Purification of mu-calpain by a novel affinity chromatography approach. New insights into the mechanism of the interaction of the protease with targets.
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 mu-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 Ca(2+)-ATPase, was coupled to a Sepharose matrix. The calmodulin-like domain of the catalytic subunit of human mu-calpain expressed in Escherichia coli was also retained by the C49-Sepharose column. Both mu-calpain and the calmodulin-like domain interacted with C49 in a Ca(2+)-dependent way and were eluted from the column by Ca(2+)-chelating agents. The finding confirmed the interaction between the calmodulin-binding domain of the plasma membrane Ca(2+)-ATPase and the calmodulin-like domain of mu-calpain. Experiments were performed to establish whether irreversibly inactivated mu-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 evident ATPase stimulation of the same pump by calmodulin. A stimulation was observed, but it was much weaker than that induced by calmodulin.
Molinari
M
M
Institute of Biochemistry, Swiss Federal Institute of Technology, Zurich.
Maki
M
M
Carafoli
E
E
D000595:Amino Acid Sequence; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002154:Calpain / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002384:Catalysis; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data
D002147:Calmodulin; D002154:Calpain; D000252:Calcium-Transporting ATPases