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EXTRA‐LYSOSOMAL PROTEOLYSIS AND EXPRESSION OF CALPAINS AND CALPASTATIN IN CULTURED THYROID CELLS
Author(s) -
KARLSSON J.O.,
NILSSON M.
Publication year - 1997
Publication title -
cell biology international
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.932
H-Index - 77
eISSN - 1095-8355
pISSN - 1065-6995
DOI - 10.1006/cbir.1997.0128
Subject(s) - calpastatin , calpain , proteolysis , blot , proteases , cytoplasm , chemistry , biochemistry , microbiology and biotechnology , enzyme , biology , gene
Proteolysis at neutral pH in the soluble fraction of cultured pig thyroid epithelial cells was examined using a synthetic calpain substrate, succinyl‐Leu‐Tyr‐7‐amino‐4‐methylcoumarin. The Ca 2+ ‐independent proteolytic activity was largely inhibited by substances known to affect cysteine‐ and metalloproteases, whereas no or little effects were obtained with inhibitors affecting serine‐ and aspartic proteases. Addition of Ca 2+ did not significantly alter the rate of substrate degradation. Biochemical separation via hydrophobic interaction chomatography and Western blotting demonstrated the presence of both m‐calpain (40% of total calpain) and μ‐calpain (60%) in confluent thyrocytes. Determination of calpastatin activity indicated a 30 times higher level of the inhibitor as compared to total calpain activity. Western blotting showed the presence of a 110kD calpastatin form with additional low mol wt forms possibly representing fragmentation products. In immunofluorescent stainings, m‐calpain had a diffuse cytoplasmic distribution whereas μ‐calpain was located both in the cytoplasm and at the cell—cell contacts. Calpastatin immunoreactivity was mainly granular and located close to the nucleus, although a fibrillar distribution was also observed. The results show the presence of all components of the calpain/calpastatin system and indicate a strict control of calpain activity in cultured thyrocytes. The different subcellular distributions of calpains and calpastatin suggests that they are compartmentalized and require mobilization to interact.