A separation method for the assay of adenylylcyclase, intracellular cyclic AMP, and cyclic-AMP phosphodiesterase using tritium-labeled substrates.
A method for the separation of cyclic AMP from adenosine and polyvalent adenine nucleotides is described. The method consists of the sequential elution of adenosine and cyclic AMP from a single column of acidic aluminum oxide (alumina) with dilute hydrochloric acid and ammonium acetate. Adenosine, adenine, xanthine, and hypoxanthine are rapidly eluted with the application of 0.005 N hydrochloric acid while cyclic AMP remains adsorbed to the alumina. A subsequent application of 0.1 M ammonium acetate elutes more than 90% of the cyclic AMP. Under these conditions, polyvalent nucleotides (AMP, ADP, and ATP) remain adsorbed to the alumina. The method permits the measurement of adenylylcyclase activity using [3H]ATP as the labeled substrate. The same technique can be used to measure the accumulation of cyclic AMP in intact cells after labeling the ATP pool with [3H]adenine. With slight modification, the technique can be used to measure the activity of cyclic-AMP phosphodiesterase using [3H]cyclic AMP as the substrate. The proposed technique provides rapid, highly reproducible assays using inexpensive, disposable columns.
Alvarez
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R
Institute of Pharmacology, Syntex Research, Palo Alto, California 94304.
Daniels
D V
DV
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000032:analysis*; D000262:Adenylyl Cyclases / Q000032:analysis*; D002460:Cell Line; D000242:Cyclic AMP / Q000032:analysis*; D006801:Humans; D007700:Kinetics; D013379:Substrate Specificity; D014316:Tritium
D014316:Tritium; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D000262:Adenylyl Cyclases