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  • Title: Kinetic analysis of agonist-receptor interactions. Model for the "irreversible" binding of choleragen to human fibroblasts.
    Author: Osborne JC, Chang PP, Moss J.
    Journal: J Biol Chem; 1982 Sep 10; 257(17):10210-4. PubMed ID: 7107601.
    Abstract:
    Current studies on receptor-ligand interactions usually employ a "binding" step followed by extensive washing to remove free ligand. This procedure, by definition, removes reversibly bound ligand from the receptor; the equations used by most workers to analyze the data, however, require an equilibrium between free and bound ligand and are not applicable given the design of most binding assays. The assay, in fact, measures binding that is slowly reversible or irreversible. Assuming that ligand receptor interaction involves two stages with a reversible step followed by an "irreversible" event, the Ka for the reversible reaction may be obtained from the rate of the irreversible step. 125I-Choleragen binding to human fibroblasts was only slowly reversible at both 0 and 37 degrees C. The two-step model was, therefore, applied experimentally to determine the Ka for the reversible step in 125I-choleragen binding to human fibroblasts at 0 degrees C (Ka = 1.9 x 10(8) M-1) and at 37 degrees C (Ka = 3.6 x 10(8) M-1). As predicted by the two-step model for ligand binding, the addition of 50 micrograms/ml of unlabeled toxin enhanced the rate of release of radioactivity at 37 degrees C; the rate of radiolabel release remained low at 0 degrees C, even with unlabeled toxin present in the medium. The rate of release of previously incorporated 125I-toxin was accelerated by 50 micrograms/ml of toxin greater than 5 micrograms/ml of toxin. The two-step model for ligand binding appears to be applicable to the study of 125I-choleragen binding to fibroblasts and should be useful, in general, for the analysis of receptor-ligand interaction.
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