High and low affinity Ca2+ binding to the sarcoplasmic reticulum: use of a high-affinity fluorescent calcium indicator.
The fluorescent calcium indicator, calcein, has been used as a high-affinity indicator of Ca2+ in the aqueous phase at physiological pH in the study of high-affinity calcium binding to sarcoplasmic reticulum (SR). The binding constant of the indicator at physiological pH is 10(3)-10(4) M-1 and increases with increasing pH. The binding mechanism of the indicator with Ca2+ and Mg2+ is described. Application of calcein as an aqueous indicator of Ca2+ binding to the SR at room temperature has revealed two classes of binding sites: one with high capacity and low affinity (ca. 820 nmol/mg protein, Kd = 1.9 mM), and another with low capacity and higher affinity (ca. 35 nmol/mg protein, Kd = 17.5 micronM). The divalent cation specificity of the low-affinity  site is low and Ca2+/Mg2+ specificity of the high-affinity site is high. Quantitative studies of the bindings indicate that the high-affinity site residues in the Ca2+ ATPase (carrier) protein and represents complexation in the active site of the carrier and that the low-affinity site residues in the nonspecific acidic binding proteins. The contribution of Donnan equilibrium effects to the measured binding is shown to be insignificant. Stopped flow kinetic studies of Ca2+ passive binding with calcein and arsenazo III dyes have demonstrated that the binding to high-affinity site is very fast and that the overall binding reaction with the low-affinity site is slow, with a time course of about 4 s. Our analysis has shown that at least part of the low-affinity acidic proteins are within the SR matrix and that Ca2+ can reach them only by transversing the membrane via the Ca2+ carrier (Ca2+ ATPase). A model of the SR is proposed that accounts for several functional properties of the organelle in terms of its known protein composition and topological organization.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism*; D002474:Cell-Free System; D005456:Fluorescent Dyes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008954:Models, Biological; D009124:Muscle Proteins / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D014966:Xanthenes
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D008024:Ligands; D008565:Membrane Proteins; D009124:Muscle Proteins; D014966:Xanthenes; D000251:Adenosine Triphosphatases; D008274:Magnesium; D002118:Calcium