Characterization of dopamine binding sites in standard preparations of brain synaptic membranes

Brain synaptic membranes, prepared according to DeRobertis and to Whittaker, were compared morphologically and biochemically. Electron microscopy revealed that DeRobertis preparations were heavily contaminated with synaptosomes, mitochondria, storage vesicles, and a variety of extraneous membrane structures; Whittaker preparations appeared to consist of synaptic membranes, with a relatively small number of presynaptic storage vesicles. Substantial dopamine β-hydroxylase, monoamine oxidase, and catecholamine-O-methyl transferase activities were present in DeRobertis preparations; Whittaker membrane preparations contained low-to-undetectable activities of these enzymes. Although dopamine bound to membranes derived from both brain cortex and corpus striatum, only membranes from the latter area contained dopamine-sensitive adenylate cyclase. Binding of dopamine to Whittaker striatal synaptic membrane preparations was relatively rapid, saturable, partially reversible, and did not chemically alter the ligand. A variety of dopamine acceptors appeared to be present in this preparation since the ed50 (from Klotz plots) of dopamine binding was 25 times that for activation of adenylate cyclase; Scatchard plots revealed both high and low affinity binding sites for dopamine; and, binding and adenylate cyclase activation studies with dopamine, carried out in the presence of fluphenazine, cocaine, pargyline, and reserpine, reveal that even the more-pure Whittaker preparations of striatal synaptic membranes contain at least three major dopamine-binding components: postsynaptic (and perhaps presynaptic as well) membrane receptors linked to adenylate cyclase, a presynaptic synaptosomal reuptake receptor, and storage vesicle sequestration.