α- l-Iduronidase, β- d-glucuronidase, and 2-sulfo- l-iduronate 2-sulfutase: preparation and characterization of radioactive substrates from heparin

Abstract

Radioactive disaccharide substrates for α- l-iduronidase, β- d-glucuronidase, and 2-sulfo- l-iduronate 2-sulfatase have been prepared from heparin by deaminative cleavage followed by reduction with NaBT 4. Six disaccharides were isolated from this reaction mixture and identified. Acid hydrolysis of the major disaccharide, O-(α- l-idopyranosyluronic acid 2-sulfate)-(1→4)-(2,5-anhydro- d-mannitol- 1-t 6-sulfate (IdAs-Ms), produced 48% of O-(α- l-idopyranosyluronic acid)-(1→4)-(2,5-anhydro- d-mannitol- 1-t 6-sulfate) (IdA-Ms) and 25% of O-(α- l-idopyranosyluronic acid)-(1→4)-2,5-anhydro- d-mannitol- 1-t. The most-sensitive substrate for determining a- l-iduronidase activity was IdA-Ms which, when incubated with leucocyte and skin-fibroblast homogenates prepared from patients having a deficiency of a- l-iduronidase (Mucopolysaccharidosis Type I; MPS-I), was hydrolysed to yield 2,5-anhydro- d-mannitol- 1-t 6-sulfate at a rate 50-times less than that found for normal control-preparations. Similarly, O-(β- d-glucopyranosyluronic acid)-(1→4)-(2,5-anhydro-D-mannitol- 1-t 6-sulfate) was degraded by whole-cell homogenates prepared from β- d-glucuronidase-deficient (Mucopolysaccharidosis, Type VII) fibroblasts, to yield 2,5-anhydro- d-mannitol- 1-t 6-sulfate at a rate 60-times less than 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-( a- l-idopyranosyluronic acid 2-sulfate)-(1→4)-2,5-anhydro- d-mannitol- 1-t. C-6 Sulfation of the anhydro- d-mannitol- 1-t residue is an important structural determinant in the mechanism of action of both a- l-iduronidase and 2-sulfo- l-iduronate 2-sulfatase on disaccharide substrates.