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Solubilization of dopamine‐D 2 receptors from synaptosomal membranes of the bovine caudate nucleus
Author(s) -
Kidrič Marjetka,
Petrović Jelena,
Šoškić V.,
Trajković Divna
Publication year - 1984
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.1476-5381.1984.tb16222.x
Subject(s) - chaps , chemistry , dithiothreitol , spiperone , receptor , centrifugation , membrane , dopamine receptor , chromatography , digitonin , caudate nucleus , biochemistry , biology , endocrinology , enzyme
1 Dopamine D 2 ‐receptors were solubilized from synaptosomal membranes of the bovine caudate nucleus using different detergents. They were labelled with [ 3 H]‐spiperone and assayed by polyethylene glycol precipitation. 2 CHAPS was found to be the best solubilizing agent among all detergents used. Optimal conditions for solubilization were: 0.25% CHAPS, 3.5 mg ml −1 protein, 25 min, 4 °C and the yield of D 2 ‐receptors was 18.6%. 3 Addition of some sulphobetain detergents increased the extent of solubilization, 125 m m NaCl and 0.25 m sucrose decreased it, while SH‐group protecting agents (2 m m dithiothreitol and 6 m m β‐mercaptoethanol), as well as MEGA‐9 and MEGA‐12 were almost ineffective. 4 —log IC 50 values for solublized dopamine D 2 ‐receptors are in linear correlation with the corresponding values for membrane‐bound receptors ( r = 0.962, slope factor 0.96) and K d value of solubilized receptors was 3.61 ± 0.94 n m , while that of membrane‐bound receptors was 1.25±0.10 n m . 5 Specific binding of [ 3 H]‐spiperone to the solubilized receptors resolved by linear sucrose density gradient centrifugation shows two maxima, one in the first several fractions from the bottom and the other with an apparent 5 value of 7.3.