DOMAIN-STRUCTURE OF CALPAIN - MAPPING THE BINDING-SITE FOR CALPASTATIN

The peptide EKLGERDDTIPPEYRELLEKKTGV was synthesized to mimic the cent ral consensus sequence of calpastatin, the specific, endogenous inhibi tor of the calpains (EC 3.4.22.17). The peptide competitively inhibits hydrolysis of casein by either micro- or milli-calpain but does not a ffect the activity of other proteases. This inhibitory peptide was pre ferentially cross-linked to milli-calpain in the presence of calcium u sing the heterobifunctional cross-linking reagent m-maleimidobenzoyl-N -hydroxysuccinimide ester. Cross-linking of the peptide was blocked by calpastatin. The site of crosslinking for the peptide within milli-ca lpain was localized using random chemical cleavage of the enzyme-pepti de complex at cysteine residues. Calpain fragments were identified as amino-terminal fragments through reactivity with a peptide-specific an tiserum or as non-amino-terminal fragments through incorporation of C- 14 from (CN)-C-14. Analysis of the control and cross-linked fragments, from experiments using both milli-calpain and micro-calpain, maps the chemical cross-linking site to cysteine-497 and localizes the binding site for the calpastatin-like peptide to this highly conserved region of domain III of calpains catalytic subunit.