Simultaneous determination of guanine nucleotides in rat brain by high-performance liquid chromatography with dual-electrochemical detection

A new, sensitive, and specific assay method for guanine nucleotides using high-performance liquid chromatography with dual-electrochemical detection was developed. GTP, GDP, GMP, and cyclic GMP were separated with reversed-phase "ion-pair" chromatography and detected by a dual-electrochemical detector. Only guanine nucleotides among all purine and pyrimidine nucleotides responded to the electrochemical detector at 0.95 V. The peak heights for these guanine nucleotides were linear at concentrations between 0.5 pmol and 1 nmol. The regional distribution of these guanine nucleotides in the rat brain was studied by this new assay method.     

Determination of cyclic nucleotide phosphodiesterase activity by high-performance liquid chromatography

A new assay for cyclic nucleotide phosphodiesterase activity by high-performance liquid chromatography with on-line radiochemical detection has been developed. The method is based on the measurement of 3H-labeled nucleoside monophosphates formed from cyclic nucleotides by the action of 3',5'-cyclic-nucleotide phosphodiesterase (PDE). The reaction products are determined from the incubation mixture after removal of the protein by injection of an aliquot into the liquid chromatograph. The detection limit with counting efficiency of 30% is 20 fmol of 3H-labeled product, which makes the method suitable for detection of low PDE activities.     

Studies on the specificity of immunohistochemical techniques for cyclic AMP and cyclic GMP.

Immunohistochemical studies employing antibodies against cyclic nucleotides indicate that cyclic AMP and cyclic GMP are localized to distinct subcellular sites. These antibodies, however, cross-react weakly with noncyclic nucleotides (eg. ATP, GTP), and therefore we investigated the speficity of the immunohistochemical technique. Slides of fetal nuclei exposed to gaseous nitrous acid demonstrated reduced immunofluorescence. The slides were then incubated with cyclic and noncyclic nucleotides, and restoration of distinct cyclic AMP and cyclic GMP staining pattern was achieved only with appropriate cyclic nucleotides. Antibodies that were used have a greater affinity for acetylated derivatives of cyclic nucleotides. By using a gas phase technique, tissue slices were acetylated and immunohistochemical staining intensity was compared with the effect of acetylation on antibody affinity for various nucleotides. Acetylation greatly increased affinity of cyclic AMP antibody for cyclic AMP but not other nucleotides, and greatly intensified cyclic AMP staining. Acetylation moderately increased affinity of cyclic GMP antibody for cyclic GMP, and moderately intensified cyclic GMP staining. Conclusion: Both nitrous acid and acetylation studies support the specificity of the immunohistochemical method for cyclic nucleotides.     

Analysis of the free nucleotide pools of mammalian tissues by high-pressure liquid chromatography

Perchloric acid extracts of tissues were neutralized with tri-N-octylamine and, after removal of ClO₄^?, subjected to preliminary purification on a Cu²⁺-loaded column of Chelex 100. A high-pressure liquid chromatographic (HPLC) anion-exchange procedure was developed and gave good resolution of the naturally occurring free nucleotides on a single column. Where heterogeneous peaks eluted, an effective supplementary analysis was achieved by reverse-phase HPLC. An HPLC paired-ion technique was also evaluated for use in nucleotide analysis. Although anion-exchange was best for overall separation of nucleotides, both reverse-phase and paired-ion chromatography gave excellent separation of cyclic nucleotides. Reduced pyridine nucleotides were detected and measured in the form of their acid-decomposition products. The recovery of nucleotides was examined throughout the described analytical techniques and shown to be quantitative.     

Structure and Energetics of Allosteric Regulation of HCN2 Ion Channels by Cyclic Nucleotides

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels play an important role in regulating electrical activity in the heart and brain. They are gated by the binding of cyclic nucleotides to a conserved, intracellular cyclic nucleotide-binding domain (CNBD), which is connected to the channel pore by a C-linker region. Binding of cyclic nucleotides increases the rate and extent of channel activation and shifts it to less hyperpolarized voltages. We probed the allosteric mechanism of different cyclic nucleotides on the CNBD and on channel gating. Electrophysiology experiments showed that cAMP, cGMP, and cCMP were effective agonists of the channel and produced similar increases in the extent of channel activation. In contrast, electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) on the isolated CNBD indicated that the induced conformational changes and the degrees of stabilization of the active conformation differed for the three cyclic nucleotides. We explain these results with a model where different allosteric mechanisms in the CNBD all converge to have the same effect on the C-linker and render all three cyclic nucleotides similarly potent activators of the channel.     

