Electrophoretic Analysis of the Major Polypeptides of the Human Erythrocyte Membrane

Abstract

The polypeptides of the human erythrocyte membrane were analyzed by polyacrylamide gel electrophoresis in 1% sodium dodecyl sulfate. Six major bands (I-VI) together make up over two-thirds of the protein staining profile. Component III (mol wt 89,000) predominates in the ghost membrane; it constitutes 30% of the protein and numbers over 106 chains/ghost. Components I and II form a slow-moving doublet (approximate mol wt 250,000) containing 25% of the protein. The molar amounts of I + II, IV (mol wt 77,500), V (mol wt 41,300), and VI (mol wt 36,200) are similar, falling in the range 3.4-4.6 × 105 chains/ghost. Four bands weee recognized in gels stained by the periodic acid-Schiff procedure. A broad Schiff-positive zone just behind the tracking dye corresponds to membrane lipids. Three bands of lower mobility are sialoglycoproteins. The most prominent of these has an apparent molecular weight of 83,500 and contains at least 57% of the sialic acid of ghosts. The Schiff-positive bands were not colored by protein stains. Sialidase treatment of ghosts selectively increased the mobilities of the sialoglycoproteins without affecting the protein-staining profile. Attempts to produce subunits from the large polypeptides by treatment with various denaturing agents were unsuccessful. Normally, no polypeptides of size less than 15,000 were seen in ghost electrophorograms. However, heating ghosts with low levels of sodium dodecyl sulfate and high levels of salt produced diffuse bands of low average molecular weight. This highly variable effect is attributed to degradation by proteinases. Components I, II, and V were solubilized by incubating ghosts at low ionic strength. Component VI was released by washing with buffered saline at concentrations above 0.1 m. Both elution procedures were rapid (15 min), complete, and selective; they were also conservative in that new bands were not created and the electrophorograms of released and retained material were complementary. The eluted material contained negligible sialic acid and no Schiff-positive lipids. Two classes of membrane protein were distinguished by their response to the elution procedures. Components I, II, V, and VI compose one class. They make up 30-35% of the protein and are tenuously related to the membrane, possibly by predominantly ionic bonds. The second class, which includes components III, IV, and the sialoglycoproteins, together with various minor components, constitutes 65-70% of the protein. These polypeptides are tightly bound; their properties may reflect participation in hydrophobic protein-protein and pro-tein-lipid interactions. © 1971, American Chemical Society. All rights reserved.