No abstract

Acidocalcisomes are acidic calcium storage organelles found in several microorganisms. They are characterized by their acidic nature, high electron density, high content of polyphosphates and several cations. Electron microscopy contrast tuned images of Herpetomonas sp. showed the presence of several electron dense organelles ranging from 100 to 300 nm in size. In addition, X-ray element mapping associated with energy-filtering transmission electron microscopy showed that most of the cations, namely Na, Mg, P, K, Fe and Zn, are located in their matrix. Using acridine orange as an indicator dye, a pyrophosphate-driven H + uptake was measured in cells permeabilized by digitonin. This uptake has an optimal pH of 6.5-6.7 and was inhibited by sodium fluoride (NaF) and imidodiphosphate (IDP), two H + -pyrophosphatase inhibitors. H + uptake was not promoted by ATP. Addition of 50 M Ca 2+ induced the release of H + , suggesting the presence of a Ca 2+ /H + countertransport system in the membranes of the acidic compartments. Na + was unable to release protons from the organelles. The pyrophosphate-dependent H + uptake was dependent of ion K + and inhibited by Na + Herpetomonas sp. immunolabeled with monoclonal antibodies raised against a Trypanosoma cruzi V-H + -pyrophosphatase shows intense fluorescence in cytoplasmatic organelles of size and distribution similar to the electron-dense vacuoles.Together, these results suggest that the electron dense organelles found in Herpetomonas sp. are homologous to the acidocalcisomes described in other trypanosomatids. They possess a vacuolar H + -pyrophosphatase and a Ca 2+ /H + antiport. However, in contrast to the other trypanosomatids so far studied, we were not able to measure any ATP promoted H + transport in the acidocalcisomes of this parasite.

ATP-dependent Ca2+ uptake was studied in a subcellular fraction from Herpetomonas sp. prepared by mechanical disruption and using 45Ca2+ as a tracer. The uptake was stimulated by Ca2+ with a K0.5 of 0.1 microm and a Hill number (nH)=2.8+/-0.4. The Ca2+-dependent ATP hydrolysis was optimal at pH 7.0 and had a Ca2+ dependence identical to uptake. The uptake was highly stimulated by oxalate whereas calmodulin had no activating effect. ATP stimulated Ca2+ uptake with a biphasic pattern that resembled the curves described for the purified preparations of rabbit sarcoplasmic reticulum. The ATP stimulation is described as the sum of two Michaelis-Menten curves with Km1=0.25+/-0.19 microm and Km2=29.6+/-6.8 microm. GTP or UTP could also promote Ca2+ uptake, but with less efficiency than ATP. Vanadate inhibited the uptake with low apparent affinity. Thapsigargin and cyclopiazonic acid were almost ineffective. The Ca2+ uptake was insensitive to H+ ionophores and to bafilomycin suggesting no participation of acidocalcisomes. The results are comparable to those obtained using cells permeabilized with digitonin and using arsenaze III as Ca2+ indicator. The Ca2+ uptake activity described here seems to belong to the endoplasmic reticulum of Herpetomonas sp. and is suitable for further studies on the mechanisms of calcium homeostasis in parasites.