Sphingosine kinase from Swiss 3T3 fibroblasts: a convenient assay for the measurement of intracellular levels of free sphingoid bases.
Sphingoid bases are important for diverse cellular processes. This paper describes the development of a simple and rapid enzymatic method to quantify mass levels of free long-chain sphingoid bases in cellular lipid extracts. The assay is based on the ability of sphingosine kinase from Swiss 3T3 fibroblasts to phosphorylate sphingoid bases, predominantly sphingosine. To quantitatively determine cellular levels of sphingosine or other long-chain sphingoid bases, it was necessary to solubilize cellular lipids by sonication and to generate standard curves in the presence of lipid extracts containing at least 50 nmol of phospholipids. The assay conditions were optimized to allow quantification of sphingosine over a broad range from 25 to 1,000 pmol. Using this method we were able to obtain reproducible measurements of free sphingoid bases in various cell types and to detect increases in intracellular sphingoid base levels after treatment with exogenous sphingosine or stimulation with fetal bovine serum.
Olivera
A
A
Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, DC 20007.
Rosenthal
J
J
Spiegel
S
S
D016475:3T3 Cells; D000818:Animals; D001923:Brain Chemistry; D002460:Cell Line; D006801:Humans; D007424:Intracellular Fluid / Q000737:chemistry; D008055:Lipids / Q000302:isolation & purification; D051379:Mice; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D012995:Solubility; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000737:chemistry; D000099157:Sphingosine Kinase
D008055:Lipids; D017853:Phosphotransferases (Alcohol Group Acceptor); D013110:Sphingosine; D000099157:Sphingosine Kinase