1,721,053 research outputs found
Biosynthesis of alpha-N-acetylglucosaminidase in cultured human kidney carcinoma cells
No full textThe biosynthesis of α-N-acetylglucosaminidase was studied in cultured human kidney carcinoma cells by (1) labeling cells with 35S-methionine, isolation of the enzyme by immunoprecipitation and analysis on gel electrophoresis of the denatured polypeptide(s) and (2) analysis of the native enzyme on linear sucrose gradient centrifugation. The enzyme is synthesized as precursor forms of apparent molecular weight 82,000-86,000. Processing of these precursors yields a polypeptide of apparent molecular weight of 80,000. The precursor-product relationship was indicated by pulse-chase as well as endocytosis experiments. Sucrose gradient centrifugation of the native enzyme shows that, extracellularly, the molecule is present with a molecular weight of 80,000; intracellularly, 80-90% of the enzyme is present with an apparent molecular weight of 240,000. We suggest that this is a polymeric form and that polymerization of α-N-acetylglucosaminidase is a late event of the maturation process
CELL-TO-CELL CONTACT BETWEEN NORMAL FIBROBLASTS AND LYMPHOBLASTS DEFICIENT IN LYSOSOMAL-ENZYMES
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Contatto cellulare tra linfoblasti di pazienti con Mucopolisaccaridosi e fibroblasti normali
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Heparan N-sulfatase: In vitro mutagenesis of potential N-glycosylation sites
No full textHeparan N-sulfatase cDNA contains five potential N-glycosylation sites at Asn positions 41, 142, 151, 264, and 413. We used site-directed mutagenesis, substituting the codon of asparagine for glutamine, to eliminate selected glycosylation sites and then performed expression studies in COS-7 cells to determine the influence on the catalytic activity, lysosomal targeting, and glycosylation-phosphorylation of the enzyme. Elimination of site 5 did not affect significantly enzyme activity; elimination of sites 2 and 4 gave a partial reduction, while elimination of sites 1 and 3 resulted in drastic reduction of catalytic activity (25 and 14%, respectively, of normal values), indicating that glycosylation of asparagine 41 and asparagine 151 is essential for catalysis and/or enzyme stability. Wild type enzyme produced in the presence of tunicamycin was also inactive, indicating that glycosylation is required for acquisition of enzyme activity and/or for enzyme stability. Metabolic labeling of each mutant cDNA, transiently transfected into COS cells, showed that enzyme from mutants N142Q, N264Q, and N413Q appeared to be properly folded, as judged by its ability to be proteolytically processed to a lower molecular weight form, while enzyme from mutants N41Q and N151Q did not reach lysosomes. These studies confirm that the five glycosylation sites of heparan N-sulfatase are all functional and show that Asn 41 and Asn 151 have a role in protein folding and/or stability. (C) 2001 Academic Press
ESTIMATION OF N-ACETYLGLUCOSAMINE-6-SULFATE SULFATASE WITH THE FLUOROGENIC SUBSTRATE 4-METHYLUMBELLIFERYL-ß-N- ACETYLGLUCOSAMINIDE-6-SULPHATE.
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