PURIFICATION AND CHARACTERIZATION OF M-CALPAIN FROM THE SKELETAL-MUSCLE OF THE AMPHIBIAN RANA-RIDIBUNDA

Calpain was purified to apparent homogeneity from the skeletal muscle of the amphibian Rana ridibunda. It is composed of two subunits of 78 and 28 kDa, respectively. The enzyme exhibits kinetic properties simil ar to those of mammalian and avian skeletal muscle m-calpains. Ca2+ re quirements for half and maximum activities are 400 mu M and 1.5 mM, re spectively. It is strongly inhibited by thiol protease inhibitors such as leupeptin, E-64, and antipain and by alkylating thiol group agents such as iodoacetic acid (IAA), iodoacetamide (IAM), and N-ethylmaleim ide (NEM). Its activity is enhanced by reduced thiols such as dithioth reitol (DTT), cysteine, and a-mercaptoethanol. The enzyme is stable in the absence of Ca2+ at 55 degrees C, it displays maximum activity at 25 degrees C, and it shows a broad pH optimum between 6.5 and 7.8. In the absence of Ca2+, various divalent cations such as Sr2+, Mn2+, and Ba2+ strongly activate, while other divalent cations such as Ni2+, Co2 +, Cd2+, Zn2+, and Cu2+ have no effect on its activity. In the presenc e of Ca2+, the cations Sr2+, Mn2+, and Ba2+ show a synergistic effect, while the cations of the other group strongly inhibit the calpain act ivity. The above data demonstrate that calpain from the skeletal muscl e of the amphibian Rana ridibunda is a neutral, Ca2+-activated thiol p rotease and that it belongs to the class of m-calpains. (C) 1994 Wiley -Liss, Inc.