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Isolation of Glycosomes from Trypanosoma cruzi

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Trypanosomatids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2116))

Abstract

Glycosomes are peroxisome-related organelles of trypanosomatids in which the glycolytic and some other metabolic pathways are compartmentalized. We describe here two methods for the purification of glycosomes from Trypanosoma cruzi for preparative purposes, differential and isopycnic centrifugation. These are two techniques that allow the separation of different cellular compartments based on their different physicochemical characteristics. The first type of centrifugation is a rapid method that does not require large inputs and allows for fractions enriched in specific cell compartments to be obtained. The second type of centrifugation is a more elaborate method, but enables highly purified cellular compartments to be isolated. The success in obtaining these purified, intact organelles critically depends on using an appropriate method for controlled rupture of the cells.

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References

  1. Fritsch A (1975) Preparative density gradient centrifugations. Beckman Instruments International S.A, Geneva

    Google Scholar 

  2. Opperdoes FR (1977) Particle-bound enzymes in the bloodstream form of Trypanosoma brucei. Eur J Biochem 76(1):21–28

    Article  CAS  Google Scholar 

  3. Pabón MA, Cáceres AJ, Gualdrón M, Quiñones W, Avilán L, Concepción JL (2007) Purification and characterization of hexokinase from Leishmania mexicana. Parasitol Res 100(4):803–810

    Article  Google Scholar 

  4. Rivero LA, Concepción JL, Quintero-Troconis E, Quiñones W, Michels PA, Acosta H (2016) Trypanosoma evansi contains two auxiliary enzymes of glycolytic metabolism: phosphoenolpyruvate carboxykinase and pyruvate phosphate dikinase. Exp Parasitol 165:7–15. https://doi.org/10.1016/j.exppara.2016.03.003

    Article  CAS  PubMed  Google Scholar 

  5. Rondón-Mercado R, Acosta H, Cáceres AJ, Quiñones W, Concepción JL (2017) Subcellular localization of glycolytic enzymes and characterization of intermediary metabolism of Trypanosoma rangeli. Mol Biochem Parasitol 216:21–29. https://doi.org/10.1016/j.molbiopara.2017.06.007

    Article  CAS  PubMed  Google Scholar 

  6. Graham JM, Rickwood D (2001) Biological centrifugation. Springer, Berlin

    Google Scholar 

  7. Claude A (1946) Fractionation of mammalian liver cells by differential centrifugation: II. Experimental procedures and results. J Exp Med 84:61–89

    Article  CAS  Google Scholar 

  8. Concepcion JL, Gonzalez-Pacanowska D, Urbina JA (1998) 3-Hydroxy-3-methyl-glutaryl-CoA reductase in Trypanosoma (Schizotrypanum) cruzi: subcellular localization and kinetic properties. Arch Biochem Biophys 352(1):114–120

    Article  CAS  Google Scholar 

  9. Fisher WD, Cline GB, Anderson NG (1964) Density gradient centrifugation in angle-head rotors. Anal Biochem 9:477–482

    Article  CAS  Google Scholar 

  10. Price CA (1982) Centrifugation in density gradients, 1st edn. Academic, Cambridge, MA

    Google Scholar 

  11. Rødahl E (1998) Ultracentrifugation. In: Encyclopedia of immunology, 2nd edn. Elsevier, Amsterdam, pp 2446–2448

    Chapter  Google Scholar 

  12. Opperdoes FR, Baudhuin P, Coppens I, De Roe C, Edwards SW, Weijers PJ, Misset O (1984) Purification, morphometric analysis, and characterization of the glycosomes (microbodies) of the protozoan hemoflagellate Trypanosoma brucei. J Cell Biol 98(4):1178–1184

    Article  CAS  Google Scholar 

  13. Taylor MB, Gutteridge WE (1987) Trypanosoma cruzi: subcellular distribution of glycolytic and some related enzymes of epimastigotes. Exp Parasitol 63(1):84–97

    Article  CAS  Google Scholar 

  14. Vertommen D, Van Roy J, Szikora JP, Rider MH, Michels PA, Opperdoes FR (2007) Differential expression of glycosomal and mitochondrial proteins in the two major life-cycle stages of Trypanosoma brucei. Mol Biochem Parasitol 158(2):189–201. https://doi.org/10.1016/j.molbiopara.2007.12.008.

