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. 1993 Dec;110(4):1291–1296. doi: 10.1111/j.1476-5381.1993.tb13958.x

Solubilization and characterization of GABAB receptor binding sites from porcine brain synaptic membranes.

M Facklam 1, N G Bowery 1
PMCID: PMC2175871  PMID: 8306067

Abstract

1. The characteristics of membrane bound GABAB receptors in pig brain are similar to those in rat brain as judged by in vitro binding experiments and sensitivity to GTP. The rank order of affinity of GABAB receptor ligands was CGP 54626 > GABA approximately (-)-baclofen >> CGP 35348 = CGP 36742 > (+)-baclofen in membranes from both species. 2. For solubilization of GABAB receptors from pig brain, washed membranes were preincubated with 5 mM MgSO4 and subsequently incubated with various detergents. 3-[(3-Cholamidopropyl)dimethyl-ammoniol]-1-propane sulphonate (CHAPS) (0.5%) proved to be the most successful, solubilizing 22.7 +/- 4.7% (mean +/- s.e. mean, n = 6) of GABAB receptors. 3. Binding of [3H]-GABA to GABAB receptors solubilized with 0.5% CHAPS exhibited similar characteristics to the binding at membrane bound receptors since, firstly, the Kd and Bmax values (around 30 nM and 450 fmol mg-1 protein, respectively) were comparable; secondly, stereospecific binding for baclofen was obtained in both forms; thirdly, the affinity for the agonists GABA and (-)-baclofen and the antagonists CGP 35348, CGP 36742 and CGP 54626 were the same; fourthly, comparable sensitivity to Ca2+ (2.5 mM) was observed and finally, a similar sensitivity to GTP was apparent. 4. Saturation experiments performed with the GABAB antagonist, [3H]-CGP 54626, indicated a higher Kd value and a lower Bmax value for solubilized (7.7 +/- 2.6 nM and 1033 +/- 41 fmol mg-1 protein, mean +/- s.e. mean, n = 3) than for membrane bound receptors (1.35 +/- 0.08 nM, 1171 +/- 20 fmol mg-1 protein, n = 3).

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Selected References

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