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.