Purification and characterization of plasma membrane-associated human sperm alpha-L-fucosidase.
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, alpha-L-fucosidase. This alpha-L-fucosidase has been solubilized and purified 8600-fold to high specific activity (35 000 U/mg protein) by affinity chromatography on agarose-C(24)-fucosylamine. To our knowledge, this is the first report concerning the purification and characterization of a mammalian plasma membrane-associated alpha-L-fucosidase. Both SDS-PAGE and Western blot analysis indicated the alpha-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 alpha-L-fucosidase isoforms (pIs 6.5 and 6.7) and a possible minor isoform (pI 6.3). Treatment of alpha-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 alpha-L-fucopyranoside indicated that sperm alpha-L-fucosidase has a pH optimum near 7, an apparent K(m) of 0.08 mM, and a V(max) of 6.8 micro mol/min/mg protein. The unusual properties of human sperm alpha-L-fucosidase argue in support of a potentially important, but presently unknown, role for this enzyme in human reproduction.
Khunsook
Sumpars
S
Department of Biological Sciences Division of Biochemical Sciences, Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania 18015, USA.
Bean
Barry S
BS
McGowan
Susan R
SR
Alhadeff
Jack A
JA
D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005643:Fucose / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification; D007700:Kinetics; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D013094:Spermatozoa / Q000166:cytology / Q000201:enzymology*; D005644:alpha-L-Fucosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D007527:Isoenzymes; D008565:Membrane Proteins; D005643:Fucose; D005644:alpha-L-Fucosidase