ATP depletion of erythrocytes is associated with discocyte-echinocyte transformation and accumulation of calcium. This study was undertaken to evaluate whether this shape transformation results from interaction of Ca2+, Mg2+, and ATP with structural proteins of the membrane. ATP, Mg2+, or Ca2+ were incorporated into ghosts during hypotonic hemolysis followed by restoration of isotonicity with NaCl and incubation. Reconstituted ghosts were examined by electron microscope after osmium tetroxide fixation followed by platinum and carbon shadowing and in-phase contrast microscope. Four ghost configurations were observed: (1) flat, discoidal electron-penetrable ghosts were produced when ghosts were prepared without any additives, or with ATP alone, Mg2+ alone, or EDTA; (2) biconcave and cup-shaped less electron-penetrable ghosts predominated when equimolar concentrations of Mg2+ and ATP (0.5-2 mM) were introduced into ghosts during hemolysis; (3) spherical, spiculated, or markedly deformed electron-dense ghosts of smaller volume were the prevalent shape forms when Ca2+ alone was introduced during hemolysis; (4) small, smooth, electron-dense spheroids were present when both Ca2+ and Mg ATP (2 mM) were added during hemolysis. The above shapes were independent of ouabain, monovalent cations (NaCl, KCl, or choline chloride), transmembrane osmotic gradient, and ghost volume. The shape effects of Ca2+ and Mg ATP were prevented by preincubation of red cells with SH-blocking agent N-ethylmaleimide (4 mM) and by repeated ghost washing, which was associated with removal of water-soluble fibrous proteins from membranes. It is concluded that the interaction of Ca2+, Mg2+, and ATP with membrane structural proteins exerts a significant role in the control of shape of human erythrocytes.
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