title: Purification and characterization of human serum N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase.
abstract: Many lysosomal enzymes are recognized and selected by a unique marker in the form of mannose 6-phosphate groups which are present exclusively on their N-linked oligosaccharides. Two enzymes act sequentially to catalyze the addition of mannose 6-phosphate groups to the proteins: N-acetylglucosamine phosphotransferase (GlcNAc phosphotransferase) and N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase (phosphodiester alpha-GlcNAcase). We report here the purification and partial characterization of phosphodiester alpha-GlcNAcase from human serum. The enzyme was purified over 600,000-fold by utilizing ammonium sulfate precipitation, fractionation on wheat germ agglutinin-Sepharose, Fe(3+)-chelating Sepharose, and Cu(2+)-chelating Sepharose, and renaturation from gel slices after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein observed after renaturation and subsequent SDS-PAGE and silver staining had an apparent molecular mass of 118 kDa, which is slightly smaller than bovine liver phosphodiester alpha-GlcNAcase (Mullis et al. (1994) J. Biol. Chem. 269, 1718-1726). Serum enzyme activity does not require Triton X-100 and is not stimulated by its addition. These results suggest that the enzyme found in serum represents a form secreted after proteolysis in the Golgi of the membrane-bound enzyme. The serum enzyme hydrolyzed UDP-GlcNAc to UDP and GlcNAc and hydrolyzed GlcNAc-P-Man alpha Me into alpha MeMan-P and GlcNAc. The enzyme had no hydrolyzing activity toward UDP-GalNAc, UDP-Glc, [6-3H]GlcNAc beta 1-3Gal beta 1-4Glc, p-nitrophenyl-alpha-N- acetylglucosaminide, p-nitrophenyl-beta-N-acetylglucosaminide, p-nitrophenyl-alpha-N-galactopyranoside, or p-nitrophenyl-beta-N- galactopyranoside. The enzyme activity was strongly inhibited by UDP-GlcNAc and GlcNAc-1-phosphate, had a pH optimum between pH 6.0 and 7.0, and was inhibited by FeCl3, FeSO4, and CuSO4. The Km values for UDP-GlcNAc and GlcNAc-P-Man alpha Me were 0.94 and 0.45 mM, respectively. Over 77% of enzyme activity remained after incubation for 10 min at 70 degrees C, demonstrating an unusual thermostability of the serum enzyme.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens 30602, USA.
authors.forename: J K
authors.initials: JK
authors.lastname: Lee
authors.forename: M
authors.initials: M
authors.lastname: Pierce
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D006801:Humans; D010727:Phosphoric Diester Hydrolases / Q000097:blood / Q000302:isolation & purification*; D013379:Substrate Specificity
chemical_list: D010727:Phosphoric Diester Hydrolases; C026645:N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase