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Determination of diadenosine 5′,5‴,-P1,P4-tetraphosphate levels in cultured mammalian cells

✍ Scribed by Jeffrey C. Baker; Myron K. Jacobson

Publisher
Elsevier Science
Year
1984
Tongue
English
Weight
846 KB
Volume
141
Category
Article
ISSN
0003-2697

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✦ Synopsis

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.

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Diadenosine 5',5"'-P,,P,-tetraphosphate (Ap,A), a n intracellular regulatory nucleotide, has been found to react with the antitumor drug cw-diamminedichloroplatinum(I1J and its aqua derivative to form a single complex. This complex has been purified by highperformance liquid chromatography and chara