Evidence for heterodimeric nature of calpain molecules as studied by cross-linking modification.

Purified calpain I and calpain II from porcine erythrocytes and kidney were cross-linked with a bifunctional reagent, disuccinimidyl suberate, and the cross-linked products were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The major product had a molecular mass of 105 kDa, while the starting materials were resolved into 80-kDa and 30-kDa subunits. The cross-linking in the presence of 2 mM Ca2+ yielded several higher-molecular-weight species. The cross-linked products were shown to contain both the 80-kDa and 30-kDa proteins by means of immunoblotting with antibodies monospecific for the respective subunits, suggesting that the original calpain molecule existed in solution as an 80-kDa plus 30-kDa heterodimer and that Ca2+ induced closer association of these heterodimeric molecules.