Purification and characterization of the major sialoglycoproteins of the rat erythrocyte membrane

The major sialoglycoproteins of the rat erythrocyte membrane were purified by hot phenol partitioning followed by cation-exchange chromatography on SP-Sephadex. Further purification was obtained by extraction with n-butanol and anion-exchange chromatography on DEAE-cellulose. The resulting sialoglycoprotein fraction was free of lipids and nonsialylated glycoproteins and gave rise to four major periodic acid-Schiff staining bands when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fastest migrating protein on these gels with an apparent molecular weight of 19,000 was purified to homogeneity by gel filtration. The amino acid and sugar compositions of these materials are reported. The protein moiety is rich in serine, threonine, and hydrophobic amino acids and the carbohydrate moiety is high in sialic acid and N-acetylgalactosamine. Most of the carbohydrate is linked O-glycosidically to serine and threonine residues, as shown by susceptibility to base-catalyzed β-elimination and concomitant reduction of serine and threonine to alanine and α-aminobutyric acid and of N-acetylgalactosamine to N-acetylgalactosaminitol in the presence of reducing agents. The significance of these data in light of the known role of the rat erythrocyte membrane sialoglycoproteins in erythropoiesis is discussed. The properties of the rat erythrocyte membrane sialoglycoproteins are compared to those of other species.