牙鲆孵化酶的分离纯化及其部分生物化学性质研究

Purification and partial biochemical characterization of hatching enzyme from Paralichthys olivaceus

利用凝胶过滤柱层析和阴离子交换柱层析技术,从牙鲆(Paralichthys olivaceus)胚胎孵化液中分离纯化出了大小约为34.8ku的牙鲆孵化酶。该酶卵膜裂解的最适反应温度为35℃,最适pH为7.0;对底物酪蛋白的米氏常数Km值为1.53mmol/L。该酶对丝氨酸蛋白酶和胰蛋白酶的特异性抑制剂非常敏感,而对其他蛋白酶抑制剂不敏感,表明该酶极可能是一种丝氨酸蛋白酶类型的胰蛋白酶。此外,该酶可浓度依赖性地被EDTA所抑制,被Cu2+所强烈抑制,被Ca2+和Mg2+所激活,对Zn2+则不敏感,表明该酶很可能是一种金属蛋白酶。

Using sephacryl S-100 column gel-filtration and DEAE-sepharose fast flow ion-exchange chroma tography, hatching enzyme （HE） from Paralichthys olivaceus （PHE） was purified. The molecular size of PHE in SDS-PAGE is about 34.8 ku, and its choriolytic activity was optimized at pH of 7.0 and temperature of 35 ℃, respectively. The Km value was 1. 53 mmol/L with the substrate of casein. The PHE was very sensitive to trypsin-specific inhibitors and serine protease-specific inhibitors but not sensitive to other protein inhibitors. The results of inhibitors on PHE imply that PHE is most probably a trypsin-type serine protease. The PHE could be inhibited by EDTA in a dose-dependent manner. Metal ions of Ca^2＋ and Mg^2＋ could greatly enhance the ehoriolytic activity of PHE, whereas Cu^2＋ could enormously inhibit the choriolytic activity of PHE. But Zn^2＋ had shown almost no inhibition on PHE activity. The results on EDTA and metal ions imply that this PHE might be a kind of metalloprotease.