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 [gamma-(32)P]ATP was studied on percoll-separated young and old human erythrocytes. After phosphorylation for 30 s 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 (50-90 s), the phosphoenzyme reached almost identical equilibrium levels in both membrane preparations. On the other hand, a single Ca(2+)-dependent radioactive band of about 150 kDa 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.
Romero
Pedro J
PJ
Laboratory of Membrane Physiology, Institute of Experimental Biology, Faculty of Sciences, UCV, Aptdo 47114, Caracas 1041A, Venezuela. romepe@hotmail.com
Salas
Valentina
V
Hernández
Concepción
C
D000252:Calcium-Transporting ATPases / Q000097:blood*; D002469:Cell Separation; D016922:Cellular Senescence / Q000502:physiology*; D002499:Centrifugation, Density Gradient; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans
D000252:Calcium-Transporting ATPases