Autolytic transition of mu-calpain upon activation as resolved by antibodies distinguishing between the pre- and post-autolysis forms.
Autolytic transition of mu-calpain upon activation as resolved by antibodies distinguishing between the pre- and post-autolysis forms.
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mu-钙蛋白酶激活后的自溶转变由区分自溶前和自溶后形式的抗体解决。
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发表时间：
1992
期刊：
影响因子：
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通讯作者：
K. Suzuki
中科院分区：
文献类型：
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作者：
T. Saido;S. Nagao;M. Shiramine;M. Tsukaguchi;H. Sorimachi;H. Murofushi;T. Tsuchiya;H. Ito;K. Suzuki
一种新的方法来观察自溶激活的哺乳动物细胞质钙蛋白酶，μ-钙蛋白酶，开发使用一组antipeptidic抗体能够区分前和后自溶形式的酶。针对合成肽产生的抗体被设计为与μ-钙蛋白酶大亚基的自溶前和自溶后形式的N-末端序列相匹配，其特异性地与钙蛋白酶的相应形式反应，而不与其它形式反应。抗体的特异性和灵敏度足以检测粗细胞裂解物中的抗原。钙蛋白酶激活的免疫化学检测的相关性被证实的观察，即蛋白水解的底物蛋白纯化的μ-钙蛋白酶在不同的pCa的自溶作为探测这些抗体和自溶之前底物蛋白水解。我们还观察到钙依赖性自溶的钙蛋白酶伴随随后的蛋白水解的底物在完整的细胞中使用的抗体。该方法将提供一种新的方法来评估的酶的生理目标，通过确定钙蛋白酶激活的局部细胞内位点。
A novel method to observe the autolytic activation of a mammalian cytoplasmic calcium protease, mu-calpain, was developed using a set of antipeptidic antibodies capable of distinguishing between the pre- and post-autolysis forms of the enzyme. Antibodies raised against synthetic peptides designed to match the N-terminal sequences of the pre- and post-autolysis forms of the mu-calpain large subunit reacted specifically with the corresponding form of calpain and not with the other. The antibodies were specific and sensitive enough to detect the antigens in crude cell lysates. The relevance of the immunochemical detection of calpain activation was confirmed by the observation that proteolysis of a substrate protein by purified mu-calpain paralleled autolysis at various pCa as probed by these antibodies and that autolysis preceded substrate proteolysis. We also observed calcium-dependent autolysis of calpain accompanying subsequent proteolysis of substrate in intact cells using the antibodies. The method will provide a novel approach to assess the physiological targets of the enzyme by determining the local intracellular sites of calpain activation.