Cyclic nucleotides, assays

Cyclic nucleotides are purinic base derivatives with powerful biological activity. It is widely accepted that cyclic nucleotides mediate many of the intracellular biochemical events triggered by neurotransmitters and hormones (1,2). Therefore, the analysis of these compounds carries special relevance in biological sciences. A wide variety of techniques has been developed for cyclic nucleotide assays including binding to phosphokinase (3,4) or to antibodies (5) activation of enzymes... 

A series of triazolophthalazines were recently reported to be PDE2 inhibitors with IC50 values as low as 0.1 nM. In this series, compound 2 demonstrated activity in chemotaxis assays. No data were reported for effects on cyclic nucleotide levels, however, efficacy in cellular assays such as the chemotaxis assay suggests the effect is related to PDE2 inhibition in the cell types studied [12]. Pyridopyrimidines such as 3 were claimed to have IC50 values less than 50 nM [13]. 

Huang, W., Zhang, Y., and Sportsman, J.R., A fluorescence polarization assay for cyclic nucleotide phosphodiesterases, /. Biomol. Screen., 7, 215, 2002. 

The assays for the enzyme synthesis of sulfogalactosyl-glycerolipid, sulfatides and galactocerebrosides were carried out as previously described respectively by Subba Rao, et al. (28) Sarlieve, et al. 05, 29), and Neskovic, et al. (30). The assay for 2, 3 cyclic nucleotide phosphohydrolase was performed according to the method of Prohaska, et al. (31). EL coli. alkaline phosphatase type III-S, 2, 3 -cAMP, and sodium deoxycholate were obtained from Sigma (St. Louis, Mo.). Protein was determined by the method of Lowry, et al. (32) with crystalline bovine serum albumin as the standard. 

For BSA assay, to 1.5 ml of buffer-BSA solution, typically 40 fi of a 1 100 dilution of rabbit anti-BSA (2.4 mg/ml) and 40 /aI of a 1 10 dilution of urease-BSA conjugate were added. For cAMP assay, 30 ju.1 of a 1 10 dilution of the (NH4)2S04 fraction of cAMP antiserum (0.5 mg/ml) and 30/al of a 1 10 dilution of enzyme-cyclic nucleotide conjugate were added to 1.0 ml of buffer or buffer-nucleotide standard. 

VM. XXXVm [13]. Assay of Cyclic Nucleotides by Radioimmunoassay Methods. A. L. Steiner. 

Assay of cyclic nucleotides by enzymatic cycling techniques Protein binding assay Protein kinase assay Radioimmunoassay... 

ASSAY OF CYCLIC NUCLEOTIDES BY ENZYMATIC CYCLING TECHNIQUES... 

The method is based upon the ability of low concentrations of cyclic nucleotides to activate protein kinases which catalyse the phosphorylation of protein substrates, such as histone, by ATP [156,157]. The extent of phosphorylation is proportional to the amount of the cyclic nucleotides. The limits of sensitivity of the method are about 0.3 pmol for cyclic AMP and 0.5 pmol for cyclic GMP. Purification on a Dowex 50 column separates the two cyclic nucleotides from each other and removes any substances, such as ATP, which might interfere with the assay. Cyclic GMP is further purified by column chromatography on aluminum oxide and Dowex 1. Cyclic AMP-activated protein kinase is prepared from bovine heart and cyclic GMP-activated protein kinase from lobster tail. 

By using specific cyclic AMP and cyclic GMP antibodies and [ I]- and [ Ijsuccinyl cyclic nucleotide tyrosine methylesters, the cyclic nucleotides can be assayed simultaneously, the precipitate being counted in a dual channel spectrometer. 

Various mammalian cells and tissues, when incubated in a solution containing adenine, rapidly concentrate this purine and convert it into 5 -AMP and subsequently into ATP. The basis of the prelabelling technique [112] is that, in tissues and cells prelabelied with radioactive adenine, the relative amounts of cyclic AMP newly formed from ATP can be determined by isolating the cyclic nucleotide and measuring the amount of radioisotope which it contains [161]. The limitation on the measurement of small amounts of cyclic AMP with an acceptable precision, i.e., the limit of sensitivity of the assay, is a function of the specific activity of the radioactive adenine utilised for the prelabelling procedure. Thus, by increasing the specific activity of the radioactive adenine proportionally smaller amounts of cyclic AMP can be measured with the same precision. 

