The Journal of Membrane Biology

, Volume 40, Issue 2, pp 117–142 | Cite as

Equilibrium binding of calcium to fragmented human red cell membranes and its relation to calcium-mediated effects on cation permeability

  • Hartmut Porzig
  • Daniel Stoffel


We have measured Ca binding to fragmented human red cell membranes under equilibrium conditions in the presence of low concentrations of EGTA-buffered, ionized Ca. The ionic strength of the assay medium was maintained at 0.16. Two high affinity Ca binding sites were identified: Site I was pH-sensitive. Its apparent dissociation constant (K′) increased from 2×10−7 to 6×10−7m as the pH was shifted from 6.8 to 6.0. Between pH 8.5 and 6.8K′ remained constant. The capacity of the same site decreased between pH 8.5 and 6.8 from 0.16 to 0.04 nmoles/mg protein. Site II was insensitive to pH changes between 8.5 and 6.0. It had aK′ value of ∼3×10−6m and a capacity of ∼0.2 nmoles/mg protein. Mg and the local anesthetic propranolol (but not tetracaine) inhibited Ca binding to site I competitively and to site II noncompetitively. The properties of the high affinity Ca membrane binding sites are consistent with the assumption that site I corresponds to the site at which Ca initiates an increase in K permeability in resealed red cell ghosts. Site II is possibly involved in the Ca-mediated resealing of red cell ghosts after osmotic hemolysis. In the presence of MgATP, only a single saturable high affinity Ca binding site was observed (K′∼6×10−7 at pH 6.8). The capacity of this site (∼1.8 nmoles/mg protein) was almost 10 times higher than the combined capacities of sites I and II under control conditions. The results are discussed in the light of inevitable but severe shortcomings due to the evaluation of binding constants from nonlinear Scatchard plots by a curve-fitting procedure.


Propranolol Dissociation Constant Assay Medium Tetracaine Scatchard Plot 
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Copyright information

© Springer-Verlag New York Inc. 1978

Authors and Affiliations

  • Hartmut Porzig
    • 1
  • Daniel Stoffel
    • 1
  1. 1.Pharmakologisches Institut der Universität BernBernSwitzerland

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