title: 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.
authors.affiliation: Department of Zoology, School of Sciences, Aristotle University of Thessaloniki, Greece.
authors.forename: N
authors.initials: N
authors.lastname: Sargianos
authors.forename: C
authors.initials: C
authors.lastname: Gaitanaki
authors.forename: I
authors.initials: I
authors.lastname: Beis
mesh_terms: D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D004795:Enzyme Stability; D007700:Kinetics; D009132:Muscles / Q000737:chemistry*; D011895:Rana ridibunda; D013696:Temperature
chemical_list: D002413:Cations, Divalent; D002154:Calpain