Measurement of Cyclic 3',5'-Adenosine Monophosphate by the Activation of Skeletal Muscle Protein Kinase

Abstract Cyclic 3',5'-adenosine monophosphate can be measured directly in crude tissue extracts by its stimulation of the rate of phosphorylation of casein catalyzed by skeletal muscle protein kinase. The reaction is carried out in the presence of tissue extract, which had been treated with and freed of trichloracetic acid, γ-32P-ATP-Mg2+, purified casein, and partly purified rabbit muscle protein kinase. The phosphorylated casein is isolated on filter paper discs. Cyclic AMP can be measured in 5 mg of tissue in the amount of 5 x 10-13 moles. Forty extracts can be assayed in 5 hours. Reproducibility of measurements made on heart, liver, skeletal muscle, and fat pad from several mammalian species varied ±5%. The validity of the assay is shown by measurements at various tissue dilutions, by the effect of cyclic nucleotide phosphodiesterase treatment, by recovery of cyclic AMP added to tissue extracts, and by increases in tissue cyclic AMP concentrations in response to the administration of glucagon and catecholamines. Other nucleotides, including cyclic GMP, in concentrations similar to or larger than those found in the tissues we have investigated, do not interfere with the measurement of cyclic AMP. The procedure is also applicable to the measurement of adenyl cyclase activity without separation of the cyclic AMP produced.