Extra-lysosomal proteolysis and expression of calpains and calpastatin in cultured thyroid cells.
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 Ca2+-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 Ca2+ did not significantly alter the rate of substrate degradation. Biochemical separation via hydrophobic interaction chromatography and Western blotting demonstrated the presence of both m-calpain (40% of total calpain) and mu-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 110 kD calpastatin form with additional low mol wt forms possibly representing fragmentation products. In immunofluorescent stainings, m-calpain had a diffuse cytoplasmic distribution whereas mu-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.
Karlsson
J O
JO
Institute of Anatomy and Cell Biology, Göteborg University, Sweden.
Nilsson
M
M
D000818:Animals; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000378:metabolism* / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors; D003593:Cytoplasm / Q000201:enzymology; D003600:Cytosol / Q000502:physiology; D004848:Epithelium / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000378:metabolism*; D013552:Swine; D013961:Thyroid Gland / Q000166:cytology / Q000502:physiology*
D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D006023:Glycoproteins; C039304:calpain inhibitors; C033668:calpastatin; D010447:Peptide Hydrolases; D002154:Calpain