Calpain I activates Ca2+ transport by the reconstituted erythrocyte Ca2+ pump.
Calpain I purified from human erythrocyte cytosol activates both the ATP hydrolytic activity and the ATP-dependent Ca2+ transport function of the Ca2(+)-translocating ATPase solubilized and purified from the plasma membrane of human erythrocytes and reconstituted into phosphatidylcholine vesicles. Following partial proteolysis of the enzyme by calpain I, both the initial rates of calcium ion uptake and ATP hydrolysis were increased to near maximal levels similar to those obtained upon addition of calmodulin. The proteolytic activation resulted in the loss of further stimulation of the rates of Ca2+ translocation or ATP hydrolysis by calmodulin as well as an increase of the affinity of the enzyme for calcium ion. However, the mechanistic Ca2+/ATP stoichiometric ratio was not affected by the proteolytic treatment of the reconstituted Ca2(+)-translocating ATPase. The proteolytic activation of the ATP hydrolytic activity of the reconstituted enzyme could be largely prevented by calmodulin. Different patterns of proteolysis were obtained in the absence or in the presence of calmodulin during calpain treatment: the 136-kDa enzyme was transformed mainly into a 124-kDa active ATPase fragment in the absence of calmodulin, whereas a 127-kDa active ATPase fragment was formed in the presence of calmodulin. This study shows that calpain I irreversibly activates the Ca2+ translocation function of the Ca2(+)-ATPase in reconstituted proteoliposomes by producing a calmodulin-independent active enzyme fragment, while calmodulin antagonizes this activating effect by protecting the calmodulin-binding domain against proteolytic cleavage by calpain.
Wang
K K
KK
Laboratory of Molecular Pharmacology, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
Roufogalis
B D
BD
Villalobo
A
A
D000408:Alamethicin / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification / Q000378:metabolism; D002147:Calmodulin / Q000494:pharmacology; D002154:Calpain / Q000494:pharmacology*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D017830:Octoxynol; D010713:Phosphatidylcholines; D010766:Phosphorylation; D011092:Polyethylene Glycols / Q000494:pharmacology; D011510:Proteolipids; D013816:Thermodynamics
D002147:Calmodulin; D008081:Liposomes; D010713:Phosphatidylcholines; D011510:Proteolipids; C020485:proteoliposomes; D000408:Alamethicin; D000001:Calcimycin; D011092:Polyethylene Glycols; D017830:Octoxynol; D002154:Calpain; D000252:Calcium-Transporting ATPases; D002118:Calcium