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Purification and characterization of two iron superoxide dismutases of Phytomonas sp. isolated from Euphorbia characias (plant trypanosomatids)

Published online by Cambridge University Press:  10 June 2004

C. MARÍN ,
I. RODRÍGUEZ-GONZÁLEZ ,
A. B. HITOS ,
M. J. ROSALES ,
M. DOLLET  and
M. SÁNCHEZ-MORENO
C. MARÍN
Affiliation:
Instituto de Biotecnología. Facultad de Ciencias. Universidad de Granada, C/ Severo Ochoa s/n. 18071 Granada, Spain
I. RODRÍGUEZ-GONZÁLEZ
Affiliation:
Instituto de Biotecnología. Facultad de Ciencias. Universidad de Granada, C/ Severo Ochoa s/n. 18071 Granada, Spain
A. B. HITOS
Affiliation:
Instituto de Biotecnología. Facultad de Ciencias. Universidad de Granada, C/ Severo Ochoa s/n. 18071 Granada, Spain
M. J. ROSALES
Affiliation:
Instituto de Biotecnología. Facultad de Ciencias. Universidad de Granada, C/ Severo Ochoa s/n. 18071 Granada, Spain
M. DOLLET
Affiliation:
CIRAD, TA 30/G, Campus International de Baillarguet 34398, Montpellier Cedex 5, France
M. SÁNCHEZ-MORENO
Affiliation:
Instituto de Biotecnología. Facultad de Ciencias. Universidad de Granada, C/ Severo Ochoa s/n. 18071 Granada, Spain
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Abstract

Two superoxide dismutases (SODI and SODII) have been purified by differential centrifugation, fractionation with ammonium sulphate followed by chromatographic separation (ionic exchange and affinity), from a plant trypanosomatid isolated from Euphorbia characias, and then characterized for several biochemical properties. Both enzymes were insensitive to cyanide but sensitive to hydrogen peroxide, properties characteristic of iron-containing superoxide dismutase. SODI had a molecular mass of approximately 66 kDa, whereas the molecular mass of SODII was approximately 22 kDa, both enzymes showing single bands. The isoelectric points of SODI and SODII were 6·8 and 3·6, respectively. The enzymatic stability persisted at least for 6 months when the sample was lyophilized and preserved at −80 °C. Digitonin titration and subcellular fractionation showed that both enzymes were in the cytoplasmic fraction, although part of SODII isoenzyme was also associated with glycosomes. We assayed these activities (SOD) in 18 trypanosomatid isolates on isoelectric focusing gels, and have demonstrated that the SOD is a biochemical marker sufficient to identify a trypanosomatid isolated from a plant as belonging to the genus Phytomonas and to distinguish between a true Phytomonas and other trypanosomatids that are capable of causing transient infections in plants.

Keywords

Phytomonasplant trypanosomessuperoxide dismutasesbiochemical purification and characterization
Type
Research Article
Information
Parasitology , Volume 129 , Issue 1 , July 2004 , pp. 79 - 86
Copyright
© 2004 Cambridge University Press

