Functional reconstitution of membrane glycoproteins into lipid vesicles using lectin precipitation. Application to the VIP receptor.
We studied the interaction of n-octyl-beta-d-glucopyranoside-solubilized VIP receptors (VIPR) with wheat germ agglutinin and found that the addition of the lectin to the detergent extract led to the formation of aggregates that could be pelleted by high speed centrifugation. Resuspension of the pellet in the presence of the competing trisaccharide, N,N', N"-triacetylchitotriose (TAC), dissociated the lectin from the complex without altering the precipitability of VIPR. The final pellet (referred to as TAC pellet) contained an average of 12% of total protein and 96% of total VIP binding activity with a typical rank order of potency for VIP-related peptides. Lipid analysis and electron microscopic examination indicated that the precipitated material was composed of lipid vesicles. VIPR molecules were shown to be integrally inserted in the liposomes because they could not be dissociated from the vesicles at pH 11 or with high salt concentration. By comparing the liposome-associated VIP binding activity in the presence and absence of detergent and by showing accessibility of VIPR to PNGase F, it was concluded that VIP binding sites were not simply trapped within the reconstituted vesicles but likely exposed at the external surface of the liposomes.
El Battari
A
A
INSERM U.260, Faculté de Médecine, 27 Bd Jean Moulin, F-13385 Marseille Cedex 5, France. Assou.Elbattari@medecine.univ-mrs.fr
Zerfaoui
M
M
Luis
J
J
Xiao
Z L
ZL
Kadi
A
A
Bellan
C
C
Lombardo
D
D
Marvaldi
J
J
D000581:Amidohydrolases / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D011232:Chemical Precipitation; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes; D007700:Kinetics; D008055:Lipids / Q000032:analysis; D008081:Liposomes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008854:Microscopy, Electron; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010741:Phospholipases A / Q000378:metabolism; D018005:Receptors, Vasoactive Intestinal Peptide / Q000378:metabolism*; D012995:Solubility; D014407:Tumor Cells, Cultured; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism; D014909:Wheat Germ Agglutinins
D003902:Detergents; D007457:Iodine Radioisotopes; D008055:Lipids; D008081:Liposomes; D008562:Membrane Glycoproteins; D018005:Receptors, Vasoactive Intestinal Peptide; D014909:Wheat Germ Agglutinins; D014660:Vasoactive Intestinal Peptide; D010741:Phospholipases A; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase