In Vitro Biosynthesis of Locust Adipokinetic Hormones: Isolation and Identification of the Bioactive Peptides and Their Prohormones

  • R. C. H. M. Oudejans
  • F. P. Kooiman
  • T. K. F. Schulz
  • A. M. Th. Beenakkers
Part of the Chromatographic Society Symposium Series book series (CSSS)


The biosynthesis of adipokinetic hormones (AKH) I and II and their precursors was studied in the migratory locust Locusta migratoria using radiolabeled amino acids in an in vitro system. The peptides from the glandular part of the corpora cardiaca (CC) were extracted and separated by reversed-phase high performance liquid chromatography (HPLC). Incorporation of the [3H] amino acids was measured by monitoring the radioactivity of the column eluate. Total time for biosynthesis and processing of the prohormones to the bioactive AKH I and II was 75 min. Only the glandular part of the CC synthesized these peptide hormones.

Protein fractions from CC were separated by gradient gel electrophoresis (PAGE or SDS-PAGE) and by two-dimensional gel electrophoresis (isoelectrofocusing followed by SDS-PAGE). After electrophoresis, proteins were transferred to nitrocellulose membranes by electroblotting and the incorporation of radiolabel was measured with an automatic linear analyzer. Time-course studies and pulse-labeling experiments revealed incorporation of radiolabel into most proteins.

By using monoclonal antibodies raised against AKH I, immunoreactive protein bands (prohormones and/or processing products) with a Mr of 72,000 and 33,000 were only detectable in the cytoplasm fraction of the glandular lobe of the CC.


