Magnesium-dependent ecto-ATPase activity in procyclic Trypanosoma brucei brucei is modulated by purine starvation.
LEITE, M.S.1; THOMAZ, R.R.1; PANIZZUTTI, R.A.2; VERCESI, A.E.3; MEYER-FERNANDES, J.R.3
Instituto de Bioquímica Médica, UFRJ - Rio de Janeiro 1; Departamento de Anatomia, Instituto de Ciências Biomédicas, UFRJ – Rio de Janeiro 2; Departamento de Patologia Clínica, UNICAMP - São Paulo 3.
Trypanosoma brucei brucei is the causative agent of livestock disease nagana that is related to African sleeping sickness in man. Parasitic protozoa, including T. brucei brucei, are unable to synthesize their own purines, relying on the salvage of preformed purines from the extracellular milieu. Ecto-ATPases are integral membrane proteins that catalyze the hydrolysis of extracellular nucleosides di- and tri-phosphate. These enzyme activities can be measured using intact cells. Previously, we described a magnesium-dependent ectonucleotide diphosphohydrolase activity in procyclic T. brucei brucei. To investigate the capacity of T. brucei brucei to obtain adenosine through sequential hydrolysis of ATP [ATP:ADP:AMP:Adenosine (ADO)] we analyzed the growth curves of cells in purine free trypanosome medium (PFTM: this medium resembles SDM79 - traditional complete medium for T. brucei brucei culture - without purines, and using dialysed fetal bovine serum) or PFTM supplemented with either 100 μM ATP or 100 μM ADO for 6 days. The growth curves with ATP or ADO were similar to that obtained with SDM79 medium, showing a doubling time about 12 h. Cells in PFTM grew less than SDM79 in all times assessed. The effects of purine starvation on ecto-ATPase activity in T. brucei brucei procyclics was investigated by growing the cells for 72 h in PFTM or PFTM supplemented with 100 μM ATP, a condition that restore growth rate to levels similar to those obtained with SDM79. This concentration of ATP represents purine repleted medium as shown by the growth curve for procyclics in PFTM. After 72 h of purine starvation, the ecto-ATPase activity had increased about 2.4-fold compared to activity of cells in purine repleted medium. We also described the sequential hydrolysis of ATP by intact cells with formation of ADP, AMP and ADO by HPLC method. Therefore T. brucei brucei procyclics respond to purine stress by increasing the ecto-ATPase activities. In this work we showed that ecto-ATPase activities can be modulated by purine starvation, suggesting the involvement of these ecto-ATPase activities in purine acquisition.
Supported by: CAPES, CNPq and FAPERJ
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