Purification and characterization of two iron superoxide dismutases of Phytomonas sp. isolated from Euphorbia characias (plant trypanosomatids).
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 degrees 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.
Marín
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Instituto de Biotecnología, Facultad de Ciencias, Universidad de Granada, C/Severo Ochoa s/n, 18071 Granada, Spain.
Rodríguez-González
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I
Hitos
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Rosales
M J
MJ
Dollet
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Sánchez-Moreno
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M
D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D028481:Euphorbia; D006861:Hydrogen Peroxide / Q000378:metabolism; D007526:Isoelectric Point; D007527:Isoenzymes; D008970:Molecular Weight; D010935:Plant Diseases / Q000469:parasitology*; D011190:Potassium Cyanide / Q000378:metabolism; D011528:Protozoan Infections / Q000469:parasitology*; D013482:Superoxide Dismutase / Q000302:isolation & purification* / Q000378:metabolism; D014351:Trypanosomatina / Q000201:enzymology* / Q000378:metabolism
D007527:Isoenzymes; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D011190:Potassium Cyanide