Skip to main content
SpringerLink
Log in

Differential energetic metabolism during Trypanosoma cruzi differentiation. II. Hexokinase, phosphofructokinase and pyruvate kinase

  • Original Article
  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Summary

The activities of hexokinase (ATP:hexose-6-phosphate transferase, E.C. 2.7.1.1), phosphofructokinase (ATP: fructose-6-phosphate 1-phosphotransferase, E. C. 2.7.1.11) and pyruvate kinase (ATP: pyruvate transferase, E.C. 2.7.1.40), and their kinetic behaviour in two morphological forms of Trypanosoma cruzi (epimastigotes and metacyclic trypomastigotes) have been studied. The kinetic responses of the three enzymes to their respective substrates were normalized to hyperbolic forms on a velocity versus substrate concentration plots. Hexokinase and phosphofructokinase showed a higher activity in epimastigotes than in metacyclics, whereas pyruvate kinase had similar activity in both forms of the parasite. The specific activity of hexokinase from epimastigotes was 102.00 mUnits/mg of protein and the apparent Km value for glucose was 35.4 μM. Metacyclic forms showed a specific activity of 55.25 mUnits/mg and a Km value of 46.3 μM. The kinetic parameters (specific activity and Km for fructose 6-phosphate) of phosphofructokinase for epimastigotes were 42.60 mUnits/mg and 0.31 mM and for metacyclics 13.97 mUnits/mg and 0.16 mM, respectively. On the contrary, pyruvate kinase in both forms of T. cruzi did not show significant differences in its kinetic parameters. The specific activity in epimastigotes was 37.00 mUnits/mg and the Km for phosphoenolpyruvate was 0.47 mM, whereas in metacyclics these values were 42.94 mUnits/mg and 0.46 mM, respectively. The results presented in this work, clearly demonstrate a quantitative change in the glycolytic pathway of both culture forms of T. cruzi.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

NNN:

Novy-Nicolle-McNeal medium

Eagle's MEM:

Eagle's Minimal Essential Medium with Earle's salts

IFCS:

heat Inactivated Fetal Calf Serum 56°C, 30 min)

Tris:

tris(hydroxymethyl) aminomethane

EDTA:

Ethylenediaminetetraacetic Acid

References

  1. Brener Z: Biology of Trypanosoma cruzi. Anna Rev Microbiol 27: 347–382, 1973

    Google Scholar 

  2. De Sonza W: Cell biology of Trypanosoma cruzi. Int Rev Cytol 86: 197–283, 1984

    Google Scholar 

  3. Hoare CA: The trypanosomes of mammals. Blackwell Scientific Publications, Oxford and Cambridge, 1972

    Google Scholar 

  4. Brener Z, Alvarenga NJ: Life cycle of T. cruzi in the vector. In American Trypanosomiasis Research. Sci Publ No 318, PAHO, Washington DC, 1976, pp 83–86

    Google Scholar 

  5. Contreras VT, Morel CM, Goldenberg S: Stage specific gene expression precedes morphological changes during Trypanosoma cruzi metacyclogenesis. Mol Biochem Parasitol 14: 83–96, 1985

    Google Scholar 

  6. Contreras VT, Salles JM, Thomas N, Morel CM, Goldenberg S: In vitro differentiation of Trypanosoma cruzi under chemically defined conditions. Mol Biochem Parasitol 16: 315–327,1985

    Google Scholar 

  7. Nagakura K, Tachibana H, Kaneda Y: Alteration of the cell surface acid phosphatase concomitant with the morphological transformation in Trypanosoma cruzi. Comp Biochem Physiol 81B: 815–817, 1985

    Google Scholar 

  8. Boné GJ, Parent G: Stearic acid, an essential growth factor for Trypanosoma cruzi. J Gen Microbiol 31: 261–266, 1963

    Google Scholar 

  9. Brun R, Jenni L: Cultivation of African and South American trypanosomes of medical or veterinary importance. Br Med Bull 41: 122–129, 1985

    Google Scholar 

  10. Adroher FJ, Lupiáñez JA, Osuna A: Influence of saccharides and sodium chloride on growth and differentiation of Trypanosoma cruzi. Cell Differ 22: 165–170, 1988

