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Abstract

  1. Top of page
  2. Abstract
  3. REFERENCES

Trypanothione reductase is thought to be important in maintaining an intracellular reducing environment in trypanosomatids. To investigate the role of trypanothione reductase we transfected Leishmania donovani and Trypanosoma cruzi with an expression vector containing the L. donovani trypanothione reductase gene and achieved over-expression of enzyme activity (10–14-fold) in transformed cells. Following treatment of L. donovani cells with the thiol-oxidizing agent diamide, the ability to regenerate dihydrotrypanothione from trypanothione disulphide was considerably enhanced in cells which over-expressed trypanothione reductase. However, the growth of transformed and control cells was equally sensitive to inhibition by nifurtimox, nitrofurazone and gentian violet, drugs that are thought to act by inducing oxidative damage. Likewise, growth of transformed and control cells were equally susceptible to inhibition by hydrogen peroxide, and control and transformed L. donovani promastigotes metabolized hydrogen peroxide at comparable rates. Thus, these experiments suggest that the ability to regenerate dihydrotrypanothione from trypanothione disulphide is not a rate-limiting step in the metabolism of hydrogen peroxide.

Abbreviations
GSH

glutathione

GSSG

glutathione disulphide

PCR

polymerase chain reaction

GspdSH

glutathionylspermidine

T(SH)2

dihydrotrypanothione

T(S)2

trypanothione disulphide

IC50

concentration which inhibits growth by 50%

Enzymes
 

Glutathione reductase (EC 1.6.4.2)

 

trypanothione reductase (EC 1.6.4.8)

 

trypanothione peroxidase (EC 1.11.1.–)

