Preparation of an affinity chromatography matrix for the selective purification of the dopamine D2 receptor from bovine striatal membranes.
A ligand affinity matrix has been developed and utilized to purify the dopamine D2 receptor approx. 2100 fold from bovine striatal membranes. 3-[2-Aminoethyl]-8-[3-(4-fluorobenzoyl)propyl]-4-oxo-1-phenyl-1,3,8- triazaspiro[4.5]decan-4-one (AES) was synthesized and used to prepare the affinity matrix by coupling to epoxy-activated Sepharose 6B (AES-Sepharose). AES (Ki approximately 1.7 nM) is similar in potency to the parent compound, spiperone (Ki approximately 0.8 nM), in competing for [3H]spiperone-binding activity. AES has no significant potency in competing for the dopamine D1 receptor as assessed by competition for [3H]SCH23390 binding (Ki greater than 1 microM). Covalent photoaffinity labeling of the dopamine D2 receptor in bovine striatal membranes with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS) was prevented by AES at nanomolar concentrations. The dopamine D2 receptor was solubilized from bovine striatal membranes using 0.25% cholate in the presence of high ionic strength, followed by precipitation and subsequent treatment with 0.5% digitonin. Nearly 100% of the [3H]spiperone-binding activity in the cholate-digitonin solubilized preparation was absorbed at a receptor-to-resin ratio of 2:1 (v/v). Dopamine D2 receptor was eluted from the affinity resin using a competing dopaminergic antagonist molecule, haloperidol. Recovery of dopamine D2 receptor activity from the affinity matrix was approx. 9% of the activity adsorbed to the resin. The [3H]spiperone-binding activity in AES-Sepharose affinity purified preparations is saturable and of high affinity (0.2 nM). Affinity-purified preparations maintain the ligand-binding characteristics of a dopamine D2 receptor as assessed by agonist and antagonist competition for [3H]spiperone binding.
Clagett-Dame
M
M
Neuroscience Research Division, Abbott Laboratories, Abbott Park, IL.
Schoenleber
R
R
Chung
C
C
McKelvy
J F
JF
D000345:Affinity Labels; D000818:Animals; D001552:Benzazepines / Q000378:metabolism; D001667:Binding, Competitive; D002417:Cattle; D002462:Cell Membrane / Q000032:analysis; D019826:Cholic Acid; D002793:Cholic Acids; D002846:Chromatography, Affinity; D003342:Corpus Striatum / Q000032:analysis*; D004072:Digitonin; D015394:Molecular Structure; D010777:Photochemistry; D011954:Receptors, Dopamine / Q000302:isolation & purification* / Q000378:metabolism; D017448:Receptors, Dopamine D2; D012685:Sepharose; D013134:Spiperone / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism
D000345:Affinity Labels; D001552:Benzazepines; D002793:Cholic Acids; D011954:Receptors, Dopamine; D017448:Receptors, Dopamine D2; C061999:3-(2-aminoethyl)-8-(3-(4-fluorobenzoyl)propyl)-4-oxo-1-phenyl-1,3,8-triazaspiro(4.5)decan-4-one; D013134:Spiperone; D012685:Sepharose; D019826:Cholic Acid; D004072:Digitonin