Characterization and immunolocalization of bovine N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase.
N-Acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase (phosphodiester alpha-GlcNAcase) has been purified 3,000-fold from bovine liver and its kinetic properties determined as described in the previous report (Mullis, K. G., Huynh, M., and Kornfeld, R. (1993) J. Biol. Chem. 269, 1718-1726). This report describes the hydrodynamic and lectin binding properties of phosphodiester alpha-GlcNAcase as well as its intracellular localization. The molecular weight of phosphodiester alpha-GlcNAcase is 204,950, as determined from density gradient centrifugation in D2O and H2O glycerol gradients and gel filtration. Enzymatically active enzyme migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 129,000, consistent with native phosphodiester alpha-GlcNAcase being a dimer. The lectin binding properties of phosphodiester alpha-GlcNAcase indicate that it contains sialylated species of both complex type N-linked oligosaccharides and O-linked oligosaccharides. In immunofluorescence studies phosphodiester alpha-GlcNAcase shows a perinuclear, Golgi localization in Vero cells as does the mid-Golgi marker alpha-mannosidase II. After exposure of the Vero cells to brefeldin A, phosphodiester alpha-GlcNAcase assumes an endoplasmic reticulum staining pattern. In contrast, in cells costained with the trans-Golgi marker wheat germ agglutinin, the wheat germ agglutinin marker assumed an endosomal network appearance after exposure to brefeldin A. These findings indicate that phosphodiester alpha-GlcNAcase is normally located within the Golgi stack, separate from the trans-Golgi and trans-Golgi network stained by wheat germ agglutinin.
Mullis
K G
KG
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Kornfeld
R H
RH
D000818:Animals; D000906:Antibodies; D002240:Carbohydrate Sequence; D002417:Cattle; D002499:Centrifugation, Density Gradient / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000201:enzymology; D007150:Immunohistochemistry / Q000379:methods; D037102:Lectins; D008099:Liver / Q000201:enzymology; D008361:Mannosidases / Q000032:analysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010727:Phosphoric Diester Hydrolases / Q000032:analysis* / Q000737:chemistry*; D011485:Protein Binding; D014709:Vero Cells
D000906:Antibodies; D037102:Lectins; D010727:Phosphoric Diester Hydrolases; C026645:N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase; D008361:Mannosidases; C038829:mannosyl-oligosaccharide 1,3 - 1,6-alpha-mannosidase