A monosaccharide is bound to the sodium pump alpha-subunit

Abstract

We have recently reported that the Na pump alpha-subunit has cytosolic-oriented oligosaccharides which were sensitive to cleavage by an enzyme specific for hydrolysis of N-linked glycans [Pedemonte et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 9789-9793]. We now describe experiments that characterize the saccharides and further substantiate our previous findings. Bovine milk galactosyltransferase has been used in conjunction with radiolabeled UDP-galactose to label N-acetylglucosamine residues on the protein. The Na pump alpha-subunit contains some O-linked carbohydrates; however, the bulk (> 80%) of the radioactivity was found in oligosaccharides sensitive to peptide:N-glycosidase F degradation but not to alkaline hydrolysis. Alkaline hydrolysis produced degradation of the protein, and the [3H]Gal radiolabeled carbohydrates remained bound to peptides and were released by subsequent peptide N-glycosidase F treatment. The exogenously galactosylated sugars cleaved by the glycosidase were analyzed by liquid chromatography and had elution volumes identical to a galactose-N-acetylglucosamine disaccharide standard. Since the galactose was exogenously added, we propose that the N-linked glycans on the alpha-subunit of the Na pump are composed of a single sugar residue, which is probably N-acetylglucosamine.