Fish Physiology and Biochemistry

, Volume 24, Issue 3, pp 259–270 | Cite as

Proteome analysis of rainbow trout (Oncorhynchus mykiss) liver proteins during short term starvation

  • S.A.M. Martin
  • P. Cash
  • S. Blaney
  • D.F. Houlihan


Basal rates of protein synthesis in the liver are similar in fed and starved trout; during starvation protein degradation must increase as the animal loses weight and the liver decreases in size. Little is known about how protein degradation is controlled in fish under various physiological circumstances. In this study proteome analysis has been used to identify proteins that are changed in abundance that may be involved in increased protein degradation in the liver of rainbow trout following a period of 14 days without food. Protein extracts from whole liver were analysed on high resolution two dimensional gels. The protein profiles from individual fish were digitised and computer software used to construct a composite reference gel. In total 780 protein spots were identified and their abundance monitored for fed and starved groups of fish. All protein spots were recorded in terms of their isolelectric point (pI), molecular weight and abundance. Twenty four proteins were found to have differences in abundance between the two groups, 8 were increased in fed fish with 16 increased in abundance as a result of food withdrawal. Twenty two protein spots were excised from gels and subjected to trypsin digestion followed by peptide separation by MALDI-TOF spectrometry. Peptide masses were used to search the GenBank data base for protein identification. Twelve of the proteins were identified on the basis of the homology of their peptide profiles to existing protein sequences. One protein, which increased in abundance under starvation conditions, was identified as cathepsin D, a lysosomal endopeptidase involved in protein degradation. Northern blot analysis of RNA isolated from liver of rainbow trout showed an increase in expression of cathepsin D reflecting either increased transcription or stability of the mRNA in starved fish, supporting the proteome evidence. Thus in starved trout an increase in lysosomal proteases may play a part in the loss of proteins.

cathepsin D gene expression mass spectrometry protein degradation proteome rainbow trout 


