Purification and further characterization of the Ca2+-activated proteinase specific for the intermediate filament proteins vimentin and desmin.
Purification and further characterization of the Ca2+-activated proteinase specific for the intermediate filament proteins vimentin and desmin.
复制标题
对中间丝蛋白波形蛋白和结蛋白具有特异性的 Ca2 激活蛋白酶的纯化和进一步表征。
DOI：
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发表时间：
1982
影响因子：
4.8
通讯作者：
Peter Traubs
中科院分区：
文献类型：
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作者：
James Nelson;Peter Traubs
从埃利希腹水瘤细胞中纯化出一种钙激活的中性蛋白酶。所用的方案已导致从埃利希腹水肿瘤细胞核后上清液中以21%的产率将酶纯化3，600倍。纯化的蛋白酶对中间丝亚基蛋白、波形蛋白和结蛋白具有高底物特异性，并且对除细胞角蛋白的60，000-道尔顿蛋白之外的其它中间丝蛋白没有显示出活性。此外，肌动蛋白，微管蛋白，肌原纤维的主要组成蛋白和几个标准蛋白质没有降解。纯化的蛋白酶的表征表明，它被激活的Ca 2+（10至100 μ M），可能是钙调蛋白的独立性和不可逆的失去活性时，在没有底物的Ca 2+的存在下孵育。该酶对波形蛋白和结蛋白的Km分别为1.7 × 10（-8）M和5.2 × 10（-7）M。该蛋白酶具有72，000道尔顿的主要亚基，其具有催化中心和29，000道尔顿的次要组分;通过凝胶渗透色谱法，其表观分子量为100，000。它需要一个还原的巯基来发挥活性，并且可以被巯基阻断剂抑制。该蛋白酶的高底物特异性表明它参与调节含有波形蛋白和结蛋白的中间丝的分布和周转。
A calcium (Ca2+)-activated, neutral proteinase has been purified from Ehrlich ascites tumor cells. The protocol used has resulted in a 3,600-fold purification of the enzyme in a yield of 21% from the Ehrlich ascites tumor cell postnuclear supernatant. The purified proteinase has a high substrate specificity for the intermediate filament subunit proteins, vimentin and desmin, and showed no activity towards other intermediate filament proteins except a 60,000-dalton protein of the cytokeratins. Also, there was no degradation of actin, tubulin, the major constituent proteins of myofibrils and several standard proteins. Characterization of the purified proteinase has shown that it is activated by Ca2+ (10 to 100 microM), is probably calmodulin-independent and irreversibly loses activity when incubated in the presence of Ca2+ without substrate. The enzyme has a Km of 1.7 x 10(-8) M for vimentin and 5.2 x 10(-7) M for desmin. The proteinase has a major subunit of 72,000 daltons which has the catalytic center and a minor component of 29,000 daltons; by gel permeation chromatography it has an apparent molecular weight of 100,000. It requires a reduced sulfhydryl group for activity and can be inhibited by sulfhydryl-blocking reagents. The high substrate specificity of the proteinase indicates that it is involved in the regulation of the distribution and turnover of vimentin- and desmin-containing intermediate filaments.