Molecular Human Reproduction, Vol. 8, No. 3, 221-227,
March 2002
© 2002 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Purification and characterization of human seminal plasma 
-L-fucosidase
1 Department of Biological Sciences and 2 Division of Biochemical Sciences, Department of Chemistry, Lehigh University, Bethlehem, PA 18015, USA
Human seminal plasma 
-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-
-aminocaproyl-fucopyranosylamine. The purified -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 -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--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 KM and Vmax values for the 4MU-Fuc substrate were determined to be 0.06 mmol/l and 92 µmol/min/mg protein respectively, using Lineweaver–Burk double reciprocal plots. Isoelectric focusing and neuraminidase treatment studies provided further evidence that the purified seminal plasma -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.
glycosidase/glycoprotein/semen/sperm
3 To whom correspondence should be addressed at: Biological Sciences, 111 Research Drive, Lehigh University, Bethlehem,PA 18015, USA. E-mail: bb\|[Oslash]\|\|[Oslash]\|{at}lehigh.edu
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