Solubilization and purification of the alpha 1-adrenergic receptor using a novel affinity resin.

Publisher Website
Google Scholar
Abstract
The highly selective .alpha.1-adrenergic receptor antagonist prazosin was used to identify binding sites having .alpha.-adrenergic specificity in rat hepatic plasma membranes. Solubilization of the membrane-bound receptors was achieved by incubation with the nonionic detergent digitonin, and binding activity was assayed by using [3H]prazosin and a polyethylene glycol precipitation technique. Only 20-30% of the total receptor pool was released by the solubilization procedure. Binding of [3H]prazosin was saturable [maximal value, 206 .+-. 8 fmol[femtomole]/mg of protein (membrane) vs. 74 .+-. 4 fmol/mg of protein (soluble)] and of high affinity [Kd, 0.6 .+-. 0.2 nM (membrane) vs. 0.8 .+-. 0.2 nM (soluble)]. To aid in purification of the receptors, an affinity resin was developed using an analog of prazosin, 2-(4-succinoylpiperazin-1-yl)-4-amino-6,7-dimethoxyquinazoline (CP 57,609; Kd, 2.7 .times. 10-7 M) immobilized via an amide linkage to agarose. The resulting resin demonstrated high affinity (Kd 3.2 .times. 10-7 M) for the solubilized receptors, as determined by competitive inhibition assay. The degree of substitution to the resin was determined by a direct radioimmunoassay using antibodies against albumin-complexed CP 57,609 and found to be 0.1-0.2 .mu.mol/ml of agarose. Affinity chromatography using the resulted in 513-fold purification in a single step. The specificity of the purified binding sites was similar to that of membrane-bound receptors. This novel affinity resin should thus provide a powerful tool for isolating the receptor protein in quantities sufficient for detailed biochemical characterization.