Apparent Ca2+ dissociation constant of Ca2+ chelators incorporated non-disruptively into intact human red cells

The Journal of Physiology
Teresa Tiffert, Virgilio L Lew

Abstract

1. A recently developed method of measuring cytoplasmic Ca2+ buffering in intact red cells was applied to re-evaluate the intracellular Ca2+ binding properties of the Ca2+ chelators benz2 and BAPTA. Incorporation of the free chelators was accomplished by incubating the cells with the acetoxymethyl ester forms (benz2 AM or BAPTA AM). The divalent cation ionophore A23187 was used to induce equilibrium distribution of Ca2+ between cells and medium. 45Ca2+ was added stepwise to cell suspensions in the presence and absence of external BAPTA. To induce full Ca2+ equilibration, the plasma membrane Ca2+ pump was inhibited either by depleting the cells of ATP or by adding vanadate to the cell suspension. 2. The properties of the incorporated chelators were assessed from the difference in cytoplasmic Ca2+ buffering between chelator-free and chelator-loaded cells, over a wide range of intracellular ionized calcium concentrations ([Ca2+]i), from nanomolar to millimolar. 3. Under the experimental conditions applied, incorporation of benz2 and BAPTA into the red cells increased their Ca2+ buffering capacity by 300-600 mumol (340 g Hb)-1. The intracellular apparent Ca2+ dissociation constants (KDi) were about 500 nM for benz2 and 800 nM for B...Continue Reading

References

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Apr 1, 1997·The Journal of Physiology·T Tiffert, V L Lew

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Citations

Dec 25, 2007·Proceedings of the National Academy of Sciences of the United States of America·Eric SoupeneFrans A Kuypers
Nov 28, 2001·The Journal of General Physiology·F Gómez-Lagunas
Oct 3, 2002·The Journal of General Physiology·Franklin M MullinsJeffrey R Balser

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