EXTRACTION OF BRAIN CAPILLARY MEMBRANE-PROTEINS USING TRITON X-114

Brain capillaries contain a great variety of membrane proteins involve d in the transport of hydrophilic nutrients or in the reception of hor monal signals. The use of Triton X-114 fractionation to purify membran e proteins according to their degree of hydrophobicity was investigate d. Analysis by polyacrylamide gel electrophoresis showed a distinct po lypeptide composition for each fraction. Most of the proteins (68%) we re solubilized by Triton X-114 and, of these proteins, the majority (7 4%) was found in the detergent-poor phase. Alkaline phosphatase which possesses a glycosyl-phosphatidylinositol anchor partitioned in the pe llet of insoluble proteins where it was enriched 2.3-fold. In contrast , gamma-glutamyltranspeptidase, the GLUT1 glucose transporter and P-gl ycoprotein, three integral membrane proteins, and p21(ras) and a 42 kD a G protein a subunit, both covalently modified by lipids, were effici ently solubilized and fractionated in the detergent-rich fraction wher e 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 i n the purification of many blood-brain barrier membrane proteins.