Size Dependence of Ghosts From Stored Erythrocytes on Calcium and Adenosine Triphosphate

Heterogeneity within ghosts prepared from stored erythrocytes was studied by multichannel particle analysis of size distributions of reconstituted ghosts. On restoration of isotonicity of hemolysates from fresh erythrocytes, ghosts were smaller than the corresponding red cells. After subsequent incubation for 15 min, they swelled to a final stable volume. The hemolysis of stored erythrocytes and restoration of isotonicity yielded ghosts similar to those from fresh cells, but, after subsequent incubation, a second population of small ghosts appeared. This population resulted from markedly diminished ghost swelling, that is attributed to decreased sodium permeability and increased membrane rigidity, causing resistance to changes in volume. Erythrocyte ATP depletion during storage of ACD blood was associated with an increase in the percentage of small ghosts. Similar ghosts were produced from fresh cells when CaCl₂ (1 x 10^-6M) was introduced into ghosts during hemolysis. The erythrocyte calcium content increased during prolonged storage from 11.3 ± 6.3 µg atoms/liter of cells (stored for 1-2 wk) to 22.0 ± 7.0 µg atoms/liter of cells, after 7-9 wk of storage. The introduction of nucleotides (2 x 10^-3M) into ghosts during hemolysis of stored cells prevented the appearance of small ghosts. The inhibitory effect was greater than predicted from stability constants of calcium nucleotide complexes, indicating the specific interaction of nucleotides with the membrane. The percentage of small ghosts from red cells stored for various times agreed with the percentage of nonviable cells derived from the literature. It is concluded that the decrease in ATP and the accumulation of calcium in stored erythrocytes induce conformational changes of membrane fibrous (contractile) proteins that results in decreases in membrane elasticity and permeability, which is in turn reflected by formation of small ghosts.