Dudkiewicz A B
Biol Reprod. 1984 May;30(4):1005-14. doi: 10.1095/biolreprod30.4.1005.
Arylsulfatase A was extracted and purified from boar epididymal sperm acrosomes. Acrosomes were extracted by sonication in 50 mM Tris-maleate buffer containing 50 mM MgCl2, pH 6.1, followed by treatment with 50 mM Tris-maleate plus 0.2% Brij-35, pH 6.1. Purification of arylsulfatase A was performed with a three-step procedure consisting of centrifugation (85,000 X g), affinity chromatography with p-aminobenzamidine-Sepharose followed by chromatography on diethyaminoethyl (DEAE) Sephadex. The specific activity of the purified enzyme was 54 mumol/h per mg protein. The purified arylsulfatase did not contain any detectable acrosin or hyaluronidase activities. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis revealed a major band with an estimated molecular weight of 65,000 daltons. Properties of arylsulfatase A, determined by hydrolysis of p-nitrocatechol sulfate, indicated that the enzyme was inhibited 46% by 3.1 microM Ag+ and had a pH optimum of 4.2. Boar acrosomal arylsulfatase A dispersed the cumulus cells of ovulated hamster and rabbit eggs as well as those of follicular pig eggs. No effect of the enzyme on the zona pellucida or the oolemma was observed.
芳基硫酸酯酶A从公猪附睾精子顶体中提取并纯化。顶体在含有50 mM MgCl2、pH 6.1的50 mM Tris-马来酸缓冲液中通过超声处理提取，随后用50 mM Tris-马来酸加0.2% Brij-35、pH 6.1处理。芳基硫酸酯酶A的纯化采用三步法，包括离心（85,000×g）、用对氨基苯甲脒-琼脂糖进行亲和层析，然后在二乙氨基乙基（DEAE）葡聚糖凝胶上进行层析。纯化酶的比活性为每毫克蛋白质54 μmol/h。纯化的芳基硫酸酯酶不含有任何可检测到的顶体蛋白酶或透明质酸酶活性。十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳显示一条主要条带，估计分子量为65,000道尔顿。通过对硫酸对硝基邻苯二酚的水解测定的芳基硫酸酯酶A的性质表明，该酶被3.1 μM Ag+抑制46%，最适pH为4.2。公猪顶体芳基硫酸酯酶A使排卵的仓鼠和兔卵以及卵泡期猪卵的卵丘细胞分散。未观察到该酶对透明带或卵细胞膜有影响。
