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 (P2M) 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 [3H]-DA binding. The specific [3H]-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 Rf value of 0.67 on native disk polyacrylamide gel and it gave one single polypeptide subunit on the SDS gel with an Rf 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 [3H]-DA to this DABP in the P2M fraction and the specific [3H]-DA to the purified DABP demonstrated a nanomolar range affinity corresponding to either D2 or D3 receptors. These data suggested that the purified DABP could be related to either D2 or D3 receptors in the brain.
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000302:isolation & purification*; D002458:Cell Fractionation / Q000379:methods; D002540:Cerebral Cortex / Q000032:analysis* / Q000378:metabolism; D003342:Corpus Striatum / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins; D008297:Male; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008970:Molecular Weight; D009419:Nerve Tissue Proteins; D051381:Rats; D011919:Rats, Inbred Strains; D011954:Receptors, Dopamine / Q000302:isolation & purification*; D013574:Synaptosomes / Q000032:analysis
D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011954:Receptors, Dopamine; D004298:Dopamine