Purification and multimeric structure of bovine N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase.
N-Acetylglucosamine-1-phosphodiester alpha-N-Acetylglucosaminidase (EC 3.1.4.45; phosphodiester alpha-GlcNAcase) catalyzes the second step in the synthesis of the mannose 6-phosphate determinant required for efficient intracellular targeting of newly synthesized lysosomal hydrolases to the lysosome. A partially purified preparation of phosphodiester alpha-GlcNAcase from bovine pancreas was used to generate a panel of murine monoclonal antibodies. The anti-phosphodiester alpha-GlcNAcase monoclonal antibody UC1 was coupled to a solid support and used to immunopurify the bovine liver enzyme 670,000-fold in two steps to apparent homogeneity with an overall yield of 14%. The purified phosphodiester alpha-GlcNAcase has a specific activity of 498 micromol of [3H]GlcNAc-alpha-phosphomannose-alpha-methyl cleaved per h per mg of protein using 0.5 mM [3H]GlcNAc-alpha-phosphomannose-alpha-methyl as substrate. The subunit structure of the enzyme was determined using a combination of analytical gel filtration chromatography, SDS-polyacrylamide gel electrophoresis, and amino-terminal sequencing. The data indicate that bovine phosphodiester alpha-GlcNAcase is a 272,000-Da complex of four identical 68,000-Da glycoprotein subunits arranged as two disulfide-linked homodimers. A soluble form of the enzyme, isolated from fetal bovine serum, showed the same subunit structure. Both forms of the enzyme reacted with a rabbit antibody raised to the amino-terminal peptide of the liver enzyme, suggesting that phosphodiester alpha-GlcNAcase is a type I membrane-spanning glycoprotein with its amino terminus in the lumen of the Golgi apparatus.
Kornfeld
R
R
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Bao
M
M
Brewer
K
K
Noll
C
C
Canfield
W M
WM
D000135:Acid Phosphatase; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004622:Embryo, Mammalian / Q000201:enzymology; D004798:Enzymes / Q000097:blood; D005944:Glucosamine / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000494:pharmacology; D007527:Isoenzymes; D008099:Liver / Q000201:enzymology; D008667:Metalloproteins / Q000378:metabolism; D010084:Oxidation-Reduction; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000187:drug effects / Q000276:immunology; D011487:Protein Conformation; D017421:Sequence Analysis; D000071681:Tartrate-Resistant Acid Phosphatase
D000911:Antibodies, Monoclonal; D004220:Disulfides; D004798:Enzymes; D006023:Glycoproteins; D007527:Isoenzymes; D008667:Metalloproteins; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase; D010727:Phosphoric Diester Hydrolases; C026645:N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase; D006026:Glycoside Hydrolases; D005944:Glucosamine