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Solubilization and characterization of GABA B receptor binding sites from porcine brain synaptic membranes
Author(s) -
Facklam M.,
Bowery N.G.
Publication year - 1993
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1993.tb13958.x
Subject(s) - gabab receptor , receptor , baclofen , chaps , chemistry , gabaa receptor , membrane , gtp' , binding site , agonist , biophysics , biochemistry , biology , enzyme
1 The characteristics of membrane bound GABA B 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 GABA B receptor ligands was CGP 54626 > GABA ∼(−)‐baclofen >> CGP 35348 = CGP 36742 > (+)‐baclofen in membranes from both species. 2 For solubilization of GABA B receptors from pig brain, washed membranes were preincubated with 5 m m MgSO 4 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 GABA B receptors. 3 Binding of [ 3 H]‐GABA to GABA B receptors solubilized with 0.5% CHAPS exhibited similar characteristics to the binding at membrane bound receptors since, firstly, the K d and B max values (around 30 n m 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 Ca 2+ (2.5 m m ) was observed and finally, a similar sensitivity to GTP was apparent. 4 Saturation experiments performed with the GABA B antagonist, [ 3 H]‐CGP 54626, indicated a higher K d value and a lower B max value for solubilized (7.7 ± 2.6 n m and 1033 ± 41 fmol mg −1 protein, mean ± s.e.mean, n = 3) than for membrane bound receptors (1.35 ± 0.08 n m , 1171 ± 20 fmol mg −1 protein, n = 3).