6] Monitoring cyclic nucleotide metabolism in intact cells by 18O labeling

Abstract

Monitoring cellular cyclic nucleotide hydrolytic rates by analysis of O labeling provides the capability to define some of the kinetic and regulatory characteristics of cyclic nucleotide phosphodiesterase and nucleotidylyl cyclase activities in intact cells. Information of this nature can be used to identify the site(s) in the cyclic nucleotide metabolic pathway affected by biological signals or chemical agents and to determine if a compartmentalized species of these activities is more significantly affected. Information regarding the dynamics of cyclic nucleotide metabolism circumvents the constraint of relying solely on measurements of cellular cyclic nucleotide steady state levels that may not be changed by the metabolic perturbation imposed just as alterations in high-energy phosphate utilization rates may not be reflected as changes in cellular ATP concentration. How the information on rates of O appearance and labeling patterns in adenine (i.e., cAMP) and guanine (i.e., cGMP) nucleotide a-phosphoryls can be used to develop these insights has been described to only a limited extent thus far, but different means of utilizing the analytical data are continuing to be developed to provide this and other information as more sophisticated ways of mathematically treating the results are developed.