By using highly specific radioactive cyclic nucleotides and the snake venom nucleotidase, the most sensitive, specific and convenient method of assay for phosphodiesterase has been evolved. 

The assay is carried out both at 1 jaM and 1 mM substrate concentrations [162]. The enzyme preparation is incubated with tritium-labelled cyclic nucleotide in the presence of Mg, the reaction is stopped by brief heating in a water bath, the 5 -nucleotide formed is converted to the nucleoside by snake venom 5 -nucleotidase, the nucleoside is separated from the remaining cyclic nucleotide by anion exchange chromatography (Dowex 2), and the radioactivity of both compounds is counted. 

The entire assay can conveniently be carried out in a glass scintillation vial [82]. The reaction volume contains Tris buffer (pH 7.5), Mg, [ H]cyclic nucleotide, and snake venom 5 -nucleotidase. After a 10-min incubation at 37°C, the reaction is stopped by the addition of a slurry of AG1-X2 anion exchange resin. The resin binds substrate cyclic nucleotides but does not bind nucleosides. Scintillation fluid is added and the amount... 

Schumm and Webb23 suggested that cyclic nucleotides can exert an influence on the posttranscriptional events of RNA processing and transport since they found that the addition of cyclic AMP or GMP stimulated the release of RNA from isolated hepatic nuclei. Subsequently, in preliminary experiments, the addition of cAMP to the cell-free system composed of cell saps of livers of control rats, but not composed of cell saps of livers of tryptophan-treated rats, caused increases in the release of labeled RNA from the liver nuclei of control rats. This response in the cell-free system to added cAMP probably reflects the in vivo concentrations of cAMP in the tissues from which the cytosol was prepared. Thus, these preliminary findings suggest that tryptophan may elevate in vivo the cAMP levels in liver cytosol, and this may be of importance in the enhanced nucleocytoplasmic translocation of mRNA in liver owing to tryptophan. However, preliminary assays of cAMP activities in the livers of control and tryptophan-treated rats have failed to reveal significant differences. 

Kingan, T. (1989). A Competitive Enzyme-linked Immunosorbent Assay Applications in the Assay of Peptides, Steroids, and Cyclic Nucleotides, Biochem. 183 283-289. 

A polyacrylamide-boronate affinity gel, Affi-gel 601 , has been used for the separation of cyclic adenosine monophosphate, cyclic guanosine monophosphate, and cyclic cytidine monophosphate from their corresponding 5-nucleotides and nucleosides. A simple direct assay of 3 5 -cyclic nucleotide phosphodiesterase activity has been developed, based on the use of the gel. 

Quantitative analysis of cyclic nucleotides in protein kinase activity can also be performed using the FAB-MIKES technique. It gives results comparable with those obtained by conventional radiometric assay for monitoring the kinetics of cyclic nucleotide phosphodiesterase activity. 

Two of the seven cyclic nucleotide phosphodiesterase gene families PDE3 and PDE4, have a high affinity of cAMP and are specifically inhibited by ci-lostamide and rolipram, respectively. Using these inhibitors in assays with 0.1 [ H]cAMP as substrate, Gao et al. (1996) found PDE3 activity... 

Assay of Ribonuclease. Several kinds of assay have been found useful for studying ribonuclease (RNAase). A spectrophotometric assay is based on the empirical, unexplained observation that there is a decrease in optical density at 300 m/ during hydrolysis of RNA. RNA can be precipitated from solutions of its degradation products by uranyl acetate in trichloroacetic acid the soluble nucleotides can be measured spectro-photometrically or chemically. A manometric assay determines the formation of secondary acid groups through release of CO2 from a bicarbonate buffer acid groups have also been determined by titration. Recently cyclic nucleotides have been used as substrates in place of nucleic acid. ... 

Kuo, J.-F., Lee, T.-P., Reyes, P. L., Walton, K. G., Donnelly, T. E., Jr., and Greengard, P., 1972, Cyclic nucleotide-dependent protein kinases. X. An assay method for the measurement of guanosine 3, 5 -monophosphate in various biological materials and a study of agents regulating its levels in heart and brain, /. Biol. Chem. 247 16. 

An optimized assay is one in which the growth conditions prior to the assay and the initial cell density in the assay produce an ODeoo of less than 0.2 in 5FOA medium lacking cyclic nucleotides and of approximately 1.2 in medium containing a high level of cAMP or cGMP (Fig. 2a). 

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