ASSAY OF ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE AND GUANOSINE 3',5'-CYCLIC MONOPHOSPHATE IN BIOLOGICAL MATERIALS BY ENZYMATIC RADIOISOTOPIC DISPLACEMENT

A simple yet precise microassay for as little as 5 μμmoles of 3′,5′-cyclic adenosine monophosphate or 3′,5′-cyclic guanosine monophosphate has been developed through a modification of the isotope dilution principle. The hydrolysis of tritium-labeled cyclic nucleotides by a uniform phosphodiesterase reaction is diminished by the addition of nonlabeled cyclic nucleotides. Cyclic nucleotides are isolated from tissue samples by perchloric acid extraction, absorption and elution from small anion-exchange columns, and lyophilization. A simple preparation from rat brain contains both 3′,5′-cyclic adenosine monophosphate and 3′,5′-cyclic guanosine monophosphate phosphodiesterase activities. Enzyme reaction mixtures include snake venom to provide an excess of 5′-nucleotidase activity. The nucleosides produced in the coupled enzyme system are separated from unreacted substrate by treatment with anion-exchange resin. The entire assay procedure of coupled enzyme incubation, resin treatment, and liquid scintillation counting is carried out in a standard plastic counting vial. Unreacted nucleotide substrates, which are absorbed by the resin, do not interact with the p-dioxane-naphthalene scintillation mixture and therefore do not interfere with the detection of unbound labeled nucleosides. The reaction is carried out to an extent previously determined to be about 30% of completion with a low concentration of tritium-labeled 3′,5′-cyclic nucleotide. Addition of unknown or standard samples of the unlabeled form of the same nucleotide diminishes the radioactivity detected in the assay. Numerical values are assigned by reference to a standard curve. The assay has been used to confirm the epinephrine-induced increase in skeletal muscle levels of 3′,5′-cyclic adenosine monophosphate. © 1968, American Chemical Society. All rights reserved.