Modes of action of hypoxanthine, inosine and inosine 5'-monophosphate on cyclic nucleotide phosphodiesterase from bovine brain

A purified bovine brain cyclic nucleotide phosphodiesterase catalyzed the hydrolysis of both cyclic AMP and cyclic GMP. Alternative substrate inhibition experiments indicated that cyclic AMP and cyclic GMP were hydrolyzed by the same enzyme and that they shared a common binding site. Inosine, inosine 5'-monophosphate and hypoxanthine competitively inhibited the hydrolysis of cyclic AMP and cyclic GMP by bovine brain cyclic nucleotide phosphodiesterase. This inhibition of cyclic nucleotide hydrolysis by the purines was affected by the pH of the mixture. The inhibition constants of inosine, inosine 5'-monophososphate and hypoxanthine when inhibiting enzymatic hydrolysis of cyclic AMP were 0.36 ± 0.05, 1.3 ± 0.2 and 3.2 ± 0.5 mM, respectively. With cyclic GMP as substrate, the inhibition constants were 0.50 ± 0.09, 1.8 ± 0.2 and 4.5 ± 0.7 mM for inosine, inosine 5'-monophosphate and hypoxantine respectively. The per cent inhibition by inosine, inosine 5'-monophosphate or hypoxanthine of the cyclic nucleotide phosphodiesterase activity was not altered by the addition of calmodulin (calcium-dependent protein activator of cyclic nucleotide phosphodiesterase) to the enzyme. The effect of calmodulin was not changed by these purine inhibitors. These results suggest that the binding site of calmodulin differed from that of the inhibitors and from that of cyclic AMP and cyclic GMP.