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  1. 1.
    A. M. Th. Beenakkers, R. E. B. Bloemen, T. A. De Vlieger, D. J. Van der Horst and W. J. A. Van Marrewijk, Insect adipokinetic hormones, Peptides, 6 (Suppl. 3):437–444 (1985).CrossRefPubMedGoogle Scholar
  2. 2.
    I. Orchard, Adipokinetic hormones — an update, J. Insect Physiol., 33:451–463 (1987).CrossRefGoogle Scholar
  3. 3.
    R. C. H. M. Oudejans, H. Voshol, T. K. F. Schulz and A. M. Th. Beenakkers, Time-resolved fluorescence immunoassay, in: “Immunological Techniques in Insect Biology”, L. I. Gilbert and T. A. Miller, eds., Springer-Verlag, New York, pp 75–92 (1988).CrossRefGoogle Scholar
  4. 4.
    J. H. B. Diederen, H. A. Maas, H. J. Pel, H. Schooneveld, W. F. Jansen and H. G. B. Vullings, Co-localization of the adipokinetic hormones I and II in the same glandular cells and in the same secretory granules of corpus cardiacum of Locusta migratoria and Schistocerca gregaria.An immunoelectron-microscopic study, Cell Tissue Res., 249:379–389 (1987).CrossRefGoogle Scholar
  5. 5.
    J. V. Stone, W. Mordue, K. E. Batley and H. R Morris, Structure of locust adipokinetic hormone, a neurohormone that regulates lipid utilization during flight, Nature, 263:207–211(1976).CrossRefGoogle Scholar
  6. 6.
    K. Siegert, P. Morgan and W. Mordue, Primary structures of locust adipokinetic hormones II, Biol Chem. Kappe Segler, 366:723–727 (1979).Google Scholar
  7. 7.
    G. Gäde, New structures of insect neuropeptides, in: “Endocrinological Frontiers in Physiological Insect Ecology”, F. Sehnal, A. Zabza and D. L. Denlinger, eds., Technical University Press, Wroclaw, pp 625–650 (1988).Google Scholar
  8. 8.
    S. Hekimi and M. O’Shea, Identification and purification of two precursors of the insect neuropeptide adipokinetic hormone, J. Neuroscl, 7:2773–2784 (1987).CrossRefGoogle Scholar
  9. 9.
    P. Moshitzky, W. J. Henzel, A. Rafaeli, J. Ramachandran and S. W. Applebaum, Synthesis of adipokinetic hormone (AKH-I) in the locust brain, Insect Biochem., 17:1133–1137(1987).CrossRefGoogle Scholar
  10. 10.
    M. H. Schaifer and B. E. Noyes, The potential of adipokinetiic hormone to teach us about neuropeptides, Bio-Essays, 7:67–71 (1987).Google Scholar
  11. 11.
    M. H. Schaifer and B. E. Noyes, The peptides, mRNA, and genes of the AKH family, in: “Molecular Entomology”, J. H. Law, ed., Alan R Liss, New York, pp 141–153 (1987).Google Scholar
  12. 12.
    R. C. H. M. Oudejans, F. P. Kooiman and A. M. Th. Beenakkers, Biosynthesis of locust adipokinetic hormones: incorporation of radiolabeled amino acids, Gen. Comp. Endocrinol, 74:306 (1989).Google Scholar
  13. 13.
    T. K. F. Schulz, F. P. Kooiman, R. C. H. M. Oudejans and A. M. Th. Beenakkers, Biosynthesis of locust adipokinetic hormones: an immunological approach, Gen. Comp. Endocrinol, 74:306 (1989).Google Scholar
  14. 14.
    D. J. Van der Horst, A. M. C. Baljet, A. M. Th. Beenakkers and E. Van Handel, Turnover of locust haemolymph diglycerides during flight and rest, Insect Biochem., 8:369–373(1978).CrossRefGoogle Scholar
  15. 15.
    T. C. C. Grace, Establishment of four strains of cells from insect tissues grown in vitro, Nature, 195:788–789(1962).CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    T. Pannabecker and I. Orchard, Regulation of adipokinetic hormone release from locust neuroendocrine tissue: participation of calcium and cyclic AMP, Brain Res., 423:13–22(1987).CrossRefGoogle Scholar
  17. 17.
    C. M. Williams, Physiology of insect diapause. IV. The brain and prothoracic glands as an endocrine system in the cecropia silkworm, BioL Bull, 103:120–138 (1952).CrossRefGoogle Scholar
  18. 18.
    J. V. Stone and W. Mordue, Isolation of granules containing adipokinetic hormone from locust corpora cardiaca by differential centrifugation, Gen. Comp. Endocrinol, 39:543–547 (1979).CrossRefGoogle Scholar
  19. 19.
    G. Gäde, G. J. Goldsworthy, G. Kegel and R Keller, Single step purification of locust adipoinetic hormones I and II by reversed-phase high-performance liquid chromatography, and amino-acid composition of the hormone II, Hoppe-Seyler’s Zeitschrift für Physiologische Chemie, 365:393–398 (1984).CrossRefGoogle Scholar
  20. 20.
    G. J. Goldsworthy, W. Mordue and J. Guthkelch, Studies on insect adipokinetic hormones, Gen. Comp. Endocrinol, 18:545–551 (1972).CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    U. K. Laemmli and M. Favre, Nutrition of the head of bacteriophage T4, J. Mol Biol., 80:575–599 (1973).CrossRefPubMedGoogle Scholar
  22. 22.
    W. N. Burnette, “Western bloting”: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A, Anal. Biochem., 112:195–203(1981).CrossRefPubMedGoogle Scholar
  23. 23.
    T. K. F. Schulz, D. J. Van der Horst, H. Amesz, H. O. Voorma and A. M. Th. Beenakkers, Monoclonal antibodies specific for apoproteins of lipophorins from the migratory locust, Arch. Insect Biochem. Physiol., 6:97–107 (1987).CrossRefGoogle Scholar
  24. 24.
    F. Wold, In vivo chemical modification of proteins (Post-translational modifications), Ann. Rev. Biochem., 50:783–814 (1981).CrossRefPubMedGoogle Scholar
  25. 25.
    A. J. Turner, Processing and metabolism of neuropeptides, Essays in Biochem., 22:69–119(1986).Google Scholar
  26. 26.
    J. M. Fisher and RH. Scheller, Prohormone processing and the secretory pathway, J. Biol Chem., 263:16515–16518 (1988).PubMedGoogle Scholar
  27. 27.
    H. Schooneveld, G. I. Tesser, J. A. Veenstra and H. M. Romberg-Privee, Adipokinetic hormone and AKH-like peptide demonstrated in the corpora cardiaca and nervous system of Locusta migratoria by immunocytochemistry, Cell Tissue Res., 230:67–76 (1983).CrossRefGoogle Scholar
  28. 28.
    H. Schooneveld, H. M. Romberg-Privee and J. A. Veenstra, Immunocytochemical differentiation between adipokinetic hormone (AKH)-like peptides in neurons and glandular cells in the corpus cardiacum of Locusta migratoria and Periplaneta americana with C-terminal and N-terminal specific antisera to AKH, Cell Tissue Res., 243:9–14(1986).CrossRefGoogle Scholar
  29. 29.
    S. Hekimi and M. O’Shea, Adipokinetic hormones in locusts: synthesis and developmental regulation, Society for Neuroscience, Abstracts of 15th Annual Meeting, Abstract nr. 282.12 (1985).Google Scholar
  30. 30.
    S. Hekimi and M. O’Shea, Neuropeptide synthesis and its regulation in an insect model system, Society for Neuroscience, Abstracts of 16th Annual Meeting, Abstract nr. 260.12 (1986).Google Scholar

Copyright information

© Plenum Press, New York 1990

Authors and Affiliations

  • R. C. H. M. Oudejans
    • 1
  • F. P. Kooiman
    • 1
  • T. K. F. Schulz
    • 1
  • A. M. Th. Beenakkers
    • 1
  1. 1.Department of Experimental ZoologyUniversity of UtrechtUtrechtThe Netherlands

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