title: alpha-L-iduronidase, beta-D-glucuronidase, and 2-sulfo-L-iduronate 2-sulfatase: preparation and characterization of radioactive substrates from heparin.
abstract: Radioactive disaccharide substrates for alpha-L-iduronidase, beta-D-glucuronidase, and 2-sulfo-L-iduronate 2-sulfatase have been prepared from heparin by deaminative cleavage followed by reduction with NaBT4. Six disaccharides were isolated from this reaction mixture and identified. Acid hydrolysis of the major disaccharide, O-(alpha-L-idopyranosyluronic acid 2-sulfate)-(1 linked to 4)-(2,5-anhydro-D-mannitol-l-t 6-sulfate (IdAs--Ms), produced 48% of O-(alpha-L-idopyranosyluronic acid)-(1 linked to 4)-(2,5-anhydro-D-mannitol-l-t 6-sulfate) (IdA--Ms) and 25% of O-(alpha-L-idopyranosyluronic acid)-(1 linked to 4)-2,5-anhydro-D-mannitol-l-t. The most-sensitive substrate for determining alpha-L-iduronidase activity was IdA--Ms which, when incubated with leucocyte and skin-fibroblast homogenates prepared from patients having a deficiency of alpha-L-iduronidase (Mucopolysaccharidosis Type I; MPS-I), was hydrolysed to yield 2,5-anhydro-D-mannitol-l-t 6-sulfate at a rate 50-times less than that found for normal control-preparations. Similarly, O-(beta-D-glucopyranosyluronic acid)-(1 linked to 4)-(2,5-anhydro-D-mannitol-l-t 6-sulfate) was degraded by whole-cell homogenates prepared from beta-D-glucuronidase-deficient (Mucopolysaccharidosis, Type VII) fibroblasts, to yield 2,5-anhydro-D-mannitol-l-t 5-sulfate at a rate 60-times less that that found for MPS-I and normal control-preparations. IdAs--Ms was degraded by 2-sulfo-L-iduronate 2-sulfatase at a rate more than 45-times greater than that found for O-(alpha-L-idopyranosyluronic acid 2-sulfate)-(1 linked to 4)-2,5-anhydro-D-mannitol-l-t. C-6 Sulfation of the anhydro-D-mannitol-l-t residue is an important structural determinant in the mechanism of action of both alpha-L-iduronidase and 2-sulfo-L-iduronate 2-sulfatase on disaccharide substrates.
mesh_terms: D002478:Cells, Cultured; D004187:Disaccharides / Q000378:metabolism*; D005966:Glucuronidase / Q000172:deficiency / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006493:Heparin; D006801:Humans; D007066:Iduronate Sulfatase / Q000378:metabolism*; D007068:Iduronidase / Q000172:deficiency / Q000378:metabolism*; D007553:Isotope Labeling; D009083:Mucopolysaccharidoses / Q000201:enzymology; D008059:Mucopolysaccharidosis I / Q000201:enzymology; D013379:Substrate Specificity; D013429:Sulfatases / Q000378:metabolism*; D014316:Tritium
chemical_list: D004187:Disaccharides; D014316:Tritium; D006493:Heparin; D013429:Sulfatases; D007066:Iduronate Sulfatase; D006026:Glycoside Hydrolases; D005966:Glucuronidase; D007068:Iduronidase