Cyclic GMP assay

For cyclic GMP, a cyclic GMP-activated protein kinase from lobster muscle is utilised. The cyclic GMP assay is somewhat less sensitive than the radioimmunoassay but has the advantage of the short time needed for the preparation of the binding protein 0.5 to 1 pmol of cyclic GMP can be estimated [155]. 

The cyclic GMP assay is carried out in a volume of 100 /u,l of 50 mM sodium acetate buffer, pH 4.0. The other reaction components are 6-10 pmol [ H]cyclic GMP, standard cyclic GMP solutions or unknown samples, and cyclic GMP binding protein. Tubes are incubated at 0°C for 75 min and the reaction mixtures are then diluted, filtered, and counted. 

The conditions for the cyclic GMP assay are the same, except that cyclic GMP samples or standards up to 20 pmol are used with the cyclic GMP-activated protein kinase preparation. 

This is the most comnonly used method of assay for cyclic GMP. Assay kits for cyclic GMP and cyclic AMP are commercially available. 

Only two other assays allow the determination of amounts as small as 0.1 pmol of cyclic GMP per tube the cycling system [141,142] and the radioimmunoassay [84,143]. The assay described above is faster and less laborious than these methods involving enzymatic cycling. 

Cyclic AMP interferes with cyclic GMP binding 15-20% only when present in a ten-fold excess 5 -AMP interferes 15% at 0.1 mM and 30% at I mM concentrations. Samples from tissues containing very low concentrations of cyclic GMP need purification prior to the assay. Small columns of Dowex 1 separate cyclic GMP from cyclic AMP with nearly quantitative recovery of both nucleotides. ATP is retained on the column and 5 -AMP is separated from cyclic GMP. Desalting procedures are essential when incubation media containing large amounts of salt must be concentrated prior to the assay. Binding is decreased 35% by 145 mM NaCl. 

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. 

Bovine serum albumin (BSA) and cyclic AMP (cAMP) are determined by a competitive binding enzyme immunoassay (315). With urease as label, an ammonia gas-sensing electrode is used to measure the amount of urease-labeled antigen bound to a double-antibody solid phase by continuously measuring the rate of ammonia produced from urea as substrate. The method yields accurate and sensitive assays for proteins (BSA less than 10 ng/mL) and antigens (cAMP less than 10 nM), with fairly good selectivity over cGMP, AMP, and GMP. 

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. 

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