1. The characteristics of Ca2+ binding to haemoglobin-free human erythrocyte membranes were investigated by using 45Ca and centrifugation partition of ‘ghosts’ from their external incubation medium. Equilibrium of ‘ghosts’ with external Ca2+ required less than 15min. 2. The binding did not vary with temperature in the range 0–37°C. 3. At pH7.4 ‘ghosts’ bound a maximum of 283μmol of Ca2+/g of ‘ghost’ protein, equivalent to 6.85×107 Ca2+ ions per cell. 4. Increasing the ionic strength from 0.01 to 0.46 diminished Ca2+ binding, as did ATP in concentrations ranging from 0 to 15mm in the incubation medium. 5. An increase of the pH from 3.0 to 9.3 caused a marked increase in the amount of Ca2+ bound. 6. Extraction of 45Ca-labelled ‘ghosts’ with chloroform–methanol showed that the distribution of Ca2+ was: 79% protein-bound, 16% lipid-bound, 5% in the aqueous phase, presumably non-bound. Most of the lipid-bound Ca2+ (about 80%) was associated with a phospholipid fraction containing phosphatidylserine, phosphoinositides and phosphatidylethanolamine, giving a molar Ca2+: phosphorus ratio of about 1:2.

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