Recent advances in the purification of the alpha 1-adrenoceptor

In order to purify the alpha-adrenergic receptor labeled with [3H]POB, antibodies against the irreversible alpha-adrenergic antagonist, phenoxybenzamine (POB), have been raised in rabbits immunized with POB conjugated to immunoglobulin. The antigen was prepared by first reacting POB with the sulfhydryl group of cysteine, after which the POB complex was coupled to thyroglobulin in the presence of water soluble carbodimide. POB-specific antibodies were first detected 10 days after the first booster injection. These antibodies display a high degree of specificity. Slight cross-reactivity occurred only with compounds structurally related to POB, such as dibenamine and nitrogen mustards, while catecholamines and alpha-adrenergic compounds not structurally related to POB did not cross-react, even when present in 100,000-fold excess. Moreover, the antibodies are still capable of recognizing POB once bound to alpha-adrenergic receptor. When membranes, prelabeled with [3H]POB, are solubilized with digitonin and incubated with anti-POB antiserum, the radioactivity can be specifically precipitated upon addition of goat anti-rabbit gamma-globulin serum. Thus these antibodies may be a useful tool in the purification of the alpha-adrenergic receptor of rat liver covalently linked to POB.