Western blot and flow cytometry analysis of <i>T. rangeli</i> incubated with the anti-326 peptide sequence of the AVP2 antibody.

<p>Panel A: <i>T. rangeli</i> and <i>T. cruzi</i> Dm 28c epimastigote proteins (50 µg/lane) were separated using 8% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. <i>Lane 1</i>, immunoblot probed with antiserum against H⁺PPase (AVP2) in the total extract of <i>T. rangeli</i>. The H⁺PPase antibody recognized a polypeptide with an apparent molecular mass of 64 kDa. <i>Lane 2</i>, immunoblot probed against the total extract of <i>T. cruzi</i>. Panel B: The intact cells of <i>T. rangeli</i> (5×10⁶ cells) were fixed in paraformaldehyde and sodium cacodylate buffer and stained with the anti-326 peptide sequence of <i>A. thaliana</i> vacuolar H⁺PPase (AVP2) produced in rabbit and an Alexa 488-conjugated anti-rabbit secondary antibody produced in mouse, with an emission in the range of 488 nm (red). Abscissa: fluorescence intensity; Ordinate: Events/Positive cells. The gray color represents the autofluorescence of the cells (without antibody).</p

Immunolocalization of <i>T. rangeli</i> H⁺PPase using the anti-326 peptide sequence of the AVP2 antibody.

<p>Upper panels: cells permeabilized with Triton X-100; bottom panels: non-permeabilized cells, panels A and D: interferential differential contrast microscopy (DIC), panels B and E: immunofluorescence using anti-326 peptide sequence of AVP2 and Alexa 488; panels C and F: labeling of nuclear DNA with DAPI (2,6-diamino phenylindole). Bars: 10 µm.</p

Effect of the pH of the reaction medium on the ecto-PPase activity of <i>T. rangeli</i>.

<p>Intact <i>T. rangeli</i> cells (10⁷ cells) were incubated in a reaction medium containing a buffer solution of 100 mM MES - HEPES - Tris with the pH adjusted from pH 6.0 to pH 9.0 with the addition of HCl and using 1 mM PPi as a substrate. The data represent the mean ± standard error using at least three different cell suspensions.</p

Ecto-PPase activity and gene expression along the <i>T. rangeli</i> proliferation.

<p>Panel A: Intact <i>T. rangeli</i> cells (10⁶ cells/ml) were inoculated in LIT culture, and the cell density was estimated daily by counting aliquots in a Neubauer chamber hemocytometer (squares); intact cells of <i>T. rangeli</i> (10⁷ cells) collected daily were incubated in a cold buffer solution of 50 mM Tris-HCl, pH 7.2, 100 mM sucrose and 20 mM KCl, using 1 mM PPi as a substrate. The data represent the average of an experiment performed in triplicate (triangles). Panel B: <i>T. rangeli</i> cells (10⁸ cells) were homogenized in TRIzol, and the total RNA was extracted. RNA samples were used to synthesize the complementary DNA (cDNA), and RT-PCR reactions were performed. The PCR products were subjected to agarose gel electrophoresis and visualized with UV light. Lines: 72 h and 120 h represent the time of parasite growth. <i>TrGAPDH</i> amplification was used as the positive control.</p

Identification and Characterization of an Ecto-Pyrophosphatase Activity in Intact Epimastigotes of <i>Trypanosoma rangeli</i>

<div><p>In this study, we performed the molecular and biochemical characterization of an ecto-enzyme present in <i>Trypanosoma rangeli</i> that is involved with the hydrolysis of extracellular inorganic pyrophosphate. PCR analysis identified a putative proton-pyrophosphatase (H⁺-PPase) in the epimastigote forms of <i>T. rangeli</i>. This protein was recognized with Western blot and flow cytometry analysis using an antibody against the H⁺-PPase of <i>Arabidopsis thaliana</i>. Immunofluorescence microscopy confirmed that this protein is located in the plasma membrane of <i>T. rangeli</i>. Biochemical assays revealed that the optimum pH for the ecto-PPase activity was 7.5, as previously demonstrated for other organisms. Sodium fluoride (NaF) and aminomethylenediphosphonate (AMDP) were able to inhibit approximately 75% and 90% of the ecto-PPase activity, respectively. This ecto-PPase activity was stimulated in a dose-dependent manner by MgCl₂. In the presence of MgCl₂, this activity was inhibited by millimolar concentrations of CaCl₂. The ecto-PPase activity of <i>T. rangeli</i> decreased with increasing cell proliferation <i>in vitro</i>, thereby suggesting a role for this enzyme in the acquisition of inorganic phosphate (Pi). Moreover, this activity was modulated by the extracellular concentration of Pi and increased approximately two-fold when the cells were maintained in culture medium depleted of Pi. All of these results confirmed the occurrence of an ecto-PPase located in the plasma membrane of <i>T. rangeli</i> that possibly plays an important role in phosphate metabolism of this protozoan.</p></div

Effect of the divalent metals on the ecto-PPase activity of <i>T. rangeli</i>.

<p>Panel A: Intact <i>T. rangeli</i> cells (10⁷ cells) were incubated in a reaction medium containing a cold buffer solution of 50 mM Tris-HCl, pH 7.2, 100 mM sucrose and 20 mM KCl, using 1 mM PPi as substrate in the absence (control) or presence of MgCl₂, MnCl₂ or CalCl₂ in a final concentration of 2 mM. Panel B: Intact <i>T. rangeli</i> cells (10⁷ cells) were incubated in the reaction medium described above, using 1 mM PPi as a substrate in the absence of a metal (control), in the presence of increasing concentrations of MgCl₂ or in the presence of 5 mM MgCl₂ and increasing concentrations of CaCl₂. The data represent the mean ± standard error, using at least three different cell suspensions. *Denotes a statistically significant difference (<i>p</i><0.05) compared with the control (no addition). **Denotes a significant difference (<i>p</i><0.05) compared with the ecto-PPase activity in the presence of 5 mM MgCl₂.</p

Effect of inhibitors on the ecto-ATPase, ecto-phosphatase and ecto-PPase activities of the intact cells of <i>T. rangeli</i>.

<p>Intact <i>T. rangeli</i> cells (10⁷ cells) were used to measure each of the ecto-enzymatic activities in accordance with sections 7 and 8 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106852#s2" target="_blank">Materials and Methods</a>). The activities are expressed as a percentage of that measured under the control conditions, i.e., without other additions. The data represent the mean ± standard error, using at least three different cell suspensions. *Denotes significant inhibition compared with the enzymatic activities of the control (no inhibitor).</p><p>Effect of inhibitors on the ecto-ATPase, ecto-phosphatase and ecto-PPase activities of the intact cells of <i>T. rangeli</i>.</p