The μ- and m-calpains are closely related Ca2+-dependent cysteine proteases having different in vitro Ca2+ requirements (Kd), of approx. 25 and 325μM respectively. The two isoforms are heterodimers of slightly different large (80kDa) subunits and an identical small (28kDa) subunit, so that the difference in Kd values must reside in the large subunits. As assayed here, these Kd values relate to the Ca2+ required for the first phase of calpain activation and do not reflect the lower Ca2+ then required by fully activated calpain. On the basis of sequence comparison and the X-ray structure of m-calpain, many m-type residues in the C-terminal EF-hand-containing domain IV were converted into the corresponding μ-type residues, but these mutations did not produce the expected decrease in Kd. In a series of hybrid (μ/m) large-subunit calpains, the Kd values decreased progressively towards that of μ-calpain as the proportion of μ-type sequence increased from 0 to 90%. Kd values cannot therefore be ascribed to one or a few specific intramolecular interactions, but reflect the global response of the whole molecule to Ca2+ binding. Nonetheless, 25% of the difference in Kd values between μ- and m-calpain can be ascribed to the N-terminal peptide of the large subunit, whereas the C-terminal EF-hand-containing domain IV accounts for 65% of the difference.

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