Proteolytic Activity of 26S Proteasomes Isolated from Muscles of the Tobacco Hornworm, Manduca sexta: Differences between Surviving Muscles and those Undergoing Developmentally Programmed Cell Death


The intersegmental muscles (ISMs) of tobacco hornworm, Manduca sexta are a well-characterised model system for examining the biochemical changes that accompany programmed cell death during development. When the ISMs become committed to die, there are dramatic increases in both the ubiquitin-expression, and ubiquitin-dependent proteolysis. Since the 26S proteasome is responsible for ATP/ubiq-uitin-dependent proteolysis in cells, we examined its enzymatic properties. Specific chymotrypsin-like proteolytic activity of 26S proteasomes isolated from ISM is four times higher than that of surviving flight muscle (FM). However, specific activity does not change between developmental stages within ISM or FM. The difference between proteolytic capacity of the two kinds of muscles is even higher when the ISM become committed to die because 26S proteasome content of ISM increases just before cell death. These observations underline the role of 26S proteasome in programmed cell death.


  1. 1.

    Bell, R. A., Joachim, F. A. (1976) Techniques for rearing laboratory colonies of tobacco hornworms and pink bollworms. Ann. Ent. Soc. Am. 69, 365–373.

    Article  Google Scholar 

  2. 2.

    Bradford, M. M. (1976) A rapid sensitive method for the quantitation of microgram quantities of protein using the principle of protein dye binding. Anal. Biochem. 71, 248–254.

    Article  Google Scholar 

  3. 3.

    Braun, B. C, Glickman, M., Kraft, R., Dahlmann, B., Kloetzel, P. M., Finley, D., Schmidt, M. (1999) The base of the proteasome regulatory particle exhibits chaperone-like activity. Nature Cell Biol 1, 221–226.

    CAS  Article  Google Scholar 

  4. 4.

    Dawson, S. P., Arnold, J. E., Mayer, N. J., Reynolds, S. E., Billett, M. A., Gordon, C, Colleaux, L., Kloetzel, P. M., Tanaka, K., Mayer, R. J. (1995) Developmental changes of the 26S proteasome in abdominal intersegmental muscles of Manduca sexta during programmed cell death. J. Biol. Chem. 270, 1850–1858.

    CAS  Article  Google Scholar 

  5. 5.

    Drexler, H. C. A. (1998) Programmed cell death and the proteasome. Apoptosis 3, 1–7.

    CAS  Article  Google Scholar 

  6. 6.

    Ephrussi, B., Beadle, A. W. (1936) A technique of transplantation for Drosophila. Am. Nat. 70, 218–225.

    Article  Google Scholar 

  7. 7.

    Haas, A. L., Baboshina, O., Williams, B., Schwartz, L. M. (1995) Co-ordinated induction of the ubiq-uitin conjugation pathway accompanies the developmentally programmed death of insect skeletal muscle. J. Biol. Chem. 270, 9407–9412.

    CAS  Article  Google Scholar 

  8. 8.

    Jones, M. E. E., Haire, M. F., Kloetzel, P.-M., Mykles, D. L., Schwartz, L. M. (1995) Changes in the structure and function of the multicatalytic proteinase (proteasome) during programmed cell death in the intersegmental muscles of the hawkmoth, Manduca sexta. Dev. Biol. 169, 436–447.

    CAS  Article  Google Scholar 

  9. 9.

    Lockshin, R. A. (1969) Programmed cell death. Activation of lysis by a mechanism involving the synthesis of protein. J. Insect Physiol. 15, 1505–1516.

    CAS  Article  Google Scholar 

  10. 10.

    Mykles, D. L. (1998) Intracellular proteinases of invertebrates: calcium-dependent and proteasome/ubiquitin-dependent systems. Int. Rev. Cytol. 184, 157–289.

    CAS  Article  Google Scholar 

  11. 11.

    Orino, E., Tanaka, K., Tamura, T., Sone, S., Ogura, T., Ichihara, A. (1991) ATP-dependent reversible association of proteasomes with multiple protein components to from 26S complexes that degrade ubiquitinated protein in human HL-60 cells. FEBS Lett. 284, 206–210.

    CAS  Article  Google Scholar 

  12. 12.

    Orlowski, R. Z. (1999) The role of ubiquitin-proteasome pathway in apoptosis. Cell Death Diff. 6, 303–313.

    CAS  Article  Google Scholar 

  13. 13.

    Samuels, R., Reynolds, S. E. (1993) Moulting fluid enzymes of the tobacco hornworm, Manduca sexta: timing of proteolytic and chitinolytic activity in relation to pre-ecdysial development. Arch. Insect Physiol. Biochem. 24, 33–44.

    CAS  Article  Google Scholar 

  14. 14.

    Schwartz, L. M. (1992) Insect muscle as a model for programmed cell death. J. Neurobiol. 23, 1312–1326.

    CAS  Article  Google Scholar 

  15. 15.

    Schwartz, L. M., Myer, A., Kosz, L., Engelstein, M., Maier, C. (1990) Activation of polyubiquitin gene expression during developmentally programmed cell death. Neuron 5, 411–419.

    CAS  Article  Google Scholar 

  16. 16.

    Schwartz, L. M., Truman, J. W. (1983) Hormonal control of rates of metamorphic development in the tobacco hornworm Manduca sexta. Dev. Biol. 99, 103–114.

    CAS  Article  Google Scholar 

  17. 17.

    Takayanagi, K., Dawson, S., Reynolds, S. E., Mayer, R. J. (1996) Specific developmental changes in the regulatory subunits of the 26S proteasome in intersegmental muscles preceding eclosion in Manduca sexta. Biochem. Biophys. Res. Com. 228, 517–523.

    CAS  Article  Google Scholar 

  18. 18.

    Voges, D., Zwickl, P., Baumeister, W. (1999) The 26S proteasome: A molecular machine designed for controlled proteolysis. Ann. Rev. Biochem. 68, 1015–1068.

    CAS  Article  Google Scholar 

  19. 19.

    Zwickl, P., Baumeister, W. (1999) AAA-ATPases at the crossroads of protein life and death. Nature Cell Biology 1, E97–E98.

    CAS  Article  Google Scholar 

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Correspondence to P. Löw.

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Dedicated to Professor János Kovács on the occasion of his 70th birthday

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Löw, P., Reynolds, S.E. & Sass, M. Proteolytic Activity of 26S Proteasomes Isolated from Muscles of the Tobacco Hornworm, Manduca sexta: Differences between Surviving Muscles and those Undergoing Developmentally Programmed Cell Death. BIOLOGIA FUTURA 52, 435–442 (2001).

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  • Manduca sexta
  • 26S proteasome
  • enzyme activity
  • intersegmental muscle
  • flight muscle