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.
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 T17 T18 T19 T20 T21 T22 T23
CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL GENE-Y GENE-Y GENE-Y GENE-N GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y
N-Acetylglucosamine-1-phosphodiester SDS polyacrylamide phosphodiester disulfide amino amino mannose 6-phosphate N [3H]GlcNAc-alpha-phosphomannose-alpha-methyl [3H]GlcNAc-alpha-phosphomannose-alpha-methyl N-acetylglucosamine-1-phosphodiester N N-Acetylglucosamine-1-phosphodiester alpha-N-Acetylglucosaminidase phosphodiester alpha-GlcNAcase phosphodiester alpha-GlcNAcase type I membrane-spanning glycoprotein phosphodiester alpha-GlcNAcase phosphodiester alpha-GlcNAcase EC 3.1.4.45 phosphodiester alpha-GlcNAcase phosphodiester alpha-GlcNAcase N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase