m-Calpain subunits remain associated in the presence of calcium

The hypothesis that calpain subunits dissociate in the presence of Ca²⁺ has been tested by methods which avoid interference by Ca²⁺-induced aggregation and large subunit autolysis. Inactive Cys¹⁰⁵Ser-m-calpain, bound either to Ni-NTA-agarose or to immobilized casein, after incubation with Ca²⁺, could be recovered in high yield as a heterodimer. Natural bovine m-calpain, after irreversible inhibition with Z-LLY-CHN₂, also bound to immobilized casein and was eluted as a heterodimer. The Ca²⁺ requirements of calpain containing a small subunit with EF-hand mutations were higher, both before and after autolysis, than those of wild-type calpain. In mixtures of wild-type and mutant enzymes, subunit exchange did not occur in the presence of Ca²⁺. The results demonstrate that the subunits in both natural and recombinant m-calpain, in the given experimental conditions, remain associated in the presence of Ca²⁺ both before and after autolysis. Copyright (C) 1998 Federation of European Biochemical Societies.