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Purification and Characterization of Plasma Membrane-Associated Human Sperm -L-Fucosidase¹

^a Department of Biological Sciences ^b Division of Biochemical Sciences, Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania 18015

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 association of a novel human sperm, -L-fucosidase. This -L-fucosidase has been solubilized and purified 8600-fold to high specific activity (35 000 U/mg protein) by affinity chromatography on agarose-C₂₄-fucosylamine. To our knowledge, this is the first report concerning the purification and characterization of a mammalian plasma membrane-associated -L-fucosidase. Both SDS-PAGE and Western blot analysis indicated the -L-fucosidase is highly purified and contains a single subunit with a molecular mass of 51 kDa. N-glycanase studies indicated the subunit contains N-glycans, and lectin blot analysis 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 did not 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 µmol/min/mg protein. The unusual properties of human sperm -L-fucosidase argue in support of a potentially important, but presently unknown, role for this enzyme in human reproduction.

¹ Supported in part by a grant for Undergraduate Education in Biological Sciences from the Howard Hughes Medical Institute. S.K. was supported as a scholar of the Thai Government Ministry of University Affairs.

² Correspondence: Barry Bean, Lehigh University, 111 Research Drive, Bethlehem, PA 18015. FAX: 610 758 4004; e-mail: bb00{at}lehigh.edu

³ Current address: Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand 40002

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