Specificity of cGMP binding to a purified cGMP-stimulated phosphodiesterase from bovine adrenal tissue

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Abstract
The binding of [3H]cGMP to purified bovine adrenal cGMP-stimulated phosphodiesterase was measured by Millipore filtration on cellulose ester filter. [3H]cGMP-binding activity was enhanced when the assay was terminated in buffer containing 70% of saturated ammonium sulfate to dilute the enzyme and wash the filters. The cGMP-binding activity was co-purified with the phosphodiesterase activity. The binding of [3H]cGMP to purified enzyme was measured in the presence or absence of the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine. 1-Methyl-3-isobutylxanthine showed linear competitive inhibition with respect to cGMP as substrate in the phosphodiesterase reaction but stimulated the [3H]cGMP-binding activity in the binding assay. The stimulatory effect appeared not to be the result of preservation from [3H]cGMP hydrolysis; no cGMP phosphodiesterase activity was measured under the cGMP-binding assay conditions in the absence or presence of the inhibitor. Half-maximal stimulation by 1-methyl-3-isobutylxanthine occurred in the 5-7 .mu.M concentration range. The specificity of binding of [3H]cGMP was investigated by adding increasing concentration of unlabeled analogs of cAMP and cGMP. The binding of [3H]cGMP (50 nM) was displaced by unlabeled cGMP and cAMP with the following potency: 50% displacement was reached at the 0.1 .mu.M cGMP range and only at 5-fold higher cAMP concentration. A comparative series of analogs (e.g., 5''-amino-5''-deoxyguanosine 3'',5''-monophosphate and 3''-amino-3''-deoxyguanosine 3'',5''-monophosphate) showed that the potencies of stimulation of cAMP phosphodiesterase activity parallels displacement curves or [3H]cGMP binding to purified enzyme with no correlation with phosphodiesterase inhibition sequences. The cGMP-binding activity is directly related to the non-catalytic (allosteric) cGMP-binidng site.

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