Protection of tyrosine aminotransferase against proteolytic digestion by nucleotide derivatives

The abilities of several nucleotides to protect tyrosine aminotransferase (L-tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) against proteolytic inactivation in vitro have been examined as part of an ongoing investigation of the role of cyclic GMP in the intracellular degradation of the hepatic enzyme. Although neither cyclic GMP nor cyclic AMP was found to exert such a protective effect, certain nucleotide analogs were observed to inhibit the inactivation of tyrosine aminotransferase by trypsin and chymotrypsin. The nucleotides which conferred the strongest protection were the dibutyryl derivatives of cyclic GMP and cyclic AMP. This phenomenon appears to require a purine nucleotide with hydrophobic substituent(s), while the cyclic phosphate is not essential. The nucleotides probably act by direct interaction with tyrosine aminotransferase as indicated by changes in kinetic properties and heat stability of the enzyme and by their failure to inhibit trypsin when other protein substrates, including another aminotransferase, were used. Dibutyryl cyclic AMP was shown to block the appearance of a characteristic 43 kDa tryptic cleavage product of tyrosine aminotransferase but not the conversion of the native 54 kDa form to a size of 50 kDa. Arguments are presented against the involvement of the protective effect in the actions of dibutyryl cyclic nucleotides on tyrosine aminotransferase in cells.     

Saturation of anti cyclic nucleotides antibodies by endogenous cyclic nucleotides.

Rabbits immunized against cyclic AMP or cyclic GMP produce antibodies which are fully saturated by their respective endogenous cyclic nucleotides. This was proved a) in comparing radioimmunological measurements of cyclic nucleotides in antiserum and the binding site concentration determined by equilibrium dialysis, b) in showing the ineffectiveness of serum phosphodiesterase to hydrolyze the cyclic AMP present in the anti-cyclic AMP antiserum. Immunological and radioimmunological implications of this phenomenon are discussed.     

Control of Neurospora crassa morphology by cyclic adenosine 3'', 5''-monophosphate and dibutyryl cyclic adenosine 3'', 5''-monophosphate.

The role of cyclic adenosine 3', 5'-monophosphate (cyclic AMP) in the control of the Neurospora asexual life cycle was studied. Endogenous cyclic AMP levels were 10 to 20 times higher in strains having the wild-type cr-1 allele than in those carrying the mutated allele. In a wild-type strain these levels remained constant throughtout the entire growth period in shaken liquid cultures, except during a short period at the beginning of the stationary growth phase. In this period a marked increase in the cyclic nucleotide level was observed. The culture of cr-1 mutant strains in the presence of cyclic AMP or its dibutyryl derivative restores some morphological properties characteristic of wild-type strains. Specifically these cyclic nucleotides stimulated the rate of mycelial elongation, as well as the differentiation of aerial hyphae.     

Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2'0 acetylation by acetic anhydride in aqueous solution.

The sensitivity of radioimmunoassays for cyclic AMP and cyclic GMP has been markedly improved to readily detect femtomole (10-15) amounts in tissue extracts by acetylating the cyclic nucleotides at the 2'0 position with acetic anhydride. Acetylation of cyclic nucleotides by acetic anhydride in aqueous solution proceeds more rapidly than the hydrolysis of acetic anhydride to acetic acid thus yielding 100% acetylated cyclic nucleotide. 2'0 substituted cyclic nucleotides have greater affinity for the antibody than the parent cyclic nucleotides because the antibody has been made to a protein conjugate coupled at the 2'0 position. This simple acetylation technique makes it possible to measure cyclic AMP and cyclic GMP in minute quantities of tissue without purification or concentration of the sample.     

