Kinetic and Equilibrium Studies of (-)¹²⁵Iodocyanopindolol Binding to β-Adrenoceptors on Human Lymphocytes: Evidence for the Existence of two Classes of Binding Sites

Abstract

Saturation experiments were performed on intact human peripheral mononuclear leucocytes (MNL) and MNL membranes with (-)125Iodocyanopindolol (125ICYP) over a large concentration range (1.5-600 pmol/l). The corresponding Scatchard plots were curvilinear suggesting two saturable classes of binding sites: A high affinity binding site (Bmax1 = 1000 +/- 400 sites/cell, Kd1 = 2.1 +/- 0.9 pmol/l for intact MNL and Bmax1 = 550 +/- 190 sites/cell, Kd1 = 4.1 +/- 0.9 pmol/l for MNL membranes) and a low affinity binding site (Bmax2 = 9150 +/- 3590 binding sites/cell, Kd2 = 440 +/- 50 pmol/l for intact MNL and Bmax2 = 11560 +/- 4690 sites/cell, Kd2 = 410 +/- 70 pmol/l for MNL membranes). Dissociation of (-)125ICYP from MNL was biphasic consisting of a slow dissociating component (dissociation rate constant k-1 = (0.5 +/- 0.2) X 10(-3) min-1 for intact MNL and k-1 = (1.0 +/- 0.1) X 10(-3) min-1 for MNL membranes) and a fast dissociating component (k-2 = (80 +/- 20) X 10(-3) min-1 for intact MNL and k-2 = (60 +/- 10) X 10(-3) min-1 for MNL membranes). In dissociation experiments started after equilibration with various (-)125ICYP concentrations k-1 and k-2 were independent of the equilibrium concentration, whereas the percentual occupancy of the slow and the fast dissociating component varied and was similar to the estimated fractional occupancy of either binding site at the same (-)125ICYP concentrations in saturation experiments. The association rate constant was in the same order of magnitude for both binding sites. These results suggest two independent classes of binding sites for (-)125ICYP on MNL.

Circannual variation in the expression of β2-adrenoceptors on human peripheral mononuclear leukocytes (MNLs)

“…The data were iteratively fitted to a model of two independent classes of binding sites (a saturable high-affinity and a nonsaturable lowaffinity binding site) using a computer programm named MULTI [9,15]. The total number of highaffinity binding sites (Bmax) and their equilibrium dissociation constant (Ka) were obtained from the curve fit as parameters of the #2-adrenoceptor on MNLs [1].…”

Defects of β2‐adrenergic signal transduction in chronic lymphocytic leukaemia: relationship to disease progression

“…Specific binding data (S ) were iteratively fitted to a two-binding site model by non-linear regression analysis using the Enzfitter program by D. Leatherborrow (Biosoft, Cambridge, UK) as described [14,21,22]. Binding data were also transformed according to Scatchard for visualization of the results [23].…”

Reovirus type 3 binds to antagonist domains of the beta-adrenergic receptor

“…In these studies, however, virus itself was not used to compete with ,B-adrenergic ligand, and it is possible that the kinetics of binding of antibody differ from those of virus or ,-adrenergic ligand. The kinetics of binding of P-adrenergic antagonists are quite complex (1,13), and it is likely that ,B-adrenergic binding by antagonists differs from that of agonist ligands (25). Our conditions were such that we were able to observe competition by reovirus for ,-adrenergic antagonist binding, suggesting utilization of a common receptor domain for virus and antagonist ligand, but these observations do not permit the conclusion that the same receptor epitopes are shared by both virus and antagonist ligand.…”