Solubilization of dopamine‐D2receptors from synaptosomal membranes of the bovine caudate nucleus

Abstract

1 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. 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 D₂-receptors was 18.6%. 3 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 β-mercaptoethanol), as well as MEGA-9 and MEGA-12 were almost ineffective. 4 —log IC₅₀ values for solublized dopamine D₂-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 nm, while that of membrane-bound receptors was 1.25±0.10 nm. 5 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 5 value of 7.3.