Novel carbohydrate-binding activity of bovine liver β-glucuronidase toward lactose/N-acetyllactosamine sequences
| Autor: | Tetsuko Kanamori, Nobuko Seno, Hiromi Sakagami, Hiroko Matsushita-Oikawa, Mayumi Komatsu, Isamu Matsumoto, Haruko Ogawa, Naoko Iida-Tanaka |
|---|---|
| Rok vydání: | 2006 |
| Předmět: |
Glycoconjugate
Carbohydrates Lactose Models Biological Biochemistry Chromatography Affinity Substrate Specificity Affinity chromatography Animals Chromatography High Pressure Liquid Glucuronidase Glycoproteins chemistry.chemical_classification Chromatography biology Sepharose Substrate (chemistry) Amino Sugars Carbohydrate Chromatography Agarose Lipid Metabolism DEAE-Cellulose Enzyme assay Enzyme Liver chemistry biology.protein Carbohydrate Metabolism Cattle Glycoprotein Protein Binding |
| Zdroj: | Glycobiology. 16:891-901 |
| ISSN: | 1460-2423 0959-6658 |
| DOI: | 10.1093/glycob/cwl016 |
| Popis: | Beta-glucuronidase is a lysosomal enzyme that plays an essential role in normal turnover of glycosaminoglycans and remodeling of the extracellular matrix components in both physiological and inflammatory states. The regulation mechanisms of enzyme activity and protein targeting of beta-glucuronidase have implications for the development of a variety of therapeutics. In this study, the effectiveness of various carbohydrate-immobilized adsorbents for the isolation of bovine liver beta-glucuronidase (BLG) from other glycosidases was tested. Beta-glucuronidase and contaminating glycosidases in commercial BLG preparations bound to and were coeluted from adsorbents immobilized with the substrate or an inhibitor of beta-glucuronidase, whereas beta-glucuronidase was found to bind exclusively with lactamyl-Sepharose among the adsorbents tested and to be effectively separated from other enzymes. Binding and elution studies demonstrated that the interaction of beta-glucuronidase with lactamyl-Sepharose is pH dependent and carbohydrate specific. BLG was purified to homogeneity by lactamyl affinity chromatography and subsequent anion-exchange high-performance liquid chromatography (HPLC). Lactose was found to activate beta-glucuronidase noncompetitively, indicating that the lactose-binding site is different from the substrate-binding site. Binding studies with biotinyl glycoproteins, lipids, and synthetic sugar probes revealed that beta-glucuronidase binds to N-acetyllactosamine/lactose-containing glycoconjugates at neutral pH. The results indicated the presence of N-acetyllactosamine/lactose-binding activity in BLG and provided an effective purification method utilizing the novel carbohydrate binding activity. The biological significance of the carbohydrate-specific interaction of beta-glucuronidase, which is different from the substrate recognition, is discussed. |
| Databáze: | OpenAIRE |
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