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Separation of cellular iron containing compounds by electrophoresis

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Abstract

High resolution separation of metalloproteins and other iron compounds based on native gel electrophoresis followed by59Fe autoradiography is described. Lysates of mouse spleen erythroid cells metabolically labeled with59Fe-transferrin were separated on 3–20% polyacrylamide gradient gels in the presence of Triton X100 and detected by autoradiography. In addition to ferritin and hemoglobin, several compounds characterized by their binding of iron under different conditions were described. Iron chelatable by desferrioxamine migrated in the region where several high-molecular weight compounds were detected by silver staining. The technique is nondissociative, allowing identification of iron compounds with the use of specific antibodies. Cellular iron transport and the action of iron chelators on specific cellular targets can be investigated in many small biological samples in parallel.

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References

  1. C. Hershko, Control of disease by selective iron depletion: a novel therapeutic strategy utilizing iron chelators,Baillieeres Clin. Haematol. 7, 965–1000 (1994).

    Article  CAS  Google Scholar 

  2. G. J. Kontoghiorghes and E. D. Weinberg, Iron: mammalian defence systems, mechanisms of disease, and chelation therapy approaches,Blood Rev. 9, 33–45 (1995).

    Article  PubMed  CAS  Google Scholar 

  3. T. A. Rouault, D. J. Haile, W. E. Downey, C. C. Philpott, C. Tang, F. Samaniego, J. Chin, I. Paul, J. B. Harford, and R. D. Klausner, An iron-sulfur cluster plays a novel regulatory role in the iron-responsive element binding protein,Biometals 5, 131–140 (1992).

    Article  PubMed  CAS  Google Scholar 

  4. J. V. Primosigh and E. D. Thomas, Studies on the partition of iron in bone marrow cells,J. Clin. Invest. 47, 1473–1482 (1968).

    Article  PubMed  CAS  Google Scholar 

  5. M. T. Nunez, I. Pinto, and J. Glass, Assay and characteristics of the iron binding moiety of reticulocyte endocytic vesicles,J. Membrane Biol. 107, 129–135 (1989).

    Article  CAS  Google Scholar 

  6. J. Weaver and S. Pollack, Low-Mr iron isolated from guinea pig reticulocytes as AMP-Fe and ATP-Fe complexes,Biochem. J. 261, 787–792 (1989).

    PubMed  CAS  Google Scholar 

  7. E. R. Giblett, C. G. Hickman, and O. Smithies, Serum transferrins,Nature 183, 1589–1590 (1959).

    Article  PubMed  CAS  Google Scholar 

  8. Y. Chen and J. Drysdale, Detection of iron binding proteins by a blotting technique,Anal. Biochem. 212, 47–49 (1993).

    Article  PubMed  CAS  Google Scholar 

  9. P. Owen, G. J. Kaczorowski, and H. R. Kaback, Resolution and identification of ironcontaining antigens in membrane vesicles from Escherichia coli,Biochemistry 19, 596–600 (1980).

    Article  PubMed  CAS  Google Scholar 

  10. B. Crowe and P. Owen, Immunochemical analysis of respiratory-chain components of Micrococcus luteus (lysodeikticus),J. Baderiol. 153, 498–505 (1983).

    CAS  Google Scholar 

  11. J. Mengaud and M. A. Horwitz, The major iron-containing protein of Legionella pneumophila is an aconitase homologous with the human iron-responsive elementbinding protein,J. Bacteriol. 175, 5666–5676 (1993).

    PubMed  CAS  Google Scholar 

  12. W. Scher, J. G. Holland, and C. Friend, Hemoglobin synthesis in murine virusinduced leukemic cells in vitro. I. Partial purification and identification of hemoglobins,Blood 37, 428–437 (1971).

    PubMed  CAS  Google Scholar 

  13. J. Margolis and K. G. Kenrick, Polyacrylamide gel electrophoresis in a continuous molecular sieve gradient,Anal. Biochem. 25, 347–362 (1968).

    Article  PubMed  CAS  Google Scholar 

  14. C. J. Eaves, G. Krystal, and A. C. Eaves, Erythropoietic cells,Biblthca Haemat. 48, 81–111 (1984).

    Google Scholar 

  15. D. Vyoral, A. Hradilek, and J. Neuwirt, Transferrin and iron distribution in subcellular fractions of K562 cells in the early stages of transferrin endocytosis,Biochim. Biophys. Acta 137, 148–154 (1992).

    Google Scholar 

  16. P. Ponka, A. Wilczynska, and H. M. Schulman, Iron utilization in rabbit reticulocytes. A study using succinylacetone as an inhibitor of heme synthesis,Biochim. Biophys. Acta 720, 96–105 (1982).

    Article  PubMed  CAS  Google Scholar 

  17. J. Sambrook, E. F. Fritsch, and T. Maniatis,Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989).

    Google Scholar 

  18. P. J. Wirth and A. Romano, Staining methods in gel electrophoresis, including the use of multiple detection methods,J. Chromatography A 698, 123–143 (1995).

    Article  CAS  Google Scholar 

  19. D. R. Richardson, P. Ponka, and D. Vyoral, Distribution of iron in reticulocytes after inhibition of heme synthesis with succinylacetone. Examination of cytoplasmic and mitochondrial intermediates involved in iron metabolism, Blood84, Suppl. 1 (1994).

  20. A. Jacobs, Low molecular weight intracellular iron transport compounds,Blood 50, 433–439 (1977).

    PubMed  CAS  Google Scholar 

  21. S. Pollack, T. Campana, and J. Weaver, Low molecular weight iron in guinea pig reticulocytes,Am. J. Hematol. 19, 75–84 (1985).

    Article  PubMed  CAS  Google Scholar 

  22. D. Hemmaplardh and E. Morgan, The mechanism of iron exchange between synthetic iron chelators and rabbit reticulocytes,Biochim. Biophys. Acta 373, 84–99 (1974).

    Article  PubMed  CAS  Google Scholar 

  23. M. J. Pippard, D. K. Johnson, and C. A. Finch, Hepatocyte iron kinetics in the rat explored with an iron chelator,Brit. J. Haematol. 52, 211–224 (1982).

    CAS  Google Scholar 

  24. O. Gabriel and D. M. Gersten, Staining for enzymatic activity after gel electrophoresis,Anal. Biochem. 203, 1–21 (1992).

    Article  PubMed  CAS  Google Scholar 

  25. U. Novak and L. Paradiso, Identification of proteins in DNA-protein complexes after blotting of EMSA gels,BioTechniques 19, 54–55 (1995).

    PubMed  CAS  Google Scholar 

  26. H. Schagger, W. A. Cramer, and G. von Jagow, Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis,Anal. Biochem. 217, 220–230 (1994).

    Article  PubMed  CAS  Google Scholar 

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Vyoral, D., Petrák, J. & Hradilek, A. Separation of cellular iron containing compounds by electrophoresis. Biol Trace Elem Res 61, 263–275 (1998). https://doi.org/10.1007/BF02789087

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  • DOI: https://doi.org/10.1007/BF02789087

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