Advertisement

Dye-labelling as a means to study ternary protein complexes by analytical ultracentrifugation: the band 3/ankyrin/aldolase complex from erythrocyte membranes

  • F. Dölle
  • D. Schubert
Biological Systems
Part of the Progress in Colloid & Polymer Science book series (PROGCOLLOID, volume 107)

Abstract

Demonstration of the formation of ternary complexes of proteins, in the presence of all constituents and binary complexes, and analysis of their stoichiometries is a difficult task. For the band 3/ankyrin/aldolase complex from erythrocyte membranes in detergent solutions, we have solved this problem by sedimentation equilibrium analysis in the analytical ultracentrifuge. Labelling of the ankyrin with a dye (fluorescein isothiocyanate) and measuring the absorbance versus radius profiles at a wavelength where only the dye absorbs allowed us to focus on the ankyrin-containing complexes. So, the different oligomers of uncomplexed band 3, the uncomplexed aldolase and the various binary complexes of band 3 and aldolase could be disregarded in the analysis. The ternary band 3/ankyrin/aldolase complex could be unambiguously detected. Its stoichiometry (band 3/ankyrin/aldolase tetramer) was found to vary between 4:1:1 and 4:1:4, depending on the abundancy of the enzyme.

Key words

Sedimentation equilibrium ternary protein complexes dye labelling band 3/ankyrin/aldolase complex 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Osborne JC, Powell GM, Brewer HB (1980) Biochim Biophys Acta 619:559–571PubMedGoogle Scholar
  2. 2.
    Mulzer K, Kampmann L, Petrasch P, Schubert D (1990) Colloid Polym Sci 268:60–64CrossRefGoogle Scholar
  3. 3.
    von Rückmann B, Huber E, Schuck P, Schubert D (1995) Prog Colloid Polym Sci 99:69–73CrossRefGoogle Scholar
  4. 4.
    Gilligan DM, Bennett V (1993) Semin Hematol 30:74–83PubMedGoogle Scholar
  5. 5.
    Lux SE, Palek J (1995) In: Handin RJ, Lux SE, Stossel TP (eds) Blood: Principles and Practice of Hematology. Lippincott, Philadelphia, pp 1701–1818Google Scholar
  6. 6.
    Low PS (1986) Biochim Biophys Acta 864:145–167PubMedGoogle Scholar
  7. 7.
    Jenkins JD, Madden DP, Steck TL (1984) J Biol Chem 259:9374–9378PubMedGoogle Scholar
  8. 8.
    Pappert G, Schubert D (1983) Biochim Biophys Acta 730:32–40PubMedCrossRefGoogle Scholar
  9. 9.
    Pinder JC, Smith KS, Pekrun A, Gratzer WB (1989) Biochem J 264:423–428PubMedGoogle Scholar
  10. 10.
    Mulzer K, Petrasch P, Kampmann L, Schubert D (1989) Stud Biophys 134:17–22Google Scholar
  11. 11.
    Schubert D, Schuck P (1991) Prog Colloid Polym Sci 86:12–22Google Scholar
  12. 12.
    Schuck P, Legrum B, Passow H, Schubert D (1995) Eur J Biochem 230:806–812PubMedCrossRefGoogle Scholar
  13. 13.
    Schuck P (1994) Prog Colloid Polym Sci 94:1–13Google Scholar
  14. 14.
    von Rückmann B, Jöns T, Dölle F, Drenckhahn D, Schubert D (1997) Biochim Biophys Acta 1325:226–234CrossRefGoogle Scholar
  15. 15.
    Huber E, Bäumert HG, Spatz-Kümbel G, Schubert D (1996) Eur J Biochem 242:293–300PubMedCrossRefGoogle Scholar
  16. 16.
    Schubert D, Boss K, Dorst H-J, Flossdorf J, Pappert G (1983) FEBS Lett 163:81–84PubMedCrossRefGoogle Scholar
  17. 17.
    Schubert D, Huber E, Lindenthal S, Mulzer K, Schuck P (1992) Prog Cell Res 2:209–217Google Scholar
  18. 18.
    Dorst H-J, Schubert D (1979) Hoppe-Seyler's Z Physiol Chem 360:1605–1618PubMedGoogle Scholar
  19. 19.
    Schuck P, Schubert D (1991) FEBS Lett 293:81–84PubMedCrossRefGoogle Scholar

Copyright information

© Dr. Dietrich Steinkopff Verlag GmbH & Co. KG 1997

Authors and Affiliations

  • F. Dölle
    • 1
  • D. Schubert
    • 1
  1. 1.Institut für Biophysikder Johann Wolfgang Goethe-UniversitätFrankfurtGermany

Personalised recommendations