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Abstract An assay for measuring 3',5'-cyclic nucleotide phosphodiesterase activity over a wide range of substrate concentrations has been described. The ratio of cyclic adenosine 3',5'-monophosphate (cyclic AMP) to cyclic guanosine 3',5'-monophosphate (cyclic GMP) hydrolysis varied several fold among crude subcellular fractions from bovine and rat heart when measured at micromolar but not millimolar substrate. In rat brain, liver or skeletal muscle, this ratio was virtually the same in crude subcellular fractions as in the homogenate when determined at either micromolar or millimolar substrate. A partially purified phosphodiesterase from the bovine heart supernatant had apparent Km values of 1 to 3 µm for cyclic GMP and 25 to 45 µm for cyclic AMP. Cyclic AMP and cyclic GMP interfered with the hydrolysis of each other in a manner predictable on the basis of their apparent Km values, suggesting that this bovine heart preparation contained only a single phosphodiesterase. However, in a crude liver supernatant cyclic GMP stimulated rather than inhibited cyclic AMP hydrolysis. A crude particulate fraction from bovine heart had two apparent Km values for cyclic AMP of about 0.8 and 25 µm. Most, if not all, of the cyclic GMP phosphodiesterase activity in this fraction had an apparent Km of about 20 µm. Data obtained using the partially purified bovine heart enzyme indicate that both cyclic AMP and cyclic GMP can serve as substrates for a single enzyme. However, differences in the ratios of hydrolysis of the two nucleotides among subcellular fractions and kinetic data suggest the existence of more than one 3',5'-cyclic nucleotide phosphodiesterase within a tissue. Whether these phosphodiesterase activities have relative substrate preferences or whether one or more might have specific substrate requirements has not been established.
Beavo et al. (Sun,) studied this question.