A new enzymatic assay for guanosine 3':5'-cyclic monophosphate and its application to the ductus deferens of the rat.

Abstract

A sensitive enzymatic procedure has been developed for the determination of guanosine 3':5'-cyclic monophosphate (cyclic GMP). It is based on the conversion of cyclic GMP to GMP by cyclic nucleotide phosphodiesterase and on the transfer of (32)P from [gamma-(32)P]ATP to GMP by the action of a specific ATP:GMP phosphotransferase (EC 2.7.4.8). The [(32)P]GDP is separated from the remaining [(32)P]ATP by enzymatic degradation of ATP by myosin and by precipitation of the (32)P(i) formed. The reaction blank, which is mostly caused by the nucleotide content of the enzymes, is doubled by about 0.1 pmol of cyclic GMP. The procedure has advantages in speed and/or accuracy over other methods in current use. Cyclic nucleotide concentrations were studied in the ductus deferens of the rat; two agents were used, carbachol and norepinephrine, which cause contraction. Incubation with 0.1 mM carbachol caused a 3-fold increase in cyclic GMP content, which was maximal about 2 min after carbachol addition. Cyclic AMP concentrations were not significantly changed. Addition of 0.01 mM norepinephrine increased cyclic GMP content by about 25% within 1 min and by 40% within 3 min; cyclic AMP concentrations were only slightly increased. A 3-min incubation with the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (0.1 mM) doubled the cyclic GMP content and increased cyclic AMP concentration by 50%.