Magic‐angle spinning ³¹P NMR spectroscopy of condensed phosphates in parasitic protozoa: visualizing the invisible

Abstract

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.

Phospholipid and glycolipid composition of acidocalcisomes of Trypanosoma cruzi

“…[5]), and assuming that these compounds are essentially concentrated in acidocalcisomes, the calculated concentration in the organelles is in the molar range (3-8 M) [1]. This is consistent with the detection of solidstate condensed phosphates by magic-angle spinning NMR techniques and with the very high electron density of acidocalcisomes in situ [10]. Other components of these organelles, such as carbohydrates or lipids could be involved in maintaining this physical configuration although this has not been investigated until now.…”

A Pyrophosphatase Regulating Polyphosphate Metabolism in Acidocalcisomes Is Essential for Trypanosoma brucei Virulence in Mice

“…To better understand the relation between polyP metabolism and its cellular role, studies were performed on the polyP composition and on the characterization of enzymes involved in the metabolism of these compounds. Short-and long-chain polyP and PP i were shown to be present in the acidocalcisomes of T. brucei bloodstream forms (BF) (12,13). The turnover of the acidocalcisome PP i pool is low (14), suggesting that PP i could be a product of polyP metabolism.…”

Bisphosphonates as Inhibitors of Trypanosoma cruzi Hexokinase

“…This is associated with the absence of a Pasteur effect in these parasites, in addition to their flexibility in utilizing glucose or amino acids as carbon and energy sources (10 -12). 3) Kinetoplastid parasites contain large stores of inorganic pyrophosphate (PP i ) and other short chain polyphosphates, which are by far the most abundant high energy compounds in these cells (13)(14)(15).…”

“…in the same buffer, preincubated with strong agitation (100 rpm) at 28°C in the presence of 50 M 228 or 302 for 6 h, and the experiment started by the addition of 10 mM 1-D-[ 13 C]glucose (Sigma); 1-ml samples were collected every 30 min, centrifuged at 35,000 ϫ g for 5 min, and the supernatants stored at Ϫ80°C until assayed by 13 C NMR. Perchloric acid extracts of the sediments (whole cells) were prepared as described before (14) and also stored at Ϫ80°C.…”

“…Perchloric acid extracts of the sediments (whole cells) were prepared as described before (14) and also stored at Ϫ80°C. 13 C NMR spectra were obtained using a 11.744 tesla Bruker ADVANCE 500 NMR spectrome-ter, which operates at 125.77 MHz for 13 C (500.13 MHz for 1 H); chemical shifts are reported with respect to external p-dioxane (66.5 ppm).…”