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Ecotoxicology

, Volume 25, Issue 1, pp 115–120 | Cite as

PIXE-electrophoresis shows starving collembolan reallocates protein-bound metals

  • Göran BengtssonEmail author
  • Jan Pallon
  • Christina Nilsson
  • Rita Triebskorn
  • Heinz-R. Köhler
Article

Abstract

One of multiple functions of metalloproteins is to provide detoxification to excess metal levels in organisms. Here we address the induction and persistence of a range of low to high molecular weight copper- and zinc binding proteins in the collembolan species Tetrodontophora bielanensis exposed to copper- and zinc-enriched food, followed by a period of recovery from metal exposure, in absence and presence of food. After 10 days of feeding copper and zinc contaminated yeast, specimens were either moved to ample of leaf litter material from their woodland stand of origin or starved (no food offered). The molecular weight distribution of metal binding proteins was determined by native polyacryl gel electrophoresis. One gel was stained with Comassie brilliant blue and a duplicate gel dried and scanned for the amount of copper and zinc by particle-induced X-ray emission. Specimens exposed to copper and recovered from it with ample of food had copper bound to two groups of rather low molecular weight proteins (40–50 kDa) and two of intermediate size (70–80 kDa). Most zinc in specimens from the woodland stand was bound to two large proteins of about 104 and 106 kDa. The same proteins were holding some zinc in metal-exposed specimens, but most zinc was found in proteins <40 kDa in size. Specimens recovered from metal exposure in presence of ample of food had the same distribution pattern of zinc binding proteins, whereas starved specimens had zinc as well as copper mainly bound to two proteins of 8 and 10 kDa in size. Thus, the induction and distribution of copper- and zinc-binding proteins depend on exposure conditions, and the presence of low molecular weight binding proteins, characteristic of metallothioneins, was mainly limited to starving conditions.

Keywords

Collembola Metal Protein PIXE Electrophoresis Starvation Metallothionein Metalloprotein PAGE 

Notes

Acknowledgments

We are grateful to Heike Reise, Senckenberg Museum for Natural Science Görlitz, for providing T. bielanensis. The study was financed by the European Environmental Research Organization (EERO), The German Academic Exchange Service (DAAD, Bonn), and the Swedish Institute (SI, Stockholm). Thanks are also due to Sten Rundgren, Lund University, and to Kåseberga-Fisk AB, Löderup, for motivational support.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

References

  1. Amiard J-C, Amiard-Triquet C, Barka S, Pellerin J, Rainbow PS (2006) Metallothioneins in aquatic invertebrates: their role in metal detoxification and their use as biomarkers. Aquat Toxicol 76:160–202CrossRefGoogle Scholar
  2. Babczyńska A, Wilczek G, Wilczek P, Szulińska E, Witas I (2011a) Metallothioneins and energy budget indices in cadmium and copper exposed spiders Agelena labyrinthica in relation to their developmental stage, gender and origin. Comp Biochem Physiol C 154:161–171Google Scholar
  3. Babczyńska A, Wilczek G, Szulińska E, Franiel I (2011b) Quantitative immunodetection of meallothioneins in relation to metals concentration in spiders from variously polluted areas. Ecotoxicol Environ Saf 74:1498–1503CrossRefGoogle Scholar
  4. Baudrimont M, Lemaire-Gony S, Ribeyre F, Metivaud J, Boudou A (1997) Seasonal variations of metallothionein concentrations in the Asiatic clam (Corbicula fluminea). Comp Biochem Physiol C 118:361–367Google Scholar
  5. Bengtsson G, Gunnarsson T (1984) A micromethod for the determination of metal ions in biological tissues by furnace atomic absorption spectrophotometry. Microchem J 29:282–287CrossRefGoogle Scholar
  6. Bengtsson G, Ek H, Rundgren S (1992) Evolutionary response of earthworms to long-term metal exposure. Oikos 63:289–297CrossRefGoogle Scholar
  7. Bertini I, Sigel A, Sigel H (2001) Handbook of metalloproteins. Marcel Dekker, New YorkGoogle Scholar
  8. Binet MRB, Ma R, McLeod CW, Poole RK (2003) Detection and characterization of zinc- and cadmium-binding proteins in Escherichia coli by gel electrophoresis and laser ablation-inductively coupled plasma-mass spectrometry. Anal Biochem 318:30–38CrossRefGoogle Scholar
  9. Bremner I, Davies NT (1975) The induction of metallothionein in rat liver by zinc injection and restriction of food intake. Biochem J 149:733–738CrossRefGoogle Scholar
  10. Capdevila M, Atrian S (2011) Metallothionein protein evolution: a miniassay. J Biol Inorg Chem 16:977–989CrossRefGoogle Scholar
  11. Dallinger R, Wieser W (1984) Molecular fractionation of Zn, Cu, Cd and Pb in the midgut gland of Helix Pomatia L. Comp Biochem Physiol C 79:125–129CrossRefGoogle Scholar
  12. Dallinger R, Berger B, Hunziger P, Kägi JHR (1997) Metallothionein in snail Cd and Cu metabolism. Nature 388:237–238CrossRefGoogle Scholar
  13. Dallinger R, Lagg B, Egg M, Schipflinger R, Chabicovsky M (2004) Cd accumulation and Cd-Metallothionein as a biomarker in Cepaea hortensis (Helcidae, Pulmonata) from laboratory exposure and metal-polluted habitats. Ecotoxicology 13:757–772CrossRefGoogle Scholar
  14. Degtyarenko K (2000) Bioinorganic motifs: towards functional classification of metalloproteins. Bioinformatics 16:851–864CrossRefGoogle Scholar
  15. den Besten PJ, Herwig HJ, Zandee DI, Voogt PA (1990) Cadmium accumulation and metallothionein-like proteins in the sea star Asterias rubens. Arch Environ Contam Toxicol 19:858–862CrossRefGoogle Scholar
  16. Engel DW, Brouwer M, Mercaldo-Allen R (2001) Effects of molting and environmental factors on trace metal body-burdens and hemocyanin concentrations in the American lobster, Homarus americanus. Mar Environ Res 52:257–269CrossRefGoogle Scholar
  17. Finger JM, Smith JD (1987) Molecular association of Cu, Zn, Cd and 210Po in the digestive gland of the squid Nototodarus gouldi. Mar Biol 95:87–91CrossRefGoogle Scholar
  18. Hashemi S, Kunwar PS, Blust R, De Boeck G (2008) Differential metallothionen induction patterns in fed and starved carp (Cyprinus carpio) during waterborne copper exposure. Environ Toxicol Chem 27:2154–2158CrossRefGoogle Scholar
  19. Hensbergen PJ, Donker MH, van Velzen MJM, Roelofs D, van der Schors RC, Hunziker PE, van Straalen NM (1999) Primary structure of a cadmium-induced metallothionein from the insect Orchesella cincta (Collembola). Eur J Biochem 259:197–203CrossRefGoogle Scholar
  20. Hensbergen PJ, Donker MH, Hunziger PE, van der Schors RC, van Straalen NM (2001) Two metal-binding peptides from the insect Orchesella cincta (Collembola) as a result of metallothionein cleavage. Insect Biochem Mol Biol 31:1105–1114CrossRefGoogle Scholar
  21. Holwerda DA (1991) Cadmium kinetics in freshwater clams. V. Cadmium-copper interaction in metal accumulation by Anodonta cygnea and characterization of the metal-binding protein. Arch Environ Contam Toxicol 21:432–437CrossRefGoogle Scholar
