A separation method for the assay of adenylylcyclase, intracellular cyclic AMP, and cyclic-AMP phosphodiesterase using tritium-labeled substrates

Anal Biochem. 1992 May 15;203(1):76-82. doi: 10.1016/0003-2697(92)90045-9.


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.

MeSH terms

  • 3',5'-Cyclic-AMP Phosphodiesterases / analysis*
  • Adenylyl Cyclases / analysis*
  • Cell Line
  • Cyclic AMP / analysis*
  • Humans
  • Kinetics
  • Substrate Specificity
  • Tritium


  • Tritium
  • Cyclic AMP
  • 3',5'-Cyclic-AMP Phosphodiesterases
  • Adenylyl Cyclases