Isolation and Analysis of Acidocalcisomes

Lysates from epimastigotes (2 × 10⁹ parasites grown in glass Erlenmeyer flasks with mild agitation) were fractionated using a high-density gradient of iodixanol to isolate acidocalcisomes, as previously described (42 (link)). The fraction containing acidocalcisomes was resuspended in intracellular buffer, and 0.1 ml was resuspended in 3-ml quartz cuvettes for acridine orange incorporation measurements, as detailed below. Additions were made as detailed in figure legends. To analyze the content of the obtained fractions, we performed immunoblotting detection using anti-HAL, anti-VP1, and anti-PPDK. A vacuolar H⁺-pyrophosphatase (PPase) assay was carried out by measuring the AMDP-sensitive P_i release activity in the isolated fractions. P_i release was quantified using a malachite green assay (43 (link)). Ammonia release in acidocalcisome-enriched vesicles was performed in the presence/absence of l-histidine using an ammonia detection kit following the manufacturer’s instructions (Sigma-Aldrich).