Studies on the use of a novel affinity matrix, sepharose amine-succinyl-amine haloperidol hemisuccinate, ASA-HHS, for purification of canine dopamine (D2) receptor.
Haloperidol Hemisuccinate (HHS) was synthesized specifically as a ligand for an affinity chromatography matrix. The affinity chromatography matrix, ASA-HHS was developed which had high affinity and capacity for dopamine D2 receptors in solubilized canine striatal preparations. ASA-HHS also demonstrated nonspecific interaction with the D2 receptor. Two fractions, which bound 3H-spiroperidol specifically, with similar one dimensional SDS-PAGE patterns could be eluted successfully with 20 microM haloperidol in only 30% of the runs. Both fractions represented 300-400 fold purification. Two dimensional IEF-PAGE analysis of one of the fractions demonstrated coelution of beta and gamma actin, alpha and beta tubulin with the 3H-spiroperidol binding sites. The pattern of the proteins eluted from ASA-HHS and the inconsistent recovery of active D2 receptors are discussed.
D000818:Animals; D002606:Charcoal; D002846:Chromatography, Affinity / Q000379:methods*; D003342:Corpus Striatum / Q000032:analysis; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D006220:Haloperidol / Q000031:analogs & derivatives*; D066298:In Vitro Techniques; D008970:Molecular Weight; D011943:Receptors, Adrenergic, beta / Q000302:isolation & purification; D011954:Receptors, Dopamine / Q000302:isolation & purification*; D017448:Receptors, Dopamine D2; D012995:Solubility; D013134:Spiperone / Q000378:metabolism; D014316:Tritium
D011943:Receptors, Adrenergic, beta; D011954:Receptors, Dopamine; D017448:Receptors, Dopamine D2; D014316:Tritium; D002606:Charcoal; D013134:Spiperone; C049001:haloperidol hemisuccinate; D006220:Haloperidol