Extraction of brain capillary membrane proteins using Triton X-114.
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.
Pouliot
J F
JF
Département de Chimie-Biochimie, Université du Québec à Montréal, Canada.
Béliveau
R
R
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis; D000469:Alkaline Phosphatase / Q000032:analysis; D000818:Animals; D001812:Blood-Brain Barrier; D015153:Blotting, Western; D001921:Brain / Q000098:blood supply*; D002196:Capillaries / Q000737:chemistry / Q000201:enzymology; D002417:Cattle; D002458:Cell Fractionation; D019204:GTP-Binding Proteins / Q000032:analysis; D051272:Glucose Transporter Type 1; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis* / Q000302:isolation & purification; D009004:Monosaccharide Transport Proteins / Q000032:analysis; D017830:Octoxynol; D015689:Oncogene Protein p21(ras) / Q000032:analysis; D011092:Polyethylene Glycols; D012995:Solubility; D005723:gamma-Glutamyltransferase / Q000032:analysis
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D051272:Glucose Transporter Type 1; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D007770:L-Lactate Dehydrogenase; D005723:gamma-Glutamyltransferase; D000469:Alkaline Phosphatase; D019204:GTP-Binding Proteins; D015689:Oncogene Protein p21(ras)