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  1. Anderson, L. and Seilhamer, J. 1997. A comparison of selected mRNA and protein abundances in human liver. Electrophoresis 18: 533–537.Google Scholar
  2. Anderson, N.L., Esquerblasco, R., Hofmann, J.P. and Anderson, N.G. 1991. A 2–dimensional gel database of rat liver proteins useful in gene regulation and drug effect studies. Electrophoresis 12: 907–930.Google Scholar
  3. Attaix, D., Combaret, L. and Taillandier, D. 1999. Mechanisms and regulation in protein degradation. Protein Metabol. & Nutr. Proc. VIII: 51–67.Google Scholar
  4. Balsco, J., Fernandez, J. and Gutierrez, J. 1991. The effects of starvation and refeeding on plasma amino-acid levels in carp, Cyprinus carpio L. 1758. J. Fish Biol. 38: 587–598.Google Scholar
  5. Blommaart, E.F., Luiken, J.J. and Meijer, A.J. 1997. Autophagic proteolysis: control and specificity. Histochem. J. 29: 365–385.Google Scholar
  6. Botbol, V. and Scornik, O.A. 1991. Measurement of instant rates of protein degradation in the livers of intact mice by the accumulation of bestatin-induced peptides. J. Biol. Chem. 266: 2151–2157.Google Scholar
  7. Brooks, S., Tyler, C.R., Carnevali, O., Coward, K. and Sumpter, J.P. 1997. Molecular characterisation of ovarian cathepsin D in the rainbow trout, Oncorhynchus mykiss. Gene 201: 45–54.Google Scholar
  8. Carnevali, O., Centonze, F., Brooks, S., Marota, J. and Sumpter, J.P. 1999. Molecular cloning and expression of ovarian cathepsin D in seabream, Sparus aurata. Biol. Reprod. 61: 785–791.Google Scholar
  9. Cash, P., Argo, E., Ford, L., Lawrie, L. and McKenzie, H. 1999. A proteomic analysis of erythromycin resistance in Streptococcus. Electrophoresis 20: 2259–2268.Google Scholar
  10. Cuervo, A.M. and Dice, J.F. 1998. Lysosomes, a meeting point of proteins, chaperones, and proteases. J. Mol. Med. 76: 6–12.Google Scholar
  11. Diatchenko, L., Lau, Y.F.C., Campbell, A.P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E.D. and Siebert, P.D. 1996. Suppression subtractive hybridization: A method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. USA 93: 6025–6030.Google Scholar
  12. Fountoulakis, M., Berndt, P., Boelsterli, U.A., Crameri, F., Winter, M., Albertini, S. and Suter, L. 2000. Two-dimensional database of mouse liver proteins: Changes in hepatic protein levels following treatment with acetaminophen or its nontoxic regioisomer 3–acetamidophenol. Electrophoresis 21: 2148–2161.Google Scholar
  13. Gassmann, M.G., Stanzel, A. and Werner, S. 1999. Growth factorregulated expression of enzymes involved in nucleotide biosynthesis: a novel mechanism of growth factor Action. Oncogene 18: 6667–6676.Google Scholar
  14. Gracey, A.Y., Troll, J.V. and Somero, G.N. 2001. Hypoxiainduced gene expression profiling in the euryoxic fish Gillichthys mirabilis Proc. Natl. Acad. Sci. USA 98: 1993–1998.Google Scholar
  15. Gutierrez, J., Perez, J., Navarro, I., Zanuy, S. and Carrillo, M. 1991. Changes in plasma glucagon and insulin associated with fasting in sea bass (Dicentrarchus-labrax). Fish Physiol. Biochem. 9: 107–112.Google Scholar
  16. Houlihan, D.F. 1991. Protein turnover in ectotherms and its relationship to energetics. In: Advances in Comparative and Environmental Physiology. pp 1–43. Edited by R. Gilles. Springer-Verlag, Berlin.Google Scholar
  17. Houlihan, D.F., Carter, C.G. and McCarthy, I.D. 1995. Protein synthesis in fish. In: Biochemistry and Molecular Biology of Fishes. Vol. 4, chapter 8. pp. 191–220. Edited by Hochachka and Mommsen. Elsevier Science B.V.Google Scholar
  18. Jensen, O.N., Wilm, M., Shevchenko, A. and Mann, M. 1999. Sample preparation methods for mass spectrometric peptide mapping directly from 2–DE gels. In: 2–D Proteome analysis Proteocols (ED. A.J. Link) pp. 513–530. Edited by A.J. Link. Humana Press, New Jersey.Google Scholar
  19. Kaivarainen, E.I., Nemova, N.N., Krupnova, M.Y. and Bondareva, L.A. 1998. The effect of toxic factors on intracellular proteinase activity in freshwater fish. Acta Veterinaria Brno 67: 309–316.Google Scholar
  20. Kanaya, S., Ujiie, Y., Hasegawa, K., Sato, T., Imada, H., Kinouchi, M., Kudo, Y., Ogata, T., Ohya, H., Kamada, H., Itamoto, K. and Katsura, K. 2000. Proteome analysis of Oncorhynchus species during embryogenesis. Electrophoresis 11: 1907–1913.Google Scholar
  21. Kawamoto, S., Matsumoto, Y., Mizuno, K., Okubo, K. and Matsubara, K. 1996. Expression profiles of active genes in human and mouse livers. Gene 174: 151–158.Google Scholar
  22. Kovacs, A.L., Laszlo, L., Fellinger, E., Jakab. A., Orosz, A., Rez, G. and Kovacs, J. 1989. Combined effects of fasting and vinblastine treatment on serum insulin level, the size of autophagiclysosomal compartment, protein content and lysosomal enzyme activities of liver and exocrine pancreatic cells of the mouse. Comp. Biochem. Physiol. B 94: 505–510.Google Scholar
