EXTRA-LYSOSOMAL PROTEOLYSIS AND EXPRESSION OF CALPAINS AND CALPASTATIN IN CULTURED THYROID CELLS

✍ Scribed by J.-O. KARLSSON; M. NILSSON

• Publisher: Elsevier Science
• Year: 1997
• Tongue: English
• Weight: 518 KB
• Volume: 21
• Category: Article
• ISSN: 1065-6995
• DOI: 10.1006/cbir.1997.0128

✦ Synopsis

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 -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 -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.