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References

REFERENCES

BÉCUWE, P., GRATEPANCHE, S., FOURMAUX, M. N., VAN BEEUMEN, J., SAMYN, B., MERCEREAU-PUIJALON, O., TOUZEL, J. P., SLOMIANNY, C., CAMUS, D. & DIVE, D. ( 1996). Characterization of iron-dependent endogenous superoxide dismutase of Plasmodium falciparum. Molecular and Biochemical Parasitology 76, 125–134.CrossRefGoogle Scholar
BERGMEYER, H. U. ( 1974). Methods of Enzymatic Analysis, 2nd Edn. Academic Press, New York.
BEYER, W. F. & FRIDOVICH, I. ( 1987). Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions. Analytical Biochemistry 161, 559–566.CrossRefGoogle Scholar
BRADFORD, M. M. ( 1976). A refined and sensitive method for the quantification of microgram quantities of protein-dye binding. Analytical Biochemistry 72, 248.CrossRefGoogle Scholar
DOCAMPO, R. ( 1995). Antioxidant mechanisms. In Biochemistry and Molecular Biology of Parasites ( ed. Marr, J. J. & Müller, M.), pp. 147–160. Academic Press, London.CrossRef
DOLLET, M. ( 1984). Plant trypanosomes case study. In Identification and Characterization of Pest Organisms ( ed. Hawksworth, D. L. ), pp. 415–426. CAB International.
DOLLET, M., LÓPEZ, G., GENTY, P. & DZIDO, J. L. ( 1979). Récherches actuelles de l'I.R.H.O. sur les dépérissements du cocotier et du palmier á huile en Amérique du Sud, associés aux protozoaires flagellés intraphloémiques (Phytomonas). Oléagineux 34, 449–451.Google Scholar
FERNANDEZ-BECERRA, C., OSUNA, A., MULLER, E., DOLLET, M. & SANCHEZ-MORENO, M. ( 1996). Characterization of Phytomonas isolated from fruits by electrophoretic isoenzymes and kinetoplast-DNA analysis. FEMS Microbiology Letters 145, 463–468.CrossRefGoogle Scholar
GUERRINI, F., SEGUR, S., GARGANI, D., TIBAYRENC, M. & DOLLET, M. ( 1992). An isoenzyme analysis of the genus Phytomonas: genetic, taxonomic and epidemiologic significance. Journal of Protozoology 39, 516–521.CrossRefGoogle Scholar
HAMMOND, D. J., GUTTERIDGE, W. E. & OPPERDOES, F. R. ( 1981). A novel location for two enzymes of de novo pyrimidine biosynthesis in trypanosomes and leishmania. FEBS Letters 128, 27–29.CrossRefGoogle Scholar
ISMAIL, S. O., SKEIKY, Y. A., BHATIA, A., OMARA-OPYENE, L. A. & GEDAMU, L. ( 1994). Molecular cloning, characterization, and expresión in E. coli of iron superoxide dismutase cDNA from Leishmania donovani chagasi. Infection and Immunity 62, 657–664.Google Scholar
ISMAIL, S. O., PARAMCHUK, W., SKEIKY, Y. A., BHATIA, A., REED, S. G., BATHIA, A. & GEDAMU, L. ( 1997). Molecular cloning and characterization of two iron superoxide dismutase cDNAs from Trypanosoma cruzi. Molecular and Biochemical Parasitology 86, 187–197.CrossRefGoogle Scholar
KABIRI, M. & STEVERDING, D. ( 2001). Identification of a developmentally regulated iron superoxide dismutase of Trypanosome brucei. Journal of Biochemistry 360, 173–177.Google Scholar
LIOCHEV, S. I. & FRIDOVICH, I. ( 1997). Lucigenin luminescence as a measure of intracellular superoxide dismutase in Escherichia coli. Proceedings of the National Academy of Sciences, USA 94, 2891–2896.CrossRefGoogle Scholar
MARÍN, C., FERNÁNDEZ-RAMOS, C., QUESADA, J. M., ENTRALA, E. & SÁNCHEZ-MORENO, M. ( 2000). Biochemical characterization of a trypanosomatid isolated from the plant Amaranthus retroflexus. Memorias do Instituto Oswaldo Cruz 95, 641–647.CrossRefGoogle Scholar
