Journal of comparative physiology

, Volume 140, Issue 3, pp 199–207

Hemocyanins in spiders

XI. The quaternary structure ofCupiennius hemocyanin
  • Jürgen Markl
Article

Summary

  1. 1.

    The hemocyanin of the lycosid spiderCupiennius salei was separated into its hexameric (16 S) and dodecameric (24 S) components, and analyzed quantitatively. The reassociation and topologic distribution of the subunits were studied.

     
  2. 2.

    There are two types of subunits. One is monomeric (5 S) and consists of 5 electrophoretically distinct bands which are, however, immunologically identical. The other is a disulphide bridged dimer (7 S) which yields 2 components upon electrophoresis or immunoelectrophoresis. The significance of this heterogeneity was not studied. The dimer is antigenically deficient with respect to the monomer.

     
  3. 3.

    Whereas the 16 S hemocyanin is composed of six monomers, 24 S hemocyanin contains 10 monomers and 1 dimer.

     
  4. 4.

    Alkaline dissociation of 24 S hemocyanin (dodecamer) into subunits passes through a heptameric state (18 S) which is composed of 5 monomers and the dimer. In the electron microscope, 16 S-like units with a seventh polypeptide attached can be distinguished.

     
  5. 5.

    Treatment of 24 S or 18 S hemocyanin with reducing agents to cleave the disulphide bridge leads to a second type of hexamer (16 S′) which is electrophoretically distinct from native hexamers (16 S), and composed of 5 monomers and one constituent polypeptide chain of the dimer.

     
  6. 6.

    Upon dialysis of a monomer/dimer mixture against neutral buffer containing 40 mM calcium, 16 S, 18 S and 24 S particles are formed. The three reconstituted hemocyanins exhibit subunit compositions identical to the native hemocyanins and the 18 S component obtained during dissociation.

     
  7. 7.

    The results suggest that the 24 S hemocyanin particle consists of two identical hexamers linked by the disulphide bridge of a dimeric subunit shared by both hexamers.

     

