Detergent-mediated reconstitution of a glycosyl-phosphatidylinositol-protein into liposomes.
A three-step detergent-mediated reconstitution has been applied to the incorporation of a glycosyl-phosphatidylinositol-protein into liposomes. The protein studied was alkaline phosphatase from bovine intestine. Liposomes prepared by dialysis were treated with various amounts of two detergents, either n-octyl beta-D-glucoside or Triton X-100. At different steps of the solubilization process, protein was added and the detergent was removed by hydrophobic resins. The most efficient reconstitutions were obtained with an octyl glucoside concentration corresponding to the onset of liposome solubilization and with a Triton X-100 concentration leading to partial solubilization of the liposomes. The involvement of the glycosyl-phosphatidylinositol anchor in alkaline phosphatase reconstitution was demonstrated by the inability of phosphoinositol-specific phospholipase-C-hydrolysed alkaline phosphatase to incorporate into liposomes. Between 70-85% of the protein associated with liposomes were anchored in the outer leaflet of the bilayer, oriented towards the outside of the liposome. The remainder was trapped within the lumen of the liposomes.
Angrand
M
M
Laboratoire de Physico-Chimie Biologique, UPRESA CNRS 5013, UCB-Lyon I, France.
Briolay
A
A
Ronzon
F
F
Roux
B
B
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides; D017261:Glycosylphosphatidylinositols / Q000737:chemistry* / Q000378:metabolism; D007413:Intestinal Mucosa / Q000201:enzymology; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism; D017830:Octoxynol; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D011510:Proteolipids / Q000737:chemistry* / Q000378:metabolism; D012995:Solubility; D010738:Type C Phospholipases / Q000378:metabolism
D003902:Detergents; D005960:Glucosides; D017261:Glycosylphosphatidylinositols; D008081:Liposomes; D011510:Proteolipids; C020485:proteoliposomes; C018619:octyl-beta-D-glucoside; D017830:Octoxynol; D000469:Alkaline Phosphatase; D010738:Type C Phospholipases; D043265:Phosphatidylinositol Diacylglycerol-Lyase