Radioligand Binding Assays for Determining Dissociation Constants of Phytohormone Receptors

2016 ◽  
pp. 23-34 ◽  
Author(s):  
Antje Hellmuth ◽  
Luz Irina A. Calderón Villalobos
Keyword(s):  
Radioligand Binding ◽  
Dissociation Constants ◽  
Binding Assays

Characterization of beta-adrenoreceptors of rainbow trout (Oncorhynchus mykiss) erythrocytes

1991 ◽  
Vol 158 (1) ◽  
pp. 199-216 ◽  
Author(s):  
S. D. Reid ◽  
T. W. Moon ◽  
S. F. Perry
Keyword(s):  
Rainbow Trout ◽  
Radioligand Binding ◽  
Dissociation Constants ◽  
Physiological Saline ◽  
Receptor Density ◽  
Binding Assays ◽  
Binding Characteristics ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Endogenous Catecholamines

Although many studies have characterized these receptors according to pharmacological criteria, this work represents only the second direct characterization of the rainbow trout beta-adrenergic receptors. Radioligand binding assays using (+/−)-4-(3-t-butylamino-2-hydroxy-propoxy)-[5,7-3H]benzimidazol-2- one ([3H]CGP 12177) and 1-[4,6-propyl 3H]dihydroalprenolol ([3H]DHA) were conducted to determine equilibrium binding times, ligand-receptor dissociation constants (KD) and binding capacities (Bmax). Furthermore, we assessed the influence of erythrocyte handling, suspension medium and endogenous catecholamines on Bmax and KD. Maximal binding was obtained when erythrocytes were handled minimally and maintained suspended in plasma rather than physiological saline. Washing and resuspending the erythrocytes, as well as the transfer of the erythrocytes into saline, significantly impaired apparent radioligand affinity and receptor density. Endogenous catecholamines, at levels considered normal for non-stressed animals, did not interfere with the radioligand binding assays, and thus eliminated the need to wash and resuspend erythrocytes. Based on the binding characteristics after intentional lysis of erythrocytes, it is shown that the total receptor population of trout erythrocytes can be estimated by propranolol-displaceable DHA binding, the density of high-affinity surface receptors can best be determined by isoproterenol-displaceable CGP binding, and the number of receptors located within the erythrocytes can be calculated from the difference between the total receptor density and the number of isoproterenol-displacable DHA binding sites. Each of these components must be considered when performing radioreceptor assays using these radioligands and this has significant implications for the interpretation of erythrocyte beta-adrenorecptor localization and mobilization.

scholarly journals Identification of V6.51L as a selectivity hotspot in stereoselective A2B adenosine receptor antagonist recognition

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xuesong Wang ◽  
Willem Jespers ◽  
Rubén Prieto-Díaz ◽  
Maria Majellaro ◽  
Adriaan P. IJzerman ◽  
...  
Keyword(s):  
Radioligand Binding ◽  
Structural Data ◽  
Binding Mode ◽  
Selective Recognition ◽  
Chiral Hplc ◽  
Binding Assays ◽  
Structural Determinants ◽  
X Ray Crystallography ◽  
Adenosine Receptor Antagonist ◽  
Energy Perturbation

AbstractThe four adenosine receptors (ARs) A1AR, A2AAR, A2BAR, and A3AR are G protein-coupled receptors (GPCRs) for which an exceptional amount of experimental and structural data is available. Still, limited success has been achieved in getting new chemical modulators on the market. As such, there is a clear interest in the design of novel selective chemical entities for this family of receptors. In this work, we investigate the selective recognition of ISAM-140, a recently reported A2BAR reference antagonist. A combination of semipreparative chiral HPLC, circular dichroism and X-ray crystallography was used to separate and unequivocally assign the configuration of each enantiomer. Subsequently affinity evaluation for both A2A and A2B receptors demonstrate the stereospecific and selective recognition of (S)-ISAM140 to the A2BAR. The molecular modeling suggested that the structural determinants of this selectivity profile would be residue V2506.51 in A2BAR, which is a leucine in all other ARs including the closely related A2AAR. This was herein confirmed by radioligand binding assays and rigorous free energy perturbation (FEP) calculations performed on the L249V6.51 mutant A2AAR receptor. Taken together, this study provides further insights in the binding mode of these A2BAR antagonists, paving the way for future ligand optimization.

Radioligand Binding Assays for Adenosine Receptors

1990 ◽  
pp. 17-55 ◽  
Author(s):  
Michael Williams ◽  
Kenneth A. Jacobson
Keyword(s):  
Adenosine Receptors ◽  
Radioligand Binding ◽  
Binding Assays

Adrenergic receptors on cerebral microvessels: pericyte contribution

1989 ◽  
Vol 256 (1) ◽  
pp. R224-R230 ◽  
Author(s):  
R. M. Elfont ◽  
P. R. Sundaresan ◽  
C. D. Sladek
Keyword(s):  
Endothelial Cells ◽  
Binding Sites ◽  
Adrenergic Receptors ◽  
Radioligand Binding ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Binding Studies ◽  
Cerebral Microvessels ◽  
Beta Adrenoceptor ◽  
Number Of Binding Sites

R224-R230, 1989.--[125I]iodocyanopindolol ([125I]ICYP) and [3H]rauwolscine were used to quantitate, respectively, the beta- and alpha 2-adrenergic receptors in freshly isolated bovine cerebral microvessels and in pericyte cultures derived from these microvessels. Morphological and immunocytochemical criteria distinguished the pericytes from endothelial cells. Competitive binding studies established the specificity of the radioligand binding. The maximal number of binding sites (Bmax) for [125I]ICYP in the pericytes constituted only 8% of that in the microvessels (3.5 +/- 1.3 vs. 44.4 +/- 6.6 fmol/mg protein). In contrast, the Bmax for [3H]rauwolscine in the pericytes was 50% of that in the microvessels (55.4 +/- 11.8 vs. 111.1 +/- 9.5 fmol/mg protein). The dissociation constants for both [125I]ICYP and [3H]rauwolscine were similar in the two preparations. No alpha 1-adrenergic receptors, as defined by the specific binding of [3H]prazosin, were identified either in the pericytes or microvessels. Overall, our results suggest that pericytes contribute minimally to the total beta-adrenoceptor number of cerebral microvessels, and thus the beta-adrenoceptors must be located predominantly on endothelial cells. However, the contribution of pericytes to the total alpha 2-adrenoceptor number of the microvessels may be substantial.

Radioligand Binding Assays in the Drug Discovery Process: Potential Pitfalls of High Throughput Screenings

2011 ◽  
Vol 51 (02) ◽  
pp. 169-173 ◽  
Author(s):  
François Noël ◽  
Dayde Mendonça-Silva ◽  
Luis Quintas
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
Radioligand Binding ◽  
Discovery Process ◽  
Drug Discovery Process ◽  
Binding Assays
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