Subunit composition of a high molecular weight oligomer: Limulus polyphemus hemocyanin

https://doi.org/10.1016/0003-9861(81)90242-3Get rights and content

Abstract

The hemocyanin of Limulus polyphemus is a 48-subunit aggregate. This 3.3 × 106-dalton oligomer is composed of structurally and functionally heterogeneous subunits. Using polyacrylamide electrophoresis J. Markl, A. Markl, W. Schartau, and B. Linzen (J. Comp. Physiol. Ser. B130,283–292, 1979) observed 12 bands; while using immunoelectrophoresis, M. Hoylaerts, G. Preaux, R. Witters, and R. Lontie (Arch. Int. Physiol. Biochem.87, 417–418, 1979) and J. Lamy, J. Lamy, J. Weill, J. Bonaventura, C. Bonaventura, and M. Brenowitz. (Arch. Biochem. Biophys.196, 324–339, 1979) observed 8 subunits. To proceed with an analysis of subunit roles in assembly it is first necessary to determine the number of distinct subunits. Refinement of the chromatographic separation procedures has led to the isolation of 8 immunologically distinct subunits as well as additional charge isomers which cannot be distinguished immunologically. Alkaline electrophoresis revealed 15 bands and isoelectric focusing up to 17. On the basis of extensive control experiments, including composit acrylamide-agarose immunoelectrophoresis and checks for conformational isomers, aggregation, proteolysis, and other types of degradation, we conclude that the electrophoretic heterogeneity of immunologically identical subunits is not artifactual. We have extended the nomenclature used by Lamy et al. (1979) to include the electrophoretic heterogeneity by using primes (′) to denote electrophoretically distinguishable subunits which are immunologically identical. A number of patterns have become apparent by correlating the results obtained by the different techniques. For example, immunologically pure subunit II, which shows 3 bands on alkaline electrophoresis, is in fact a mixture of electrophoretically distinct subunits II, II′, II″. Except for subunits II, II′, and II″ immunoelectrophoretically identical subunits are typically homogeneous on sodium dodecyl sulfate-gels. However, slight differences in the apparent molecular weight are observed on high-resolution gels between immunologically unrelated subunits. The immunological identity and electrophoretic differences suggest that the charge isomers which are immunologically identical have similar antigenic surfaces. If a charge substitution is not in a critical location, we would expect the electrophoretically distinct but immunologically identical subunits to have identical assembly roles. Comparison of the results for Limulus hemocyanin with the hemocyanin of related species Eurypelma californicum and Androctanus australis, which have 7 and 8 immunologically distinct subunits, respectively, suggests that the calcium-mediated aggregation from 24 to 48 subunits of Limulus does not require more extensive subunit complexity.

References (30)

  • B. Sullivan et al.

    J. Biol. Chem

    (1976)
  • J. Lamy et al.

    Arch. Biochem. Biophys

    (1979)
  • R.W. Blakesley et al.

    Anal. Biochem

    (1977)
  • K.A. Ferguson

    Metabolism

    (1964)
  • E. Gould et al.

    Anal. Biochem

    (1975)
  • J.S. Tung et al.

    Biochem. Biophys. Res. Commun

    (1971)
  • B. Salvato et al.

    Comp. Biochem. Physiol

    (1978)
  • K. Imai

    J. Biol. Chem

    (1974)
  • J. Lamy et al.

    Arch. Biochem. Biophys

    (1979)
  • B. Sullivan et al.
  • M. Bijlholt et al.

    Eur. J. Biochem

    (1979)
  • E. Antonini et al.

    Annu. Rev. Biophys. Bioeng

    (1977)
  • M. Brouwer et al.

    Biochemistry

    (1978)
  • J. Markl et al.

    J. Comp. Physiol. Ser. B

    (1979)
  • M. Johnson et al.

    Biochemistry

    (1978)
  • Cited by (42)

    • Molecular basis of the Bohr effect in arthropod hemocyanin

      2008, Journal of Biological Chemistry
      Citation Excerpt :

      During dialysis, a small amount of precipitate was formed and was separated by low speed centrifugation. The individual subunits were purified according to a published method (17). Subunit II was used in this study because of its well-characterized allosteric properties (18).

    • Preliminary crystallographic studies of Limulus polyphemus hemocyanin subunits IIIa, IIIb and IV

      2002, Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
    • SDS-induced Phenoloxidase Activity of Hemocyanins from Limulus polyphemus, Eurypelma californicum, and Cancer magister

      2001, Journal of Biological Chemistry
      Citation Excerpt :

      Three of the eight bands of dissociated L. polyphemus hemocyanin appeared to be capable of O-diphenoloxidase activity. Several authors have detected heterogeneous subunit types of Limulushemocyanin (25, 34-39). Subunits LpII, IIIA, IIIB, and IV are present in four copies; subunit I is present in three copies; subunit V and VI are present in two copies; and subunit IIA is present in one copy (40).

    • Mass spectrometric characterization of Limulus polyphemus hemocyanin

      1999, Biochemical and Biophysical Research Communications
    View all citing articles on Scopus
    View full text