[16] An automated chromatographic assay for adenylate cyclase and cyclic 3′,5′-AMP phosphodiesterase

Abstract

The establishment of cyclic 3', 5'-AMP (cAMP) as a second messenger in numerous hormonal regulations has increased the need for efficient assays for adenylate cyclase and for cyclic 3', 5'-AMP phosphodiesterase. Adenylate cyclase preparations normally contain various enzymatic activities, such as ATPase, phosphodiesterase, and other phosphatases; therefore, numerous by-products other than cAMP are formed during the assay. The success of the procedure depends on the complete separation of cAMP, which often amounts to as low as 0.01 to 0.1% of the ATP substrate, from other adenine nucleotides. Adenylate cyclase was first measured by an assay which employed coupled enzyme systems. The automated procedure described in this chapter was developed to provide a convenient means for assaying adenylate cyclase activity in “solubilized” preparation from bovine brain; the same technique has proved adaptable for the assay of phosphodiesterase activity.