Analytical determination of receptor-ligand dissociation constants of two populations of receptors from displacement curves.

Almagor, Hagai; Levitzki, Alexander

doi:10.1073/pnas.87.17.6482

Cited by 6 publications

(3 citation statements)

References 5 publications

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“…In this paper we analysed multisite protein kinetics in response to rapid changes in ligand concentrations. The model for multisite protein kinetics for variable number of bound ligands was developed for two physiological cases: when the concentration of ligand is much higher [ 55 , 56 ] and when it is comparable with the concentration of the multisite protein [ 57 ]. The results obtained by the proposed model allow for an accurate interpretation of the experimental data for the concentration of multisite proteins such as CaM, TnC, CaN and other Ca 2+ dependent secondary messengers regulated by Ca 2+ ions.…”

Section: Discussionmentioning

confidence: 99%

Kinetic regulation of multi-ligand binding proteins

et al. 2016

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BackgroundSecond messengers, such as calcium, regulate the activity of multisite binding proteins in a concentration-dependent manner. For example, calcium binding has been shown to induce conformational transitions in the calcium-dependent protein calmodulin, under steady state conditions. However, intracellular concentrations of these second messengers are often subject to rapid change. The mechanisms underlying dynamic ligand-dependent regulation of multisite proteins require further elucidation.ResultsIn this study, a computational analysis of multisite protein kinetics in response to rapid changes in ligand concentrations is presented. Two major physiological scenarios are investigated: i) Ligand concentration is abundant and the ligand-multisite protein binding does not affect free ligand concentration, ii) Ligand concentration is of the same order of magnitude as the interacting multisite protein concentration and does not change. Therefore, buffering effects significantly influence the amounts of free ligands. For each of these scenarios the influence of the number of binding sites, the temporal effects on intermediate apo- and fully saturated conformations and the multisite regulatory effects on target proteins are investigated.ConclusionsThe developed models allow for a novel and accurate interpretation of concentration and pressure jump-dependent kinetic experiments. The presented model makes predictions for the temporal distribution of multisite protein conformations in complex with variable numbers of ligands. Furthermore, it derives the characteristic time and the dynamics for the kinetic responses elicited by a ligand concentration change as a function of ligand concentration and the number of ligand binding sites. Effector proteins regulated by multisite ligand binding are shown to depend on ligand concentration in a highly nonlinear fashion.

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Section: Discussionmentioning

confidence: 99%

Kinetic regulation of multi-ligand binding proteins

et al. 2016

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“…In previous studies, exact mathematical descriptions have been presented for ligand binding displacement curves, both in the case of a single (Horovitz & Levitzki, 1987) and in the case of multiple (Almagor & Levitzki, 1990) receptor classes.…”

Section: Discussionmentioning

confidence: 99%

An exact general analysis of ligand binding displacement and saturation curves

Zoelen¹,

Kramer²,

Reen³

et al. 1993

Biochemistry

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Quantitative analysis of a ligand-protein interaction relates binding to the free concentration of ligand molecules in solution. A theoretical analysis is presented herein, by which intermolecular interactions can be described as a function of the added concentrations of ligand molecules. Following this analysis, ligand binding displacement and saturation curves can be converted directly into a linear form, even when nonradioactively labeled ligands are used to detect the ligand-protein interaction. From the linearities obtained, relevant binding parameters, including the binding dissociation constant, can be calculated. On the basis of this analysis, binding parameters have been characterized for the interaction between biotin-protein A and immunoglobulins, using ELISA-type detection, and for the interaction of a fluorescently labeled fatty acid with a specific fatty acid binding protein.

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“…The ratio between specific binding of a-BH*NT(2-13) and e2-BH*K2NN is given in the last column . ation constants of a two-site model, for which a singlesite model fails to describe the binding data, is 7 (Almagor and Levitzki, 1990) . Accordingly, Scatchard plots describing the binding of the a-radiolabeled analogues of NT(2-13) or [Lys 2 ]neuromedin to newborn mouse brain (Figs .…”

Section: Specific Bindingmentioning

confidence: 99%

Radiolabeled Ligands Specific for the G Protein‐Coupled State of Neurotensin Receptors

Gaudriault

Zsürger

Vincent

1996

Journal of Neurochemistry

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Radiolabeled analogues of neuromedin N have been prepared by acylation of the α, ε1, and ε2 amino groups of [Lys2]neuromedin N (Lys‐Lys‐Pro‐Tyr‐Ile‐Leu) either with the 125I‐labeled Bolton‐Hunter reagent or with N‐succinimidyl[2,3‐3H]propionate. The binding properties of the purified analogues toward newborn mouse brain homogenate or toward membranes of cells transitorily (COS) or permanently (AA1) transfected with the cloned rat brain neurotensin receptor cDNA were evaluated and compared with those of radiolabeled neurotensin. The α‐modified analogue of [Lys2]neuromedin N behaves exactly like neurotensin in these binding experiments, whereas the ε1‐ and ε2‐modified analogues selectively recognize the fraction of neurotensin binding sites that is sensitive to GTPγS. The proportion of neurotensin receptors coupled to GTP binding proteins is ∼50% in membranes of newborn mouse brain or of AA1 cells that respond to neurotensin by an increase of the intracellular inositol trisphosphate concentration. By contrast, membranes of transitorily transfected COS cells that do not respond to neurotensin exhibit very low levels of GTP‐sensitive receptors labeled with the ε1‐ or ε2‐modified analogues. These radiolabeled peptides offer new tools to selectively detect active neurotensin receptors.

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Analytical determination of receptor-ligand dissociation constants of two populations of receptors from displacement curves.

Cited by 6 publications

References 5 publications

Kinetic regulation of multi-ligand binding proteins

Kinetic regulation of multi-ligand binding proteins

An exact general analysis of ligand binding displacement and saturation curves

Radiolabeled Ligands Specific for the G Protein‐Coupled State of Neurotensin Receptors

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