Association of Rho(D) polypeptides with the membrane skeleton in Rho(D)-positive human red cells.
The Rho(D) antigen was recently identified as a 28,000 to 33,000 m.w. polypeptide expressed on the surface of human Rho(D)+ cells. We now show that 70 to 80% of the Rho(D) polypeptides remain firmly associated with the membrane skeleton (detergent-insoluble matrix) obtained after treatment of isolated membranes with Triton X-100. The same treatment solubilized most of the major sialoglycoprotein, glycophorin A. The membrane skeleton-bound Rho(D) polypeptides were not solubilized by procedures that dissociated spectrin, actin, and glyceraldehyde-3-phosphate dehydrogenase from the membrane. Affinity-purified 125I-labeled anti-Rho(D) antibodies bound to intact Rho(D)+ cells, Rho(D)+ membranes, and isolated membrane skeletons from Rho(D)+ cells, but not to Rho(D)- cells. The binding to Rho(D)+ cells was competitively inhibited efficiently by Rho(D)+ membranes and weakly by Rho(D)- membranes. When isolated unsealed Rho(D)+ and Rho(D)- membranes were labeled by lactoperoxidase-catalyzed iodination and solubilized in Triton X-100, Rho(D) polypeptides were immune precipitated only from Rho(D)+ membranes.
D000936:Antigen-Antibody Complex / Q000378:metabolism; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D011232:Chemical Precipitation; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D006021:Glycophorins / Q000276:immunology; D006801:Humans; D007518:Isoantibodies / Q000276:immunology; D010455:Peptides / Q000097:blood* / Q000276:immunology; D012204:Rh-Hr Blood-Group System / Q000032:analysis* / Q000276:immunology; D012462:Saline Solution, Hypertonic
D000936:Antigen-Antibody Complex; D006021:Glycophorins; D007518:Isoantibodies; D010455:Peptides; C033242:RH-antibodies; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen; D012462:Saline Solution, Hypertonic; D004492:Edetic Acid