50] cAMP analogs used to study low-Km, hormone-sensitive phosphodiesterase

Abstract

The determination of cyclic nucleotide analog I50 values for phosphodiesterases is a relatively simple method to study interactions between the enzyme and cyclic nucleotide analogs. This approach allows a large number of derivatives to be tested for preliminary information concerning hydrolysis. To conclude that the I50 values is a measure of analog hydrolysis requires that the mechanism of inhibition of [3H]cAMP hydrolysis is competitive. It is possible that some analogs act as noncompetitive inhibitors. Provided the enzyme preparation is pure with respect to phosphodiesterases, the type of inhibition can be determined. When it is important to determine if an analog is hydrolyzed, the complementary method of measuring direct hydrolysis can be used. For the low-Km phosphodiesterase and the analogs studied here, relatively low I50 values are correlated with analog hydrolysis while relatively high I50 values are correlated with the absence of detectable hydrolysis. For analogs such as N6-benzoyl- and N6-monobutyryl-cAMP the method of determining I50 values provides information that is not obtainable by direct hydrolysis. For example, neither of these analogs appear to be hydrolyzed but N6-benzoyl-cAMP has a lower I50 value and therefore more readily interacts with the low-Km phosphodiesterase. This analog or other ones may be useful in cAMP analog affinity chromatography for purification of phosphodiesterases. The method for directly determining cAMP analog hydrolysis measures the disappearance of the substrate instead of appearance of the product. However, the method is very sensitive since some, but not all, cAMP analogs have lower activation constants than cAMP does. Therefore, analogs can be tested for hydrolysis at concentrations as low as 10-50 nM and small changes in analog concentration can be directed. The methods presented have not only provided information concerning the mechanisms and structural requirements for hydrolysis but the analog specificities for various phosphodiesterases can be used as one of the determinants of analog potency in intact cells. Furthermore, the correlation of analog I50 values as an indication of hydrolysis with the effects of insulin on analog-stimulated intact cell responses provides information concerning the mechanism of insulin action. Pitfalls. Since cAMP analog preparations may be contaminated with cAMP, it is advantageous to purify the analog before determining direct hydrolysis. A method using Sephadex G-25 is presented elsewhere in this volume.(ABSTRACT TRUNCATED AT 400 WORDS)