Premium
Purification of an endogenous 68 kD inhibitor of calcium‐activated neutral proteinase (CANP) from bovine brain: Immunoblot identification and characterization
Author(s) -
Banik N. L.,
Chakrabarti A. K.,
Hogan E. L.
Publication year - 1990
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/jnr.490250115
Subject(s) - calpastatin , trypsin inhibitor , egta , calpain , microbiology and biotechnology , antiserum , papain , biochemistry , chemistry , endogeny , polyclonal antibodies , trypsin , gel electrophoresis , enzyme inhibitor , kunitz sti protease inhibitor , biology , calcium , enzyme , antibody , organic chemistry , immunology
A calcium‐activated neutral proteinase (CANP)‐specific endogenous inhibitor (calpastatin) was purified from bovine brain by successive column chromatography. The purified inhibitor exhibited a major band on sodium dodecylsulfate polyacrylamide gel electrophoresis with an approximate molecular weight of 68 kD. The polyclonal antisera raised to the inhibitor strongly reacted with the 68 kD protein band. Two lightly stained bands approximately 55–68 kD and 120–130 kD were also recognized by the inhibitor antiserum. The inhibitor specifically inhibited CANP activity and the half‐maximal inhibition was found with 75 ng of calpastatin per 1 μg of CANP in a final volume of 125 μl. Cathepsin B and papain were not inhibited by the inhibitor, while trypsin and chymotrypsin were inhibited to some extent. The inhibitor formed a complex with CANP and the inactive complex was dissociated into active fractions of enzyme and calpastatin in the presence of EGTA.