Solubilization of dopamine-D2 receptors from synaptosomal membranes of the bovine caudate nucleus.
Dopamine D2-receptors were solubilized from synaptosomal membranes of the bovine caudate nucleus using different detergents. They were labelled with [3H]-spiperone and assayed by polyethylene glycol precipitation. 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 degrees C and the yield of D2-receptors was 18.6%. Addition of some sulphobetain detergents increased the extent of solubilization, 125 mM NaCl and 0.25 M sucrose decreased it, while SH-group protecting agents (2 mM dithiothreitol and 6 mM beta-mercaptoethanol), as well as MEGA-9 and MEGA-12 were almost ineffective. -log IC50 values for solubilized dopamine D2-receptors are in linear correlation with the corresponding values for membrane-bound receptors (r = 0.962, slope factor 0.96) and Kd value of solubilized receptors was 3.61 +/- 0.94 nM, while that of membrane-bound receptors was 1.25 +/- 0.10 nM. Specific binding of [3H]-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 S value of 7.3.
D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002421:Caudate Nucleus / Q000032:analysis*; D002499:Centrifugation, Density Gradient; D002793:Cholic Acids; D003902:Detergents; D006358:Hot Temperature; D066298:In Vitro Techniques; D008566:Membranes / Q000032:analysis; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011954:Receptors, Dopamine / Q000032:analysis* / Q000378:metabolism; D012995:Solubility; D013134:Spiperone / Q000378:metabolism; D013574:Synaptosomes / Q000032:analysis*
D002793:Cholic Acids; D003902:Detergents; D009419:Nerve Tissue Proteins; D011954:Receptors, Dopamine; D013134:Spiperone; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate