MEASUREMENT OF CYCLIC-GMP AND CYCLIC-AMP PHOSPHODIESTERASES

Methods are described for measuring phosphodiesterases for cGMP and cAMP in the range of activity yielding 10-12 to 10-8 mol of product. The 5′-GMP formed is measured by conversion to GDP with guanylate kinase. Amounts of GDP greater than 10-10 mol are measured directly with an enzyme system which results in stoichiometric oxidation of NADH. This is either determined by the decrease in fluorescence or the excess NADH is destroyed with acid and the NAD+ measured by its fluorescence in strong NaOH. With smaller amounts of GDP, sensitivity is amplified 1000-fold with the succinic thiokinase-pyruvate kinase cycle. In the case of cAMP diesterase, larger amounts of 5′-AMP are measured in the same way as 5′-GMP, except that adenylate kinase is substituted for guanylate kinase. With smaller amounts, the 5′-AMP is converted to ATP, and sensitivity is amplified with the adenylate kinase-pyruvate kinase cycle. As little as 20 ng dry weight of average brain is sufficient for accurate assay of the diesterase activity toward either cAMP or cGMP. When there is danger of significant destruction of AMP or GMP by tissue 5′-nucleotidase, this is prevented by adding GMP to the cAMP reagent, AMP to the cGMP reagent, or 5′-UMP to either reagent. © 1979.