Formyl peptide chemotaxis receptors on the rat neutrophil: Experimental evidence for negative cooperativity
- 1 January 1985
- journal article
- research article
- Published by Wiley in Journal of Cellular Biochemistry
- Vol. 27 (4), 359-375
- https://doi.org/10.1002/jcb.240270406
Abstract
To examine the existence of negative cooperativity among formyl peptide chemotaxis receptors, steady-state binding of f Met-Leu-[3H]Phe to viable rat neutrophils and their purified plasma membranes was measured and the data were subjected to statistical analysis and to computer curve fitting using the NONLIN computer program. Curvilinear, concave upward Scatchard plots were obtained. NONLIN and statistical analyses of the binding data indicated that a two-saturable-sites model was preferable to a one-saturable-site model and statistically valid by the F-test (P < 0.1). In addition, Hill coefficients of 0.80 ± 0.02 were obtained. Kinetic dissociation experiments using purified plasma membranes showed evidence of site-site interactions of the destabilizing type (negative cooperativity). Thus, unlabeled f Met-Leu-Phe accelerated the dissociation of f Met-Leu-[3H]Phe under conditions where no rebinding of radioligand occurred. The rate of dissociation of f Met-Leu-[3H]Phe from the plasma membranes was dependent on the fold excess of unlabeled f Met-Leu-Phe used in the dilution medium; at the highest concentration tested (10,000-fold excess), the dissociation rate was more than double the dissociation rate seen with dilution alone, In addition, occupancy-dependent affinity was ascertained directly by studying the effect of increasing fractional receptor saturation with labeled ligand on the dissociation rate of the receptor-bound labeled ligand. These data showed that the f Met-Leu-[3H]Phe dissociation rate was dependent on the degree of binding site occupancy over the entire biologically relevant range of formyl peptide concentrations. Furthermore, monitoring of the time course of dissociation of the receptor/f Met-Leu-[3H]Phe complex as a function of receptor occupancy revealed that receptor affinity for f Met-Leu-Phe remained occupancy-dependent during the entire time of dissociation examined (up to 10 min). Finally, the average affinity profile of the equilibrium binding data demonstrated a 60% decrease in receptor affinity in changing from the high affinity to the low affinity conformation.Keywords
This publication has 35 references indexed in Scilit:
- The oligopeptide chemotactic factor receptor on human polymorphonuclear leukocyte membranes exists in two affinity statesBiochemical and Biophysical Research Communications, 1982
- Substance P binds to the formylpeptide chemotaxis receptor on the rabbit neutrophilBiochemical and Biophysical Research Communications, 1981
- The non-stoichiometric floating receptor model for hormone sensitive adenylyl cyclaseJournal of Theoretical Biology, 1976
- Cooperativity in ligand binding: A new graphic analysisBiochemical and Biophysical Research Communications, 1975
- β-Adrenergic receptors: Evidence for negative cooperativityBiochemical and Biophysical Research Communications, 1975
- Binding of insuling and other hormones to non-receptor materials: Saturability, specificity and apparent “negative cooperativity”Biochemical and Biophysical Research Communications, 1975
- Negative co-operativity in clustered receptors as a possible basis for membrane actionJournal of Theoretical Biology, 1974
- Statistical estimations in pharmacokineticsJournal of Pharmacokinetics and Biopharmaceutics, 1974
- Insulin interactions with its receptors: Experimental evidence for negative cooperativityBiochemical and Biophysical Research Communications, 1973
- On the nature of allosteric transitions: A plausible modelJournal of Molecular Biology, 1965