Utilization of spectral absorption for measurement of adenylate cyclase activity

The purpose of this study was to improve our previously described enzymatic fluorometric assay of the adenylate cyclase activity. Using physicochemica l characteristics of NADPH, of which a 0.1 mmol/L solution would have an op tical density of 0.627, we measured the adenylate cyclase activity by the s pectral absorption of NADPH. The assay consists of two parts: pharmacologic al modulation of adenylate cyclase and measurement of newly synthesized cyc lic AMP. The latter part involves four steps: enzymatic destruction of nonc yclic adenine nucleotides and phosphorylated metabolites, conversion of cyc lic AMP to ATP, amplification of ATP by enzymatic cycling, and measurement of NADPH with spectral absorption, which is generated in proportion to init ial cyclic AMP levels. This new assay was tested in membrane preparations m ade from rat hearts in comparison with the previously described fluorometri c assay. We obtained identical results by spectrophotometry and fluorometry with high reproducibility. Because the fluorometric assay possesses a high sensitivity while the spectrophotometric method is advantageous because of its wide analytical range of cyclic AMP measurement, combination of fluoro metric and spectrophotometric methods may offer a convenient way to measure the adenylate cyclase activities in Various samples. (C) 2000 Wiley-Liss, Inc.