Immunoelectrophoretic analysis of membrane glycoproteins in cultured human endothelial cells.
Human endothelial cells isolated from umbilical cords were solubilized in Triton X-100 and examined by crossed immunoelectrophoresis using rabbit antiserum against endothelial cells. Endogenous labelling of the endothelial cell proteins with 14C-mannose followed by crossed immunoelectrophoresis and autoradiography revealed about 10 immunoprecipitates. Four of these endothelial cell glycoproteins were labelled by lactoperoxidase catalyzed iodination and thus were surface located. Three of the surface located glycoproteins showed reduced electrophoretic mobility after incubation of the endothelial cells with neuraminidase and were therefore sialoglycoproteins. Amphiphilicity of endothelial cell glycoproteins was studied by crossed hydrophobic interaction immunoelectrophoresis with phenyl-Sepharose in the intermediate gel. Amphiphilic proteins also show increasing electrophoretic migration velocity with decreasing concentration of Triton X-100 in the first dimension gels. Five of the endothelial cell glycoproteins were shown to be amphiphilic using these two techniques. Two monoclonal antibodies against the platelet glycoprotein complex IIb-IIIa and glycoprotein IIIa, respectively, reacted with the same precipitate of endothelial cells. When a polyclonal antibody against the platelet glycoprotein complex IIb-IIIa was incorporated into the intermediate gel the position of two endothelial cell precipitates were lowered. One of these was a sialoglycoprotein.
Børsum
T
T
Institute for Surgical Research, University of Oslo, Norway.
D000818:Animals; D002250:Carbon Radioisotopes; D002478:Cells, Cultured; D003902:Detergents; D004730:Endothelium, Vascular / Q000032:analysis* / Q000166:cytology; D005353:Fibronectins / Q000276:immunology; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007123:Immunoelectrophoresis, Two-Dimensional; D007136:Immunoglobulins / Q000276:immunology; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins / Q000032:analysis*; D051379:Mice; D009439:Neuraminidase; D017830:Octoxynol; D010980:Platelet Membrane Glycoproteins / Q000032:analysis; D011092:Polyethylene Glycols; D011817:Rabbits; D012795:Sialoglycoproteins / Q000032:analysis
D002250:Carbon Radioisotopes; D003902:Detergents; D005353:Fibronectins; D007106:Immune Sera; D007136:Immunoglobulins; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins; D010980:Platelet Membrane Glycoproteins; D012795:Sialoglycoproteins; D011092:Polyethylene Glycols; D017830:Octoxynol; D009439:Neuraminidase