A monosaccharide is bound to the sodium pump alpha-subunit.

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Biology

Background:

• Previous work indicated cytosolic-oriented oligosaccharides on the Na pump alpha-subunit sensitive to N-linked glycan hydrolysis.
• Further characterization of these saccharides is needed to confirm their structure and linkage.

Purpose of the Study:

• To characterize the N-linked glycans on the Na pump alpha-subunit.
• To determine the composition and linkage of these oligosaccharides.

Main Methods:

• Enzymatic labeling using bovine milk galactosyltransferase and radiolabeled UDP-galactose.
• Degradation studies using peptide:N-glycosidase F and alkaline hydrolysis.
• Analysis of released carbohydrates by liquid chromatography.

Main Results:

• The majority (>80%) of radioactivity was associated with oligosaccharides sensitive to peptide:N-glycosidase F but resistant to alkaline hydrolysis.
• Liquid chromatography analysis revealed exogenously galactosylated sugars identical to a galactose-N-acetylglucosamine disaccharide standard.
• Alkaline hydrolysis degraded the protein, with radiolabeled carbohydrates remaining bound to peptides until released by peptide N-glycosidase F.

Conclusions:

• The N-linked glycans on the Na pump alpha-subunit are primarily sensitive to peptide:N-glycosidase F, suggesting N-linkage.
• The data support the presence of a single sugar residue, likely N-acetylglucosamine, as the N-linked glycan on the Na pump alpha-subunit.