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

The μ - and m-calpains are closely related Ca 2+ -dependent cysteine proteases having different in vitro Ca 2+ requirements ( K d ), of approx. 25 and 325 μ M 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 Ca 2+ required for the first phase of calpain activation and do not reflect the lower Ca 2+ 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 Ca 2+ 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.