tissues, levels of SPP varied between 0.5 and 6 pmol/mg wet wt. The lowest levels were found in heart and testes, while brain contained the highest levels. The method was adapted easily to measure minute amounts of SPP present in various cultured cell types. The amount of SPP in cell extracts was proportional to the cell number and varied between 0.04 and 2 pmol/106 cells. Concurrent measurements of sphingosine levels revealed that its concentration was significantly higher than SPP in most cells and tissues. Furthermore, with this assay we were able to measure increases in intracellular SPP levels in rat pheochromocytoma PC12 cells after treatment with exogenous sphingosine or with nerve growth factor which stimulates sphingosine kinase activity.

Keywords

sphingosine 1-phosphate
sphingosine
mass level determination

Cited by (0)

This work was supported by Research Grant 1RO1 GM43880 from the National Institutes of Health. The authors thank Drs. Ana Olivera and Sheldon Milstien for helpful suggestions and discussions during these studies.

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To whom correspondence should be addressed at Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, 353 Basic Science Building, 3900 Reservoir Road NW, Washington, DC 20007. Fax: 202-687-7186.