title: Synthesis, characterization and inhibitory activities of (4-N3[3,5-3H]Phe10)PKI(6-22)amide and its precursors: photoaffinity labeling peptides for the active site of cyclic AMP-dependent protein kinase.
abstract: PKI(6-22)amide is a 17 residue peptide corresponding to the active portion of the heat-stable inhibitor of cAMP-dependent protein kinase. The peptide is a potent (Ki = 1.6 nM), competitive inhibitor of the enzyme. The photoreactive peptide analog (4-azidophenylalanine10)PKI(6-22)amide was synthesized in both its non-radiolabeled and tritiated forms by chemical modification of precursor peptides that were prepared by stepwise solid-phase synthesis. (4-Amino[3,5-3H]phenylalanine10)PKI(6-22)amide, the precursor for the radiolabeled arylazide peptide, was obtained by catalytic reduction of the corresponding peptide containing the 3,5-diiodo-4-aminophenylalanine residue at position 10. The purified PKI peptides were analyzed by HPLC, amino acid analysis, and u.v. spectra. In the dark, (4-azidophenylalanine10)PKI(6-22)amide inhibited the catalytic subunit of cAMP-dependent protein kinase with a Ki value of 2.8 nM. The photoreactivity of the arylazide peptide was demonstrated by time-dependent u.v. spectral changes on exposure to light. Photolysis of the catalytic subunit (4-azido[3,5-3H]phenylalanine10)PKI(6-22)amide complex resulted in specific covalent labeling of the enzyme. The data indicate that this peptide is a useful photoaffinity labeling reagent for the active site of the protein kinase.
authors.affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, GA.
authors.forename: B M
authors.initials: BM
authors.lastname: Katz
authors.forename: L J
authors.initials: LJ
authors.lastname: Lundquist
authors.forename: D A
authors.initials: DA
authors.lastname: Walsh
authors.forename: D B
authors.initials: DB
authors.lastname: Glass
mesh_terms: D000345:Affinity Labels / Q000138:chemical synthesis* / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002352:Carrier Proteins / Q000138:chemical synthesis* / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000494:pharmacology; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000138:chemical synthesis; D010770:Phosphotransferases / Q000032:analysis; D010782:Photolysis; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000345:Affinity Labels; D000596:Amino Acids; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D047428:Protein Kinase Inhibitors; C061071:protein kinase inhibitor (6-22)amide, 4-azidophenylalanine(10)-; C009722:4-aminophenylalanine; D010649:Phenylalanine; D010770:Phosphotransferases; D011494:Protein Kinases