Tulsiani D R, Skudlarek M D, Orgebin-Crist M C
Department of Obstetrics and Gynecology, Vanderbilt School of Medicine, Nashville, Tennessee 37232-2633.
J Cell Biol. 1989 Sep;109(3):1257-67. doi: 10.1083/jcb.109.3.1257.
During the course of a study of glycoprotein processing mannosidases in the rat epididymis, we have made an intriguing discovery regarding the presence of a novel alpha-D-mannosidase on the rat sperm plasma membranes. Unlike the sperm acrosomal "acid" mannosidase which has a pH optimum of 4.4, the newly discovered alpha-D-mannosidase has a pH optimum of 6.2, and 6.5 when assayed in sperm plasma membranes and intact spermatozoa, respectively. In addition, the two enzymes show different substrate specificity. The acrosomal alpha-D-mannosidase is active mainly towards synthetic substrate, p-nitrophenyl alpha-D-mannopyranoside, whereas the sperm plasma membrane alpha-D-mannosidase shows activity mainly towards mannose-containing oligosaccharides. Evidence is presented which suggest that the sperm plasma membrane alpha-D-mannosidase is different from several processing mannosidases previously characterized from the rat liver. The newly discovered alpha-D-mannosidase appears to be an intrinsic plasma membrane component, since washing of the purified membranes with buffered 0.4 M NaCl did not release the enzyme in soluble form. The enzyme requires nonionic detergent (Triton X-100) for complete solubilization. The enzyme is activated by Co2+ and Mn2+. However, Cu2+ and Zn2+ are potent inhibitors of the sperm plasma membrane alpha-D-mannosidase. At a concentration of 0.1 mM, these divalent cations caused nearly complete inactivation of the sperm enzyme. In addition methyl-alpha-D-mannoside, methyl-alpha-D-glucoside, mannose, 2-deoxy-D-glucose, and D-mannosamine are inhibitors of the sperm surface alpha-D-mannosidase. The physiological role of the newly discovered enzyme is not yet known. Several published reports in three species, including the rat, suggest that the sperm surface alpha-D-mannosidase may have a role in binding to mannose-containing saccharides presumably present on the zona pellucida.
在对大鼠附睾中糖蛋白加工甘露糖苷酶的研究过程中，我们有了一个有趣的发现，即大鼠精子质膜上存在一种新型α-D-甘露糖苷酶。与精子顶体的“酸性”甘露糖苷酶（其最适pH为4.4）不同，新发现的α-D-甘露糖苷酶的最适pH为6.2，在精子质膜和完整精子中检测时分别为6.5。此外，这两种酶表现出不同的底物特异性。顶体α-D-甘露糖苷酶主要对合成底物对硝基苯基α-D-甘露吡喃糖苷有活性，而精子质膜α-D-甘露糖苷酶主要对含甘露糖的寡糖有活性。有证据表明，精子质膜α-D-甘露糖苷酶与先前从大鼠肝脏中鉴定出的几种加工甘露糖苷酶不同。新发现的α-D-甘露糖苷酶似乎是一种内在的质膜成分，因为用0.4M NaCl缓冲液洗涤纯化的膜并没有以可溶形式释放该酶。该酶需要非离子去污剂（Triton X-100）才能完全溶解。该酶被Co2+和Mn2+激活。然而，Cu2+和Zn2+是精子质膜α-D-甘露糖苷酶的有效抑制剂。在0.1mM的浓度下，这些二价阳离子几乎使精子酶完全失活。此外，甲基-α-D-甘露糖苷、甲基-α-D-葡萄糖苷、甘露糖、2-脱氧-D-葡萄糖和D-甘露糖胺是精子表面α-D-甘露糖苷酶的抑制剂。新发现的酶的生理作用尚不清楚。包括大鼠在内的三个物种的几篇已发表报告表明，精子表面α-D-甘露糖苷酶可能在与推测存在于透明带上的含甘露糖的糖类结合中起作用。
