Assay of Adenylyl Cyclase Activity by Ion-exchange High-performance Liquid Chromatography

Adenylyl cyclase(AC)activity was determined by directly quantifying the product cAMP with ion-exchange HPLC. When AC reaction was terminated, the reaction mixture was kept at 0 degrees for 2 h to remove residual ATP utilizing the action of ATP-hydrolyzing enzymes existed in the crude AC preparation of membrane pellet. Papaverine was removed by the dichloromethane extraction, and cAMP was quantified by chromatography on a WAX-1 HPLC column with baseline-resolution. It was found that cAMP was linearly formed within 10 min of the reaction, and the amount of cAMP formed was proportional to the amount of enzyme. Mouse brain AC activity determined by this method was 42 nmol/min per gram of protein, similar to that obtained by the classical [(3)H] labeled ATP method. This ion-exchange HPLC was more rapid and convenient.