CALCIUM PUMP PHOSPHOENZYME FROM YOUNG AND OLD HUMAN RED CELLS

Authors

  • Pedro J. Romero,

    Corresponding author
    1. Laboratory of Membrane Physiology, Institute of Experimental Biology, Faculty of Sciences, UCV, Aptdo. 47114, Caracas, 1041A, Venezuela
    Search for more papers by this author
  • Valentina Salas,

    1. Laboratory of Membrane Physiology, Institute of Experimental Biology, Faculty of Sciences, UCV, Aptdo. 47114, Caracas, 1041A, Venezuela
    Search for more papers by this author
  • Concepción Hernández

    1. Laboratory of Membrane Physiology, Institute of Experimental Biology, Faculty of Sciences, UCV, Aptdo. 47114, Caracas, 1041A, Venezuela
    Search for more papers by this author

To whom correspondence should be addressed: Tel.: +58(212) 751 0766 ext. 222; Fax: +58(212) 753 7087; E-mail: romepe@hotmail.com

Abstract

An increase in intracellular Ca2+ occurs during ageing of human erythrocytes in vivo. The aged cells show a reduced capacity for active Ca2+ extrusion. Such a defect may arise from pump proteolysis, due to calpain activation by the raised intracellular Ca2+. To test this possibility, Ca2+ pump phosphorylation by [γ-32P]ATP was studied on percoll-separated young and old human erythrocytes. After phosphorylation for 30s with Ca2+, the amount of phosphoenzyme produced by the young cell membranes was 50% that of the old cells. With Ca2+ plus La3+, 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 Ca2+-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 Ca2+ pump units during cell ageing, thus indicating that the reduced Ca2+ pumping activity of aged cells does not arise from pump proteolysis.

Ancillary