Purification and kinetic parameters of bovine liver N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase.
The enzyme N-acetylglucosamine phosphodiester alpha-N-acetylglucosaminidase (phosphodiester alpha-GlcNAcase) catalyzes the second step in the formation of the mannose 6-phosphate targeting signal on lysosomal enzyme oligosaccharides by removing GlcNAc residues from GlcNAc-alpha-P-mannose moieties, which are formed in the first step by UDP-N-acetyl-glucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase). Phosphodiester alpha-GlcNAcase, a membrane-bound enzyme, has been purified about 3,000-fold from bovine liver to apparent homogeneity using detergent solubilization, fractionation on DEAE-cellulose, affinity chromatography on lectin-Sepharose columns, gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme migrated as 129- and 121-kDa species on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since both bands had the same amino-terminal sequence, the smaller species is presumed to be derived from the larger by proteolysis. Kinetic analysis of bovine phosphodiester alpha-GlcNAcase with enzymatically synthesized artificial and biological substrates indicates that phosphodiester alpha-GlcNAcase requires GlcNAc-alpha-P R for substrate and that when R contains the Man alpha 1,2Man linkage the substrate binding is most effective. Unlike GlcNAc-phosphotransferase, bovine phosphodiester alpha-GlcNAcase does not require a protein recognition determinant on lysosomal enzyme substrates.
Mullis
K G
KG
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Huynh
M
M
Kornfeld
R H
RH
D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity / Q000379:methods; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007202:Indicators and Reagents; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity
D002413:Cations, Divalent; D007202:Indicators and Reagents; D010727:Phosphoric Diester Hydrolases; C026645:N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase