Assay for soluble dopamine receptors by the precipitation method

Abstract

Receptors with high affinity for neuroleptics (D2-type dopamine receptors) were solubilized from dog striatum using 1% digitonin. Soluble receptors were labelled using [3H]spiperone, and the bound 3H-ligand was separated from the free [3H]spiperone by molecular sieving on the Sephadex G-50 columns. This paper presents a polyethylene glycol (PEG) precipitation method designed to replace this column method. IC50 values for both neuroleptics and dopamine agonists, as well as dissociation constants for [3H]spiperone were similar for both methods. The precipitation method yielded a receptor density of 64 fmol/mg protein whereas the molecular sieving technique yielded a value of 194 fmol/mg protein. The qualitative similarities between the methods validates using the rapid precipitation method for monitoring the receptor during various stages of its purification.