Sivaprasadarao A, Boudjelal M, Findlay J B
Department of Biochemistry and Molecular Biology, University of Leeds, U.K.
Biochem J. 1994 Aug 15;302 ( Pt 1)(Pt 1):245-51. doi: 10.1042/bj3020245.
The membrane receptor for retinol-binding protein (RBP) has been solubilized from human placental brush-border membranes with octyl-beta-glucoside, Nonidet P-40 and CHAPS. A method, based on the preferential precipitation of 125I-RBP-receptor complex with poly(ethylene glycol) 8000, was developed in order to measure the RBP-binding activity in the detergent extracts. The receptor was fairly stable (4 degrees C, 7 days) in octyl-beta-glucoside and Nonidet P-40, but quickly lost activity in CHAPS. The detergent-solubilized form retained all the properties characteristic of the membrane-bound protein, except for a small decrease in affinity for RBP (3- and 7-fold in Nonidet P-40 and octyl-beta-glucoside respectively). The receptor was isolated using recombinant RBP coupled to Reacti-Gel 6X affinity matrix. The purified material contained major and minor protein species of 63 and 55 kDa respectively on SDS/PAGE.
视黄醇结合蛋白（RBP）的膜受体已通过辛基-β-葡萄糖苷、诺乃洗涤剂P-40和3-[(3-胆酰胺丙基)二甲基铵]-1-丙磺酸（CHAPS）从人胎盘刷状缘膜中溶解出来。为了测量去污剂提取物中的RBP结合活性，开发了一种基于用聚乙二醇8000优先沉淀125I-RBP-受体复合物的方法。该受体在辛基-β-葡萄糖苷和诺乃洗涤剂P-40中相当稳定（4℃，7天），但在CHAPS中很快失去活性。除了对RBP的亲和力略有下降（在诺乃洗涤剂P-40和辛基-β-葡萄糖苷中分别下降3倍和7倍）外，去污剂溶解形式保留了膜结合蛋白的所有特性。使用与Reacti-Gel 6X亲和基质偶联的重组RBP分离该受体。纯化的物质在十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（SDS/PAGE）上分别含有63 kDa和55 kDa的主要和次要蛋白质种类。
