IMMUNOCYTOCHEMICAL LOCALIZATION AND BIOCHEMICAL-CHARACTERIZATION OF ANOVEL PLASMA MEMBRANE-ASSOCIATED, NEUTRAL PH OPTIMUM ALPHA-L-FUCOSIDASE FROM RAT TESTIS AND EPIDIDYMAL SPERMATOZOA

1. Immunocytochemical and biochemical techniques have been used to loc alize and characterize a novel plasma membrane-associated, neutral-pH- optimum alpha-L-fucosidase from rat spermatozoa. Light and electron mi croscopy 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 al so 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-f ucosidase activity was associated with the 48 000 g pellet. This pelle t-associated activity could be solubilized with 0.5 M NaCl but not wit h 0.5% Triton X-100, suggesting that fucosidase is peripherally associ ated with membranes. Sucrose-density-gradient centrifugation of sperm homogenates indicated that fucosidase was enriched in the plasma membr ane-enriched fraction. Analysis of alpha-L-fucosidase on intact epidid ymal sperm indicated that the enzyme was active, displayed linear kine tics and had a pH-activity curve (with an optimum near 7) which was co mparable to that of fucosidase from epididymal sperm extracts. These r esults further suggest that fucosidase is associated with plasma membr anes, and that its active site is accessible to fucoconjugates. Eviden ce 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 w hich approx. 90% of the fucosidase activity on intact sperm could be r eleased from the sperm by gentle shaking with 0.5 M NaCl. Isoelectric focusing indicated that the NaCl-solubilized epididymal sperm fucosida se appears to have one major and one miser isoform with pIs near 7.2 a nd 5.2, respectively. SDS/PAGE and Western blotting indicated that the NaCl-solubilized extract of epididymal sperm contains two protein ban ds of 54 and 50 kDa which were highly immunoreactive with the IgG frac tion of anti-fucosidase antibodies. Although the function of the novel sperm fucosidase is not known, its specific localization to the plasm a membrane of the region of the rat sperm head involved in sperm-egg b inding and its high enzymic activity at neutral pH on intact sperm sug gest that this enzyme may have a role in sperm-egg interactions.