A study of Ca2+-ATPase in red cell aging

Abstract

Calcium homeostasis is important for cell functions. Elevation of intracellular calcium is commonly observed in aged erythrocytes. This change in calcium level may be due to alternation of Ca2+-ATPase activity of erythrocyte. In this study, we looked into the changes of regulation of Ca2+-ATPase during aging processes of erythocyte. Affinity of Ca2+-ATPase towards ATP was found increased significantly with age while there was no significant difference between the maximal activities of the enzyme. In addition, response of the enzyme towards protein inhibitor, i.e. PMCAI was found largely increased whereas there was a decrease in affinity towards oleic acid in aged erythrocytes. The response and affinity of the enzyme towards oleic acid in aged erythrocytes. The response and affinity of the enzyme towards calmodulin was found unchanged during aging. It was also found that activation effect of controlled proteolysis on Ca2+-ATPase in aged erythrocyte was higher. In erythrocyte upon storage, similar trend of changes of regulation of Ca2+-ATPase was observed. The notion that Ca2+-ATPase has undergone changes during aging in properties and structure was further illustrated in the immunoblotting study. More 40kDa fragment was found in aged erythrocyte. The pattern of proteolysis was observed to be different in out-dated bank blood too. From out findings, we propose the mechanism of erythrocyte aging as follows. During erythrocyte aging, membrance barrier against calcium is gradually loosened. The response of Ca2+-ATPase towards PMCAI is much increased while towards calmodulin does not change much. This leads to a decrease capacity of the enzyme to restore intracellular calcium to original level. In addition, lipid content of cell also decreases during aging, thus availability of acid phospholipids to the enzyme may decrease. The elevated level of calcium also increases the activity of calpain and other proteases that act on various other proteins including Ca2+-ATPase. The ATP-binding site of the enzyme becomes more exposed with gradual increase in susceptible sites for further proteolysis. Finally, extensive proteolysis causes the permanent inactivation of the Ca2+-ATPase and the lost of calcium pump capacity. All factors of above may act together that account for the significant decrease in Ca2+-ATPase activity and the increased in cell calcium observed in aged and outdated storage erythrocyte.