Purification and characterization of calcium-activated neutral proteinase from calf thymus

A calcium-activated neutral proteinase (CANP) was prepared from the soluble fraction of calf thymus and purified to electrophoretical homogeneity. The purified proteinase was shown to consist of two subunits, each of 80 kDa, in contrast to rabbit skeletal muscle calpain which was shown to consist of 80 kDa and 30 kDa subunits. The calcium requirement for 50% activation was 0.55 mM, indicating that this enzyme belongs to the low calcium sensitive type CANP, named mCANP or Calpain II. Optimal conditions of enzyme activity towards 0.8% casein as substrate are pH 7.5, a calcium concentration of 1.5 mM, the presence of an SH-reducing agent and an incubation temperature of 30 degrees C. The enzyme is inhibited by Zn2+, p-chloromercuribenzoate and N-ethylmaleimide.