A comparison of intact human red blood cells and resealed and leaky ghosts with respect to their interactions with surface labelling agents and proteolytic enzymes.
Resealed ghosts and intact red blood cells were directly compared with respect to their interactions with surface proteins by 4.4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) and by pyridoxal phosphate-borohydride (as seen after sodium dodecylsulfate/acrylamide gel electrophoresis) was substantially the same in cells and resealed ghosts under conditions in which a relatively small change would be apparent. In each membrane system, DIDS labels a protein component of apparent molecular weight 95 000 and pyridoxal phosphate labels the same protein plus three glucoprotein components. The sensitivity of surface proteins and of DIDS and pyridoxal phosphate-labelled sites to pronase was also similar in the cells and resealed ghosts. The glycoproteins were digested, in each case, and the 95 000 (molecular weight) protein was largely split into two proteins of apparent molecular weights 65 000 and 35 000, with both portions containing DIDS and pyridoxal phosphate in the presence of hemoglobin was similar to the labelling of intact cells, provided that the pyridoxal phosphate was present on both the outside and inside of the cells. Virtually all of the major protein components visible by staining on acrylamide gels were labelled. It is concluded that none of the probes could detect any substantial differences in reactivity of proteins of the outer surface of the membrane protein conformation or arrangement occur as a consequence of lysis and resealing of ghosts, that are detectable by the reported procedures.
D001798:Blood Proteins / Q000032:analysis; D001894:Borohydrides; D002462:Cell Membrane / Q000648:ultrastructure*; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000648:ultrastructure*; D006023:Glycoproteins / Q000097:blood; D006801:Humans; D008970:Molecular Weight; D011402:Pronase; D011732:Pyridoxal Phosphate; D013267:Stilbenes; D013395:Sucrose; D013451:Sulfonic Acids; D013861:Thiocyanates
D001798:Blood Proteins; D001894:Borohydrides; D006023:Glycoproteins; D013267:Stilbenes; D013451:Sulfonic Acids; D013861:Thiocyanates; D013395:Sucrose; D011732:Pyridoxal Phosphate; D011402:Pronase