phosphorus-radioisotopes and sphingosine-1-phosphate

phosphorus-radioisotopes has been researched along with sphingosine-1-phosphate* in 7 studies

Other Studies

7 other study(ies) available for phosphorus-radioisotopes and sphingosine-1-phosphate

ArticleYear
A practical process for the preparation of [(32)P]S1P and binding assay for S1P receptor ligands.
    Applied radiation and isotopes : including data, instrumentation and methods for use in agriculture, industry and medicine, 2015, Volume: 102

    Sphingosine-1-phosphate receptors (S1PRs) are important regulators of vascular permeability, inflammation, angiogenesis and vascular maturation. Identifying a specific S1PR PET radioligand is imperative, but it is hindered by the complexity and variability of current for binding affinity measurement procedures. Herein, we report a streamlined protocol for radiosynthesis of [(32)P]S1P with good radiochemical yield (36-50%) and high radiochemical purity (>99%). We also report a reproducible procedure for determining the binding affinity for compounds targeting S1PRs in vitro.

    Topics: Binding, Competitive; Ligands; Lysophospholipids; Phosphorus Radioisotopes; Receptors, Lysosphingolipid; Sphingosine

2015
A rapid assay for assessment of sphingosine kinase inhibitors and substrates.
    Analytical biochemistry, 2011, Apr-15, Volume: 411, Issue:2

    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

2011
An assay system for measuring the acute production of sphingosine 1-phosphate in intact monolayers.
    Analytical biochemistry, 2007, Dec-15, Volume: 371, Issue:2

    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

2007
Assaying sphingosine kinase activity.
    Methods in enzymology, 2000, Volume: 311

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

2000
Enzymatic method for measurement of sphingosine 1-phosphate.
    Methods in enzymology, 2000, Volume: 312

    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

2000
Sphingosine 1-phosphate as a major bioactive lysophospholipid that is released from platelets and interacts with endothelial cells.
    Blood, 2000, Nov-15, Volume: 96, Issue:10

    The serum-borne lysophospholipid mediators sphingosine 1-phosphate (Sph-1-P) and lysophosphatidic acid (LPA) have been shown to be released from activated platelets and to act on endothelial cells. In this study, we employed the repeated lipid extraction (under alkaline and acidic conditions), capable of detecting Sph-1-P, LPA, and possibly structurally similar lysophospholipids, whereby a marked formation of [(32)P]Sph-1-P, but not [(32)P]LPA, was observed in [(32)P]orthophosphate-labeled platelets. Platelet Sph-1-P release, possibly mediated by protein kinase C, was greatly enhanced in the presence of albumin, which formed a complex with Sph-1-P. This finding suggests that platelet Sph-1-P may become accessible to depletion by albumin when its transbilayer movement (flipping) across the plasma membrane is enhanced by protein kinase C. Although human umbilical vein endothelial cells expressed receptors for both Sph-1-P and LPA, Sph-1-P acted much more potently than LPA on the cells in terms of intracellular Ca(++) mobilization, cytoskeletal reorganization, and migration. The results suggest that Sph-1-P, rather than LPA, is a major bioactive lysophospholipid that is released from platelets and interacts with endothelial cells, under the conditions in which critical platelet-endothelial interactions (including thrombosis, angiogenesis, and atherosclerosis) occur. Furthermore, albumin-bound Sph-1-P may account for at least some of the serum biological activities on endothelial cells, which have been ascribed to the effects of albumin-bound LPA, based on the similarities between LPA and serum effects.

    Topics: Biological Transport; Blood Platelets; Calcium Signaling; Cell Movement; Chromatography, Thin Layer; Cytoskeleton; Endothelium, Vascular; Fetal Blood; Humans; Lysophospholipids; Phosphates; Phosphorus Radioisotopes; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophosphatidic Acid; Receptors, Lysophospholipid; RNA, Messenger; Serum Albumin; Sphingosine

2000
Activation of endothelial cell phospholipase D by sphingosine and sphingosine-1-phosphate.
    American journal of respiratory cell and molecular biology, 1994, Volume: 11, Issue:2

    We have investigated the activation of phospholipase D (PLD) by sphingosine and its derivatives in bovine pulmonary artery endothelial cells (BPAEC) prelabeled with [32P]orthophosphate or [32P]lyso phospholipids. Sphingosine, in a dose- and time-dependent manner, stimulated the hydrolysis of [32P]phosphatidylcholine (PC) resulting in the production of [32P]phosphatidic acid (PA), suggesting PLD activation. In the presence of ethanol (150 mM), the accumulation of [32P]phosphatidylethanol was also observed. The sphingosine-induced stimulation of PLD activity was not affected by treatment with the protein kinase C (PKC) inhibitor staurosporine or by down-regulation of PKC with TPA and was independent of extracellular Ca2+, suggesting that the PLD activation was independent of PKC and Ca2+. Chelation of intracellular Ca2+ with BAPTA actually potentiated the sphingosine-stimulated [32P]PC hydrolysis. Furthermore, the activation of PLD by sphingosine was not abolished by treatment of BPAEC with either cholera or pertussis toxin, indicating noninvolvement of toxin-sensitive G-proteins. In addition to hydrolysis of [32P]PC, sphingosine also stimulated PLD-mediated hydrolysis of [32P]phosphatidylethanolamine and [32P]phosphatidylinositol. Among the various sphingoid compounds, in addition to sphingosine, only sphingosine-1-phosphate (Sph-1-P) activated the endothelial cell PLD. The effect of sphingosine and Sph-1-P on PA phosphatase (PA Pase) activity was tested using [3H]glycerol-labeled PA. The Mg(2+)-independent and membrane-associated PA Pase activity was inhibited by sphingosine (IC50 = 200 microM) but not by Sph-1-P. This implies that sphingosine and Sph-1-P share a similar PLD-stimulating property but differ in their PA Pase inhibitory activity.

    Topics: Animals; Calcium; Cattle; Cell Survival; Cells, Cultured; Deoxyglucose; Egtazic Acid; Endothelium, Vascular; Enzyme Activation; Kinetics; Lysophospholipids; Phosphates; Phosphatidic Acids; Phospholipase D; Phospholipids; Phosphorus Radioisotopes; Protein Kinase C; Pulmonary Artery; Ribonucleotides; Sphingosine; Tetradecanoylphorbol Acetate

1994
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