Abstract
A two-step assay for adenylate and guanylate cyclase is described utilizing α- 32P-labeled ATP or GTP as substrate and involving purification of the resulting 32P-labeled cAMP or cGMP by sequential chromatography on Dowex 50 and alumina. The Dowex 50 chromatography is performed in acid, 50 m m HCl for cGMP and 10 m m HClO 4 for cAMP, and achieves complete separation from the radiochemical impurities in the substrate which are responsible for blank. The cAMP or cGMP peaks are collected directly onto alumina columns and, under acid conditions, are completely retained by the alumina. After washing the alumina with water, the 32P-labeled cAMP or cGMP is eluted with 0.2 m imidazole buffer and counted. The method delivers blanks amounting to .0005% of the substrate radioactivity, high recoveries, and excellent reproducibility.
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