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The protease core of the muscle‐specific calpain, p94, undergoes Ca 2+ ‐dependent intramolecular autolysis
Author(s) -
Rey Michelle A,
Davies Peter L
Publication year - 2002
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(02)03722-5
Subject(s) - autolysis (biology) , calpain , protease , chemistry , enzyme , proteases , gene isoform , biochemistry , mutant , intramolecular force , stereochemistry , gene
Limb girdle muscular dystrophy type 2A is linked to a skeletal muscle‐specific calpain isoform known as p94. Isolation of the intact 94‐kDa enzyme has been difficult to achieve due to its rapid autolysis, and uncertainty has arisen over its Ca 2+ ‐dependence for activity. We have expressed a C‐terminally truncated form of the enzyme that comprises the protease core (domains I and II) along with its insertion sequence, IS1, and N‐terminal leader sequence, NS. This 47‐kDa p94I‐II mini‐calpain was stable during purification. In the presence of Ca 2+ , p94I‐II cleaved itself within the NS and IS1 sequences. Mapping of the autolysis sites showed that NS and IS1 have the potential to be removed without damage to the protease core. Ca 2+ ‐dependent autolysis must be an intramolecular event because the inactive p94I‐II C129S mutant was not cleaved by incubation with wild‐type p94I‐II. In addition, the rate of autolysis of p94I‐II was independent of the concentration of the enzyme.