  22. Irving H, Williams RJP (1948) Order of stability of metal complexes. Nature 162:746–747CrossRefGoogle Scholar
  23. Janssen H, Dallinger R (1991) Diversification of cadmium-binding proteins due to different levels of contamination in Arion lusitanicus. Arch Environ Contam Toxicol 20:132–137CrossRefGoogle Scholar
  24. Jiménez I, Gotteland M, Zarzuelo A, Uauy R, Speisky H (1997) Loss of the metal binding properties of metallothionein induced by hydrogen peroxide and free radicals. Toxicology 120:37–46CrossRefGoogle Scholar
  25. Kägi JHR (1991) Overview of metallothionein. Methods Enzymol 205:613–626CrossRefGoogle Scholar
  26. Nejmeddine A, Sautiere P, Dhainaut-Courtois N, Baert J-L (1992) Isolation and characterization of a Cd-binding protein from Allolobophora caliginosa (Annelida, Oligochaeta): distinction from metallothioneins. Comp Biochem Physiol Pharmacol Toxicol Endocrinol 101:601–605CrossRefGoogle Scholar
  27. Nielsen JL, Abildtrup A, Christensen J, Watson P, Cox A, McLeod CW (1998) Laser ablation inductively coupled plasma-mass spectrometry in combination with gel electrophoresis: a new strategy for speciation of metal binding serum proteins. Spectrochim Acta Part B 53:339–345CrossRefGoogle Scholar
  28. Nordberg GF, Fowler BA, Nordberg M, Friberg LT (2007) Handbook on the toxicology of metals. Academic Press, Amsterdam BostonGoogle Scholar
  29. O’Halloran TV, Culotta VC (2000) Metallochaperones, an intracellular shuttle service for metal ions. J Biol Chem 275:25057–25060CrossRefGoogle Scholar
  30. Palacios Ò, Atrian S, Capdevila M (2011) Zn- and Cu-thioneins: a functional classification for metallothioneins? J Biol Inorg Chem 16:991–1009CrossRefGoogle Scholar
  31. Roegener J, Lutter P, Reinhardt R, Blüggel M, Meyer HE, Anselmetti D (2003) Ultrasensitive detection of unstained proteins in acrylamide gels by native UV fluorescence. Anal Chem 75:157–159CrossRefGoogle Scholar
  32. Rogival D, van Campenhout K, Goenaga Infante H, Hearn R, Scheirs J, Blust R (2007) Induction and metal speciation of metallothionein in wood mice (Apodemus sylvaticus) along a metal pollution gradient. Environ Toxicol Chem 26:506–514CrossRefGoogle Scholar
  33. Sato M, Bremner I (1993) Oxygen free radicals and metallothionein. Free Radic Biol Med 14:325–337CrossRefGoogle Scholar
  34. Serafim MA, Company RM, Bebianno MJ, Langston WJ (2002) Effect of temperature and size on metallothionein synthesis in the gill of Mytilus galloprovincialis exposed to cadmium. Mar Environ Res 54:361–365CrossRefGoogle Scholar
  35. Stürzenbaum SR, Winters C, Galay M, Morgan AJ, Kille P (2001) Metal ion trafficking in earthworms. Identification of a cadmium-specific metallothionein. J Biol Chem 276:34013–34018CrossRefGoogle Scholar
  36. Susnea I, Bernevic B, Wicke M, Ma L, Liu S, Schellander K, Przybylski M (2013) Application of MALDI-TOF-mass spectrometry to proteome analysis using stain-free gel electrophoresis. In: Cai Z, Liu S (eds) Applications of MALDI-TOF spectroscopy, Topics in current chemistry 331. Springer, Berlin, pp 37–54Google Scholar
  37. Sutherland DEK, Stillman MJ (2011) The „magic numbers“of metallothionein. Metallomics 3:444–463CrossRefGoogle Scholar
  38. Tainer JA, Roberts VA, Getzoff ED (1991) Metal-binding sites in proteins. Curr Opin Biotechnol 2:582–591CrossRefGoogle Scholar
  39. Ulrich W, Fiera C (2010) Environmental correlates of body size distributions of European springtails (Hexapoda: Collembola). Glob Ecol Biogeogr 19:905–915CrossRefGoogle Scholar
  40. Weiss W, Weiland F, Görg A (2009) Protein detection and quantitation technologies for gel-based proteome analysis. Proteomics Methods Mol Biol 564:59–82CrossRefGoogle Scholar
  41. Yamamura M, Mori T, Suzuki KT (1981) Metallothionein induced in the earthworm. Experentia 37:1187–1189CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Göran Bengtsson
    • 1
    Email author
  • Jan Pallon
    • 2
  • Christina Nilsson
    • 2
  • Rita Triebskorn
    • 3
    • 4
  • Heinz-R. Köhler
    • 3
  1. 1.Department of EcologyUniversity of LundLundSweden
  2. 2.Department of Nuclear PhysicsLund Institute of TechnologyLundSweden
  3. 3.Animal Physiological Ecology, Institute of Evolution and EcologyUniversity of TübingenTübingenGermany
  4. 4.Transfer Center Ecotoxicology and EcophysiologyRottenburgGermany

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