In Vitro Biosynthesis of Locust Adipokinetic Hormones: Isolation and Identification of the Bioactive Peptides and Their Prohormones
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.
KeywordsHigh Performance Liquid Chromatography Incubation Medium Migratory Locust Corpus Cardiaca Immunoreactive Protein Band
Unable to display preview. Download preview PDF.
- 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
- 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.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.S. Hekimi and M. O’Shea, Identification and purification of two precursors of the insect neuropeptide adipokinetic hormone, J. Neuroscl, 7:2773–2784 (1987).Google Scholar
- 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.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.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.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
- 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).PubMedCrossRefGoogle Scholar
- 25.A. J. Turner, Processing and metabolism of neuropeptides, Essays in Biochem., 22:69–119(1986).Google Scholar
- 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.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.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