N. Sargianos et al., PURIFICATION AND CHARACTERIZATION OF M-CALPAIN FROM THE SKELETAL-MUSCLE OF THE AMPHIBIAN RANA-RIDIBUNDA, The Journal of experimental zoology, 269(2), 1994, pp. 95-105
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.