Demonstration of calcium-dependent proteases (calpains) and thyroglobulin proteolysis in hog thyroid cytosol.
Ca2+-dependent neutral proteases in hog thyroid cytosol were found to digest thyroglobulin. The protease activity was divided into two peaks by DEAE-cellulose column chromatography. Peak I was eluted at 0.2 M NaCl and required only a micromolar range of Ca2+ for its 50% activation, while peak II, which was eluted at about 0.4 M NaCl, displayed little activity until the Ca2+ concentration was increased at more than 10(-4) M. Among various inhibitors used, thiol protease inhibitors (leupeptin, E-64 and monoiodoacetic acid) were the most effective, whereas a calmodulin antagonist (trifluoperazine) and serine protease inhibitors (phenylmethyl-sulfony-fluoride and pepstatin A) were not effective, indicating that these Ca2+-dependent proteases corresponded to calpains 1 and 2. Among the substrates tested, casein was the best and thyroglobulin was also a good for calpain 2. By using immunoblotting procedure with anti-thyroglobulin antibody, it has been found that calpain 2 degrades thyroglobulin to yield 67 K and 46 K thyroglobulin and further that it also degrades 40 K thyroglobulin.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013552:Swine; D013954:Thyroglobulin / Q000378:metabolism*; D013961:Thyroid Gland / Q000201:enzymology*
D011480:Protease Inhibitors; D013954:Thyroglobulin; D002154:Calpain; D002118:Calcium