Ca binding to the human red cell membrane: Characterization of membrane preparations and binding sites

Summary Inside out and right side out vesicles were used to study the sidedness of Ca binding to the human red cell membrane. It was shown that these vesicles exhibited only a limited permeability to Ca, enabling the independent characterization of Ca binding to the extracellular and cytoplasmic membrane surfaces. Ca binding was studied in 10 mM Tris HCl at pH 7.4, 22±2°C and was shown to be complete in under 5 min. Scatchard plots were made from Ca binding data obtained at free Ca concentrations in the range of 10^–6 to 10^–3M. Under these conditions inside out vesicles exhibit two independent binding sites for Ca with association constants of 1×10⁵ and 6×10³ M^–1, and right side out vesicles exhibit three independent binding sites with association constants of 2×10⁵, 1.4×10⁴ and 3×10²M^–1. Upon the addition of 0.1M KCl a third, high affinity site was found on inside out vesicles with an association constant of 3×10⁵, (in 0.1 M KCl). Ca binding to inside out vesicles increased nearly linearly with pH in the, range of pH 4 to pH 11, while binding to right side out vesicles remained practically unchanged in the range of pH 7 to pH 9. Progressive increase of the ionic strength of the medium by the addition of K, Mg or Tris decreased Ca binding to inside out vesicles as did the addition of ATP. Comparison of a series of cation competitors for Ca binding sites on inside out vesicles at 0.003 mM Ca showed that La was the most effective competitor of all while Cd was the most effective divalent cation competitor of those tested. Our findings suggest that the effects of low concentrations of Ca at the inner surface of the red cell membrane are mediated primarily through Ca binding to site 1 (and, possibly site 2) of inside out vesicles of which there are approximately 1.6×10⁵ per equivalent cell.