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Bijlholt MMC, Bruggen EFJ van, Bonaventura J (1979) Dissociation and reassembly ofLimulus polyphemus hemocyanin. Eur J Biochem 95:399–405Google Scholar
  2. Czichos-Tiedt S (1975) Versuche zur Reiningung und Charakterisierung des Hämocyanins aus der SpinneCupiennius salei Keyserling. Thesis. Universität MüchenGoogle Scholar
  3. Herick JL, Smith AJ (1968) Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch Biochem Biophys 126:155–164Google Scholar
  4. Holde KE van, Bruggen EFJ van (1971) The hemocyanins. In: Timasheff SN, Fasman GD (eds) Subunits in biological systems. Dekker. New York, pp 1–53Google Scholar
  5. Hoylaerts M, Préaux G, Witters R, Lontie R (1979) Immunological heterogeneity of the subunits ofLimulus polyphemus haemocyanin. Arch Int Physiol Biochim 87:417–418Google Scholar
  6. Jeffrey PD (1979) Hemocyanin from the Australian freshwater crayfishCherax destructor. Electron microscopy of native and reassembled molecules. Biochemistry 12:2508–2513Google Scholar
  7. Jeffrey PD, Andrews PR (1980) Application of calculated sedimentation ratios in the specification of models for protein dimers, trimers, tetramers and pentamers. Biophys Chem 11:61–70Google Scholar
  8. Johnson ML, Yphantis DA (1978) Subunit association and heterogeneity ofLimulus polyphemus hemocyanin. Biochemistry 17:1448–1455Google Scholar
  9. Klarman A, Gottlieb J, Daniel E (1979) Quaternary structure and arrangement of subunits in hemocyanin from the scorpionLeirus quinquestriatus. Biochemistry 18:2239–2244Google Scholar
  10. Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T 4. Nature (London) 227:680–685Google Scholar
  11. Lamy J, Lamy J, Baglin M-C, Weill J (1977) Scorpion hemocyanin subunits: properties, dissociation, association. In: Bannister JV (ed) Structure and function of haemocyanin. Springer, Berlin Heidelberg New York, pp 37–49Google Scholar
  12. Lamy J, Lamy J, Weill J, Markl J, Schneider H-J, Linzen B (1979a) Hemocyanins in spiders. VII. Immunological comparison of the subunits ofEurypelma californicum hemocyanin. Hoppe-Seyler's Z Physiol Chem 360:889–895Google Scholar
  13. Lamy J, Lamy J, Weill J (1979b) Arthropod hemocyanin structure. Isolation of eight subunits in the scorpion. Arch Biochem Biophys 193:140–149Google Scholar
  14. Lamy J, Lamy J, Weill J, Bonaventura J, Bonaventura C, Brenowitz M (1979c) Purification and immunological identification of the dissociation products of nativeLimulus polyphemus andTachypleus tridentatus hemocyanins. Arch Biochem Biophys 196:324–339Google Scholar
  15. Lamy J, Lamy J, Sizaret P-Y, Weill J (1981) Quaternary structure ofAndroctonus australis hemocyanin. In: Lamy J (ed) Structure, active site, and function of invertebrate oxygen binding proteins. Dekker, New York, in pressGoogle Scholar
  16. Loewe R, Schmid R, Linzen B (1977) Subunit association and oxygen binding properties in spider hemocyanins. In: Bannister JV (ed) Structure and function of haemocyanin. Springer. Berlin Heidelberg New York, pp 50–54Google Scholar
  17. Lontie R, Witters R (1973) Haemocyanin. In: Eichhorn GL (ed) Inorganic biochemistry, vol 1. Elsevier, Amsterdam, pp 344–358Google Scholar
  18. Markl J, Kempter B (1981) Subunit heterogeneity in arthropod hemocyanins. In: Lamy J (ed) Structure, active site, and function of invertebrate oxygen binding proteins. Dekker, New York, in pressGoogle Scholar
  19. Markl J, Schmid R, Czichos-Tiedt S, Linze B (1976) Haemocyanins in spiders. III. Chemical and physical properties of the proteins inDugesiella andCupiennius blood. Hoppe-Seyler's Z Physiol Chem 357:1713–1725Google Scholar
  20. Markl J, Markl A, Schartau W, Linzen B (1979a) Subunit heterogeneity in arthropod hemocyanins. I. Chelicerata. J Comp Physiol 130:283–292Google Scholar
  21. Markl J, Hofer A, Bauer G, Markl A, Kempter B, Brenzinger M, Linzen B (1979b) Subunit heterogeneity in arthropod hemocyanins. II. Crustacea. J Comp Physiol 133:167–175Google Scholar
  22. Markl J, Strych W, Schartau W, Schneider H-J, Schöberl P, Linzen B (1979c) Hemocyanins in spiders. VI. Comparison of the polypeptide chains ofEurypelma californicum hemocyanin. Hoppe-Seyler's Z Physiol Chem 360:639–650Google Scholar
  23. Markl J, Savel A, Decker H, Linzen B (1980) Hemocyanins in spiders. IX. Homogeneity, subunit composition and the basic oligomeric structure ofEurypelma californicum hemocyanin. Hoppe-Seyler's Z Physiol Chem 361:649–660Google Scholar
  24. Markl J, Decker H, Savel A, Linzen B (1981) Homogeneity, subunit heterogeneity, and quaternary structure ofEurypelma hemocyanin. In: Lamy J (ed) Structure, active site, and function of invertebrate oxygen binding proteins Dekker, New York, in pressGoogle Scholar
  25. Murray AC, Jeffrey PD (1974) Hemocyanin from the Australian freshwater crayfish,Cherax destructor. Subunit heterogeneity. Biochemistry 13:3667–3671Google Scholar
  26. Pilz I, Goral K, Hoylaerts M, Witters R, Lontie R (1980) Studies by small-angle X-ray scattering of the quaternary structure of the 24 S-component of the haemocyanin ofAstacus leptodactylus in solution. Eur J Biochem 105:539–543Google Scholar
  27. Schaick EJM van, Schutter WG, Gaykema WPJ, Bruggen EFJ van, Hol WGJ (1981) The crystal structure of the hemocyanin hexamer fromPanulirus interruptus at 5 Å resolution. In: Lamy J (ed) Structure, active site and function of invertebrate oxygen binding proteins. Dekker. New York, in pressGoogle Scholar
  28. Schepman AMH (1975) X-ray diffraction and electron microscopy. Thesis, Rijksuniversiteit GroningenGoogle Scholar
  29. Schmid R (1976) Chemisch-physikalische Untersuchungen an Spinnenhänmocyaninen und ihren Untereinheiten. Thesis, Universität MünchenGoogle Scholar
  30. Schneider H-J, Markl J, Schartau W, Linzen B (1977) Hemocyanins in spiders. IV. Subunit heterogeneity ofEurypelma (Dugesiella) hemocyanin and separation of polypeptide chains. Hoppe-Seyler's Z Physiol Chem 358:1133–1141Google Scholar
  31. Schutter WG, Bruggen EFJ van, Bonaventura J, Bonaventura C, Sullivan B (1977) Structure, dissociation and reassembly ofLimulus polyphemus hemocyanin. In: Bannister JV (ed) Structure and function of haemocyanin. Springer, Berlin Heidelberg New York, pp 13–21Google Scholar
  32. Siezen RJ, Bruggen EFJ van (1974) Structure and properties of hemocyanins. XII. Electron microscopy of dissociation products ofHelix pomatia α-heamocyanin: Quaternary structure. J Mol Biol 90:77–89Google Scholar
  33. Sullivan B, Bonaventura J, Bonaventura C, Godette G (1976) Hemocyanin of the horseshoe crab,Limulus polyphemus. Structural differentiation of the isolated components. J Biol Chem 251:7644–7648Google Scholar
  34. Weeke B (1973) Crossed immunoelectrophoresis. Scand J Immunol 2:47–56Google Scholar
  35. Wibo M (1966) Recherches sur les hémocyanines des arthropodes: constantes de sédimentation et aspects morphologiques. Thesis, Université Catholique de LouvainGoogle Scholar

Copyright information

© Springer-Verlag 1980

Authors and Affiliations

  • Jürgen Markl
    • 1
  1. 1.Zoologisches Institut der Universität MünchenMünchen 2Federal Republic of Germany

Personalised recommendations