Purification and partial characterization of hatching protease of the sea urchin, Strongylocentrotus purpuratus.
Purification and partial characterization of hatching protease of the sea urchin, Strongylocentrotus purpuratus.
复制标题
海胆 Strongylocentrotus purpuratus 孵化蛋白酶的纯化和部分表征。
DOI：
10.1016/0003-9861(77)90551-3
复制
发表时间：
1977
影响因子：
3.9
通讯作者：
D. Barrett
中科院分区：
文献类型：
--
作者：
B. F. Edwards;W. Allen;D. Barrett
孵化海胆（Strongylocentrotus purpuratus）囊胚上方的上清液含有粗孵化蛋白酶，其分子量、溶解度、电荷和密度均不均匀。它需要尿素处理（6m，22°C，6h）以将其在消化其底物（受精包膜）时产生的异质片段群与酶解离。然后可以用二乙氨基乙基纤维素、硫酸铵和 Sephadex G-100 将其纯化 340 倍。所得制剂根据凝胶排阻色谱、十二烷基硫酸钠凝胶电泳和热灭活标准均质，具有以下特性：比活度 = 1.44 U mg−1(1.44 μmol min−1mg−1)；kcat= 0.72 s-1；分子量=29,000；二甲基化酪蛋白的活化能 = 12.9 kcal mol−1；Km= 0.93mgml−1 二甲基化酪蛋白。纯酶在 pH 7 至 9、0.5mNaCl、10mmCa2+ 和 42 °C 下具有最佳活性。纯化使酶对冷冻和解冻的稳定性降低，并使其在 37 °C 下的热失活速率增加 100 倍。
The supernatant above hatched sea urchin (Strongylocentrotus purpuratus) blastulae contains crude hatching protease, which is heterogeneous in molecular weight, solubility, charge, and density. It requires urea treatment (6m, 22 °C, 6 h) to dissociate from the enzyme the heterogeneous population of fragments it has generated in digesting its substrate, the fertilization envelope. It can then be purified 340-fold by diethylaminoethyl-cellulose, ammonium sulfate, and Sephadex G-100. The resulting preparation, homogeneous by the criteria of gel exclusion chromatography, sodium dodecyl sulfate gel electrophoresis, and thermal inactivation, has the following properties: specific activity = 1.44 U mg−1(1.44 μmol min−1mg−1);kcat= 0.72 s-1; molecular weight = 29,000; energy of activation = 12.9 kcal mol−1on dimethylated casein;Km= 0.93mgml−1dimethylated casein. The pure enzyme is optimally active at pH 7 to 9, 0.5mNaCl, 10 mmCa2+, and 42 °C. Purification renders the enzyme less stable to freezing and thawing and increases the rate of its thermal inactivation at 37 °C by 100-fold.