Two distinct Ca2+ proteases (calpain I and calpain II) purified concurrently by the same method from rat kidney.

Two molecular species of calpain (Ca2+-dependent cysteine proteinases) were concurrently purified from rat kidney, both to homogeneity. Calpain I and calpain II having low and high Ca2+ requirements, respectively, were clearly separated on DEAE-cellulose chromatography at pH 7.5, and thereafter they were purified by separate but almost identical procedures which included (NH4)2SO4 fractionation and successive chromatographies on TSK-Gel G 3000 SWG, blue Sepharose CL-6B, and DEAE-Bio-Gel A. The purification folds and activity yields were 6170-fold and 17.8% for calpain I and 4160-fold and 11.9% for calpain II. Ca2+ concentrations for half-maximal activation were 2 microM for calpain I and 200 microM for calpain II. The specific activity of calpain II on casein as the substrate was more than twice higher than that of calpain I. Both enzymes are heterodimers, each composed of 80,000-Da and 25,000-Da subunits. The amino acid compositions of calpain I and calpain II are very similar but not identical. Calpain II is more acidic (pI 4.6) than calpain I (pI 5.3). This paper is the first to describe parallel isolation and characterization of low and high Ca2+-requiring proteases from one single nonmuscular tissue.