Solubilization of human erythrocyte membrane glycoproteins and separation of the MN glycoprotein from a glycoprotein with I, S, and A activity

Abstract

Human erythrocyte membrane glycoproteins are recovered in high yields in the aqueous phase after extraction of the membranes with a mixture of chloroform-methanol. Sodium dodecyl sulfate-acrylamide gel electrophoresis of the aqueous phase reveals the presence of three main glycoprotein components, glycoproteins I, II, and III, and one additional periodic acid-Schiff positive component, without contamination by other membrane proteins. Their apparent molecular weights correspond to 58 000, 37 000, 24 000, and 50 000, respectively. The blood group antigens I, Ss, and MN are recovered in the aqueous phase in high yields and with marked increase in their specific activities. The aqueous phase also contains moderate amounts of A and B and small amounts of H antigenic activities. Further separation of the glycoproteins of the aqueous phase was achieved by gel filtration on Sephadex G-100 columns in the presence of 1% dodecyl sulfate. The M and N antigenic activities were found in glycoprotein I. N activity as tested with a rabbit antiserum was also observed in glycoprotein III. The highest activities for A, I, and S antigens were found in glycoprotein III. The chloroform-methanol phase does not contain glycoproteins; only the blood group antigens A, B, and H are found in this phase. The interphase, which contains most of the membrane proteins except the main glycoproteins, shows only weak ABH, I, Ss, and MN activities. The data indicate that the major glycoprotein of the human erythrocyte membrane, glycoprotein I, is the so-called MN glycoprotein, and that the I and S antigenic activities are associated with glycoprotein III, a minor glycoprotein with an apparent molecular weight of 24 000. Glycoprotein III is also associated with the A antigenic activity.