Calpain subunits remain associated during catalysis.

The Ca2+ dependent cysteine proteases, calpains are heterodimers containing a large (ca. 80 kDa) catalytic subunit and a 25-30 kDa small subunit. Whether calpains remain dimers while catalyzing hydrolysis of protein substrates has been controversial. Now by doing subunit co-immunoprecipitation, we provide direct evidence to resolve this argument. In the presence of Ca2+ concentrations which permit catalytic activity, both subunits of either m- or mu-calpain are co-immunoprecipitated by monoclonal antibodies directed against a single subunit. Furthermore, both subunits can be co-immunoprecipitated during calpain-catalyzed proteolysis of the substrate casein. These results indicate that both major calpain isozymes maintain their heterodimeric form during the catalytic cycle. Thus, small subunit might have a direct role in regulating the physiologic function of either major calpain isozyme.