Calcium binding by human erythrocyte membranes

Abstract

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.