Abstract
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) |
|---|---|
| Pages (from-to) | 602 |
| Number of pages | 1 |
| Journal | Annals of Biomedical Engineering |
| Volume | 19 |
| Issue number | 5 |
| State | Published - 1991 |
| Externally published | Yes |
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- Biomedical Engineering
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Analysis of ligand, receptor, and G-protein interaction in the n-formyl peptide receptor of the human neutrophil. / Fay, S. P.; Posner, Richard G; Domalewski, M. D.; Sklar, L. A.
In: Annals of Biomedical Engineering, Vol. 19, No. 5, 1991, p. 602.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Analysis of ligand, receptor, and G-protein interaction in the n-formyl peptide receptor of the human neutrophil
AU - Fay, S. P.
AU - Posner, Richard G
AU - Domalewski, M. D.
AU - Sklar, L. A.
PY - 1991
Y1 - 1991
N2 - 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.
AB - 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.
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M3 - Article
VL - 19
SP - 602
JO - Annals of Biomedical Engineering
JF - Annals of Biomedical Engineering
SN - 0090-6964
IS - 5
ER -