Cyclic AMP (cAMP)-dependent protein kinase catalyzes the transfer of the γ-phosphate of ATP to serine or threonine hydroxyl groups in various protein substrates. The addition of cAMP greatly enhances the velocity of the phosphotransferase reaction. The activity of the enzyme is determined by measuring the amount of 32P transferred from [γ-32P] ATP to protamine or histone. The labeled protein substrate is separated from the assay mixture by precipitation with trichloroacetic acid and collected on glass fiber filters. Assays are carried out in the presence and absence of cAMP to evaluate the activation of the enzyme by the cyclic nucleotide. One unit of protein kinase activity is defined as that amount of enzyme necessary to catalyze the transfer of 1 nmole of 32P from [γ-32P] ATP to protamine per minute at 30°C. A unit of binding activity is equivalent to the binding of 1 nmole of cAMP. Specific activity is expressed as units per milligram of protein.
ASJC Scopus subject areas
- Molecular Biology