Purification to homogeneity of Charonia lampas alpha-fucosidase by using sequential ligand-affinity chromatography.
An alpha-fucosidase from the liver of the marine gastropod Charonia lampas was purified to homogeneity using a procedure that included cation-exchange and gel-filtration chromatography, chromatofocusing and a final series of affinity-chromatography steps which involved the following gel-immobilized ligands: N-(5-carboxy-1-pentyl)-1,5-dideoxy-1,5-imino-L-fucitol, N-(5-carboxy-1-pentyl)-2-acetamido-1,5-imino-1,2,5-trideoxy-D-glucitol and thio-beta-D-galactoside. The enzyme was found to be a tetrameric glycoprotein with a native Mr of 208,000, and to exist in a number of isoforms displaying pI values in the range 6.0-6.4. Substrate-specificity studies using a number of fucosylated oligosaccharides of the lacto-N and lacto-N-neo series and a synthetic disaccharide confirmed that the enzyme catalyses the hydrolysis of a broad range of fucosidic linkages, and established the following hierarchy of susceptibility: Fuc alpha 2Gal beta 4Glc much much greater than Fuc alpha 6GlcNAc greater than Fuc alpha 2Gal beta 4GlcNAc greater than Gal beta 3(Fuc alpha 4)GlcNAC much much greater than Gal beta 4(Fuc alpha 3)GlcNAc. Similar relative rates of hydrolysis were also demonstrated using biantennary oligosaccharide alditols as substrates which contained fucose linked either alpha 3 or alpha 6 to the N-acetylglucosaminitol residue of the chitobiosyl core.
Department of Biochemistry, G. D. Searle & Co., University of Oxford, U.K.
T D
TD
Butters
P
P
Scudder
J
J
Rotsaert
S
S
Petursson
G W
GW
Fleet
F W
FW
Willenbrock
G S
GS
Jacob
D000818:Animals; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007525:Isoelectric Focusing; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008974:Mollusca / Q000201:enzymology*; D009844:Oligosaccharides / Q000378:metabolism; D010306:Parotid Gland / Q000737:chemistry; D011134:Polysaccharides / Q000378:metabolism; D005644:alpha-L-Fucosidase / Q000302:isolation & purification*
D009844:Oligosaccharides; D011134:Polysaccharides; D005644:alpha-L-Fucosidase