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Calpastatin simultaneously binds four calpains with different kinetic constants
Author(s) -
Hanna Rachel A.,
Garcia-Diaz Beatriz E.,
Davies Peter L.
Publication year - 2007
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/j.febslet.2007.05.035
Subject(s) - calpastatin , calpain , proteolysis , chemistry , protease , proteases , kinetics , enzyme , surface plasmon resonance , calcium , biochemistry , biophysics , biology , quantum mechanics , physics , materials science , organic chemistry , nanoparticle , nanotechnology
Calpastatin is the endogenous, specific protein inhibitor of the calcium‐dependent protease, calpain. Using an active site knock‐out m‐calpain mutant we have studied the enzyme's calcium‐dependent binding to calpastatin by surface plasmon resonance without the complication of proteolysis. Calpastatin was capable of simultaneously binding four molecules of calpain. Its four inhibitory domains (CAST1, 2, 3, and 4) were individually expressed in Escherichia coli and the kinetics of their interaction with calpain was separately compared. Their K d values ranged from picomolar to nanomolar in the order CAST1 > 4 > 3 > 2. They have similar k on values but the k off values ranged over three orders of magnitude and can account for the differences in affinity.