Determination of diadenosine 5',5''',-P1,P4-tetraphosphate levels in cultured mammalian cells.
A simple method for measuring the cellular content of diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) in cultured mammalian cells is described. Ap4A was rapidly extracted by dissolving cell monolayers using 0.1 N NaOH. It was separated from the bulk of cellular components in a single step by selective adsorption to a highly specific boronate affinity resin. Subpicomole amounts were quantified by a luciferin-luciferase bioluminescence assay performed in the presence of alkaline phosphatase and venom phosphodiesterase. The selectivity of this assay for Ap4A in cultured mouse cells was established by high-performance liquid chromatography. This method allows the routine measurement of subpicomole amounts of Ap4A derived from a single dish of cells.
D000227:Adenine Nucleotides / Q000032:analysis*; D000818:Animals; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D015226:Dinucleoside Phosphates; D005347:Fibroblasts / Q000032:analysis; D049454:Firefly Luciferin; D008156:Luciferases; D008163:Luminescent Measurements; D051379:Mice; D008807:Mice, Inbred BALB C; D008832:Microchemistry; D013539:Simian virus 40
D000227:Adenine Nucleotides; D015226:Dinucleoside Phosphates; C020733:diadenosine tetraphosphate; D049454:Firefly Luciferin; D008156:Luciferases