REFERENCES

  1. Top of page
  2. Abstract
  3. REFERENCES
  • 1
    World Health Organization (1990) Chagas disease: frequency and geographical distribution, Weekly Epidemiol. Res. 65, 257264.
  • 2
    Ashford, R. W., Desjeux, P. & de Raadt, P. (1992) Estimation of population at risk of infection and number of cases of leishmaniasis, Parasitol. Today 8, 104105.
  • 3
    Gustafsson, L. L., Beerman, B. & Abdi, Y. A. (1987) Handbook of drugs for tropical parasitic diseases, Taylor & Francis Ltd, London .
  • 4
    Shames, S. L., Fairlamb, A. H., Cerami, A. & Walsh, C. T. (1986) Purification and characterization of trypanothione reductase from Crithidia fasciculata, a newly discovered member of the family of disulphide-containing flavoprotein reductases, Biochemistry 25, 35193526.
  • 5
    Henderson, G. B. & Fairlamb, A. H. (1987) Trypanothione metabolism: a chemotherapeutic target in trypanosomatids, Parasitol. Today 3, 312315.
  • 6
    Fairlamb, A. H., & Cerami, A. (1992) Metabolism and functions of trypanothione in the kinetoplastida, Annu. Rev. Microbiol. 46, 695729.
  • 7
    Shames, S. L., Kimmel, B. E., Peoples, O. P., Agabian, N. & Walsh, C. T. (1988) Trypanothione reductase of Trypanosoma congolense: gene isolation, primary sequence determination, and comparison to glutathione reductase, Biochemistry 27, 50145019.
  • 8
    Sullivan, F. X. & Walsh, C. T. (1991) Cloning, sequencing, overproduction and purification of trypanothione reductase from Trypanosoma cruzi, Mol. Biochem. Parasitol. 44, 145148.
  • 9
    Field, H., Cerami, A. & Henderson, G. B. (1992) Cloning, sequencing, and demonstration of polymorphism in trypanothione reductase from Crithidia fasciculata, Mol. Biochem. Parasitol. 50, 4756.
  • 10
    Aboagye-Kwarteng, T., Smith, K. & Fairlamb, A. H. (1992) Molecular characterization of the trypanothione reductase gene from Crithidia fasciculata and Trypanosoma brucei: comparison with other flavoprotein disulphide oxidoreductases with respect to substrate specificity and catalytic mechanism, Mol. Microbiol. 6, 30893099.
  • 11
    Taylor, M. C. (1992) The trypanothione reductase gene of Leishmania donovani, Ph.D. thesis, University of London.
  • 12
    Hunter, W. N., Bailey, S., Habash, J., Harrop, S. J., Helliwell, J. R., Aboagye-Kwarteng, T., Smith, K. & Fairlamb, A. H. (1992) Active site of trypanothione reductase: a target for rational drug design. J. Mol. Biol. 227, 322333.
  • 13
    Henderson, G. B., Fairlamb, A. H., Ulrich, P. & Cerami, A. (1987) Substrate specificity of the flavoprotein trypanothione reductase from Crithidia fasciculata, Biochemistry 26, 30233027.
  • 14
    Krauth-Siegel, R. L., Enders, B., Henderson, G. B., Fairlamb, A. H. & Schirmer, R. H. (1987) Trypanothione reductase from Trypanosoma cruzi. Purification and characterization of the crystalline enzyme, Eur. J. Biochem. 164, 123128.
  • 15
    Bailey, S., Smith, K., Fairlamb, A. H. & Hunter, W. N (1993) Substrate interactions between trypanothione reductase and N1-glutathionylspermidine disulphide at 0.28-nm resolution, Eur. J. Biochem. 213, 6775.
  • 16
    Mattock, N. M. & Peters, W. (1975) The experimental chemotherapy of leishmaniasis. II: the activity in tissue culture of some antiparasitic and antimicrobial compounds in clinical use, Ann. Trop. Med. Parasitol. 69, 359371.
  • 17
    Hakerkorn, A. (1979) The effect of nifurtimox on experimental infections with Trypanosomatidae other than Trypanosoma cruzi, Zentralbl. Bakteriol. Mikrobiol. Hyg. I. Abt. Orig. 244, 331338.
  • 18
    Docampo, R. & Moreno, S. N. J. (1984) in Free radicals in biology, pp. 243288, Academic Press, New York .
  • 19
    Docampo, R. (1990) Sensitivity of parasites to free radical damage by antiparasitic drugs, Chem. Biol. Interact. 73, 127.
  • 20
    Docampo, R., Moreno, S. N. J., Gadelha, F. R., De Souza, W. & Cruz, F. S. (1988) Prevention of Chagas' disease resulting from blood transfusion by treatment of blood: toxicity and mode of action of gentian violet, Biomed. Environ. Sci. 1, 406413.
  • 21
    Docampo, R., Moreno, S. N. J. & Cruz, F. S. (1988) Enhancement of the cytotoxicity of crystal violet against Trypanosoma cruzi in the blood by ascorbate, Mol. Biochem. Parasitol. 27, 241248.
  • 22
    Henderson, G. B., Fairlamb, A. H. & Cerami, A. (1987) Trypanothione dependent peroxide metabolism in Crithidia fasciculata and Trypanosoma brucei, Mol. Biochem. Parasitol. 24, 3945.
  • 23
    Penketh, P. G., Kennedy, W. P. K., Patton, C. L. & Sartorelli, A. C. (1987) Trypanosomatid hydrogen peroxide metabolism, FEBS Lett. 221, 427431.
  • 24
    Penketh, P. G. & Klein, R. A. (1986) Hydrogen peroxide metabolism in Trypanosoma brucei, Mol. Biochem. Parasitol. 20, 111121.
  • 25
    Kelly, J. M., Ward, H. W., Miles, M. A. & Kendall, G. (1992) A shuttle vector which facilitates the expression of transfected genes in Trypanosoma cruzi and Leishmania, Nucleic Acids Res. 15, 39633969.
  • 26
    Kendall, G., Wilderspin, A. F., Ashall, F., Miles, M. A. & Kelly, J. M. (1990) Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase does not conform to the ‘hotspot’ topogenic signal model, EMBO J. 9, 27512758.
  • 27
    Blaxter, M. L., Miles, M. A. & Kelly, J. M. (1988) Specific serodiagnosis of visceral leishmaniasis using a Leishmania donovani antigen identified by expression cloning, Mol. Biochem. Parasitol. 44, 6372.
  • 28
    Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, Plainsville , NY .
  • 29
    Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principal of protein-dye binding, Anal. Biochem. 72, 248254.
  • 30
    Shim, H., & Fairlamb, A. H. (1988) Levels of polyamines, glutathione and glutathione-spermidine conjugates during growth of the insect trypanosomatid Crithidia fasciculata, J. Gen. Microbiol. 134, 807817.
  • 31
    Homan-Muller, J. W. T., Weening, R. S. & Roos, D. (1975) Production of hydrogen peroxide by phagoctytizing human granulocytes, J. Lab. Clin. Med. 85, 198207.
  • 32
    Kosower, N. S., & Kosower, E. M. (1987) Formation of disulphides with diamide, Methods Enzymol. 143, 264270.
  • 33
    Docampo, R., Moreno, S. N. J., Muniz, R. P. A., Cruz, F. S. & Mason, R. P. (1983) Light-enhanced free radical formation and trypanocidal action of gentian violet (crystal violet), Science 220, 12921295.
  • 34
    Reske, K., Cruz, F. S. & Docampo, R. (1986) Photosensitization by the trypanocidal agent crystal violet. Type I versus type II reactions, Chem. Biol. Interact. 58, 161172.
  • 35
    Ter Kuile, B. H. & Opperdoes, F. R. (1993) Uptake and turnover of glucose in Leishmania donovani, Mol. Biochem. Parasitol. 60, 313322.
  • 36
    Lohrer, H. & Krauth-Siegel, R. L. (1990) Purification and characterisation of lipoamide dehydrogenase from Trypanosoma cruzi, Eur. J. Biochem. 194, 863869.
  • 37
    Bowman, I. B. R. (1974) Intermediary metabolism of pathogenic flagellates, Ciba Found. Symp. 20, 255271.
  • 38
    Marr, J. J. (1980) Carbohydrate metabolism in leishmania, Biochemistry and physiology of protozoa, 2nd edn, vol. 3 (Levandowsky, M. & Hutner, S. H., eds) Academic Press, New York .
  • 39
    Le Trang, N., Meshnick, S. R., Kitchener, K., Eaton, J. W. & Cerami, A. (1983) Iron containing superoxide dismutase from Crithidia fasciculata: purification, characterization, and similarity to leishmanial and trypanosomal enzymes, J. Biol. Chem. 258, 125130.
  • 40
    Boveris, A. & Stoppani, A. O. (1977) Hydrogen peroxide generation in Trypanosoma cruzi, Experientia 33, 13061308.
  • 41
    Henderson, G. B., Ulrich, P., Fairlamb, A. H., Rosenberg, I., Pereira, M., Sela, M. & Cerami, A. (1988) “Subversive” substrates for the enzyme trypanothione disulphide reductase: alternative approach to chemotherapy of Chagas disease, Proc. Natl Acad. Sci. USA 85, 53745378.
  • 42
    Gadelha, F. R., Moreno, S. N. J., De Souza, W., Cruz, F. S. & Docampo, R. (1989) The mitochondrion of Trypanosoma cruzi is a target of crystal violet toxicity, Mol. Biochem. Parasitol. 34, 117126.
  • 43
    Awad, S., Henderson, G. B., Cerami, A., & Held, K. D. (1992) Effects of trypanothione on the biological activity of irradiated transforming DNA, Int. J. Radiat. Biol. 62, 401407.