Purification of μ-calpain by a novel affinity chromatography approach. New insights into the mechanism of the interaction of the protease with targets

Abstract

A calmodulin-binding motif is a common structural feature of a number of calpain substrates (1). Since a calmodulin-like domain has been identified in both subunits of the calpain molecule, the proposal was made that the domain(s) would recognize the calmodulin-binding motifs of the substrates prior to the enzymatic modification by calpain. In keeping with the proposal, a successful attempt to purify μ-calpain from human erythrocytes was made by using an affinity chromatography approach in which the synthetic peptide C49, containing the calmodulin-binding domain of the plasma membrane Ca2+- ATPase, was coupled to a Sepharose matrix. The calmodulin-like domain of the catalytic subunit of human μ-calpain expressed in Escherichia coli was also retained by the C49-Sepharose column. Both μ-calpain and the calmodulin- like domain interacted with C49 in a Ca2+-dependent way and were eluted from the column by Ca2+-chelating agents. The finding confirmed the interaction between the calmodulin-binding domain of the plasma membrane Ca2+-ATPase and the calmodulin-like domain of μ-calpain. Experiments were performed to establish whether irreversibly inactivated μ-calpain or its expressed C-terminal portion containing the calmodulin-like domain could activate the hydrolysis of ATP by the plasma membrane Ca2+ pump, in keeping with the evident ATPase stimulation of the same pump by calmodulin. A stimulation was observed, but it was much weaker than that induced by calmodulin.