Resolution and partial characterization of the major plasma membrane sialoglycoproteins of Novikoff tumor cells

The sialic acid residues of the plasma membrane glycoprotiens were specifically radiolabeled by oxidation with NaIO₄ followed by reduction with NaB[³H]₄. Surface-labeled glycoproteins were resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and visualized by fluorography. The major surface-labeled glycoproteins were designated GP-240, GP-120, GP-92, GP-48, and GP-25, the numerical designation being their apparent molecular weight x 10^-3 estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate in 7.5% gels. These glycoproteins were not labeled by D-galactose oxidase/NaB[³H]₄, a method that introduces a tritium label into nonreducing terminal D-galactose and/or 2-acetamido-2-deoxy-D-galactose residues of their heterosaccharide moieties, indicating that the presentation of these monosaccharide residues was not suitable for binding of the enzyme. The radiolabeled glycoproteins were quantitatively solubilized in 0.5% Nonidet P-40 and subjected to affinity chromatography on Sepharose-conjugated Ricinus communis agglutinins I or II or soybean agglutinin. Most of the radiolabeled glycoproteins were bound to the Sepharose-conjugated Ricinus communis lectins and were eluted with lactose; however, no radiolabeled glycoproteins were bound to Sepharose-conjugated soybean agglutinin. After treatment of the cells with neuraminidase, GP-120 and GP-92 bound to Sepharose-conjugated soybean agglutinin, indicating exposure of nonreducing terminal 2-acetamido-2-deoxy-D-galactose on the heterosaccharide moieties of these glycoproteins. Information regarding the surface-labeling and affinity chromatography of the plasma membrane glycoproteins allowed differentiation of five classes of glycoproteins exhibiting structural differences in the nonreducing termini of their heterosaccharide moieties.