High field31 P nuclear magnetic resonance spectroscopy showed that inorganic pyrophosphate (P 2 O 7 4؊ ) is more abundant than ATP in Trypanosoma cruzi, the causative agents of Chagas' disease. These results were confirmed by specific analytical assays, which showed that in epimastigotes, the concentrations of inorganic pyrophosphate and ATP were 194.7 ؎ 25.9 and 37.6 ؎ 5.5 nmol/mg of protein, respectively, and for the amastigote form, the corresponding concentrations were 358.0 ؎ 17.0 and 36.0 ؎ 1.9 nmol/mg of protein. High performance liquid chromatographic analysis of perchloric acid extracts of epimastigotes labeled for 3 h with 32 P-orthophosphate showed a significant incorporation of the precursor into inorganic pyrophosphate. Inorganic pyrophosphate was not uniformly distributed in T. cruzi but was shown by 31 P-NMR and chemical analysis to be particularly associated with acidocalcisomes, organelles shown previously to contain large amounts of phosphorus and various elements. Electron microscopy analysis of pyrophosphatase-treated permeabilized epimastigotes showed disappearance of the electron density of the acidocalcisomes. Nonmetabolizable analogs of pyrophosphate, currently used for the treatment of bone resorption disorders, selectively inhibited the proliferation of intracellular T. cruzi amastigotes and produced a profound suppression in the number of circulating trypomastigotes in mice with an acute infection of T. cruzi, offering a potentially new route to chemotherapy.Infections caused by Trypanosoma cruzi are among the most widespread parasitic diseases in Latin America and are responsible for heavy socioeconomic losses. There is therefore considerable interest in developing novel chemotherapeutic approaches, based on unique aspects of the structure and metabolism of this parasite. T. cruzi develops intracellularly in its vertebrate hosts and is confronted during its life cycle with drastic changes in its microenvironment. Survival through such complex environmental changes requires appropriate reserves of carbon and energy sources, as well as signaling species, such as Ca 2ϩ . Recent work has led to the identification of an acidic calcium pool (acidocalcisome) in this organism (1-4) that contains most of its cellular Ca 2ϩ , together with large amounts of magnesium, sodium, zinc, and phosphorus (3). However, the precise chemical nature of the phosphorus compound(s) in these vacuoles was not determined.In this work, we used 31 P nuclear magnetic resonance (NMR) spectroscopy to investigate the nature of the acidocalcisomal phosphorus.31 P NMR has been extensively used in the past to study the energy metabolism of several microorganisms, as well as that of living vertebrate tissues (5, 6). It has been particularly useful in the identification of phosphorus-based storage compounds, such as polyphosphates (7,8), and sugar phosphates and diphosphates (9, 10). Our results indicate that pyrophosphate is more abundant than ATP in the replicating forms of the parasite and is partly located in acidocalci...

