Purification and properties of a secreted and developmentally regulated alpha-L-fucosidase from Dictyostelium discoideum.
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.
Schopohl
D
D
Max-Planck-Institut für Biochemie, Martinsried, Federal Republic of Germany.
Müller-Taubenberger
A
A
Orthen
B
B
Hess
H
H
Reutter
W
W
D000818:Animals; D015153:Blotting, Western; D002853:Chromatography, Liquid; D000242:Cyclic AMP / Q000378:metabolism; D004023:Dictyostelium / Q000201:enzymology* / Q000254:growth & development; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008856:Microscopy, Fluorescence; D009844:Oligosaccharides / Q000378:metabolism; D013379:Substrate Specificity; D005644:alpha-L-Fucosidase / Q000302:isolation & purification / Q000378:metabolism*
D009844:Oligosaccharides; D000242:Cyclic AMP; D005644:alpha-L-Fucosidase