Purification and characterization of an O-GlcNAc selective N-acetyl-beta-D-glucosaminidase from rat spleen cytosol

Glycosylation of nuclear and cytoplasmic proteins by O-linked N-acetylglucosamine (O-GlcNAc) monosaccharides is an abundant, ubiquitous, and transient post-translational modification. To characterize enzymes involved in removal of these sugars, a neutral and cytoplasmic N-acetyl-beta-D-glucosaminida...

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Bibliographic Details
Published in:The Journal of biological chemistry 1994-07, Vol.269 (30), p.19321-19330
Main Authors: DONG, D. L.-Y, HART, G. W
Format: Article
Language:English
Subjects:
Acetylglucosaminidase - isolation & purification
Acetylglucosaminidase - metabolism
Amino Acid Sequence
Analytical, structural and metabolic biochemistry
Animals
beta-N-Acetylhexosaminidases - metabolism
Biological and medical sciences
Cell Compartmentation
Chromatography
Cytosol - enzymology
Enzymes and enzyme inhibitors
Fundamental and applied biological sciences. Psychology
Hydrogen-Ion Concentration
Hydrolases
Molecular Sequence Data
Molecular Weight
Rats
Spleen - enzymology
Subcellular Fractions - enzymology
Substrate Specificity
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Summary:Glycosylation of nuclear and cytoplasmic proteins by O-linked N-acetylglucosamine (O-GlcNAc) monosaccharides is an abundant, ubiquitous, and transient post-translational modification. To characterize enzymes involved in removal of these sugars, a neutral and cytoplasmic N-acetyl-beta-D-glucosaminidase (O-GlcNAcase) with strong selectivity for O-GlcNAc-synthetic glycopeptides has been purified over 22,000-fold from rat spleen homogenate. The purified O-GlcNAcase has two major polypeptides of apparent M(r) = 54,000 (alpha subunit) and M(r) = 51,000 (beta subunit). Enzyme activity sediments at M(r) = 106,000 on sucrose gradients, indicating that the native O-GlcNAcase is an alpha beta heterodimer. The O-GlcNAcase also shows substantially stronger relative activity against O-GlcNAc-synthetic glycopeptides than other hexosaminidases. Unlike acidic lysosomal hexosaminidases, O-GlcNAcase is not inhibited by GalNAc or its analogs, has no other detectable glycosidase activities, and does not cross-react with antibodies against acidic hexosaminidases. Subcellular fractionation and latency studies demonstrate the cytoplasmic and nucleoplasmic localization of the enzyme and its ubiquitous presence in tissues. These studies suggest that O-GlcNAcase is involved in the regulated removal of O-GlcNAc from O-GlcNAc-bearing glycoproteins in the nucleoplasmic and cytoplasmic compartments of cells.
ISSN:0021-9258
1083-351X
DOI:10.1016/s0021-9258(17)32170-1