    Google Scholar 

  11. Funayama S, Funayama S, Ito I, Veiga LA: Trypanosoma cruzi: Kinetic properties of glucose 6-phosphate dehydrogenase. Exp Parasitol 43: 376–381, 1977

    Google Scholar 

  12. Wood DE: Trypanosoma cruzi: Fatty acid metabolism in vitro. Exp Parasitol 37: 60–66, 1975

    Google Scholar 

  13. Wood DE, Schiller EL: Trypanosoma cruzi: Comparative fatty acid metabolism of the epimastigotes and trypomastigotes in vitro. Exp Parasitol 38: 202–207, 1975

    Google Scholar 

  14. Adroher FJ, Osuna A, Lupiáñez JA: Fructose 1,6-bisphosphatase activity in two Trypanosoma cruzi morphological forms. J Parasitol 73: 438–441, 1987

    Google Scholar 

  15. Lupiáñez JA, Adroher FJ, Vargas AM, Osuna A: Differential behaviour of glucose 6-phosphate dehydrogenase in two morphological forms of Trypanosoma cruzi. Int J Biochem. 19: 1085–1089, 1987

    Google Scholar 

  16. Adroher FJ, Osuna A, Lupiáñez JA: Differential energetic metabolism during Trypanosoma cruzi differentiation. I. Citrate synthase, NADP-isocitrate dehydrogenase and succinate dehydrogenase. Arch Biochem Biophys 267: 252–261,1988

    Google Scholar 

  17. Gutteridge WE: Trypanosoma cruzi: Recent biochemical advances. Trans R Soc Trop Med Hyg 75: 484–492, 1981

    Google Scholar 

  18. Cannata JJB, Cazzulo JJ: The aerobic fermentation of glucose by Trypanosoma cruzi. Comp Biochem Physiol 79B: 297–308,1984

    Google Scholar 

  19. Bowman IBR, Tobie EJ, Von Brand T: CO2 fixation studies with the culture form of Trypanosoma cruzi. Comp Biochem Physiol 9: 105–114, 1963

    Google Scholar 

  20. Cazzulo JJ, Franke de Cazzulo BM, Engel JC, Cannata JJB: End products and enzyme levels of aerobic glucose fermentation in trypanosomatids. Mol Biochem Parasitol 16:329–343,1985

    Google Scholar 

  21. Rogerson GW, Gutteridge WE: Catabolic metabolism in Trypanosoma cruzi. Int J Parasitol 10: 131–135, 1980

    Google Scholar 

  22. Opperdoes FR, Borst P: Localization of nine glycolytic enzymes in a microbody-like organelle in Trypanosoma brucei: the glycosome. FEBS Lett 80: 360–364, 1977

    Google Scholar 

  23. Opperdoes FR, Borst P, Bakker S, Leene W: Localization of glycerol-3-phosphate oxidase in the mitochondrion and NAD+-linked glycerol-3-phosphate dehydrogenase in the microbodies of the bloodstream form of Trypanosoma brucei. Eur J Biochem 76: 29–39, 1977

    Google Scholar 

  24. Opperdoes FR: Compartmentation of carbohydrate metabolism in trypanosomes. Annu Rev Microbiol 41: 127–152,1987

    Google Scholar 

  25. Taylor MB, Berghausen H, Heyworth P, Messenger N, Rees LJ, Gutteridge WE: Subcellular localization of some glycolytic enzymes in parasitic flagellated protozoa. Int J Biochem 11: 117–120,1980

    Google Scholar 

  26. Cannata JJB, Valle E, Docampo R, Cazzulo JJ: Subcellular localization of phosphoenolpyruvate carboxykinase in the trypanosomatids Trypanosoma cruzi and Crithidia fasciculata. Mol Biochem Parasitol 6: 151–160, 1982

    Google Scholar 

  27. Cannata JJB, Cazzulo JJ: Glycosomal and mitochondrial malate dehydrogenases in epimastigotes of Trypanosoma cruzi. Mol Biochem Parasitol 11: 37–49, 1984

