A fluorescent method for the demonstration of cyclic 3',5'-AMP phosphodiesterase activity in polyacrylamide gel.

The importance of adenosine 3’,5’-monophosphate (cyclic AMP) as a mediator for hormonal action needs no emphasis [I] . The level of CAMP is regulated by both the synthetic enzyme adenylate cyclase and the hydrolytic enzyme CAMP phosphodiesterase. While many useful methods have been developed for the biochemical assay of CAMP phosphodiesterase .activity [2-61, only a few of them have been adapted for the localization of its activity in gel electrophoresis. Thus, an elegant but involved fluorescent method on starch gel was developed by Monn and Christiansen [7]. This method utilizes NADH in a cyclic coupling procedure and requires the addition of three auxiliary enzymes. Hrapchak and Rasmussen [8] , on the other hand, used a gel method developed by Bikle for staining this enzyme. Only one enzyme, 5’-nucleotidase, is added tb hydrolyze the primary product 5’-AMP to adenosine and Pi, which is trapped as a white precipitate of calcium phosphate. Both of these methods are indirect and pose problems in their application to tissue extracts where other nucleotide hydrolyzing enzymes are unavoidably present. Recently, a new fluorescent analog of cyclicAMP, 1 ,N6 etheno-2-aza-adenosine 3’,5’-phosphate (cyclic 2-aza+AMP) was synthesized in our laboratory [9] and found to be an efficient substrate for CAMP phosphodiesterase [lo] . The adaptation of this new substrate for the direct fluorescent visualization of CAMP phosphodiesterase should be possible without the addition of any other enzyme. The present communication reports the new fluorescent method