Identification and Characterization of an Ecto-Pyrophosphatase Activity in Intact Epimastigotes of Trypanosoma rangeli

Abstract

In this study, we performed the molecular and biochemical characterization of an ecto-enzyme present in Trypanosoma rangeli that is involved with the hydrolysis of extracellular inorganic pyrophosphate. PCR analysis identified a putative proton-pyrophosphatase (H+-PPase) in the epimastigote forms of T. rangeli. This protein was recognized with Western blot and flow cytometry analysis using an antibody against the H+-PPase of Arabidopsis thaliana. Immunofluorescence microscopy confirmed that this protein is located in the plasma membrane of T. rangeli. 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 T. rangeli decreased with increasing cell proliferation in vitro, 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 T. rangeli that possibly plays an important role in phosphate metabolism of this protozoan.

Genome‐wide Identification and Structural Analysis of Pyrophosphatase Gene Family in Cotton

“…are highly conserved with >80% pairwise similarities. There are at least six homologous genes in rice that encode V‐PPase, five in corn, four in tobacco, three in wheat, and two each in A. thaliana , barley, grape, and nectarine (Fukuda et al, 2004; Fonseca‐de‐Souza et al, 2014). We have obtained the GhVP gene by RACE and RT‐PCR techniques, and demonstrated a strong hydrophobicity for GhVP, which showed 14 transmembrane regions as assessed by bioinformatics analysis.…”