Purification of an endogenous 68 kD inhibitor of calcium‐activated neutral proteinase (CANP) from bovine brain: Immunoblot identification and characterization

Purification of an endogenous 68 kD inhibitor of calcium‐activated neutral proteinase (CANP)... 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Neuroscience Research Wiley

Purification of an endogenous 68 kD inhibitor of calcium‐activated neutral proteinase (CANP) from bovine brain: Immunoblot identification and characterization

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Publisher
Wiley
Copyright
Copyright © 1990 Wiley‐Liss, Inc.
ISSN
0360-4012
eISSN
1097-4547
D.O.I.
10.1002/jnr.490250115
Publisher site
See Article on Publisher Site

Abstract

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.

Journal

Journal of Neuroscience ResearchWiley

Published: Jan 1, 1990

References

  • Purification of a calcium‐activated neutral proteinase from bovine brain
    Banik, Banik; Hogan, Hogan; Jenkins, Jenkins; McDonald, McDonald; McAlhaney, McAlhaney; Sostek, Sostek
  • Calcium‐stimulated proteolysis in myelin: Evidence for a Ca 2+ ‐stimulated neutral proteinase associated with purified myelin of rat CNS
    Banik, Banik; McAlhaney, McAlhaney; Hogan, Hogan
  • Calcium‐activated neutral proteinase in rat brain myelin and subcellular fractions
    Chakrabarti, Chakrabarti; Yoshida, Yoshida; Powers, Powers; Singh, Singh; Hotan, Hotan; Banik, Banik
  • Calcium‐activated neutral proteinase of human brain: Subunit structure and enzymatic properties of multiple molecular forms
    Vitto, Vitto; Nixon, Nixon
  • Myelin associated calpain II
    Yangisawa, Yangisawa; Sato, Sato; O'Shannessy, O'Shannessy; Quarles, Quarles; Suzuki, Suzuki; Miyatake, Miyatake

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