Diadenosine 5',5"'-P1,P3-triphosphate in eukaryotic cells: identification and quantitation

A highly sensitive enzymatic assay for diadenosine 5',5"'-P1,P3-triphosphate (Ap3A) has been established on the basis of the coupled luminescence assay for diadenosine 5',5"'-P1,P4-tetraphosphate (A. Ogilvie (1981) Anal. Biochem. 115, 302-307). Snake venom phosphodiesterase splits Ap3A into AMP plus ADP which can be measured in a luminescence reaction containing pyruvate kinase, phosphoenolpyruvate and luciferin-luciferase. The procedure is linear with Ap3A levels ranging from 0.1 to 2 pmol. The assay has been used to measure Ap3A in various eukaryotic cells after ion-exchange chromatography and high-performance liquid chromatography of acidic extracts of the cells. The level of diadenosine triphosphate was higher in all instances than the level of diadenosine tetraphosphate. When growing in the abdominal cavity of mice, Ehrlich ascites tumor cells contained high amounts of Ap3A (0.1 nmol/10(6) cells), allowing direct optical determination in the HPLC chromatography. The quantitative measurement of Ap3A with the luminescence assay gave identical results. Ap3A extracted from Ehrlich cells was also chromatographed with authentic nucleotide in two thin-layer systems providing additional proof for the existence of Ap3A in biological material.