Characterization of a human digestive tract-specific calpain, nCL-4, expressed in the baculovirus system
Characterization of a human digestive tract-specific calpain, nCL-4, expressed in the baculovirus system
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DOI：
10.1006/abbi.1998.1021
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发表时间：
1999-02-01
影响因子：
3.9
通讯作者：
Suzuki, K
中科院分区：
文献类型：
--
作者：
Lee, HJ;Tomioka, S;Suzuki, K
人nCL-4是一种消化道特异性钙蛋白酶，使用杆状病毒表达系统，以30 K作为完全活性形式稳定产生，30 K是普遍存在的钙蛋白酶的调节亚基。nCL-4只有在与30 K共表达时才显示活性。表达的异源二聚体重组nCL-4经连续柱层析纯化至接近均一。纯化的nCL-4显示出钙依赖性活性（50%最大活性时的钙浓度（K-a）：0.125 mM），spact为21 U/mg，这与普遍存在的钙蛋白酶不同。nCL-4表现出钙依赖性自溶，但nCL-4的裂解模式明显不同于普遍存在的钙蛋白酶。虽然它被亮抑酶肽、E-64和钙蛋白酶抑制素抑制，并且像其他普遍存在的钙蛋白酶一样在7.3时表现出最适pH，但其最适温度要低得多。当在COS-7细胞中过表达时，观察到明显的不对称的胞核和/或核染色，而不是典型的胞质染色。此外，通过免疫荧光分析在大鼠胃组织中检测到nCL-4的翻译产物。总之，人nCL-4类似于普遍存在的钙蛋白酶在一些酶的性质和相互作用的30 K的活性。这是第一次报告的生化和酶的性质的一个完全活跃的组织特异性钙蛋白酶物种表达的杆状病毒系统。(C)北京：科学出版社.
Human nCL-4, a digestive tract-specific calpain, was stably produced with 30K, a regulatory subunit for ubiquitous calpain as a fully active form using the baculovirus-expression system. nCL-4 showed an activity only when it was coexpressed with 30K, Expressed heterodimeric recombinant nCL-4 was purified to near homogeneity by sequential column chromatographies. Purified nCL-4 showed a calcium-dependent activity (calcium concentration at 50% maximum activity (K-a): 0.125 mM) with a sp act of 21 U/mg, which is distinct from those of ubiquitous calpains. nCL-4 exhibited calcium-dependent autolysis, but the cleavage pattern of nCL-4 was clearly different from ubiquitous calpains. Although it was inhibited by leupeptin, E-64, and calpastatin, and exhibited an optimal pH at 7.3 like other ubiquitous calpains, its optimal temperature was much lower. When overexpressed in COS-7 cells, clear asymmetric juxtanuclear, and/or nuclear staining rather than typical cytoplasmic staining was observed. Moreover, a translation product of nCL-4 was detected in rat stomach tissue by immunofluorescence analysis. In conclusion, human nCL-4 resembles ubiquitous calpain in some enzymatic properties and interacts with 30K for its activity. This is the first report on biochemical and enzymatic properties of a fully active tissue-specific calpain species expressed in the baculovirus system. (C) 1999 Academic Press.