Characterization of a human digestive tract-specific calpain, nCL-4, expressed in the baculovirus system.
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 (Ka): 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.[1]References
- Characterization of a human digestive tract-specific calpain, nCL-4, expressed in the baculovirus system. Lee, H.J., Tomioka, S., Kinbara, K., Masumoto, H., Jeong, S.Y., Sorimachi, H., Ishiura, S., Suzuki, K. Arch. Biochem. Biophys. (1999) [Pubmed]
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