Assay of Cyclic 3′,5′-Nucleotide Phosphodiesterase in Tissue Homogenates 1

Abstract

Summary

Factors are described that affect the assay of cyclic 3′,5′-nucleotide phosphodiesterase in tissue homogenates. Enzyme activity was measured by chromatographic separation of cyclic AMP-³H from labeled derivatives using thin-layer plates of PEI-cellulose. The method separates cyclic AMP, 5′-AMP, and secondary metabolites formed in significant quantities by homogenates of toad bladders. Recovery of radioactivity for all phases of the procedure was excellent —only 0.3 to 0.4% was lost during ether extraction and 98% of radioactivity applied to the thin-layer plates was recovered in the five zones scraped from the plates. In the absence of added 5′-AMP, no 5′-AMP-³H accumulated; the major labeled products were adenosine-³H and inosine-³H. Thus methods for assay of phosphodiesterase that depend solely on the rate of accumulation of 5′-AMP or do not separate cyclic AMP from adenosine would not be valid under these circumstances. Formation of remote labeled derivatives can be avoided by use of short periods of incubation and isotope trapping with unlabeled 5′-AMP, the product of the phosphodiesterase reaction. Use of 5′-AMP, however, is safe only during short periods of incubation because of gradual formation of an inhibitor (or removal of an activator) in the presence of added 5′-AMP.