CALCIUM PUMP PHOSPHOENZYME FROM YOUNG AND OLD HUMAN RED CELLS

An increase in intracellular Ca 2+ occurs during ageing of human erythrocytes in vivo . The aged cells show a reduced capacity for active Ca 2+ extrusion. Such a defect may arise from pump proteolysis, due to calpain activation by the raised intracellular Ca 2+ . To test this possibility, Ca 2+ pump phosphorylation by [γ‐ 32 P]ATP was studied on percoll‐separated young and old human erythrocytes. After phosphorylation for 30s with Ca 2+ , the amount of phosphoenzyme produced by the young cell membranes was 50% that of the old cells. With Ca 2+ plus La 3+ , in contrast, the phosphoenzyme level was nearly the same in both preparations. After a prolonged phosphorylation period (50–90s), the phosphoenzyme reached almost identical equilibrium levels in both membrane preparations. On the other hand, a single Ca 2+ ‐dependent radioactive band of about 150kDa was apparent in both preparations after acidic electrophoresis. Likewise, Western blotting using 5F10 monoclonal antibody also detected a single band of similar molecular weight. These results demonstrate that there is no alteration in either molecular mass or number of active Ca 2+ pump units during cell ageing, thus indicating that the reduced Ca 2+ pumping activity of aged cells does not arise from pump proteolysis.