Fluorescence-based assay of sphingosine kinases.
Sphingosine kinase enzymatic activity is commonly measured using radiolabeled substrates, with thin-layer chromatography and/or solvent extraction needed to detect the reaction product sphingosine-1-phosphate. We developed a fluorescence-based assay, using a sphingosine derivative labeled with a 7-nitrobenz-2-oxa-1,3-diazole moiety (15-NBD-Sph). Separation of substrate (15-NBD-Sph) from product (the corresponding phosphate) is achieved by extraction with chloroform/methanol at pH 8.5. The phosphate derivative is recovered by >98% in the aqueous phase and is directly detected and quantified by its fluorescence. 15-NBD-Sph is readily phosphorylated by human and murine sphingosine kinases 1 and 2. The suitability of the assay for measuring the activity of the kinases, both in the purified state and when contained in lysates of mammalian cells, was demonstrated. The present method is a convenient alternative to the radiometric assays and is particularly suited to the search for inhibitors of sphingosine kinases.
Billich
Andreas
A
Novartis Research Institute Vienna, Brunner Strasse 59, A-1235 Vienna, Austria. andreas.billich@pharma.novartis.com
Ettmayer
Peter
P
D000818:Animals; D002457:Cell Extracts; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005453:Fluorescence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008246:Lysophospholipids / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D015394:Molecular Structure; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000099157:Sphingosine Kinase
D002457:Cell Extracts; D004791:Enzyme Inhibitors; D008246:Lysophospholipids; D017853:Phosphotransferases (Alcohol Group Acceptor); D013110:Sphingosine; D000099157:Sphingosine Kinase; C060506:sphingosine 1-phosphate