Rapid assays for adenylate cyclase and 3',5' cyclic AMP phosphodiesterase activities. Simultaneous measurements of other pathways of ATP catabolism

This paper deals with a rapid assay of adenylate cyclase activity and 3′,5′ cyclic AMP phosphodiesterase activity which permits simultaneous measurements of other pathways of ATP catabolism. The separation of all the adenylic nucleotides was obtained by an electrophoresis on cellulose acetate 15 min at 50 V/cm with a fluorescent buffer pH 8.6. Before electrophoresis, the incubation sample was added with a carrier solution of nucleotides to allow their localization under uv light, by fluorescence inhibition. Each fraction was cut and dissolved in Bray's liquid for scintillation counting. This assay method is rapid, reliable, and sensitive, it is suitable either for research or for routine and clinical purposes.     

Preparation of cyclic nucleotide antisera with thyroglobulin-cyclic nucleotide conjugates

Antisera to cyclic AMP and cyclic GMP were obtained by immunizing rabbits with antigens prepared by conjugating the 2'0-succinyl derivative of the cyclic nucleotides to thyroglobulin. The cyclic nucleotide-thyroglobulin conjugates were injected intradermally into multiple sites on the backs of the animals. This immunization procedure resulted in the production of antiserum in four of five animals capable of binding at a final serum dilution of greater than 1:10,000, 20% of the corresponding iodinated cyclic nucleotide derivative added. The antisera were also highly specific. The antiserum for cyclic AMP had a 2500-fold or greater relative affinity for cyclic AMP than other nucleotides or nucleosides, while that for cyclic GMP had a 5000-fold or greater affinity for 2'0 acetylated nucleotides or nucleosides except for acetylated cyclic IMP. The obstacles to measuring cyclic nucleotides, particularly cyclic GMP, in tissues were also overcome by refining and simplifying the methods for iodination, purification and assay. Furthermore, a "disequilibrium" incubation was developed as an alternative to the acetylation method to increase the sensitivity of the radioimmunoassay. Thus, the levels of both cyclic GMP and cyclic AMP can be determined rapidly and easily in the same tissue sample.     

Assay for adenylate cyclase and cyclic nucleotide phosphodiesterases and the preparation of high specific activity 32-P-labeled substrates.

Simple one step assay methods for adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) and cyclic nucleotide phosphodiesterases (3',5'-cyclic nucleotide 5'-nucleotidohydrolase EC 3.1.4.17) have been developed. [alpha-32-P] ATP is used as the substrate for adenylate cyclase. Acid-heat destruction of [32-P] ATP remaining after the cyclase reaction followed by Zn-Ba treatment quantitatively leaves cyclic [32-P] AMP in the supernatant essentially free from other 32-P-containing compounds. This assay method requires no corrections for recovery and routinely yields blank values less than 0.03 per cent. If higher sensitivity is desired, a simple 5 min alumina column step can be introduced into the procedure which quantitatively elutes cyclic [32-P] AMP directly into a liquid scintillation vial and lowers the blank values to less than 0.002 per cent. This method is rapid and easily performed, without sacrificing high reliability, specificity, or sensitivity. One step phosphodiesterase assays are easily accomplished using 32-P-labeled cyclic nucleotides as substrates. Descending paper chromatography of the reaction mixture on individual 2 cm wide paper strips gives a complete and quantitative separation of all possible products including [5'-32-P] AMP and [5'-32-P] GMP from their respective 32-P-labeled 3',5'-cyclic nucleotides in 1-2 h. The paper strips are cut, inserted in scintillation vials without scintillant and the 32-P-products determined by Cerenkov counting. Low blank values of less than 0.5 per cent and the use of high specific activity 32-P-labeled cyclic nucleotide substrates make this method the most reliable and most sensitive phosphodiesterase assay described to date. Because of the simplicity, specificity, and high sensitivity obtainable with these assay methods using 32-P-labeled substrates, we have also devised simple conditions for the preparation and purification of [alpha-32-P] ATP, cyclic [32-P] AMP and cyclic [32-P] GMP with specific activities in excess of 100 Ci/mmol. These high specific activity 32-Plabeled cyclic nucleotides are important for these new assay methods and are also useful to follow purification recovery of endogenous cyclic AMP and cyclic GMP from biological materials before protein binding or radioimmunological isotope displacement assays when performed in the femtomole range.     

