A rapid method for the assay of guanylate cyclase.
An extremely rapid and sensitive assay for guanylate cyclase utilizing [alpha-32P]-GTP has been developed. It involves incubation of 5-100 mug of enzyme protein with 1 mM [alpha-32P]-GTP in 40 mM Tris HC1 buffer (pH 7.4) containing 3-3 mM MnSO2, 10 mM theophylline and 1 mM cyclic GMP. The reaction is terminated by addition of EDTA, and [32P]-cyclic GMP formed is isolated by sequential chromatography on Dowex-50-H+ and alumina. Recovery of 75-85% of [3H]-cyclic GMP and a blank of 0.001-0.003% of added [32P]-GTP was routinely obtained. The [32P] radioactivity isolated was shown to be cyclic GMP by a variety of techniques. The assay has also been shown to be applicable for a variety of tissues.
D000311:Adrenal Glands / Q000201:enzymology; D000818:Animals; D001921:Brain / Q000201:enzymology; D002852:Chromatography, Ion Exchange; D006160:Guanosine Triphosphate; D006162:Guanylate Cyclase / Q000032:analysis*; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008168:Lung / Q000201:enzymology; D008722:Methods; D008832:Microchemistry; D009206:Myocardium / Q000201:enzymology; D009928:Organ Specificity; D010727:Phosphoric Diester Hydrolases; D010761:Phosphorus Radioisotopes; D011863:Radioimmunoassay; D051381:Rats
D010761:Phosphorus Radioisotopes; D006160:Guanosine Triphosphate; D010727:Phosphoric Diester Hydrolases; D006162:Guanylate Cyclase