Purification and properties of a secreted and developmentally regulated alpha-L-fucosidase from Dictyostelium discoideum
| Autor: | D Schopohl, Annette Müller-Taubenberger, B Orthen, H Hess, W Reutter |
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| Předmět: |
Size-exclusion chromatography
Blotting Western Oligosaccharides Biochemistry Dictyostelium discoideum Substrate Specificity Extracellular Cyclic AMP Animals Dictyostelium Molecular Biology Ammonium sulfate precipitation chemistry.chemical_classification Gel electrophoresis alpha-L-Fucosidase Molecular mass biology Hydrolysis Fast protein liquid chromatography Cell Biology Hydrogen-Ion Concentration biology.organism_classification Molecular biology Enzyme chemistry Microscopy Fluorescence Electrophoresis Polyacrylamide Gel Chromatography Liquid |
| Zdroj: | Europe PubMed Central |
| Popis: | During its development the eukaryotic microorganisms Dictyostelium discoideum secretes an alpha-L-fucosidase (EC 3.2.1.51). In cells of the growth phase almost no alpha-L-fucosidase activity is detectable. The activity increases steadily up to the aggregation stage and accumulates also in the extracellular medium. The developmental regulation is mediated by pulsatile cAMP signals. The alpha-L-fucosidase was purified from extracellular medium. The isolation procedure started with concentration of the enzyme by batchwise anion-exchange chromatography and ammonium sulfate precipitation, followed by Sephacryl S-300 gel filtration and further purification by fast protein liquid chromatography on Mono Q, phenyl-Superose, and finally Superose 12. The purified preparation was found to be essentially free of activities of six other glycosidases also secreted by D. discoideum. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme showed one major band with an apparent molecular mass of 62 kilodalton. Gel filtration of the enzyme on a Superose 12 column was consistent with an active monomer. A monoclonal antibody was produced, which recognizes a carbohydrate epitope shared by all lysosomal enzymes in D. discoideum. The pH optimum of the alpha-L-fucosidase is at 3.7. The apparent Michaelis constant for p-nitrophenyl alpha-L-fucoside as substrate is 1.2 mM. The enzyme catalyzes preferentially the hydrolysis of alpha 1----6GlcNAc but also of alpha 1----2Gal and alpha 1----3Glc fucosyl linkages. |
| Databáze: | OpenAIRE |
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