High resolution31 P nuclear magnetic resonance spectra at 303.6 MHz (corresponding to a 1 H resonance frequency of 750 MHz) have been obtained of perchloric acid extracts of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major, the causative agents of African sleeping sickness, Chagas' disease, and leishmaniasis. Essentially complete assignments have been made based on chemical shifts and by direct addition of authentic reference compounds. The results indicate the presence of high levels of short chain condensed polyphosphates: di-, tri-, tetra-, and pentapolyphosphate.31 P NMR spectra of purified T. brucei, T. cruzi, and L. major acidocalcisomes, calcium and phosphorus storage organelles, indicate that polyphosphates are abundant in these organelles and have an average chain length of 3.11-3.39 phosphates. In the context of the recent discovery of several pyrophosphate-utilizing enzymes in trypanosomatids, the presence of these inorganic polyphosphates implies a critical role for these molecules in these parasites and a potential new route to chemotherapy.Infections caused by trypanosomatid parasites continue to be among the most widespread diseases in developing nations, and the increasing fraction of immunocompromised individuals in the global population further contributes to morbidity and economic loss. New chemotherapeutic leads based on unique aspects of parasite biology are therefore of great interest, particularly in the case of emerging resistance to traditional treatments.One approach to the discovery of new drug targets is to identify metabolic pathways in parasites that are essential for parasite survival but do not have an equivalent in the host. Then, one can pursue specific inhibitors of such metabolic activities as possible means of controlling the parasites without damaging the host. NMR spectroscopy has already been used by several groups to investigate the carbohydrate and/or phosphate metabolism of protozoa, including Crithidia fasciculata (1), Entamoeba histolytica (2), Trypanosoma cruzi (3, 4), Trypanosoma brucei (5, 6), Leishmania pifanoi (7), Babesia microti (8), and the free-living ciliate Tetrahymena pyriformis (9). In Tetrahymena, large levels of pyrophosphate were found, and more recently we have found similarly large levels of pyrophosphate in T. cruzi (10). These observations are of interest since a number of protozoa are already known to utilize pyrophosphate in addition to ATP in their high energy phosphate metabolic processes. For example, E. histolytica possesses a pyrophosphatedependent phosphofructokinase (11), a pyruvate-phosphate dikinase (12), a phosphoenolpyruvate carboxytransphosphorylase (13), and a pyrophosphate-acetate kinase (14). Several of these pyrophosphate-utilizing enzymes have also been discovered in other parasitic protists (see, e.g., the discussion in Ref. 15), and more recently a pyruvate-phosphate dikinase (16) and a plantlike vacuolar proton-translocating pyrophosphatase (17-19) were discovered in many trypanosomatids as well.An unusual character...

We report a simple new nuclear magnetic resonance (NMR) spectroscopic method to investigate order and dynamics in phospholipids in which inter-proton pair order parameters are derived by using high resolution 13C cross-polarization/magic angle spinning (CP/MAS) NMR combined with 1H dipolar echo preparation. The resulting two-dimensional NMR spectra permit determination of the motionally averaged interpair second moment for protons attached to each resolved 13C site, from which the corresponding interpair order parameters can be deducted. A spin-lock mixing pulse before cross-polarization enables the detection of spin diffusion amongst the different regions of the lipid molecules. The method was applied to a variety of model membrane systems, including 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/sterol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/sterol model membranes. The results agree well with previous studies using specifically deuterium labeled or predeuterated phospholipid molecules. It was also found that efficient spin diffusion takes place within the phospholipid acyl chains, and between the glycerol backbone and choline headgroup of these molecules. The experiment was also applied to biosynthetically 13C-labeled ergosterol incorporated into phosphatidylcholine bilayers. These results indicate highly restricted motions of both the sterol nucleus and the aliphatic side chain, and efficient spin exchange between these structurally dissimilar regions of the sterol molecule. Finally, studies were carried out in the lamellar liquid crystalline (L alpha) and inverted hexagonal (HII) phases of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). These results indicated that phosphatidylethanolamine lamellar phases are more ordered than the equivalent phases of phosphatidylcholines. In the HII (inverted hexagonal) phase, despite the increased translational freedom, there is highly constrained packing of the lipid molecules, particularly in the acyl chain region.

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We report the results of a solid-state 31 P nuclear magnetic resonance (NMR) spectroscopic investigation of the acidocalcisome organelles from Trypanosoma brucei (bloodstream form), Trypanosoma cruzi and Leishmania major (insect forms). The spectra are characterized by a broad envelope of spinning sidebands having isotropic chemical shifts at V V0, 3 37 and 3 321 ppm. These resonances are assigned to orthophosphate, terminal (K K) phosphates of polyphosphates and bridging (L L) phosphates of polyphosphates, respectively. The average polyphosphate chain length is V V3.3 phosphates. Similar results were obtained with whole L. major promastigotes.31 P NMR spectra of living L. major promastigotes recorded under conventional solution NMR conditions had spectral intensities reduced with respect to solution-state NMR spectra of acid extracts, consistent with the invisibility of the solid-state phosphates. These results show that all three parasites contain large stores of condensed phosphates which can be visualized by using magic-angle spinning NMR techniques. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.