No abstract

Previous assays for cytidine 3', 5'-cyclic monophosphate (cyclic CMP) have been criticized as being ambiguous. Here a modified RIA protocol, in which the production of assay components has been optimized and a novel trilayer chromatography column separation introduced which successfully separates cyclic CMP from compounds, endogenous to living tissues, which cross-react with anti-cyclic CMP sera, is described. The assay is capable of assaying cyclic CMP between 0.1 and 5 pmol, can be increased in sensitivity by means of an additional acetylation step, and enables the separation of cyclic CMP, cyclic AMP and cyclic GMP so that all three can be estimated in a single sample.

The enzyme cytidylyl cyclase catalyses the conversion of cytidine 5'-triphosphate into cytidine 3',5'-cyclic monophosphate, a third naturally occurring cyclic nucleotide currently under investigation to assign a biochemical function. Quantitation of the activity of this enzyme has been carried out by the positive-ion fast-atom bombardment mass spectrometric analysis of the enzyme incubation mixture after the reaction has been terminated. The data obtained are in good agreement with those obtained from the conventional radiometric and radioimmunoassays of the same enzyme preparations. The advantage of the mass spectrometer-based assay is the facility for multiple component monitoring. Thus, the production of the cytidine diphosphates and monophosphates, and the production of four cytidine 3',5'-cyclic monophosphate analogues as side-products, were simultaneously estimated. The identities of two of the side-products, 2'-O-glutamyl- and 2'-O-aspartyl-cytidine-3',5'-cyclic monophosphate, and of the cytidine 3',5'-cyclic monophosphate product, were confirmed by mass-analysed ion kinetic energy spectra from the collision-induced dissociation of the protonated molecules.