Determination of 3′,5′-Adenosine Monophosphate with a Method Based on a Radioactive Phosphate Exchange Reaction

A method has been developed for detecting 3',5'-cyclic adenylic acid in biological fluids by converting the nucleotide enzymatically to ATP and measuring the latter by a radioactive phosphate exchange reaction with the coupled enzymes phosphoglycerate kinase-glyceraldehyde phosphate dehydrogenase. The substrate requirements of the enzymes used and an efficient fractionation method based on precipitation of interfering substances with zinc sulfate-barium hydroxide made the determination specific for 3',5'-AMP. The method detected as little as 6 x 10-12 moles of 3',5'-cyclic adenylate and was effectively applied to the detection of the nucleotide in tissues and in urine.