Gamete Biology

Purification and Characterization of Plasma Membrane-Associated Human Sperm -L-Fucosidase

Abstract

Detergent and salt extraction studies, as well as cytochemical localization with fluorescein isothiocyanate-bovine serum albumin-L-fucose, have provided further evidence for the plasma membrane localization of a novel human sperm -L-fucosidase. This -L-fucosidase has been solubilized and purified 8600-fold to high specific activity (35,000 units/mg protein) by affinity chromatography on agarose-C₂₄-fucosylamine. This is the first report on the purification and characterization of a mammalian plasma membrane-associated -L-fucosidase. SDS-PAGE and Western blotting indicated the -L-fucosidase is highly purified, and contains a single subunit with M_r of 51 kDa. N-Glycanase studies indicated the subunit contains N-glycans, and lectin blotting detected the presence of mannose, but no terminal galactose or sialic acid, residues. Isoelectric focusing indicated the presence of two major -L-fucosidase isoforms (pIs 6.5 and 6.7) and a possible minor isoform (pI 6.3). Treatment of -L-fucosidase with neuraminidase didn't change its isoform profile, providing further evidence for the enzyme's lack of sialic acid residues. Kinetic analysis with 4-methylumbelliferyl -L-fucopyranoside indicated that sperm -L-fucosidase has a pH optimum near 7, an apparent K_M of 0.08 mM and a V_max of 6.8 mmol/min/mg protein. The unusual properties of human sperm -L-fucosidase argue for a potentially important, but presently unknown, role for this enzyme in human reproduction.