Peptide-based affinity labeling of adenosine cyclic monophosphate-dependent protein kinase

Peptide-based affinity-labeling experiments can be used to identify residues and domains in protein kinases that are involved in peptide substrate recognition. The catalytic subunit of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase presents a good model system for the study of kinase active sites. The enzyme is readily purified in milligram quantities from bovine heart, and a synthetic peptide substrate is available (kemptide; Leu-Arg-Arg-Ala-Ser-Leu-Gly), which is phosphorylated as efficiently as natural protein substrates. The general experimental strategy for peptide-based affinity labels and photoaffinity labels consists of three stages. To measure the extent of labeling, an inactivation assay is carried out. Free catalytic subunit is incubated with various concentrations of the label and for the photoactive peptides, the samples are photolyzed at an appropriate wavelength. To distinguish specific from nonspecific affinity labeling, the experiments are repeated in the presence of substrates or substrate analogs. Aliquots are withdrawn and assayed for phosphotransferase activity using the spectrophotometric assay. To distinguish specific from nonspecific affinity labeling, the experiments are repeated in the presence of substrates or substrate analogs. If the inactivation is directed to the active site, the catalytic subunit should be protected by the addition of Mg adenosine triphosphate (ATP), Mg-β, γ-methylene-ATP, and kemptide.