Characterization of a human digestive tract-specific calpain, nCL-4, expressed in the baculovirus system

Citation
Hj. Lee et al., Characterization of a human digestive tract-specific calpain, nCL-4, expressed in the baculovirus system, ARCH BIOCH, 362(1), 1999, pp. 22-31
Citations number
33
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
ISSN journal
0003-9861 → ACNP
Volume
362
Issue
1
Year of publication
1999
Pages
22 - 31
Database
ISI
SICI code
0003-9861(19990201)362:1<22:COAHDT>2.0.ZU;2-I
Abstract
Human nCL-4, a digestive tract-specific calpain, was stably produced with 3 0K, a regulatory subunit for ubiquitous calpain as a fully active form usin g the baculovirus-expression system. nCL-4 showed an activity only when it was coexpressed with 30K, Expressed heterodimeric recombinant nCL-4 was pur ified to near homogeneity by sequential column chromatographies. Purified n CL-4 showed a calcium-dependent activity (calcium concentration at 50% maxi mum activity (K-a): 0.125 mM) with a sp act of 21 U/mg, which is distinct f rom those of ubiquitous calpains. nCL-4 exhibited calcium-dependent autolys is, but the cleavage pattern of nCL-4 was clearly different from ubiquitous calpains. Although it was inhibited by leupeptin, E-64, and calpastatin, a nd exhibited an optimal pH at 7.3 like other ubiquitous calpains, its optim al temperature was much lower. When overexpressed in COS-7 cells, clear asy mmetric juxtanuclear, and/or nuclear staining rather than typical cytoplasm ic staining was observed. Moreover, a translation product of nCL-4 was dete cted in rat stomach tissue by immunofluorescence analysis. In conclusion, h uman nCL-4 resembles ubiquitous calpain in some enzymatic properties and in teracts 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.