Structures of the complex glycans found on the beta-subunit of (Na,K)-ATPase.
(Na,K)-ATPase is an integral membrane protein responsible for maintaining the Na+ and K+ ion concentration gradients across the plasma membranes of cells. All active (Na,K)-ATPase preparations consist of two subunits, designated alpha and beta. The alpha-subunit is the catalytic subunit and contains the cardiac glycoside binding site. In contrast, the physiological function of the beta-subunit remains unclear although it appears to be involved in the processes of folding, membrane insertion, and stabilization of the alpha-subunit. Previous work has determined the amino acid sequence and disulfide bond arrangements for the beta-subunit from both lamb and dog kidney. In this report, we describe the isolation and structural characterization of the glycan moieties of the beta-subunit from both lamb and dog kidney (Na,K)-ATPase. The three glycosylation sites of these beta-subunits were fractionated using reverse phase chromatography after cleavage of the polypeptide chain with trypsin and thermolysin. Glycopeptides derived from each glycosylation site were analyzed by matrix-assisted laser desorption ionization mass spectrometry. The mass spectrometry results indicated that the predominant glycoforms at the three glycosylation sites of these beta-subunits were a combination of the tetraantennary glycan form and the unusual glycan form of tetraantennary with a limited number of repeating N-acetyllactosamine units. These results further define the covalent structure for the beta-subunit from both lamb and dog kidney (Na,K)-ATPase and suggest that the beta-subunit may be derived from an adhesion molecule.
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0575.
M J
MJ
Treuheit
C E
CE
Costello
T L
TL
Kirley
D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D004285:Dogs; D007668:Kidney / Q000201:enzymology*; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry* / Q000302:isolation & purification; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000302:isolation & purification; D014357:Trypsin
D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D011134:Polysaccharides; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase