title: The detection of associated erythrocyte membrane proteins by cross-linking and surface radiolabelling.
abstract: Membrane proteins of intact erythrocytes were cross-linked using the cleavable homobifunctional reagent, 3,3'-dithiobis(propionimidate) and the non-cleavable heterobifunctional reagent, 4-azidobenzimidate. Subsequently, the exposed cross-linked membrane proteins were radiolabelled by the lactoperoxidase method. Cross-linked and radiolabelled membrane proteins were analyzed by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis in which resolution in the second dimension was preceded by mercaptoethanol treatment. Complexes so formed by 3,3'-dithiobis(propionimidate) were cleavable, whilst components cross-linked by 4-azidobenzimidate were uncleavable. Three complexes were found. Two of these contained protein III as the only radiolabelled component. It was assumed that the first (Mr approx. 200000) represents a dimer and the second (Mr approx. 300000) an oligomer of protein III. The third complex, which was identified after cross-linking with 4-azidobenzimidate, consisted of PAS II and a low molecular weight (lipid-like) component. Since PAS I (dimer of a PAS II) was not associated with this component it was concluded that PAS I and PAS II are situated in different environments of the erythrocyte membrane.
mesh_terms: D001345:Autoradiography; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000032:analysis*; D004912:Erythrocytes / Q000032:analysis*; D006801:Humans; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000032:analysis*
chemical_list: D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D008565:Membrane Proteins