  23. Krupnova, M.Y., Vysotskaya, R.U. and Ruokolainen, T.P. 1985. Variation in lysosome enzyme-activity in trout during fasting. Ukr. Biokhim. Zh. 57: 62–65.Google Scholar
  24. Kultz, D. and Somero, G.N. 1996. Differences in protein patterns of gill epithelial cells of the fish Gillichthys mirabilis after osmotic and thermal acclimation. J. Comp. Physiol. B 166: 88–100.Google Scholar
  25. Lahm, H.W. and Langen, H. 2000. Mass spectrometry: A tool for the identification of proteins separated by gels. Electrophoresis 21: 2105–2114.Google Scholar
  26. Liang, P. and Pardee, A.B. 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257: 967–971.Google Scholar
  27. Lockhart, D.J. and Winzeler, E.A. 2000. Genomics, gene expression and DNA arrays. Nature 405: 827–836.Google Scholar
  28. McLaughlin, R.L., Ferguson, M.M. and Noakes, D.L.G. 1995. Tissue concentrations of RNA and protein for juvenile brook trout (Salvelinus fontinalis): Lagged responses to fluctuations in food availability. Fish Physiol. Biochem. 14: 459–469.Google Scholar
  29. McMillan, D.N. and Houlihan, D.F. 1988. The effect of refeeding on tissue protein-synthesis in rainbow trout. Physiol. Zool. 61: 429–441.Google Scholar
  30. Meijer, A.J., Blommaart, E.F.C., Dubbelhuis, P.F. and van Sluijters, D.A. 1999. Regulation of hepatic nitrogen metabolism. Protein Metabol. & Nutr. Proc. VIII: 155–175.Google Scholar
  31. Mitch, W.E. and Goldberg, A.L. 1996. Mechanisms of disease - Mechanisms of muscle wasting - The role of the ubiquitinproteasome pathway. New Engl. J. Med. 335: 1897–1905.Google Scholar
  32. Mommsen, T.P., French, C.J. and Hochachka, P.W. 1980. Sites and patterns of protein and amino acid utilization during the spawning migration of salmon. Can. J. Zool. 58: 1785–1799.Google Scholar
  33. Moon, T.W. 1998. Glucagon: From hepatic binding to metabolism in teleost fish. Comp. Biochem. Physiol. B 121: 27–24.Google Scholar
  34. Navarro, I., Blasco, J., Banos, N. and Gutierrez, J. 1997. Effects of fasting and feeding on plasma amino acid levels in brown trout. Fish Physiol. Biochem. 16: 303–309.Google Scholar
  35. Nemova, N.N. and Sidorov, V.S. 1984. Lysosomal proteinases during embryogensis of the salmon (Salmo salar). J. Evol. Biochem. Physiol. 20: 371–375.Google Scholar
  36. Pamer, E. and Cresswell, P. 1998. Mechanisms of MHC class I - Restricted antigen processing. Ann. Rev. Immunol. 16: 323–358.Google Scholar
  37. Peragon, J., Barroso, J.B., Garcia-Salguero, L., Aranda, F., dela Higuera, M. and Lupianez, J.A. 1999. Selective changes in the protein-turnover rates and nature of growth induced in trout liver by long-term starvation followed by re-feeding. Mol. Cell. Biochem. 201: 1–10.Google Scholar
  38. Perkins, D.N., Pappin, D.J.C., Creasy, D.M. and Cottrell, J.S. 1999. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20: 3551–3567.Google Scholar
  39. Raj, L., Vivekanand, P., Das, T.K., Badam, E., Fernandes, M., Finley, R.L., Brent, R., Appel, L.F., Hanes, S.D. and Weir, M. 2000. Targeted localized degradation of Paired protein in Drosophila development. Curr. Biol. 10: 1265–1272.Google Scholar
  40. Ricker, W.E. 1979. Growth rates and models. In: Fish Physiology, Vol. VIII, Bioenergics and Growth. pp. 677–743. Edited by Hoar, Randalland Brett, Academic Press, London.Google Scholar
  41. Schmidt, M., Krol, T., Renne, U. and Panicke, L. 2000. Lysosomal proteolytic activity in the liver of growing mice. Archiv für Tierzucht - Arch. Animal Breed. 43: 363–374.Google Scholar
  42. Scornik, O.A. and Botbol, V. 1987. Protein metabolism and liver growth. In: Lysosomes, their role in protein breakdown. pp. 445–484. Edited by H. Glaumann and F.J. Ballard. Academic press, London (UK).Google Scholar
  43. Seglen, P.O. and Bohley, P. 1992. Autophagy and other vacuolar protein degradation mechanisms. Experimentia 48: 158–172.Google Scholar
  44. Shevchenko, A., Wilm, M., Vorm, O. and Mann, M. 1996. Mass spectrometric sequencing of proteins from silver stained polyacrylamide gels. Anal. Chem. 68: 850–858.Google Scholar
  45. Solomon, V., Lecker, S.H. and Goldberg, A.L. 1998. The N-end rule pathway catalyzes a major fraction of the protein degradation in skeletal muscle. J. Biol. Chem. 273: 25,216–25,222.Google Scholar
  46. Walton, M.J. and Cowey, C.B. 1982. Aspects of intermediary metabolism in salmonid fish. Comp. Biochem. Phys. B 73: 59–79.Google Scholar
  47. Waterlow, J.C., Garlick, P.J. and Millward, D.J. 1978. Protein turnover in mammalian tissues and in the whole body. Elsevier North Holland, Amsterdam.Google Scholar
  48. Wilm, M., Shevchenko, A., Houthaeve, T., Breit, S., Schweigerer, L., Fotsis,. T. and Mann, M. 1996. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379: 466–469.Google Scholar

Copyright information

© Kluwer Academic Publishers 2001

Authors and Affiliations

  • S.A.M. Martin
  • P. Cash
  • S. Blaney
  • D.F. Houlihan

There are no affiliations available

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