phosphorus-radioisotopes and sphingosine-kinase

Sphingosine kinases (SphKs) catalyze the transfer of phosphate from adenosine triphosphate (ATP) to sphingosine to generate sphingosine 1-phosphate (S1P), an important bioactive lipid molecule that mediates a diverse range of cell signaling processes. The conventional assay of SphK enzymatic activity uses [γ-(32)P]ATP and sphingosine as substrates, with the radiolabeled S1P product recovered by organic extraction, displayed by thin layer chromatography, and quantified by liquid scintillation counting. Although this assay is sensitive and accurate, it is slow and labor-intensive; thus, it precludes the simultaneous screening of more than a few inhibitor compounds. Here we describe a 96-well assay for SphKs that is rapid and reproducible. Our method, which takes advantage of the limited solubility of S1P, detects radioactive S1P adhering to the plate by scintillation proximity counting. Our procedure obviates extraction into organic solvents, postreaction transfers, and chromatography. Furthermore, our assay enables assessment of both inhibitors and substrates, and it can detect endogenous SphK activity in cell and tissue extracts. The SphK kinetic parameter, K(m), and the K(i) values of inhibitors determined with our assay and the conventional assay were indistinguishable. These results document that our assay is well-suited for the screening of chemical libraries of SphK inhibitors.

Topics: Adenosine Triphosphate; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Enzyme Inhibitors; High-Throughput Screening Assays; Kinetics; Lysophospholipids; Phosphorus Radioisotopes; Phosphotransferases (Alcohol Group Acceptor); Scintillation Counting; Small Molecule Libraries; Sphingosine; Substrate Specificity

Sphingosine kinase (SK) is a signaling enzyme that phosphorylates sphingosine to produce sphingosine 1-phosphate. Sphingosine and sphingosine 1-phosphate (S1P) belong to a class of bioactive sphingolipid metabolites that are critical in a number of cellular processes, yet often have opposing biological functions. The intracellular localization of sphingosine kinase has been demonstrated in multiple studies to be a critical aspect of its signaling function. To date, assays of sphingosine kinase activity have been developed for measuring activity in lysates, where the effects of localization are lost. Here we outline a system in which the rate of production of S1P can be measured in intact cells using exogenously added radiolabeled ATP instead of tritiated sphingosine. The surprising ability of ATP to enter unpermeabilized monolayers is one aspect that makes this assay simple, efficient, and inexpensive, yet sensitive enough to measure endogenous enzyme activity. The assay is well behaved in terms of kinetics and substrate dependence. Overall, this assay is ideal for future studies to identify changes in S1P production in intact cells such as those that result from the differential intracellular targeting of sphingosine kinase.

Topics: Adenosine Triphosphate; Cells, Cultured; Chromatography, Thin Layer; HeLa Cells; Humans; Kinetics; Lysophospholipids; Phosphorus Radioisotopes; Phosphotransferases (Alcohol Group Acceptor); Sensitivity and Specificity; Sphingosine; Unilamellar Liposomes

Topics: Lysophospholipids; Phosphorus Radioisotopes; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Topics: 3T3 Cells; Adenosine Triphosphate; Animals; Cell Line; HL-60 Cells; Humans; Kidney; Lysophospholipids; Mice; PC12 Cells; Phospholipids; Phosphorus Radioisotopes; Phosphotransferases (Alcohol Group Acceptor); Radioisotope Dilution Technique; Rats; Recombinant Proteins; Sphingosine; Transfection; Tumor Cells, Cultured