Peptide-based photoaffinity label of the catalytic subunit of the cyclic AMP-dependent protein kinase

摘要: A photoaffinity label of the catalytic subunit cAMP-dependent protein kinase was prepared from amino acid L-p-benzoyl-Phe. The acids L- and D-p-benzoyl-Phe were synthesized p-aminobenzophenone. Using solid-phase peptide synthesis, these incorporated into substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly, with enantiomers replacing phosphorylatable Ser. diastereomeric peptides separated by reverse phase HPLC. analogs tested as competitive inhibitors bovine heart. They performed equally well, K/sub i/'s on order 100..mu..M. However when photolyzed in presence enzyme, it found that only containing L-benzoyl-Phe caused photoinactivation. photoinactivation appeared to be time- concentration-dependent. solution 2..mu..M enzyme retained 10% activity after 2 minutes photolysis 100..mu..M peptide; contrast, 90% active D-benzoyl-Phe under identical conditions. Radiolabelled used establish binding stoichiometry enzyme; results showed labelling specific (approximately 1:1). Experiments are currently being determine site kinase.