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.
Department of Physiology, Duke University Medical Center, Durham, North Carolina 27710.
J A
JA
Gingrich
N
N
Amlaiky
S E
SE
Senogles
W K
WK
Chang
R D
RD
McQuade
J G
JG
Berger
M G
MG
Caron
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