A highly sensitive adenylate cyclase assay

A highly sensitive adenylate cyclase assay method has been developed which employs sequential chromatography on columns of Dowex cation ex-change resin and aluminum oxide. With the use of [a-“‘PIATP as substrate, this method permits the nearly complete separation of cyclic [“‘PIAMP formed from the substrate and other “P-containing compounds, i.e., “P in the assay blanks was barely detectable. In comparative studies, this method was found to be considerably more sensitive than previously reported methods. The high sensitivity of this method permits detection of the small amounts of cyclic AMP formed at low enzyme concentrations or at early time points in kinetic studies. Adenylate cyclase, the enzyme that catalyzes the formation of cyclic adenosine 3’,5’-monophosphate (cyclic AMP) from ATP, may be assayed in vitro by measuring the production of radioactively labeled cyclic AMP from the substrate [oI-~~P]ATP. A particular problem in assaying this enzyme results from the fact that only a minute fraction, usually less tha