A monosaccharide is bound to the sodium pump alpha-subunit.
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.
Pedemonte
C H
CH
Department of Physiology, University of Pennsylvania, Philadelphia 19104-6085.
Kaplan
J H
JH
D000117:Acetylglucosamine / Q000378:metabolism; D000818:Animals; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D004285:Dogs; D005690:Galactose / Q000378:metabolism; D006031:Glycosylation; D007679:Kidney Medulla / Q000201:enzymology; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000378:metabolism*; D011485:Protein Binding; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D009005:Monosaccharides; D000254:Sodium-Potassium-Exchanging ATPase; D000117:Acetylglucosamine; D005690:Galactose