title: Trypanosoma cruzi: effects of heat shock on ecto-ATPase activity.
abstract: In this work, we demonstrate that Trypanosoma cruzi Y strain epimastigotes exhibit Mg2+-dependent ecto-ATPase activity that is stimulated by heat shock. When the epimastigotes were incubated at 37°C for 2h, the ecto-ATPase activity of the cells was 43.95±0.97 nmol Pi/h×10(7) cells, whereas the ecto-ATPase activity of cells that were not exposed to heat shock stress was 16.97±0.30 nmol Pi/h×10(7) cells. The ecto-ATPase activities of cells, that were exposed or not exposed to heat shock stress had approximately the same Km values (2.25±0.26 mM ATP and 1.55±0.23 mM ATP, respectively) and different Vmax values. The heat-shocked cells had higher Vmax values (54.38±3.07 nmol Pi/h×10(7) cells) than the cells that were not exposed to heat shock (19.38±1.76 nmol Pi/h×10(7) cells). We also observed that the ecto-phosphatase and ecto-5'nucleotidase activities of cells that had been incubated at 28°C or 37°C were the same. Interestingly, cycloheximide, an inhibitor of protein synthesis, suppressed the heat shock effect of ecto-ATPase activity on T. cruzi. The Mg2+-dependent ecto-ATPase activity from the Y strain (high virulence) was approximately 2-fold higher than that of Dm28c (a clone with low virulence). In addition, these two strains presented different responses to heat shock with regard to their ecto-ATPase activities; Y strain epimastigotes had a stimulation of 2.52-fold while the Dm28c strain had a 1.71-fold stimulation. In this context, the virulent trypomastigote form of T. cruzi, Dm28c, had an ecto-ATPase activity that was more than 7-fold higher (66.67±5.98 nmol Pi/h×10(7) cells) than that of the insect epimastigote forms (8.91±0.76 nmol Pi/h×10(7) cells). This difference increased to approximately 10-fold when both forms were subjected to heat shock stress (181.14±16.48 nmol Pi/h×10(7) cells for trypomastigotes and 16.71±1.17 nmol Pi/h×10(7) cells for epimastigotes at 37°C). The ecto-ATPase activity of a plasma membrane-enriched fraction obtained from T. cruzi epimastigotes was not increased by heat treatment, which suggested that cytoplasmic components had an influence on enzyme activation by heat shock stress.
authors.affiliation: Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro-UFRJ, Brazil.
authors.forename: Naira Lígia Lima
authors.initials: NL
authors.lastname: Giarola
authors.forename: Elmo Eduardo
authors.initials: EE
authors.lastname: de Almeida-Amaral
authors.forename: Itallo
authors.initials: I
authors.lastname: Collopy-Júnior
authors.forename: André Luiz
authors.initials: AL
authors.lastname: Fonseca-de-Souza
authors.forename: David
authors.initials: D
authors.lastname: Majerowicz
authors.forename: Lisvane Silva
authors.initials: LS
authors.lastname: Paes
authors.forename: Katia C
authors.initials: KC
authors.lastname: Gondim
authors.forename: José Roberto
authors.initials: JR
authors.lastname: Meyer-Fernandes
mesh_terms: D015720:5'-Nucleotidase / Q000235:genetics / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D015153:Blotting, Western; D017301:Ca(2+) Mg(2+)-ATPase / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D003513:Cycloheximide / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D018869:Heat-Shock Response / Q000187:drug effects / Q000502:physiology*; D006358:Hot Temperature / Q000009:adverse effects*; D006801:Humans; D006868:Hydrolysis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013997:Time Factors; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics
chemical_list: D011500:Protein Synthesis Inhibitors; D000255:Adenosine Triphosphate; D003513:Cycloheximide; D015720:5'-Nucleotidase; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; C027201:ectoATPase