A brain synaptic dopamine‐binding protein: Isolation and partial characterization

A dopamine‐binding protein (DABP) has been purified from the rat brain cortex to homogeneity. Solubilization of the DABP from the synaptosomal membranes (P 2M ) by cholic acid, subsequent agarose gel filtration of the cholic acid extract to separate phospholipids from the DABP, and lastly DA affinity chromatography successfully resulted in a purified DABP with approximately 0.006% yield in protein concentration and 0.03% yield in specific [ 3 H]‐DA binding. The specific [ 3 H]‐DA binding of the purified DABP was 117 fmol/mg protein/10 min with a 4.6‐fold purification compared with the whole homogenate. The purified DABP had an R f value of 0.67 on native disk polyacrylamide gel and it gave one single polypeptide subunit on the SDS gel with an R f value of 0.63. The apparent molecular weight of this single subunit was estimated to be 34.5 kilodaltons. The elution patterns from either DA‐ or ADTN‐affinity (2‐amino‐6, 7‐dihydroxy‐1,2,3,4‐tetrahydronaphthalene‐affinity) columns indicated that this DABP had higher affinity for DA agonists than for DA antagonists. Photoaffinity labeling of [ 3 H]‐DA to this DABP in the P 2M fraction and the specific [ 3 H]‐DA to the purified DABP demonstrated a nanomolar range affinity corresponding to either D 2 or D 3 receptors. These data suggested that the purified DABP could be related to either D 2 or D 3 receptors in the brain.