The large-scale extraction and partial purification of endogenous 3',5'-cyclic UMP, 3',5'-cyclic IMP and 3',5'-cyclic dTMP are described. Rat liver, kidney, heart, spleen and lung tissues were subjected to a sequential purification procedure involving freeze-clamping, perchlorate extraction, alumina and Sephadex ion-exchange chromatography and preparative electrophoresis. The samples thus obtained co-chromatographed with authentic cyclic UMP, cyclic IMP and cyclic dTMP on t.l.c. and h.p.l.c. and the u.v. spectra of the extracted samples were identical with those of the standards. Fast atom bombardment of the three cyclic nucleotide standards yielded mass spectra containing a molecular protonated ion in each case; mass-analysed ion kinetic-energy spectrometry ('m.i.k.e.s') of these ions produced a spectrum unique to the parent cyclic nucleotide. The extracted putative cyclic UMP, cyclic IMP and cyclic dTMP each produced a m.i.k.e.s. identical with that obtained with the corresponding cyclic nucleotide standard. Rat liver, heart, kidney, brain, intestine, spleen, testis and lung protein preparations were each found capable of the synthesis of cyclic UMP, cyclic IMP and cyclic dTMP from the corresponding nucleoside triphosphate, of the hydrolysis of these cyclic nucleotides and of their binding, with the exception that cyclic dTMP was not synthesized by the kidney preparation.

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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.

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