THE PLASMA-MEMBRANE CALCIUM-PUMP IS THE PREFERRED CALPAIN SUBSTRATE WITHIN THE ERYTHROCYTE

The activation of calpain in normal human erythrocytes incubated in th e presence of Ca2+ and the Ca2+ ionophore A23187 led to the decline of the Ca2+-dependent ATPase activity of the cells. Preloading of the er ythrocyte with an anticalpain antibody prevented the decline. The pump was also inactivated by applied to isolated erythrocyte plasma membra nes. The decline of the pump activity corresponded to the degradation of the pump protein and was inversely correlated to the amount of the natural inhibitor of calpain, calpastatin, present in the cells. In er ythrocytes containing only 50% of the normal level the degradation sta rted at a concentration of Ca2+ significantly lower than in normal cel ls. A comparison of the concentrations of Ca2+ required for the degrad ation of a number of erythrocyte membrane proteins showed that the Ca2 + pump and band 3 were the most sensitive. All other membrane proteins tested were attacked at higher levels of intracellular Ca2+. Thus, th e degradation of the Ca2+ pump protein may be a simple and sensitive m eans to monitor calpain activation in vivo. Furthermore, the results h ave shown that the calpastatin level correlated directly with the amou nt of activable calpain and with the concentration of Ca2+ required to trigger the activation process.