MENARA, A., DOLLET, M., GARGANI, D. & LOUISE, C. ( 1988). Culture in vitro sur cellules d'invertébrés des Phytomonas sp. (Trypanosomatidae) associés au hartrot, maladie du cocotier. Comptes Rendus de l'Académie des Sciences, Paris, série III 307, 597–602.Google Scholar
MIRALLES, D., MARÍN, C., MAGÁN, R., FERNÁNDEZ-RAMOS, C., ENTRALA, E., CORDOVA, O., VARGAS, F. & SÁNCHEZ-MORENO, M. ( 2002). In vitro culture and biochemical characterization of six trypanosome isolates from Peru and Brazil. Experimental Parasitology 102, 23–29.CrossRefGoogle Scholar
PAOLETTI, F. & MOCALI, A. ( 1990). Determination of superoxide dismutase activity by purely chemical system based on NAD(P)H oxidation. Methods in Enzymology. 186, 209–265.CrossRefGoogle Scholar
PARAMCHUCK, W. J., ISMAIL, S. O., BHATIA, A. & GEDAMU, L. ( 1997). Cloning, characterization and overexpression of two iron superoxide dismutase cDNAs from Leishmania chagasi: role in pathogenesis. Molecular and Biochemical Parasitology 90, 203–221.Google Scholar
PARSONS, M., FURUYA, T., PAL, S. & KESSLER, P. ( 2001). Biogenesis and function of peroxisomes and glycosomes. Molecular and Biochemical Parasitology 115, 19–28.CrossRefGoogle Scholar
PODLIPAEV, S. ( 2001). The more insect trypanosomatids under study-the more diverse Trypanosomatidae appears. International Journal for Parasitology 31, 648–652.CrossRefGoogle Scholar
QUESADA, J. M., ENTRALA, E., FERNÁNDEZ-RAMOS, C., MARÍN, C. & SÁNCHEZ-MORENO, M. ( 2001). Phytomonas spp.: superoxide dismutase in plant trypanosomes. Molecular and Biochemical Parasitology 115, 123–127.CrossRefGoogle Scholar
SÁNCHEZ-MORENO, M., FERNÁNDEZ-BECERRA, C., FERNÁNDEZ-RAMOS, C., LUQUE, F., RODRIGUEZ-CABEZAS, M. N., DOLLET, M. & OSUNA, A. ( 1998). Trypanosomatid protozoa in plants of southern Spain: characterization by analysis of isoenzymes, kinetoplast DNA, and metabolic behavior. Parasitology Research 84, 354–361.CrossRefGoogle Scholar
SANCHEZ-MORENO, M., FERNANDEZ-BECERRA, C., MASCARO, C., ROSALES, M. J., DOLLET, M. & OSUNA, A. ( 1995). Isolation, in vitro culture, ultrastructure study and characterization by lectin-agglutination tests of Phytomonas isolated from tomatoes (Lycopersicon esculentum) and cherimoyas (Annona cherimolia) in southeastern Spain. Parasitology Research 81, 575–581.CrossRefGoogle Scholar
STEIGER, R. F., OPPERDOES, F. R. & BONTEMPS, J. ( 1980). Subcellular fractionation of Trypanosoma brucei blood-stream forms with special reference to hydrolases. European Journal of Biochemistry 105, 163–175.CrossRefGoogle Scholar
TEMPERTON, N. J., WILKINSON, S. R. & KELLY, J. M. ( 1996). Cloning of an Fe-superoxide dismutase gene homologue from Trypanosoma cruzi. Molecular and Biochemical Parasitology 76, 339–343.CrossRefGoogle Scholar
TEIXEIRA, M. M. G., SERRANO, M. G., NUNES, L. R., CAMPANER, M., BUCK, G. A. & CAMARGO, E. P. ( 1996). Trypanosomatidae: a spliced-leader-derived probe specific for the genus Phytomonas. Experimental Parasitology 84, 311–319.CrossRefGoogle Scholar
UTTARO, A. D., MIRKIN, N., RIDER, M. H., MICHELS, P. A. M. & OPPERDOES, F. R. ( 1999). Phytomonas sp. as a model of Trypanosomatid metabolism and drug. Memorias do Instituto Oswaldo Cruz 94 (Suppl. II), C19.Google Scholar
UTTARO, A. D., SÁNCHEZ-MORENO, M. & OPPERDOES, F. R. ( 1997). Genus-specific biochemical markers for Phytomonas spp. Molecular and Biochemical Parasitology 90, 337–342.CrossRefGoogle Scholar