Pouliot J F, Béliveau R
Département de Chimie-Biochimie, Université du Québec à Montréal, Canada.
Biochem Mol Biol Int. 1994 Dec;34(6):1075-84.
Brain capillaries contain a great variety of membrane proteins involved in the transport of hydrophilic nutrients or in the reception of hormonal signals. The use of Triton X-114 fractionation to purify membrane proteins according to their degree of hydrophobicity was investigated. Analysis by polyacrylamide gel electrophoresis showed a distinct polypeptide composition for each fraction. Most of the proteins (68%) were solubilized by Triton X-114 and, of these proteins, the majority (74%) was found in the detergent-poor phase. Alkaline phosphatase which possesses a glycosyl-phosphatidylinositol anchor partitioned in the pellet of insoluble proteins where it was enriched 2.3-fold. In contrast, gamma-glutamyltranspeptidase, the GLUT1 glucose transporter and P-glycoprotein, three integral membrane proteins, and p21ras and a 42 kDa G protein alpha subunit, both covalently modified by lipids, were efficiently solubilized and fractionated in the detergent-rich fraction where they were enriched 3.5-, 4.8-, 4.4-, 4.5- and 4.7-fold, respectively. Triton X-114 fractionation could therefore be used as a first step in the purification of many blood-brain barrier membrane proteins.
脑毛细血管包含多种参与亲水性营养物质运输或激素信号接收的膜蛋白。研究了使用Triton X-114分级分离法根据膜蛋白的疏水性程度来纯化它们。聚丙烯酰胺凝胶电泳分析显示每个级分都有独特的多肽组成。大多数蛋白质（68%）可被Triton X-114溶解，在这些蛋白质中，大部分（74%）存在于去污剂贫乏相中。具有糖基磷脂酰肌醇锚定的碱性磷酸酶分配在不溶性蛋白质沉淀中，在那里它富集了2.3倍。相比之下，γ-谷氨酰转肽酶、GLUT1葡萄糖转运蛋白和P-糖蛋白这三种整合膜蛋白，以及p21ras和42 kDa G蛋白α亚基（两者均被脂质共价修饰），在富含去污剂的级分中被有效溶解和分级，在那里它们分别富集了3.5倍、4.8倍、4.4倍、4.5倍和4.7倍。因此，Triton X-114分级分离法可作为纯化许多血脑屏障膜蛋白工作的第一步。
