Origins of the difference in Ca2+ requirement for activation of μ- and m-calpain Available to Purchase

The μ- and m-calpains are closely related Ca²⁺-dependent cysteine proteases having different in vitro Ca²⁺ requirements (K_d), 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 K_d values must reside in the large subunits. As assayed here, these K_d 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 K_d. In a series of hybrid (μ/m) large-subunit calpains, the K_d values decreased progressively towards that of μ-calpain as the proportion of μ-type sequence increased from 0 to 90%. K_d 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 K_d 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.