Purification and characterization of multicatalytic proteinase from eggs of the ascidian Halocynthia roretzi.
Purification and characterization of multicatalytic proteinase from eggs of the ascidian Halocynthia roretzi.
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海鞘类 Halocynthia roretzi 卵中多催化蛋白酶的纯化和表征。
DOI：
10.1093/oxfordjournals.jbchem.a122649
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发表时间：
1989
影响因子：
2.7
通讯作者：
S. Ishii
中科院分区：
文献类型：
--
作者：
Y. Saitoh;H. Yokosawa;K. Takahashi;S. Ishii
通过 DEAE 纤维素和羟基磷灰石柱色谱以及 Sepharose 6B 凝胶过滤等程序，从海鞘类 Halocynthia roretzi 卵中纯化出一种多催化（高分子量）蛋白酶。通过圆盘聚丙烯酰胺凝胶电泳、等电聚焦、沉降速度和凝胶过滤判断，纯化的酶似乎是均质的。通过凝胶过滤估计酶的分子量为610,000。等电点和沉降系数(S20,w)分别为6.2和22.8S。该酶在SDS-聚丙烯酰胺凝胶电泳上显示出数条分子量在25,000～33,000之间的蛋白质条带，在电镜下显示出由多个亚基组成的圆柱或环状结构，表明该酶以由多种蛋白质成分组成的大分子存在。该酶具有胰凝乳蛋白酶样和胰蛋白酶样活性，最适pH均为7.0。糜抑素及其类似物、钙蛋白酶抑制剂 I 和弹性蛋白抑制这两种活性，而亮肽素和抗痛素仅抑制后者。前一种活性受到低浓度 SDS 或脂肪酸的刺激，而后者则不然。因此，从海鞘卵中纯化的酶的特性与来自哺乳动物组织的多催化蛋白酶的特性相似。
A multicatalytic (high-molecular-weight) proteinase has been purified from eggs of the ascidian Halocynthia roretzi by a procedure including column chromatographies on DEAE-cellulose and hydroxylapatite and gel filtration on Sepharose 6B. The purified enzyme seemed to be homogeneous, as judged by disc-polyacrylamide gel electrophoresis, isoelectrofocusing, sedimentation velocity, and gel filtration. The molecular weight of the enzyme was estimated to be 610,000 by gel filtration. The isoelectric point and the sedimentation coefficient (S20,w) were 6.2 and 22.8S, respectively. The enzyme showed several protein bands with molecular weight ranging from 25,000 to 33,000 on SDS-polyacrylamide gel electrophoresis and a cylindrical or ring-like structure composed of several subunits under the electron microscope, indicating that the enzyme exists as a large molecule consisting of several protein components. The enzyme exhibited chymotrypsin-like and trypsin-like activities whose pH optima were both 7.0. Chymostatin and its analog, calpain inhibitor I, and elastatinal inhibited both activities, whereas leupeptin and antipain only inhibited the latter. The former activity was stimulated by a low concentration of SDS or fatty acid, whereas the latter was not. Thus, the properties of the enzyme purified from ascidian eggs are similar to those of multicatalytic proteinases from mammalian tissues.