Summary
Two-hexameric (2×6) hemocyanins from the brachyuran crabsCancer pagurus andCallinectes sapidus, the freshwater crayfishAstacus leptodactylus and the lobsterHomarus americanus were isolated and dissociated into native subunits.
The subunits of each hemocyanin were analyzed by electrophoresis and immunology. Three immunologically distinct subunit types, which were termedα,β andγ, could be identified in each case. They were isolated preparatively, and interspecifically correlated. Subunitα is subdivided into several electrophoretically distinct isoforms which are immunologically closely related (Astacus) or identical (other species). InAstacus andCancer one of these isoforms was shown to dimerize and to act as inter-hexamer bridge. It represents a fourth subunit type termedα′. A fifth, ‘diffuse’ component, which in PAGE migrated at the position of a dimer, was identified in the crossed immunoelectrophoretic patterns as denatured hemocyanin.
A common feature of the four hemocyanins is the presence of 4 copies ofβ and 8 copies ofα/γ within the 2×6 particles. Theα:γ ratio is 4:4 in the two Astacidea and 6:2 in the two Brachyura.α′ exists in 2 copies inAstacus andCancer which means that a single dimerα′-α′ is present in a two-hexamer. This leaves 2 monomericα copies inAstacus and 4 inCancer.
Every subunit from the four species except ofAstacus α′-α′ was capable to form hexamers in reassembly experiments. If subunit combinations were tested, hetero-hexamers were formed preferentially. Two-hexamers were reconstituted only in the presence of all subunit types and the native subunit stoichiometry was required to obtain twohexamers in considerable yields. Factors limiting 2×6 reassembly are discussed.
Authentic 2×6 molecules ofAstacus, Homarus andCancer hemocyanin were immunolabeled with subunit-specific antibody fragments (Fab) or IgG molecules, and the resulting immuno complexes were studied in the electron microscope. A topological model of the quaternary structure of decapod 2×6 hemocyanins is derived, showing the position of each copy of the four subunit types. In this model, the inter-hexamer bridgeα′-α′ is surrounded by twoβ and twoγ subunits forming the central core of the dodecamer. Two additionalβ and two additionalγ subunits form the periphery together with oneα subunit occupying the peripheral short edges of each hexameric half structure. The model is discussed with respect to the current literature.
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Abbreviations
- PAGE :
-
polyacrylamide gel electrophoresis
- SDS :
-
sodium dodecyl sulfate
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This paper is decicated to Professor Dr. Bernt Linzen
Preliminary accounts of this work have been presented in the proceedings of a symposium at Tutzing 1985. Linzen B (ed) (1986) Invertebrate oxygen carriers. Springer, Berlin Heidelberg New York. This also includes: Stöcker et al. 1986; Markl et al. 1986) and in a review article (Markl 1986)
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Stöcker, W., Raeder, U., Bijlholt, M.M.C. et al. The quaternary structure of four crustacean two-hexameric hemocyanins: immunocorrelation, stoichiometry, reassembly and topology of individual subunits. J Comp Physiol B 158, 271–289 (1988). https://doi.org/10.1007/BF00695326
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DOI: https://doi.org/10.1007/BF00695326