Immunocytochemical localization and biochemical characterization of a novel plasma membrane-associated, neutral pH optimum α-l-fucosidase from rat testis and epididymal spermatozoa

Abstract

1. Immunocytochemical and biochemical techniques have been used to localize and characterize a novel plasma membrane-associated, neutral-pH-optimum alpha-L-fucosidase from rat spermatozoa. Light and electron microscopy specifically localized the fucosidase on the plasma membrane of the convex region of the principal segment of testicular and cauda epididymal sperm heads. Immunoreactivity for alpha-L-fucosidase was also detected in the Golgi apparatus of spermatocytes and spermatids but no immunoreactivity was observed in the acrosome. 2. Fractionation of epididymal sperm homogenates indicated that over 90% of the alpha-L-fucosidase activity was associated with the 48,000 g pellet. This pellet-associated activity could be solubilized with 0.5 M NaCl but not with 0.5% Triton X-100, suggesting that fucosidase is peripherally associated with membranes. Sucrose-density-gradient centrifugation of sperm homogenates indicated that fucosidase was enriched in the plasma membrane-enriched fraction. Analysis of alpha-L-fucosidase on intact epididymal sperm indicated that the enzyme was active, displayed linear kinetics and had a pH-activity curve (with an optimum near 7) which was comparable to that of fucosidase from epididymal sperm extracts. These results further suggest that fucosidase is associated with plasma membranes, and that its active site is accessible to fucoconjugates. Evidence that most of the fucosidase is associated with the exterior of the plasma membrane came from studies in which intact sperm had fucosidase activity comparable to that of sperm sonicates, and from studies in which approx. 90% of the fucosidase activity on intact sperm could be released from the sperm by gentle shaking with 0.5 M NaCl. Isoelectric focusing indicated that the NaCl-solubilized epididymal sperm fucosidase appears to have one major and one minor isoform with pIs near 7.2 and 5.2, respectively. SDS/PAGE and Western blotting indicated that the NaCl-solubilized extract of epididymal sperm contains two protein bands of 54 and 50 kDa which were highly immunoreactive with the IgG fraction of anti-fucosidase antibodies. Although the function of the novel sperm fucosidase is not known, its specific localization to the plasma membrane of the region of the rat sperm head involved in sperm-egg binding and its high enzymic activity at neutral pH on intact sperm suggest that this enzyme may have a role in sperm-egg interactions.

A molecular sensor that allows a gut commensal to control its nutrient foundation in a competitive ecosystem

“…Permease activity was quantitated by monitoring the uptake of L- H]fucose by intact logarithmic-phase cells (12). ␣-LFucosidase activity in culture supernatants was assayed at pH 7 in 0.1 M sodium phosphate, by using 1 mM 4-methylumbelliferyl-␣-L-fucopyranoside as the substrate (21).…”

Determination of glycosidase activity in porcine oviductal fluid at the different phases of the estrous cycle

“…Glycosidase activities were assayed, as we have previously described (Aviles et al 1996, Abascal et al 1998, using 4-methylumbelliferyl-glucopyranosides as substrates. Briefly, stock solutions for substrates (0.1 M) were prepared in purified water and kept at K80 8C until use.…”

Immunocytochemical and biochemical detection of alpha-L-fucosidase in Trypanosoma cruzi

“…Immunocytochemical techniques and detection of enzymatic activity were used to demonstrate that at least 50% of the enzyme is associated with the cytoplasmic and plasma membranes. Although most of the a-L-fucosidase activity of virtually all mammalian tissues is in the soluble fraction, membrane-associated a-Lfucosidase has been described in rat testis and epididymal spermatozoa (14,29), in human sperm plasma membrane (15), and in human erythrocyte membranes (16).…”

“…The enzyme is also present in body fluids and in the plasma membrane, as reported for sperm cells (14,15) or human erythrocytes (16).…”

“…The a-L-fucosidase from the membrane fraction of epimastigotes has a neutral pH optimum (pH 6.0-7.5), having no activity at acidic pH, in contrast to a-L-fucosidases isolated from lysosomes. A more neutral pH optimum was described for a-L-fucosidases from Corynebacterium, pH 8. foetus, pH 7.0 (32), and for the enzymes from rat sperm and erythrocytes (14,16).…”