Characterization of membrane glycoproteins involved in attachment of microfilaments to the microvillar membrane.
We have characterized a novel integral membrane glycoprotein, from intestinal microvilli, with a relative molecular mass (Mr) of 140 000 as measured by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). This glycoprotein has been purified to homogeneity, and specific antibodies have been prepared to localize it immunocytochemically and to determine its topological organization with respect to the membrane bilayer. This protein's major features are: (1) in contrast to other major glycoproteins of the microvillus membrane it cannot be quantitatively extracted by detergents; (2) treatment of the core residue (an insoluble fraction which remains after Triton X-100 treatment of purified microvilli) with low-ionic-strength buffer in the presence of chelating agents promotes partial solubilization of the amphipathic glycoprotein; (3) controlled proteolysis with papain, using right-side-out sealed vesicles derived from microvilli, results in solubilization of the protein. Once solubilized by papain, the protein co-migrates (on SDS-PAGE) with the protein obtained by dialysis but, unlike the low-ionic-strength form, it does not bind detergents or exhibit hydrophilic properties. These observations are consistent with the protein having a small hydrophobic domain that anchors it to the microvillar membrane. Most of these features were reported some years ago for aminopeptidase, but this newly described protein has a distinct behaviour with respect to its association with microfilaments. We have demonstrated that the 110K protein, a major cytoskeletal protein of the lateral bridges, will bind to the glycoprotein in vitro. These observations suggest that this 140K polypeptide is a transmembrane protein and may provide attachment sites in vivo for cytoskeletal proteins.
D003599:Cytoskeleton / Q000032:analysis*; D006023:Glycoproteins / Q000032:analysis* / Q000276:immunology; D007413:Intestinal Mucosa / Q000276:immunology; D007421:Intestine, Small / Q000032:analysis*; D008565:Membrane Proteins / Q000032:analysis* / Q000276:immunology; D008871:Microvilli / Q000032:analysis; D008970:Molecular Weight; D009994:Osmolar Concentration; D010206:Papain / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D006023:Glycoproteins; D008565:Membrane Proteins; D014357:Trypsin; D010206:Papain