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. 1977 Apr;18(1):3–22. doi: 10.1016/S0006-3495(77)85592-6

High and low affinity Ca2+ binding to the sarcoplasmic reticulum: use of a high-affinity fluorescent calcium indicator.

V C Chiu, D H Haynes
PMCID: PMC1473279  PMID: 15667

Abstract

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.

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Selected References

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  1. Carvalho A. P. Binding of cations by microsomes from rabbit skeletal muscle. J Cell Physiol. 1966 Feb;67(1):73–83. doi: 10.1002/jcp.1040670109. [DOI] [PubMed] [Google Scholar]
  2. Carvalho A. P., Leo B. Effects of ATP on the interaction of Ca++, Mg++, and K+ with fragmented sarcoplasmic reticulum isolated from rabbit skeletal muscle. J Gen Physiol. 1967 May;50(5):1327–1352. doi: 10.1085/jgp.50.5.1327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Caswell A. H., Pressman B. C. Kinetics of transport of divalent cations across sarcoplasmic reticulum vesicles induced by ionophores. Biochem Biophys Res Commun. 1972 Oct 6;49(1):292–298. doi: 10.1016/0006-291x(72)90043-5. [DOI] [PubMed] [Google Scholar]
  4. Chevallier J., Butow R. A. Calcium binding to the sarcoplasmic reticulum of rabbit skeletal muscle. Biochemistry. 1971 Jul 6;10(14):2733–2737. doi: 10.1021/bi00790a012. [DOI] [PubMed] [Google Scholar]
  5. Duggan P. F., Martonosi A. Sarcoplasmic reticulum. IX. The permeability of sarcoplasmic reticulum membranes. J Gen Physiol. 1970 Aug;56(2):147–167. doi: 10.1085/jgp.56.2.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fiehn W., Migala A. Calcium binding to sarcoplasmic membranes. Eur J Biochem. 1971 May 28;20(2):245–248. doi: 10.1111/j.1432-1033.1971.tb01387.x. [DOI] [PubMed] [Google Scholar]
  7. Haynes D. H. 1-Anilino-8-naphthalenesulfonate: a fluorescent indicator of ion binding electrostatic potential on the membrane surface. J Membr Biol. 1974 Jul 12;17(3):341–366. doi: 10.1007/BF01870191. [DOI] [PubMed] [Google Scholar]
  8. Ikemoto N., Bhatnager G. M., Gergely J. Fractionation of solubilized sarcoplasmic reticulum. Biochem Biophys Res Commun. 1971 Sep 17;44(6):1510–1517. doi: 10.1016/s0006-291x(71)80257-7. [DOI] [PubMed] [Google Scholar]
  9. Ikemoto N. The calcium binding sites involved in the regulation of the purified adenosine triphosphatase of the sarcoplasmic reticulum. J Biol Chem. 1974 Jan 25;249(2):649–651. [PubMed] [Google Scholar]
  10. Inesi G., Scarpa A. [Fast kinetics of adenosine triphosphate dependent Ca 2+ uptake by fragmented sarcoplasmic reticulum]. Biochemistry. 1972 Feb 1;11(3):356–359. doi: 10.1021/bi00753a008. [DOI] [PubMed] [Google Scholar]
  11. MARTONOSI A., FERETOS R. SARCOPLASMIC RETICULUM. I. THE UPTAKE OF CA++ BY SARCOPLASMIC RETICULUM FRAGMENTS. J Biol Chem. 1964 Feb;239:648–658. [PubMed] [Google Scholar]
  12. MacLennan D. H., Holland P. C. Calcium transport in sarcoplasmic reticulum. Annu Rev Biophys Bioeng. 1975;4(00):377–404. doi: 10.1146/annurev.bb.04.060175.002113. [DOI] [PubMed] [Google Scholar]
  13. Martonosi A., Halpin R. A. Sarcoplasmic reticulum. X. The protein composition of sarcoplasmic reticulum membranes. Arch Biochem Biophys. 1971 May;144(1):66–77. doi: 10.1016/0003-9861(71)90455-3. [DOI] [PubMed] [Google Scholar]
  14. Meissner G. ATP and Ca2+ binding by the Ca2+ pump protein of sarcoplasmic reticulum. Biochim Biophys Acta. 1973 Apr 16;298(4):906–926. doi: 10.1016/0005-2736(73)90395-7. [DOI] [PubMed] [Google Scholar]
  15. Meissner G., Conner G. E., Fleischer S. Isolation of sarcoplasmic reticulum by zonal centrifugation and purification of Ca 2+ -pump and Ca 2+ -binding proteins. Biochim Biophys Acta. 1973 Mar 16;298(2):246–269. doi: 10.1016/0005-2736(73)90355-6. [DOI] [PubMed] [Google Scholar]
  16. Meissner G., Fleischer S. Characterization of sarcoplasmic reticulum from skeletal muscle. Biochim Biophys Acta. 1971 Aug 13;241(2):356–378. doi: 10.1016/0005-2736(71)90036-8. [DOI] [PubMed] [Google Scholar]
  17. Meissner G., Fleischer S. Characterization of sarcoplasmic reticulum from skeletal muscle. Biochim Biophys Acta. 1971 Aug 13;241(2):356–378. doi: 10.1016/0005-2736(71)90036-8. [DOI] [PubMed] [Google Scholar]
  18. Meissner G. Isolation and characterization of two types of sarcoplasmic reticulum vesicles. Biochim Biophys Acta. 1975 Apr 21;389(1):51–68. doi: 10.1016/0005-2736(75)90385-5. [DOI] [PubMed] [Google Scholar]
  19. Ostwald T. J., MacLennan D. H. Isolation of a high affinity calcium-binding protein from sarcoplasmic reticulum. J Biol Chem. 1974 Feb 10;249(3):974–979. [PubMed] [Google Scholar]
  20. Vale M. G., Carvalho A. P. Utilization of X-537A to distinguish between intravesicular and membrane-bound calcium ions in sarcoplasmic reticulum. Biochim Biophys Acta. 1975 Dec 1;413(2):202–212. doi: 10.1016/0005-2736(75)90104-2. [DOI] [PubMed] [Google Scholar]
  21. WILBRANDT W., ROSENBERG T. The concept of carrier transport and its corollaries in pharmacology. Pharmacol Rev. 1961 Jun;13:109–183. [PubMed] [Google Scholar]
  22. Worsfold M., Peter J. B. Kinetics of calcium transport by fragmented sarcoplasmic reticulum. J Biol Chem. 1970 Nov 10;245(21):5545–5552. [PubMed] [Google Scholar]
  23. Yamada S., Tonomura Y. Reaction mechanism of the Ca 2+ -dependent ATPase of sarcoplasmic reticulum from skeletal muscle. VII. Recognition and release of Ca 2+ ions. J Biochem. 1972 Aug;72(2):417–425. doi: 10.1093/oxfordjournals.jbchem.a129917. [DOI] [PubMed] [Google Scholar]
  24. Yamamoto T., Tonomura Y. Reaction mechanism of the Ca++ -dependent ATPase of sarcoplasmic reticulum from skeletal muscle. I. Kinetic studies. J Biochem. 1967 Nov;62(5):558–575. doi: 10.1093/oxfordjournals.jbchem.a128706. [DOI] [PubMed] [Google Scholar]

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