Solubilization of dopamine D-1 receptors with a zwitterionic detergent DCHAPS and their reconstitution.
1. Dopamine D-1 receptors of the bovine caudate nucleus were solubilized with different detergents. They were labelled with [3H]SCH 23390 and assayed by filtration through PEI-coated glass fibre filters and Sephadex G-50 columns. 2. DCHAPS was the best solubilizer among all detergents used and at 0.075% DCHAPS, 10 mg/ml protein, 30 min, 4 degrees C, gave the yield of 48.7%. 3. Reconstitution of solubilized receptors was performed using SM-2 Bio-Beads. Phosphatidylcholine did not improve reconstitution suggesting that DCHAPS solubilized sufficient amounts of the membrane phospholipids. 4. Loss of affinity of solubilized receptors to [3H]SCH 23390 binding was reversible. Apparent Kd values of 0.36 +/- 0.02, 21.3 +/- 3.2 and 0.77 +/- 0.05 nM were obtained for membrane-bound, solubilized and reconstituted receptors, respectively.
Soskić
V
V
Department of Chemistry, Faculty of Science, University of Belgrade, Yugoslavia.
Joksimović
J
J
D000818:Animals; D001552:Benzazepines / Q000378:metabolism; D001667:Binding, Competitive; D001923:Brain Chemistry; D002417:Cattle; D002793:Cholic Acids; D003902:Detergents; D011869:Radioligand Assay; D011954:Receptors, Dopamine / Q000737:chemistry* / Q000378:metabolism; D017447:Receptors, Dopamine D1; D012995:Solubility
D001552:Benzazepines; D002793:Cholic Acids; D003902:Detergents; D011954:Receptors, Dopamine; D017447:Receptors, Dopamine D1; C050286:3-((3-deoxycholamidopropyl)dimethylammonio)-1-propane