Ca²⁺- and Mg-ATP-dependent shape change of human erythrocyte ghosts and Triton shells

Human erythrocyte membranes (ghosts) prepared from fresh blood changed in shape from spherical to crenated, when suspended in 10^-7-10^-6 M Ca²⁺-EGTA buffers. Although the ghosts from long-stored ACD blood (10 weeks) were less sensitive to 10^-7-10^-6 M Ca²⁺, the ghosts obtained from this blood after it had been preincubated with adenine and inosine for 3 h at 37 °C were highly sensitive to Ca²⁺. When these highly sensitive ghosts were incubated in 10 mM Tris-Cl buffer (pH 7.4) or 1 mM MgCl₂ (pH 7.4) at 0 °C, they gradually lost Ca²⁺ sensitivity within 60 min, but they recovered Ca²⁺ sensitivity again after re-incubation with 2 mM Mg-ATP for 20 min at 37 °C followed by washing with 1 mM MgCl₂ (pH 7.4). The shape of these highly Ca²⁺-sensitive ghosts immediately changed from crenate to disc on addition of 1 mM Mg-ATP even at 6 °C in the presence of 10^-7-10^-6 M Ca²⁺. A similar shape change was also observed when ghosts treated with 0.5% Triton X-100 (Triton shells) were used. Triton shells from fresh blood ghosts or from long-stored blood ghosts which had been preincubated with 2 mM Mg-ATP for 20 min at 37 °C shrank immediately in the presence of 10^-6 M Ca²⁺ and then swelled on addition of 1 mM Mg-ATP. The specificity to ATP and the dependency on ATP concentration are in agreement with those of the ghost shape change at step 2 (Jinbu, Y. et al., Biochem biophys res commun 112 (1983) 384-390) [18]. These results suggest that cytoskeletal protein phosphorylation enhances sensitivity to Ca²⁺ and induces erythrocyte shape change in the presence of physiological concentrations of ATP and Ca²⁺.