Decreased Ca pump ATPase activity associated with increased density in human red blood cells

Red blood cells (rbcs) from five different normal humans were separated according to density using a simple procedure. The procedure involved centrifugation for 30 minutes in small glass tubes in the absence of any density gradient medium. This produced a column of rbcs arranged according to their density. Samples of the top 8% of the columns and bottom 8% of the columns were removed from the tubes with a micropipet. From each donor, samples of the least and most dense cells, respectively, were pooled from multiple tubes for each donor and designated "top" and "bottom" cells. These top and bottom cells were compared with unselected (total) cells from the same subjects, respectively. Top cells were larger and bottom cells were smaller than total cells. ATPase activities were operationally defined and measured in saponin lysates of these rbcs. The Ca pump ATPase (both in the calmodulin-activated and calmodulin-independent states [achieved by addition of compound 48/80]) of the top cells exhibited greater activity, and the Ca pump ATPase of bottom cells exhibited lower activity than total cells. It was suggested that loss of Ca pump ATPase activity is associated with rbc aging and may be a determinant of rbc life span. A mechanism for the loss of Ca pump ATPase activity was suggested. This speculative mechanism is based upon selective proteolysis of the Ca pump ATPase by the Ca-activated protease, calpain.