Saturation binding radioligand concentrations

Estimating the Bmax value is technically difficult since it basically is an exercise in estimating an effect at infinite drug concentration. Therefore, the accuracy of the estimate of Bmax is proportional to the maximal levels of radioligand that can be used in the experiment. The attainment of saturation binding can be deceiving when the ordinates are plotted on a linear scale, as they are in Figure 4.2. 

Bylund, D. B., Murrin, L. C. Radioligand saturation binding experiments over large concentration ranges. Life Sci. 2000, 67, 287-2911. 

Fig. 1. Saturation binding of a cannabinoid receptor ligand to brain membranes. Shown are the results of a typical saturation assay of [ H]SR141716A (a CBi-selective antagonist) in rat cortex membranes. Various concentrations (0.05-5.5 nAf) of radioligand were incubated in assay buffer with 0.1% BSA and rat cortex membrane homogenates (20 jig/tube) for 1 h at 30°C in the absence (total DPM) and presence (nonspecific DPM) of 5 pM unlabeled SR141716A. Specific DPM and nonspecific DPM shown are the mean of three values measured in consecutive assay tubes in the same rack. Specific DPM were calculated by subtracting mean nonspecific DPM from mean total DPM at each concentration of [ H]SR141716A.

Saturation binding experiments are performed under equihbrium conditions with increasing concentrations of radioligand in the absence and presence of saturating concentration unlabeled competitor to determine total and nonspecific binding, respectively (Fig. 4). 

Figure 4 Saturation binding. Increasing radioligand concentrations results in saturation of specific binding sites.

Without any calcium added, specific binding increased with the radioligand concentration, but saturation was not reached and meaningful kinetics could not be calculated. None of the known RyR ligands like ryanodine, calmodulin, methylxanthines, ATP, cADP-ribose, nor dantrolene affected flubendiamide binding. 

RRAs are based on the specific and saturable binding of a radioligand (L) to a biological receptor or enzyme (R) at equilibrium. By specific it is meant that L interacts with a single R. Saturability implies that at some concentration of L there is no further... 

Direct quantitation of receptor concentrations and dmg—receptor interactions is possible by a variety of techniques, including fluorescence, nmr, and radioligand binding. The last is particularly versatile and has been appHed both to sophisticated receptor quantitation and to dmg screening and discovery protocols (50,51). The use of high specific activity, frequendy pH]- or p lj-labeled, dmgs bound to cmde or purified cellular materials, to whole cells, or to tissue shces, permits the determination not only of dmg—receptor saturation curves, but also of the receptor number, dmg affinity, and association and dissociation kinetics either direcdy or by competition. Complete theoretical and experimental details are available (50,51). 

Two main types of assay are usually undertaken, namely, competition and saturation assays. The former is used to measure the binding affinity of an unlabelled compound. A range of concentrations of the compound are mixed with a constant concentration of a carrier-free receptor specific radioligand and... 

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