title: Purification and characterization of human seminal plasma alpha-L-fucosidase.
abstract: Human seminal plasma alpha-L-fucosidase (EC 3.2.1.51) has been purified 7100-fold to very high purity and specific activity (83,000 nmol/min/mg protein) by affinity chromatography on agarose-epsilon-aminocaproyl-fucopyranosylamine. The purified alpha-L-fucosidase appeared to contain a single subunit of 56-57 kDa (as determined by SDS-PAGE and Western analysis). Lectin blotting and N-glycanase treatment studies indicated that this subunit is N-glycosylated and contains sialic acid residues. Human seminal plasma alpha-L-fucosidase was shown to contain three multimeric forms of 110, 236 and 314 kDa respectively (as determined by Sephadex G-200 chromatography) and therefore probably exists in dimeric, tetrameric and hexameric forms. Kinetic analysis with the 4-methylumbelliferyl-alpha-L-fucopyranoside (4MU-Fuc) substrate indicated a broad acidic optimum (pH 4.0-4.5) with a second neutral optimum (pH 6.4-7.4) with 60-80% of maximal activity. Apparent K(M) and V(max) values for the 4MU-Fuc substrate were determined to be 0.06 mmol/l and 92 micromol/min/mg protein respectively, using Lineweaver-Burk double reciprocal plots. Isoelectric focusing and neuraminidase treatment studies provided further evidence that the purified seminal plasma alpha-L-fucosidase is a sialoglycoprotein with several isoforms between pI values 5-7. The acidic isoforms between pI values 5-6 appear to be related chemically to the more neutral isoforms by sialic acid residues since neuraminidase treatment converted the former into the latter isoforms.
authors.affiliation: Department of Biological Sciences, Lehigh University, Bethlehem, PA 18015, USA.
authors.forename: Sumpars
authors.initials: S
authors.lastname: Khunsook
authors.forename: Jack A
authors.initials: JA
authors.lastname: Alhadeff
authors.forename: Barry S
authors.initials: BS
authors.lastname: Bean
mesh_terms: D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008297:Male; D012661:Semen / Q000201:enzymology*; D013379:Substrate Specificity; D005644:alpha-L-Fucosidase / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D005644:alpha-L-Fucosidase