Optimisation of conditions for solubilisation of the bovine dopamine D2 receptor.
Solubilisation of the dopamine D2 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 [3H]spiperone binding assay incorporating a polyethylene glycol precipitation step. Solubilisation 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 [3H]spiperone from the soluble receptor by competing compounds correlated closely with displacement from the membrane-bound receptors. [3H]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 D2 receptors.
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000032:analysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D019826:Cholic Acid; D002793:Cholic Acids; D003342:Corpus Striatum / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011954:Receptors, Dopamine / Q000302:isolation & purification* / Q000378:metabolism; D017448:Receptors, Dopamine D2; D012965:Sodium Chloride; D012995:Solubility; D013134:Spiperone / Q000378:metabolism; D014461:Ultracentrifugation
D002793:Cholic Acids; D011954:Receptors, Dopamine; D017448:Receptors, Dopamine D2; D012965:Sodium Chloride; D013134:Spiperone; D019826:Cholic Acid