    Google Scholar 

  28. Grace TDC: Establishment of four strains of cells from insect tissues grown in vitro. Nature 195: 788–789, 1962

    Google Scholar 

  29. Osuna A, Jiménez-Ortiz A, Lozano J: Medios de cultivo para la obtención de formas metacíclicas de Trypanosoma cruzi. Rev Ibér Parasitol 39: 129–133, 1979

    Google Scholar 

  30. Joshi MD, Jagannathan V: Hexokinase. I. Brain. Methods Enzymol 9: 371–375, 1966

    Google Scholar 

  31. Ling KH, Paetkau V, Marcus F, Lardy HA: Phosphofructokinase. I. Skeletal muscle. Methods Enzymol 9: 425–429, 1966

    Google Scholar 

  32. Valentine WN, Tanaka KR: Pyruvate kinase: clinical aspects. Methods Enzymol 9: 468–473, 1966

    Google Scholar 

  33. Fersht A: Enzyme structure and mechanism. 2nd edn. WH Freeman & Co., Reading and San Francisco, 1985

    Google Scholar 

  34. Atkins GL, Nimmo IA: A comparison of seven methods for fitting the Michaelis-Menten equation. Biochem J 149: 775–777,1975

    Google Scholar 

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

    CAS  PubMed  Google Scholar 

  36. Bradford MM: 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, 1976

    Article  CAS  PubMed  Google Scholar 

  37. Bergmeyer HU, Bernt E: D-glucose: Determination with glucose oxidase and peroxidase. In: HU Bergmeyer (ed). Methods of enzymatic analysis. Verlag-Chemie Academic Press, New York, 1974, pp 1205–1215

    Google Scholar 

  38. Urbina JA, Crespo A: Regulation of energy metabolism in Trypanosoma (Schizotrypanum) cruzi. I. Hexokinase and phosphofructokinase. Mol Biochem Parasitol 11: 225–239, 1984

    Google Scholar 

  39. Visser N, Opperdoes FR: Glycolysis in Trypanosoma brucei. Eur J Biochem 103: 623–632, 1980

    Google Scholar 

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

    Google Scholar 

  41. Tetley L, Coombs GH, Vickerman K: The distribution of cell organelles in Leishmania amastigotes as shown by three-dimensional reconstruction. Parasitology 87: xxxvi, 1983

    Google Scholar 

  42. Racagni GE, Machado de Domenech EE: Characterization of Trypanosoma cruzi hexokinase. Mol Biochem Parasitol 9: 181–188, 1983

    Google Scholar 

  43. Aguilar Z, Urbina JA: The phosphofructokinase of Trypanosoma (Schizotrypanum) cruzi: purification and kinetic mechanism. Mol Biochem Parasitol 21: 103–111, 1986

    Google Scholar 

  44. Taylor M, Gutteridge WE: The regulation of phosphofructokinase in epimastigote Trypanosoma cruzi. FEBS Lett 201:262–266,1986

    Google Scholar 

  45. Cáceres O, Fernándes JF: Glucose metabolism, growth and differentiation of Trypanosoma cruzi. Rev Brasil Biol 36: 397–410,1976

    Google Scholar 

  46. Lehmann DL: Comparative utilization of carbohydrates by culture forms of Trypanosoma (Schizotrypanum) cruzi and T. ranarum. Ann Trop Med Parasitol 57: 232–234, 1963

    Google Scholar 

  47. Michels PAM: Compartmentation of glycolysis in trypanosomes: a potential target for new trypanocidal drugs. Bio Cell 64: 157–164, 1988

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Departamento de Parasitología, Facultad de Farmacia, Universidad de Granada, 18071, Granada, Spain

    Francisco-Javier Adroher & Antonio Osuna

  2. Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Granada, 18001, Granada, Spain

    José A. Lupiáñez

Authors
  1. Francisco-Javier Adroher
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Antonio Osuna
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. José A. Lupiáñez
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Adroher, FJ., Osuna, A. & Lupiáñez, J.A. Differential energetic metabolism during Trypanosoma cruzi differentiation. II. Hexokinase, phosphofructokinase and pyruvate kinase. Mol Cell Biochem 94, 71–82 (1990). https://doi.org/10.1007/BF00223564

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00223564

Key words

Search

Navigation