Cyclic nucleotide content of tobacco BY-2 cells.

The cyclic nucleotide content of cultured tobacco bright yellow-2 (BY-2) cells was determined, after freeze-killing, perchlorate extraction and sequential chromatography, by radioimmunoassay. The identities of the putative cyclic nucleotides, adenosine 3',5'-cyclic monophosphate (cyclic AMP), guanosine 3',5'-cyclic monophosphate (cyclic GMP) and cytidine 3',5'-cyclic monophosphate (cyclic CMP) were unambiguously confirmed by tandem mass spectrometry. The potential of BY-2 cell cultures as a model system for future investigations of cyclic nucleotide function in higher plants is discussed.

Ion-pair liquid chromatography-electrospray mass spectrometry for the analysis of cyclic nucleotides.

An LC-MS method has been developed combining ion-pair chromatography with an electrospray interface linking microbore and capillary HPLC to mass spectrometry. Separation of cyclic nucleotides on C18 reversed-phase columns, using tetrabutylammonium bromide as an ion pairing agent was evaluated with different mobile phase compositions. It was found that low ion-pairing agent concentration (50-500 microM) used in combination with low flow-rates (5-10 microl min(-1)) allowed the system to operate for up to several days without observing a reduced signal caused by source pollution. The loss of sensitivity expected in ion-pair chromatography could be remedied by using a 2-propanol coaxial sheath flow. Optimal conditions for negative ion electrospray resulted in a linear detection response in the femtomole to picomole range. Using biological samples this method was evaluated and compared with a classical ion-suppression RP-HPLC method using UV detection.

Assay of soluble guanylate cyclase activity by isocratic high-performance liquid chromatography.

A HPLC method alternative to labelled or unlabelled procedures was developed for the assay of guanylate cyclase (GC) activity. The substrate (GTP) and the product (cGMP) of the enzymatic reaction were separated in the isocratic mode on a muBondapak C18 column. The activity of GC was linearly dependent on the amount of cGMP produced in the presence of sodium nitroprusside. This approach was applied to follow the purification of GC from bovine lung and to evaluate its stability in different storage conditions.

Estimation of cytidylyl cyclase activity and monitoring of side-product formation by fast-atom bombardment mass spectrometry.

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.

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.

The identification of cyclic nucleotides from living systems using collision-induced dissociation of ions generated by fast atom bombardment mass spectrometry.

Extracts derived from rat liver and Phaseolus leaves are shown, by collision-induced dissociation of [MH]+ ions generated by fast atom bombardment mass spectrometry, to contain cytidine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate respectively, and not the 2',3'-cyclic isomers. Interference peaks, expected to be common to all mass-analysed ion kinetic energy spectra of ions generated by the fast atom bombardment process from glycerol-based matrices are identified. It is shown that unequivocal identification of cytidine 3',5'-cyclic monophosphate can be made at the microgram level. Attempts to derive a quantitative procedure based on using different cyclic nucleotides as internal standards were unsuccessful due to the poor solubility of these compounds in the matrix system.

Enzyme immunoassay for cytidine 3',5'-cyclic monophosphate (cyclic CMP).

An enzyme immunoassay for cytidine 3',5'-cyclic monophosphate (cyclic CMP) is presented. This assay is based upon the principles of competitive reaction and the double antibody solid phase method. Succinyl cyclic CMP-human serum albumin conjugate was injected into rabbits. Specific anti-cyclic CMP antibodies were incubated with a mixture of succinyl cyclic CMP labeled with beta-D-galactosidase and standard or sample cyclic CMP that had been succinylated prior to assay. The antibody-bound beta-D-galactosidase-cyclic CMP conjugate was separated from that of free with a second antibody, anti-rabbit immunoglobulin G, that was immobilized to a polystyrene ball. Then, activity of the enzyme on the solid phase was fluorometrically determined. When cyclic CMP contents in biological materials were estimated, acid extracts were partially purified by Dowex 1 x 8 formate column chromatography. The present immunoassay allows the detection of as little as 0.5 fmol of cyclic CMP with practically no interference from other cyclic nucleotides and cytidine analogs. By use of the enzyme immunoassay technique, we determined the amounts of cyclic CMP in various tissues of rats. They were found to be as little as 0.24-0.51 pmol/g wet weight, which was roughly 3000 to 20,000 and 100 to 500 times less than those of cyclic AMP and cyclic GMP, respectively.

Relationship between the levels of cyclic cytidine 3':5'-monophosphate, cyclic guanosine 3':5'-monophosphate, and cyclic adenosine 3':5'-monophosphate in urines and leukocytes and the type of human leukemias.

Cyclic cytidine 3':5'-monophosphate (cyclic CMP), cyclic guanosine 3':5'-monophosphate (cyclic GMP), and cyclic adenosine 3':5'-monophosphate (cyclic AMP) contents of leukocytes and urines of leukemic patients have been investigated. We have studied four types of leukemia: acute myeloblastic leukemia; chronic myelocytic leukemia; acute lymphoblastic leukemia; and chronic lymphocytic leukemia. As controls, the cyclic nucleotide content of leukocytes and urines of healthy volunteers and patients with solid tumors selected for their normal hemogram has been determined. It has also been measured in phytohemagglutinin-stimulated lymphocytes. Our data show that: (a) the concentration of cyclic CMP is always lower than that of cyclic GMP or cyclic AMP; (b) in urines, the concentrations of the three nucleotides are higher in patients than in healthy volunteers, the greatest differences being observed between the cyclic CMP concentrations of acute leukemia patients and controls; and (c) in white blood cells, cyclic AMP concentration is lower in leukemic than in normal cells. The cyclic GMP concentration is the same everywhere except in monoblastic cells and leukocytes from solid tumor patients. High cyclic CMP levels are associated only with acute leukemia, whether myeloblastic, monoblastic, or lymphoblastic, a fact which suggests that cyclic CMP could be a biochemical marker of hematopoietic stem cell malignancy.

Changes in cyclic nucleotide levels and phosphodiesterase and protein kinase activities in mitogenically stimulated 3T3 fibroblasts.

Activities of cyclic-nucleotide-hydrolysing enzymes cAMP-, cCMP- and cGMP phosphodiesterase, the intracellular concentrations of cAMP, cCMP, and cGMP, and the activity of the cAMP-dependent protein kinase were studied in serum-starved 3T3 cells stimulated to proliferate by serum. Within 1 and 2 min after stimulation the activities of cAMP- and cGMP phosphodiesterase were unaffected while the concentration of cGMP was raised and that of cAMP lowered, suggesting increased synthesis of cGMP and simultaneously reduced synthesis of cAMP. 48 h after stimulation, when the cells multiplied rapidly, both the cAMP phosphodiesterase and the cCMP phosphodiesterase were reduced. Evidence was also obtained that cAMP-dependent protein kinase is important for expressing the cAMP effect in the 3T3 cells.