Optimisation of Conditions for Solubilisation of the Bovine Dopamine D2 Receptor

Publisher Website
Google Scholar
Abstract
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.