Chromatographic separation of cyclic guanosine 3′,5′-monophosphate from guanylate cyclase reaction mixtures

Abstract

Guanylate cyclase can be assayed by measuring the formation of labeled cyclic guanosine 3′,5′-monophosphate (cGMP) from labeled guanosine 5′-triphosphate (GTP) in a manner analogous to the assay of adenylate cyclase (1,2,3). The separation of labeled cGMP from unreacted substrate must be complete due to the very low activity of guanylate cyclase in tissue samples. Typically 0.01–0.2% of the substrate is converted to cGMP. Therefore, any contamination with residual substrate would result in a high blank counting rate in the absence of tissue. In addition, labeled cGMP must be separated from unknown radiochemical impurities present in commercial preparations of GTP or generated during the incubation with tissue by the action of interfering enzymes. These compounds may cochromatograph with cGMP and increase the blank counting rate. These problems have been investigated for the separation of cAMP from adenylate cyclase reaction mixtures (4), but have not been specifically evaluated for the assay of guanylate cyclase. We compared three chromatographic separation procedures: alumina alone, sequential chromatography on Dowex AG 50W-X4 (H +) and alumina, and finally sequential chromatography on alumina and Dowex AG 1-X8 (formate).