Optimisation of Conditions for Solubilisation of the Bovine Dopamine D 2 Receptor

Solubilisation of the dopamine D 2 receptor from a membrane preparation of bovine corpus striatum using cholate and NaCl was independently optimised with regard to cholate (0.2%, wt/vol), NaCl (1.5 M ), and membrane protein (4 mg/ml) concentrations. A maximum solubilisation yield of 58% was obtained and receptors were measured using a [ 3 H]spiperone binding assay incorporating a polyethylene glycol precipitation step. Soiubilisation was confirmed by ultracentrifugation studies, passage of the receptor through fine‐pore filters, increased thermolability, and by retention of the prelabelled receptor on gel filtration. The soluble receptor showed saturability and reversibility of binding. Displacement of [ 3 H]spiperone from the soluble receptor by competing compounds correlated closely with displacement from the membrane‐bound receptors. [ 3 H]‐Spiperone binding was found to be pH‐dependent, with maximum binding occurring at pH 7.8. A comparison of solubilisation was made with six other agents both with and without added NaCl and it was concluded that the cholate/ NaCl solubilisation system provides an efficient, inexpensive, and reliable method for the preparation of functional bovine dopamine D 2 receptors.