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.
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.