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The major glycoprotein of the human erythrocyte membrane has been isolated by treatment with lithium di-iodosalicylate and found to be a single polypeptide chain with a molecular weight of about 50,000. This molecule, which is 60% carbohydrate and 40% protein, carries multiple blood-group antigens, the receptors for influenza viruses, and various plant agglutinins. Four unique carbohydrate-containing peptides (a-i, a-2, a-3, and jB) are produced by tryptic digestion of the isolated glycoprotein; their order in the molecule has been determined by sequential tryptic digestion of intact erythrocyte membranes and partially digested glycoprotein fragments. Cleavage of the native protein with cyanogen bromide produces five fragments; two of these (C-5 and C-i) contain most of the carbohydrate in the molecule and are derived from the N-terminal half of the polypeptide chain. The nonpolar amino acids of this glycoprotein are located predominantly in the C-terminal fragment (C-2).Phytohemagglutinin conjugated to ferritin has been used to map the distribution of glycoprotein receptors over the surfaces of intact erythrocytes by freezeetching and electron microscopy. This label localizes to sites on the membrane that overlie the intramembranous particles. These findings suggest that the glycoprotein is oriented at the cell surface with its oligosaccharide-rich N-terminal end exposed to the exterior, while its C-terminal segment interacts with other components in the interior of the membrane to form intramembranous particles.Glycoproteins comprise about 10% of the total protein of the human erythrocyte membrane (1). Their carbohydrate moieties are antigenic determinants (2, 3) and receptors for viruses and plant agglutinins (4), and their sialic acid residues are responsible for most of the negative charge at the cell surface (5).Glycoproteins have been isolated from erythrocyte membranes with various solvents, such as phenol (6), butanol (7), pyridine (8), sodium dodecyl sulfate (SDS) (9), or formic acid (10 This report summarizes studies on the properties of the major glycoprotein of the human erythrocyte membrane extracted from membranes by a new procedure with lithium di-iodosalicylate as a dissociating agent. A water-soluble glycoprotein has been isolated in high yield that has the chemical properties and biological activities characteristic of the native membrane-bound molecules. This glycoprotein has been partially characterized after tryptic digestion and cyanogen bromide cleavage, and its location in the membrane has been determined by electron microscopy. MATERIALS AND METHODSHuman blood was obtained fresh from donors, and the erythrocytes were washed three times in phosphate-buffered saline (pH 7.4) before preparation of ghost membranes by the procedure of Dodge et al. (11). Glycoprotein was extracted from freeze-dried ghost membranes with 0.3 M lithium diiodosalicylate and purified by phosphocellulose chromatography (12). Samples were electrophoresed in acrylamide gels containing Tris-glycine (pH 8.4), a...