From the one-carbon amide formamide to RNA all the steps are prebiotically possible

Formamide provides the raw material and the reaction leads connecting hydrogen cyanide HCN chemistry with higher complexity molecular structures. Formamide is liquid between 4 and 210 °C and, upon heating in the presence of one of several catalysts, affords nucleic bases, acyclonucleosides, carboxylic acids and aminoacids. In formamide in the presence of a source of phosphate, nucleosides are non-fastidiously phosphorylated in every position of the sugar residue, also yielding cyclic nucleotides. Guanine 3′,5′ cyclic nucleotide monophosphates polymerize to oligonucleotides, up to 30 nucleotides long. Adenine 3′,5′ cyclic nucleotide monophosphate reacts similarly but less efficiently. Preformed oligonucleotides may undergo terminal ligation in the absence of enzymes, thus allowing the formation of abiotically obtained long RNA chains.     

A simple ultrasensitive method for the assay of cyclic AMP and cyclic GMP in tissues.

A simple and rapid method for a highly sensitive radioimmunoassay of cyclic AMP and GMP is described. The method is based on the observation that the affinity of the cyclic nucleotide antibodies for the 2′-0-succinyl or acetyl derivatives is considerably greater than that for the nonacylated cyclic nucleotides. With the present method, 3–10 fmoles of cyclic AMP and cyclic GMP can be assayed using commercially available antisera against cyclic AMP or cyclic GMP. A reproducible conversion of the cyclic nucleotides in aqueous samples to the 2′-0-acylated derivative is brought about by a simple one-step addition of premixed reagents containing either succinic anhydride or acetic anhydride and triethylamine. The time required for succinylation or acetylation of 100 samples is less than 5 min using this technique. Tissue extracts after purification through anion exchange resins do not interfere with the acylation. After acylation, labeled antigen and antiserum are added and incubated at 4°C for 15 hr. The bound antingen is separated from the unbound antigen by a simple ethanol precipitation using bovine serum albumin to ensure complete precipitation. The specificity of the method has been validated by the following criteria: (1) treatment of the samples with purified phosphodiesterase results in complete loss of cyclic nucleotide immunoreactivity, (2) cyclic nucleotide content was a linear function of the tissue weight, and (3) in one instance using bovine rod outer segments, identical values of cyclic GMP were obtained with the present method and a purely physical method (high pressure liquid chromatography).     

Separation of cyclic AMP and cyclic GMP from thymidine,electrolytes and polyvalent nucleotides in tissue samples

Cyclic AMP and cyclic GMP can be separated from thymidine and its possible metabolites, electrolytes, and polyvalent nucleotides using columns of acidic alumina. Electrolytes and thymidine are not adsorbed on acidic alumina at pH 4.4 while cyclic nucleotides and polyvalent nucleotides are adsorbed at this pH. Cyclic AMP and cyclic GMP are eluted together from acidic alumina with 0.2 M ammonium formate (pH 6.0) and the polyvalent nucleotides remain adsorbed. The cyclic nucleotides are separated by chromatography on Dowex AG 1 X 8 resin. Recovery is 60–64% for cyclic AMP and cyclic GMP isolated from renal tissue samples. This methodology permits the separation of tritiated thymidine from cyclic nucleotides which are present in tissue preparations used in studies on the role of cyclic nucleotides in cellular growth.     

Separation of cyclic AMP and cyclic GMP from thymidine, electrolytes and polyvalent nucleotides in tissue samples.

Cyclic AMP and cyclic GMP can be separated from thymidine and its possible metabolites, electrolytes, and polyvalent nucleotides using columns of acidic alumina. Electrolytes and thymidine are not adsorbed on acidic alumina at pH 4.4 while cyclic nucleotides and polyvalent nucleotides are adsorbed at this pH. Cyclic AMP and cyclic GMP are eluted together from acidic alumina with 0.2 M ammonium formate (pH 6.0) and the polyvalent nucleotides remain adsorbed. The cyclic nucleotides are separated by chromatography on Dowex AG 1 X 8 resin. Recovery is 60--64% for cyclic AMP and cyclic GMP isolated from renal tissue samples. This methodology permits the separation of tritiated thymidine from cyclic nucleotides which are present in tissue preparations used in studies on the role of cyclic nucleotides in cellular growth.     

