Affinity chromatography of the D1 dopamine receptor from rat corpus striatum.
The D1 dopamine receptor from rat corpus striatum has been purified 200-250-fold by using a newly developed biospecific affinity chromatography matrix based on a derivative of the D1 selective antagonist SCH 23390. This compound, (RS)-5-(4-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benz azepin-7-o l (SCH 39111), possesses high affinity for the D1 receptor and, when immobilized on Sepharose 6B through an extended spacer arm, was able to adsorb digitonin-solubilized D1 receptors. The interaction between the solubilized receptor and the affinity matrix was biospecific. Adsorption of receptor activity could be blocked in a stereoselective fashion [SCH 23390 greater than SCH 23388; (+)-butaclamol greater than (-)-butaclamol]. The elution of [3H]SCH 23390 activity from the gel demonstrated similar stereoselectivity for antagonist ligands. Agonists eluted receptor activity with a rank order of potency consistent with that of a D1 receptor [apomorphine greater than dopamine greater than (-)-epinephrine much greater than LY 171555 greater than serotonin]. SCH 39111-Sepharose absorbed 75-85% of the soluble receptor activity, and after the gel was washed extensively, 35-55% of the absorbed receptor activity could be eluted with 100 microM (+)-butaclamol with specific activities ranging from 250 to 450 pmol/mg of protein. The affinity-purified receptor retains the ligand binding characteristics of a D1 dopamine receptor. This affinity chromatography procedure should prove valuable in the isolation and molecular characterization of the D1 dopamine receptor.
Gingrich
J A
JA
Department of Physiology, Duke University Medical Center, Durham, North Carolina 27710.
Amlaiky
N
N
Senogles
S E
SE
Chang
W K
WK
McQuade
R D
RD
Berger
J G
JG
Caron
M G
MG
D000818:Animals; D002462:Cell Membrane / Q000032:analysis; D002846:Chromatography, Affinity; D003342:Corpus Striatum / Q000032:analysis*; D008024:Ligands; D051381:Rats; D011954:Receptors, Dopamine / Q000032:analysis* / Q000302:isolation & purification; D012995:Solubility
D008024:Ligands; D011954:Receptors, Dopamine