We have used a combination of spectrofluorometric and flow cytometric methods to characterize the interaction of fluorescent formyl peptide ligands with cell surface receptors. Using commercially available fluorescent microbeads as calibration standards, a family of fluoresceinated formyl peptides (N-formyl-met-leu-(phe)n-lys-fluorescein, n = 1-3), and digitoninpermeabilized human neutrophils, we were able to examine both equilibrium and kinetic aspects of ligand binding. Equilibrium studies showed that GTP[S] caused a loss of binding affinity of approximately two orders of magnitude, from approximately 0.04 nM (LRG) to -3 nM (LR), resp. Kinetic studies revealed that this change in affinity was due to principally an increase in the dissociation rate constant from -1 × 10-3 sec-1 (LRG) to approximately 1 × 10-1 sec-1 (LR). In contrast the association rate constants in the presence and absence of guanine nuclcotide (-3 × 107 sec-1 M-1) were statistically indistinguishable, and close to the diffusion limit. In the presence of guanine nuclocotide (LR), the kinetic data were adequately fit by a single step reversible model. However, in the absence of guanine nucleotide, while a large fraction of the receptors has essentially instantaneous access to G proteins, a substantial fraction is initially uncoupled from G proteins and only has access to them over a period of minutes. The binding data are consistent with the idea that those receptors with rapid access to the G proteins may be physically pre-coupled to the receptors in permeabilized neutrophil preparations even in the absence of the peptide ligand. Quenching of the fluorescein of the shorter peptides (n = 1-2) upon binding suggests that the pocket is large enough to contain at least five, but no more than six amino acids, while pH-dependent intensity measurements suggest that the mechanism of quenching is dependent upon the position of fluorescein within the pocket.
|Original language||English (US)|
|Number of pages||1|
|Journal||Annals of Biomedical Engineering|
|State||Published - 1991|
ASJC Scopus subject areas
- Biomedical Engineering