Radioimmunoassay of cytidine 3',5'-cyclic monophosphate: unambiguous assay by means of an optimized protocol incorporating a trilayer column separation to obviate cross-reactivity problems.

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.

Cytidylate cyclase: development of assay and determination of kinetic properties of a cytidine 3',5'-cyclic monophosphate-synthesizing enzyme.

A method is described for the separation of cytidine 3',5'-cyclic monophosphate (cyclic CMP) from cytidine tri-, di- and mono-phosphates and from cytidine 3',5'-cyclic pyrophosphate, cytidine 2'-monophosphate-3',5'-cyclic monophosphate, cytidine 2'-O-aspartyl-3',5'-cyclic monophosphate and cytidine monophosphate, compounds previously shown to be the result of putative cytidylate cyclase activity. This separation, involving elution of a novel bilayer column of QAE-Sephadex and alumina with 0.03 M-HCl, has been incorporated into an assay protocol to determine the enzyme-catalysed conversion of radiolabelled CTP to cyclic CMP. By this assay, cytidylate cyclase activity has been shown to be present in rat lung, spleen, ovary, testes, brain, stomach, liver, heart and kidney preparations; the activity was of a similar order in each tissue and had a sharp pH optimum of 7.0-7.5. The liver preparation had a Vmax. of 1.2 nmol of cyclic CMP formed/min per mg, and a Km of 220 microM-CTP, and although active in the absence of added cations, it was stimulated by Fe2+ and Mn2+ ions. In several of the tissues examined, the cytidylate cyclase activity was inversely proportional to age of the animals.

Cytidylate cyclase activity: identification of cytidine 3',5'-cyclic monophosphate and four novel cytidine cyclic phosphates as biosynthetic products from cytidine triphosphate.

Identification of cytidine 3',5'-cyclic monophosphate (cyclic CMP) as one of the products resulting from the incubation of dialysed cell-free preparations from rat brain, liver and kidney with cytidine 5'-triphosphate (CTP) is described. The non-acidic precipitable products after incubation of the tissue preparations with unlabelled, with 14C-single labelled, and with 14C- and 32P-dual labelled CTP were examined by thin-layer chromatography and high-pressure liquid chromatography, isotopic ratio determination, UV absorbance spectrophotometry, selective hydrolysis with nucleotidase, phosphodiesterase and acid, and by fast atom bombardment mass spectrometry with mass-analysed ion kinetic energy spectrum scanning. In addition to cyclic CMP and unchanged CTP, the products of the reaction were found to include cytidine monophosphate (CMP) and cytidine diphosphate (CDP) together with four novel cytidine compounds identified as cytidine 3',5'-cyclic pyrophosphate, cytidine 2'-monophosphate 3',5'-cyclic monophosphate, cytidine 2'-O-aspartyl-3',5'-cyclic monophosphate and cytidine 2'-O-glutamyl-3',5'-cyclic monophosphate. The evidence presented constitutes conclusive proof of the natural occurrence of cytidylate cyclase activity; the four novel cytidine cyclic phosphates described provide a feasible explanation of the discrepancies in previous reports which have led to the controversy which exists concerning the existence of cytidylate cyclase activity.

Extraction, purification and identification of cytidine 3',5'-cyclic monophosphate from rat tissues.

The large-scale extraction and purification to homogeneity of cyclic CMP and its unequivocal identification are described. Rat liver, kidney, heart, spleen and lung tissues were subjected to a sequential purification procedure involving freeze-clamping, perchlorate extraction, alumina and boronate column chromatography, polyacrylamide-gel column electrophoresis and high-voltage paper electrophoresis. The purified sample co-chromatographed with authentic cyclic CMP on t.l.c. and high-pressure liquid chromatography and was positive in a cyclic CMP radio-immunoassay. The u.v., i.r. and p.m.r. spectra were each essentially identical with those of authentic cyclic CMP. Fast-atom bombardment of authentic cyclic CMP yielded a mass spectrum containing a molecular protonated ion: mass-ion-kinetic-energy scanning of this ion produced a spectrum unique to 3',5'-cyclic CMP. The extracted nucleotide produced an identical mass-ion-kinetic-energy spectrum.