Purification and characterization of m‐calpain from the skeletal muscle of the amphibian Rana ridibunda

Abstract

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 similar to those of mammalian and avian skeletal muscle m-calpains. Ca2+ requirements for half and maximum activities are 400 microM and 1.5 mM, respectively. 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-ethylmaleimide (NEM). Its activity is enhanced by reduced thiols such as dithiothreitol (DTT), cysteine, and 2-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 presence of Ca2+, the cations Sr2+, Mn2+, and Ba2+ show a synergistic effect, while the cations of the other group strongly inhibit the calpain activity. The above data demonstrate that calpain from the skeletal muscle of the amphibian Rana ridibunda is a neutral, Ca(2+)-activated thiol protease and that it belongs to the class of m-calpains.

The Calpain-Calpastatin System and the Calcium Paradox in the Isolated Perfused Pigeon Heart

“…The millimolar form of calpain, isolated from the pigeon myocardium by ion exchange chromatography (Fig. 1), is activated by high Mn 2+ and Ba 2+ concentration (Table 1), similarly to m-calpain from other sources [29,32], but differently to others [28,33] is considered to be necessary for enzyme and inhibitor interaction [36][37]. Furthermore, a functionally active fraction of the endogenous inhibitor, known to be associated with membranes [38][39], could be released by structural changes of sarcolemmal phospholipids and carbohydrates, which are observed during Ca 2+ depletion [40][41].…”

“…Calpain activity was measured as a release of peptides from alkali denatured casein, as described by Ishiura et al [28], with slight modifications [29]. The standard assay mixture (0.5 ml) contained 50 mM imidazole-HCl buffer, pH 7.4, 10 mM 2-mercaptoethanol, 5 mg/ml alkali denatured casein, 5 mM CaCl 2 and 20-100 µl of each sample.…”