The assay of cyclic AMP (cAMP) is based on the rate of casein phosphorylation catalyzed by skeletal muscle protein kinase which is dependent upon the cyclic nucleotide concentration. The reaction is carried out in the presence of trichloroacetic acid-treated tissue extract, [γ-32P] ATP, purified casein, and partly purified rabbit skeletal muscle protein kinase. cAMP dependent protein kinase is partially purified by a minor modification of the method reported by Walsh. This procedure is rapid and yields an enzyme preparation with a reproducible, highly sensitive activation by cAMP. Fresh, unfrozen muscle is ground twice in a chilled meat grinder, weighed, and homogenized with 2.5 volumes of ice-mold 4 mM EDTA, pH 7.0, in a Waring blender. All subsequent operations are carried out at 0–4%. The homogenate is centrifuged at 13,000 g for 20 minutes. The supernatant fraction is adjusted to pH 5.5 with 1 N acetic acid, and the precipitate is removed by centrifugation.
ASJC Scopus subject areas
- Molecular Biology