Peptide-based photoaffinity label of the catalytic subunit of the cyclic AMP-dependent protein kinase
A photoaffinity label of the catalytic subunit of the cAMP-dependent protein kinase was prepared from the amino acid L-p-benzoyl-Phe. The amino acids L- and D-p-benzoyl-Phe were synthesized from p-aminobenzophenone. Using solid-phase peptide synthesis, these were incorporated into the cAMP-dependent kinase substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly, with the amino acid enantiomers replacing the phosphorylatable Ser. The diastereomeric peptides were separated by reverse phase HPLC. The peptide substrate analogs were tested as competitive inhibitors of the catalytic subunit of the cAMP-dependent protein kinase from bovine heart. They performed equally well, with K/sub i/'s on the order of 100..mu..M. However when photolyzed in the presence of the enzyme, it was found that only the peptide containing L-benzoyl-Phe caused photoinactivation. The photoinactivation appeared to be time- and concentration-dependent. A solution of 2..mu..M enzyme retained only 10% activity after 2 minutes of photolysis with 100..mu..M L-benzoyl-Phe peptide; in contrast, the enzyme was 90% active after photolysis with the D-benzoyl-Phe peptide under identical conditions. Radiolabelled L-benzoyl-Phe peptide was used to establish the binding stoichiometry of peptide to enzyme; these results showed that the photoaffinity labelling was specific (approximately 1:1). Experiments are currently being performed to determine the site of labelling on the kinase.
- Research Organization:
- Rockefeller Univ., New York, NY
- OSTI ID:
- 5283219
- Report Number(s):
- CONF-8606151-
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 45:6; ISSN FEPRA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
AMINO ACIDS
ANIMALS
BIOCHEMICAL REACTION KINETICS
BODY
CARBOXYLIC ACIDS
CARDIOVASCULAR SYSTEM
CATTLE
CHROMATOGRAPHY
DOMESTIC ANIMALS
ENZYME INHIBITORS
ENZYMES
HEART
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLING
LIQUID COLUMN CHROMATOGRAPHY
MAMMALS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANS
PEPTIDES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RUMINANTS
SEPARATION PROCESSES
STOICHIOMETRY
TIME DEPENDENCE
TRACER TECHNIQUES
TRANSFERASES
VERTEBRATES