Complete analysis of cellular nucleotides by two-dimensional thin layer chromatography

We describe methods for the complete analysis of cellular nucleotides from as few as 10(6) 32Pi-labeled cells in a simple 2-day experiment. Nucleotides are extracted with acid, neutralized, and resolved by two-dimensional thin layer chromatography on polyethyleneimine cellulose. In the first dimension the nucleotides are separated based on the negative charge of their phosphate groups (i.e. cyclic, mono-, di, and triphosphates) and in the second dimension on their content of nucleobases (i.e. Ura, Cyt, Thy, Gua, and Ade). Because the separation is logical, one can predict the chromatographic migration of most nucleotides. By running standards we have determined the chromatographic location of over 90 biologically important nucleotides, nucleotide derivatives, and modified nucleotides from tRNA. We also developed a set of enzymatic and chemical methods to be used in conjunction with the chromatographic separations for verifying the identity of nucleotides and characterizing novel nucleotides. In this paper we use these methods to analyze and inventory the nucleotide content of Salmonella typhimurium in balanced log phase growth. Other potential uses of the method are also described.     

Uptake of Cyclic AMP by Natural Populations of Marine Bacteria

The major objective of this study was to describe the mechanism(s) of cyclic AMP uptake by natural populations of marine bacteria. A second objective was to determine whether this uptake could contribute to the intracellular regulatory pool of cyclic AMP. Using high-specific-activity ³²P-labeled cyclic AMP, we found several high-affinity uptake systems. The highest-affinity system had a half-saturation constant of <10 pM. This system was extremely specific for cyclic nucleotides, particularly cyclic AMP. It appeared to meet the criteria for active transport. Uptake of cyclic AMP over a wide concentration range (up to 2 μM) showed multiphasic kinetics, with half-saturation constants of 1 nM and greater. These lower-affinity systems were much less specific for cyclic nucleotides. Although much of the labeled cyclic AMP taken up by the high-affinity systems was metabolized, some remained as intact cyclic AMP within the cells during 1 h of incubation. This suggests that at least some of the bacteria use cyclic AMP dissolved in seawater to augment their intracellular pools.     

Quantitative profiling of nucleotides and related phosphate-containing metabolites in cultured mammalian cells by liquid chromatography tandem electrospray mass spectrometry

A method has been developed for the quantitative profiling of over twenty nucleotides and related phosphorylated species using ion-pair reversed-phase liquid chromatography hyphenated to negative ion tandem electrospray mass spectrometry. The influence of mobile phase pH and ion-pairing agent concentration were assessed to optimise separation and peak shapes. Full quantitative analysis was obtained for the nucleotides by reference to structurally related calibration standards. The developed method was applied to profile changes in nucleotides and related compounds in monolayer cultured Chinese hamster ovary (CHO) cells expressing the beta(2) adrenoceptor when exposed to pharmacological stimuli. These experiments demonstrate the potential of the LC-MS/MS method to detect changes in nucleotide drug targets as well as the simultaneous monitoring of levels of other nucleotides.     

Tansley Review No. 106

For three decades, hypotheses relating to the occurrence and function of cyclic nucleotides in higher plants have been highly controversial. Although cyclic nucleotides had been shown to have key regulatory roles in animals and bacteria, investigations with higher plants in the 1970s and early 1980s were criticized on the basis of (i) a lack of specificity of effects apparently elicited by cyclic nucleotides, (ii) the equivocal identification of putative endogenous cyclic nucleotides and (iii) ambiguity in the identification of enzymes connected with cyclic nucleotide. More recent evidence based on more rigorous identification procedures has demonstrated conclusively the presence of cyclic nucleotides, nucleotidyl cyclases and cyclic nucleotide phosphodiesterases in higher plants, and has identified plant processes subject to regulation by cyclic nucleotides. Here we review the history of the debate, the recent evidence establishing the presence of these compounds and their role; future research objectives are discussed.