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  • Title: Differences in the distribution of O-sulphate groups of cell-surface and secreted heparan sulphate produced by human neuroblastoma cells in culture.
    Author: Hampson IN, Kumar S, Gallagher JT.
    Journal: Biochim Biophys Acta; 1983 Sep 22; 763(2):183-90. PubMed ID: 6225467.
    Abstract:
    Confluent cultures of a human neuroblastoma cell line (CHP100) were incubated for 48 h with D-[1-3H]glucosamine and sodium [35S]sulphate. Radioactive glycosaminoglycans were analysed in the growth medium, rapid trypsin digest of the cell monolayer and a 1% (w/v) Triton/0.5 M NaOH extract of the final cell pellet. Sulphated glycosaminoglycans co-chromatographed when eluted by NaCl gradient from DEAE-cellulose. The medium contained mainly chondroitin sulphates, whereas the cell surface was enriched in heparan sulphate. Heparan sulphate was isolated as chondroitinase ABC-resistant material and treated with nitrous acid. Analysis of the scission products on Bio-Gel P-10 yielded fragments varying in size from single disaccharides to glycans consisting of nine disaccharide units. Cell-surface and medium heparan sulphate had respectively 52% and 54% N-sulphated glucosamine residues distributed in similar patterns along the polymer chain. The N:O-sulphate ratio of neuroblastoma heparan sulphate was 1.1:1. Analysis by high-voltage electrophoresis of di- and tetrasaccharide products produced by nitrous acid treatment showed that the distribution of 'O'-sulphate groups differed strikingly between heparan sulphates from the medium and cell-surface compartments. A di-O-sulphated tetrasaccharide was identified in both heparan sulphate species. The absence of detectable amounts of 35[S]sulphate associated with fragments larger than tetrasaccharide supports the close topographical association of N-sulphate and O-sulphate groups.
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