Preparation and properties of an improved photoaffinity ligand for the N-formyl peptide receptor.
Biochim Biophys Acta
; 882(3): 271-80, 1986 Jul 16.
Article
in En
| MEDLINE
| ID: mdl-3015224
ABSTRACT
A new superior photoaffinity ligand for the N-formyl peptide receptor was prepared by derivatization of N-formyl-Met-Leu-Phe-Lys with a commercially available heterobifunctional crosslinking agent. The product, N-formyl-Met-Leu-Phe-N epsilon-(2-(p-azidosalicylamido)ethyl-1,3'- dithiopropionyl)-Lys was readily synthesized and radiolabelled, and had increased specificity and stability as compared to previously used photoaffinity ligands. The ligand rapidly associated with the receptor with high affinity (Kd = 0.28 nM). Once bound, it was virtually non-dissociable (in the absence of photolysis) in an experimental time-frame (t1/2 (off) = 154 min). The covalent incorporation of the photoaffinity ligand into the receptor upon irradiation was complete within 5 min, minimizing cell damage, and the efficiency of covalent incorporation was approx. 40%. The derivative had enhanced biological activity, having an ED50 for superoxide anion production of 0.23 nM, 27-fold lower than its parent peptide. This derivative of the N-formyl peptide was useful for up to 3 months after radiolabelling, showing a progressive decline in specific activity during storage in the dark at 4 degrees C. The enhanced stability, reproducibility and solubility of the photoaffinity ligand is expected to aid in the purification of the N-formyl peptide receptor and will prove a useful tool for analysing receptor-mediated processes.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
Azides
/
Affinity Labels
/
Receptors, Immunologic
/
N-Formylmethionine Leucyl-Phenylalanine
Limits:
Humans
Language:
En
Journal:
Biochim Biophys Acta
Year:
1986
Type:
Article