A high-performance liquid chromatography assay of brain adenylate cyclase using [3H]ATP as substrate

A sensitive, reproducible assay for adenylate cyclase is described which separates labeled cyclic AMP from ATP and other nucleotides by high-performance liquid chromatography (HPLC) on reverse-phase columns. The technique utilizes [3H]ATP as substrate, and the principal compound contaminating the [3H]cyclic AMP peak, adenosine, is removed by incubation of assay tubes with small amounts of adenosine deaminase. The HPLC elution utilizes high resolution (3 microns) short (10 cm) C-18 columns for increased resolution and decreased flow rates. Since cyclic AMP elutes at 4 min following injection, this procedure can easily process large numbers of samples per day when combined with automated techniques of sample injection and collection.