    Article  PubMed  Google Scholar 

  15. Beaufay H, Jacques P, Baudhuin P, Sellinger OZ, Berthet J, de Duve C (1964) Tissue fractionation studies. 18. Resolution of mitochondrial fractions from rat liver into three distinct populations of cytoplasmic particles by means of density equilibrium in various gradients. Biochem J 92(1):184–205

    Article  CAS  Google Scholar 

  16. Antonenkov VD, Hiltunen JK (2012) Transfer of metabolites across the peroxisomal membrane. Biochim Biophys Acta 1822(9):1374–1386. https://doi.org/10.1016/j.bbadis.2011.12.011

    Article  CAS  PubMed  Google Scholar 

  17. Gualdron-López M, Vapola MH, Miinalainen IJ, Hiltunen JK, Michels PA, Antonenkov VD (2012) Channel-forming activities in the glycosomal fraction from the bloodstream form of Trypanosoma brucei. PLoS One 7(4):e34530. https://doi.org/10.1371/journal.pone.0034530

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Quiñones W, Urbina JA, Dubourdieu M, Luis Concepción J (2004) The glycosome membrane of Trypanosoma cruzi epimastigotes: protein and lipid composition. Exp Parasitol 106(3–4):135–149

    Article  Google Scholar 

  19. Quiñones W, Cáceres AJ, Ruiz MT, Concepción JL (2015) Glycosomal membrane proteins and lipids from Leishmania mexicana. Comp Biochem Physiol B Biochem Mol Biol 182:27–36. https://doi.org/10.1016/j.cbpb.2014.11.012

    Article  CAS  PubMed  Google Scholar 

  20. Lee JC, Timasheff SN (1981) The stabilization of proteins by sucrose. J Biol Chem 256(14):7193–7201

    CAS  PubMed  Google Scholar 

  21. Aman RA, Wang CC (1986) An improved purification of glycosomes from the procyclic trypomastigotes of Trypanosoma brucei. Mol Biochem Parasitol 21(3):211–220

    Article  CAS  Google Scholar 

  22. CAMARGO EP (1964) Growth and differentiation in Trypanosoma cruzi. I. Origin of metacyclic trypanosomes in liquid media. Rev Inst Med Trop Sao Paulo 6:93–100

    CAS  PubMed  Google Scholar 

  23. Cáceres AJ, Portillo R, Acosta H, Rosales D, Quiñones W, Avilan L, Salazar L, Dubourdieu M, Michels PA, Concepción JL (2003) Molecular and biochemical characterization of hexokinase from Trypanosoma cruzi. Mol Biochem Parasitol 126(2):251–262

    Article  Google Scholar 

  24. Bergmeyer HU (1983) Methods of enzymatic analysis. Academic, New York, NY

    Google Scholar 

  25. Lowry OH, Rosbrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    CAS  Google Scholar 

  26. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Laboratorio de Enzimología de Parásitos, Facultad de Ciencias, Universidad de Los Andes, Mérida, Venezuela

    Héctor Acosta & Wilfredo Quiñones

Authors
  1. Héctor Acosta
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  2. Wilfredo Quiñones
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Corresponding author

Correspondence to Wilfredo Quiñones .

Editor information

Editors and Affiliations

  1. School of Biological Sciences, University of Edinburgh, Edinburgh, UK

    Paul A. M. Michels

  2. School of Applied Sciences, University of Huddersfield, Huddersfield, UK

    Michael L. Ginger

  3. Faculty of Biology, Technion–Israel Institute of Technology, Haifa, Israel

    Dan Zilberstein

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Acosta, H., Quiñones, W. (2020). Isolation of Glycosomes from Trypanosoma cruzi. In: Michels, P., Ginger, M., Zilberstein, D. (eds) Trypanosomatids. Methods in Molecular Biology, vol 2116. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0294-2_37

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  • DOI: https://doi.org/10.1007/978-1-0716-0294-2_37

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0293-5

  • Online ISBN: 978-1-0716-0294-2

  • eBook Packages: Springer Protocols

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