A highly sensitive enzymatic assay for diadenosine 5',5"'P',P3&phosphate
(Ap3A) has been established on the basis of the coupled luminescence. assay for diadenosine 5',5"'-P',P4-tetraphosphate (A. Ogilvie (1981) Anal. Biochem. 115, 302-307). Snake venom phosphodiesterase splits Ap,A into AMP plus ADP which can be measured in a luminescence reaction containing pyruvate kinase, phosphoenolpyruvate and luciferin-luciferase. The procedure is linear with Ap,A levels ranging from 0.1 to 2 pmol. The assay has been used to measure Ap,A 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 Ap& (0. I nmol/106 cells), allowing direct optical determination in the HPLC chromatography. The quantitative measurement of Ap3A with the luminescence assay gave identical results. Ap,A extracted from Ehrlich cells was also chromatographed with authentic nucleotide in two thin-layer systems providing additional proof for the existence of Ap& in biological material.