Purification and kinetic studies of an alpha-L-fucosidase of Venus mercenaria

Abstract

An alpha-L-fucosidase activity has been isolated from the liver (hepatopancreas) of the common edible clam, Venus mercenaria, and has been purified approximately 300-fold (11% yield) by affinity chromatography on agarose-epsilon-amino-caproylfucosamine. Isoelectric focusing profiles were heterogeneous, revealing several isoenzymes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of a single subunit of Mr 50,000. The purified enzyme preparation contained only trace amounts of other alpha- and beta-D-glycosidases tested. In addition to p-nitrophenyl alpha-L-fucopyranoside, the enzyme hydrolyzed natural substrates such as fucose-containing milk pentasaccharides, thyroglobulin glycopeptides, human salivary glycoproteins, and blood-group-active glycosphingolipids. The enzyme preparation had a broad pH optimum range between 4.5 and 5.5. The apparent Km value with respect to p-nitrophenyl alpha-L-fucopyranoside was 0.26mM.