| 摘要: |
| 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。 |
| 关键词: 牙鲆(Paralichthys olivaceus) 孵化酶 卵膜裂解活性 丝氨酸蛋白酶 金属蛋白酶 |
| DOI： |
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| 基金项目:国家教育部留学回国人员基金项目(980418) |
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| Purification and partial biochemical characterization of hatching enzyme from Paralichthys olivaceus |
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| Abstract: |
| Using sephacryl S-100 column gel-filt ration and DEAE-sepharose fast flow ion-exchange chromatography, hatching enzyme(HE) from Paralichthys olivaceus( PHE) was purified. The molecular size of PHE in SDS-PAGE is about 34.8ku, 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 at rypsin-type serine protease. The PHE could be inhibited by EDTA in a dose-dependent manner. Metal ions of Ca2+ and Mg2+ could greatly enhance the choriolytic activity of PHE, whereas Cu2+ could enormously inhibit the choriolytic activity of PHE. But Zn2+ 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. |
| Key words: Paralichthys olivaceus hatching enzyme choriolytic activity serine protease metalloprotease |
