title: A real-time high-throughput fluorescence assay for sphingosine kinases.
abstract: Sphingosine kinases (SphKs), of which there are two isoforms, SphK1 and SphK2, have been implicated in regulation of many important cellular processes. We have developed an assay for monitoring SphK1 and SphK2 activity in real time without the need for organic partitioning of products, radioactive materials, or specialized equipment. The assay conveniently follows SphK-dependent changes in 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled sphingosine (Sph) fluorescence and can be easily performed in 384-well plate format with small reaction volumes. We present data showing dose-proportional responses to enzyme, substrate, and inhibitor concentrations. The SphK1 and SphK2 binding affinities for NBD-Sph and the IC50 values of inhibitors determined were consistent with those reported with other methods. Because of the versatility and simplicity of the assay, it should facilitate the routine characterization of inhibitors and SphK mutants and can be readily used for compound library screening in high-throughput format.
authors.affiliation: Department of Biochemistry and Molecular Biology and the Massey Cancer Center, Virginia Commonwealth University School of Medicine, Richmond, VA 23298.
authors.forename: Santiago
authors.initials: S
authors.lastname: Lima
authors.affiliation: Department of Biochemistry and Molecular Biology and the Massey Cancer Center, Virginia Commonwealth University School of Medicine, Richmond, VA 23298.
authors.forename: Sheldon
authors.initials: S
authors.lastname: Milstien
authors.affiliation: Department of Biochemistry and Molecular Biology and the Massey Cancer Center, Virginia Commonwealth University School of Medicine, Richmond, VA 23298.
authors.forename: Sarah
authors.initials: S
authors.lastname: Spiegel
mesh_terms: D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis*; D013110:Sphingosine / Q000737:chemistry*; D000099157:Sphingosine Kinase
chemical_list: D005456:Fluorescent Dyes; D017853:Phosphotransferases (Alcohol Group Acceptor); D013110:Sphingosine; D000099157:Sphingosine Kinase; C000594250:sphingosine kinase 2, human
keywords: NBD-sphingosine; fluorescence sphingosine kinase assay; red emission excitation shift; sphingosine kinase 1; sphingosine kinase 2