用两性离子去污剂DCHAPS增溶多巴胺D-1受体及其重组。

Solubilization of dopamine D-1 receptors with a zwitterionic detergent DCHAPS and their reconstitution.

Soskić V, Joksimović J

Department of Chemistry, Faculty of Science, University of Belgrade, Yugoslavia.

Int J Biochem. 1992 Apr;24(4):585-91. doi: 10.1016/0020-711x(92)90331-t.

Abstract

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.

摘要

用不同的去污剂溶解牛尾状核的多巴胺D-1受体。用[³H]SCH 23390对其进行标记，并通过PEI包被的玻璃纤维滤器和葡聚糖G-50柱过滤进行测定。2. 在所有使用的去污剂中，DCHAPS是最佳增溶剂，在0.075% DCHAPS、10 mg/ml蛋白质、30分钟、4℃的条件下，产率为48.7%。3. 使用SM-2生物珠对溶解的受体进行重组。磷脂酰胆碱并未改善重组效果，这表明DCHAPS溶解了足够量的膜磷脂。4. 溶解的受体对[³H]SCH 23390结合的亲和力丧失是可逆的。膜结合受体、溶解受体和重组受体的表观解离常数（Kd）值分别为0.36±0.02、21.3±3.2和0.77±0.05 nM。