The human placental transferrin receptor: reconstitution into liposomes and electron microscopy.
Human transferrin receptor was isolated from Triton X-100 solubilized placental plasma membranes by a rapid one-step chromatographic procedure based on immunoadsorption of the receptor-transferrin complex on anti-transferrin Sepharose and lectin-affinity on wheat germ agglutinin. Following exchange of Triton X-100 with CHAPS or n-octylglucoside, the purified receptor was incorporated into egg phosphatidylcholine liposomes upon detergent removal by dialysis (lipid/protein ratio 15:1 to 45:1 (w/w)). Reconstitution of the receptor was confirmed by trypsin cleavage to dissociate the large extracellular receptor domain from the liposomal membranes. Electron micrographs of the receptor-lipid recombinants negatively stained with sodium sillicotungstate, showed that the receptor molecules distributed very inhomogeneously on the liposomes, most receptors being clustered. Single copies of the receptor were seen as elongate structures (5 x 10 nm) oriented with their long axis parallel to the liposome surface and separated from this by a 2-3 nm gap. This result provides evidence for a narrow connecting link between the globular extracellular receptor domain and the membrane spanning segment.
Demant
E J
EJ
Department of Biochemistry, Panum Institute, University of Copenhagen, Denmark.
Christiansen
K
K
Tranum-Jensen
J
J
D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D008081:Liposomes; D008854:Microscopy, Electron; D017830:Octoxynol; D010920:Placenta / Q000378:metabolism*; D011092:Polyethylene Glycols; D011247:Pregnancy; D011487:Protein Conformation; D011990:Receptors, Transferrin / Q000737:chemistry / Q000302:isolation & purification* / Q000648:ultrastructure; D014357:Trypsin
D008081:Liposomes; D011990:Receptors, Transferrin; D011092:Polyethylene Glycols; D017830:Octoxynol; D014357:Trypsin