Journal of Comparative Physiology B

, Volume 162, Issue 8, pp 665–680

Quaternary and subunit structure of Calliphora arylphorin as deduced from electron microscopy, electrophoresis, and sequence similarities with arthropod hemocyanin

  • Jürgen Markl
  • Thorsten Burmester
  • Heinz Decker
  • Anette Savel-Niemann
  • J. Robin Harris
  • Michaela Süling
  • Ulrike Naumann
  • Klaus Scheller
Article

DOI: 10.1007/BF00301616

Cite this article as:
Markl, J., Burmester, T., Decker, H. et al. J Comp Physiol B (1992) 162: 665. doi:10.1007/BF00301616

Summary

Arylphorin was purified from larvae of the blowfly Calliphora vicina and studied in its oligomeric form and after dissociation at pH 9.6 into native subunits. In accordance with earlier literature, it was electrophoretically shown to be a 500 kDa hexamer (1×6) consisting of 78 kDa polypeptides (= subunits). Electron micrographs of negatively stained hexamers show a characteristic curvilinear, equilateral triangle of 12 nm in diameter (top view) and a rectangle measuring 10×12 nm (side view). Alternatively, particles in the top view orientation exhibit a roughly circular shape 12 nm in diameter. Crossed immunoelectrophoresis revealed the presence of a major subunit type; the nature of a very minor and a third immunologically separated component remains unclear. A novel 2×6 arylphorin particle was detected and isolated. It comprises less than 10% of the total arylphorin material and shows a long, narrow interhexamer bridge in the electron microscope. An arylphorin dissociation intermediate identified as a trimer (1/2×6) was isolated; its possible quaternary structure is discussed on the basis of electron micrographs. The epitope of monoclonal antibody Ec-7 directed against tarantula (Eurypelma californicum) hemocyanin subunit d and also reactive to Calliphora arylphorin was traced to a highly conserved peptide of 27 amino acids localized in the center of the protein. The primary structure of Calliphora arylphorin as published in our preceding paper (Naumann and Scheller 1991) is compared in detail to the sequences of spider and spiny lobster hemocyanin. This revealed a basic framework of 103 strictly conserved amino acids. Isofunctional exchanges are proposed for another 76 positions. On the basis of these similarities, and the published three-dimensional model of spiny lobster hemocyanin, a detailed model of the quaternary structure of Calliphora arylphorin is presented. A second larval storage protein previously termed protein II was purified from Calliphora hemolymph. It was demonstrated to be a 500 kDa hexamer of 83 kDa subunits. In the electron microscope it shows a cubic view 9 nm in length with a large central hole and a rectangular view (9×10 nm) with a large central cavity. A morphologically very similar hemolymph protein was detected in Drosophila melanogaster larvae. From its structural appearance it is uncertain whether protein II belongs to the hemocyanin superfamily or not.

Key words

HemocyaninArylphorinLarval serum proteinsEvolutionArthropods

Abbreviations

FPLC

fast performance liquid chromatography

HPLC

high performance liquid chromatography

LSP

Larval serum protein

PAGE

Polyacrylamide gel electrophoresis

SDS

Sodium dodecyl sulphate

Tris

Tris-(hydroxymethyl)-aminomethane

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • Jürgen Markl
    • 1
  • Thorsten Burmester
    • 2
  • Heinz Decker
    • 3
  • Anette Savel-Niemann
    • 3
  • J. Robin Harris
    • 1
  • Michaela Süling
    • 1
  • Ulrike Naumann
    • 2
  • Klaus Scheller
    • 2
  1. 1.Institut für ZoologieUniversität MainzMainzFederal Republic of Germany
  2. 2.Theodor Boveri Institut (Biozentrum)Universität WürzburgWürzburgFederal Republic of Germany
  3. 3.Zoologisches InstitutUniversität MünchenMünchen 2Federal Republic of Germany