GTPyS assay

The biological activity of the compounds was assessed in two binding assays using [ H]-CP 55,940 (see Table 6.36) and [ H]-rimonabant (data not shown), and their functional activity determined using a GTPyS assay. Upon... 

ECso =1.4 and 1500 nM in GTPyS assays using cloned and S receptors, respectively... 

Table 3 Agonist activity of 14-pyridylacryloylaminodihydromorphinones and codeinones in the [35S]GTPyS assay ...

Table 5 Activity of 14-aminomorphindole and derivatives in binding and [35S]GTPyS assays...

Figure 3 Enhancement by brucine of ACh potency at Mi receptors in functional assays in membranes. Brucine (100 pM) increased the potency of ACh to stimulate [35S]GTPyS binding to G proteins in mi CHO cell membranes. In this experiment, the EC50 value for ACh decreased from 2.8 pM ( ) to 0.9 pM ( ) without significantly affecting the basal response or maximal stimulation. (From Ref. 9.)...

Figure 2 Effect of pertussis toxin treatment on the regulation of basal [35S]GTP-yS binding by (2S,3R)TMT-L-Tic in hDOR/CHO cell membranes. Chinese hamster ovary cells stably expressing the human delta opioid receptors (hDOR/CHO) were treated in the presence ( ) or absence ( ) of 50 ng/mL pertussis toxin for 18 h. The cells were washed and cell membranes prepared as previously described [40]. Membranes were incubated with appropriate concentrations of (2S,3R)TMT-L-Tic in the presence of 0.1 nM [35S]GTPyS (1,250 Ci/mmol) in 1.0 mL of assay buffer (25 mM Tris, 150 mM NaCl, 50 iM GDP, 2.5 mM MgCl2, 1 mM EDTA, 30 pM bestatin, 10 pM captopril, pH = 7.4). After 90 min incubation at 30°C, the reaction was terminated by rapid filtration. The filters were washed with 25 mM Tris/120 mM NaCl, pH 7.4, and bound radioactivity was measured by liquid scintillation spectrophotometry.

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