The Transmembrane Proteins in the Plasma Membrane of Normal Human Erythrocytes

Abstract

Abstract The arrangement of proteins in the normal human erythrocyte membrane was studied using lactoperoxidase-catalyzed iodination. The outer membrane surface of both intact erythrocytes and resealed ghosts was labeled with externally added lactoperoxidase. The cytoplasmic membrane surface was probed by sealing lactoperoxidase inside the resealed ghosts. Following iodination, the membrane components were separated on sodium dodecyl sulfate acrylamide gels, and the distribution of radioactive iodide into the various molecular weight classes was determined. The same components appear to be labeled on the external surface of resealed ghosts as are labeled in intact erythrocytes: a 90,000 molecular weight component (Protein 3), the major glycoprotein (periodate fuchsin sulfite (PAS)I), and two minor glycoproteins with apparent molecular weights of 40,000 (PAS II) and 27,000 (PAS III). However, PAS II is more extensively labeled in resealed ghosts than in intact erythrocytes, suggesting that alteration of the membrane has occurred during the course of the preparation of membranes and their resealing. Iodination of the internal surface of resealed ghosts reveals that all of the components labeled on the exterior surface also appear to be labeled at the cytoplasmic surface of the membrane. In addition, spectrin also is labeled at the inner surface of resealed ghosts. Separation of the partially purified glycoproteins by sodium dodecyl sulfate gel electrophoresis indicates that the region of PAS II is actually composed of at least two components, designated PAS II' and PAS II. The slower moving component, PAS II', appears to be labeled at both surfaces of the membrane, whereas the faster moving component, PAS II, which also stains more intensely with periodate fuchsin sulfite, is labeled only at the cytoplasmic surface of the membrane. Treatment of intact erythrocytes with pronase hydrolyzes PAS II, indicating that this component also spans the membrane. The differences in labeling between the major glycoprotein, PAS I, and PAS II indicate that these components are not identical, as has been suggested by others. Thus all five components detectable on the external surface of the normal human erythrocyte span the membrane and can be labeled on the interior surface.