Atp disodium salt

Both ATP disodium salt (Sigma, St. Louis MO) and ADP sodium salt (Sigma, St. Louis MO) were dissolved in purified MQ water to make 100 mM stock solutions. ATPγS tetralithium (ATPγS, Tocris, Bristol UK) was dissolved in MQ water to make a 50 mM stock. ATP, ATPγS and ADP stocks were diluted to 1 mM (ATP, ATPγS) or 5 mM (ADP) in ASW for injection^{52 (link)}. For the pH effects experiment, 1 mM ATP was adjusted to pH 8.2 with NaOH. BzATP triethylammonium (Tocris, Bristol UK) was dissolved in MQ water to make a 10 mM stock and diluted to 300 uM in ASW for injection⁸⁴ . Oxidized ATP (oATP) (EMD Millipore, Burlington, MA) was dissolved in MQ water to make a 1 mM stock and then diluted to 100uM in ASW for injection^{85 (link)}. PPADS sodium salt (Cayman Chemical, Ann Arbor, MI) was dissolved in ASW to create a 50 mM stock and diluted to 1 mM for injection^{86 (link)}.

To evaluate ATP production with l-Ala as their sole energy source, the parasites (approximately 5 × 10⁷ cells per ml) were starved as described above and recovered or not (negative control) by incubation for 1 h in the presence of 5 mM His or Pro (as positive controls) or 5 mM l-Ala. The intracellular concentration of ATP in each sample was determined before and after recovery by using a luciferase assay according to the manufacturer’s instructions (Sigma). ATP concentrations were estimated by using a calibration curve (ATP disodium salt, Sigma); luminescence (λ570 nm) was detected using a SpectraMax i3 plate reader (Molecular Devices, Sunnyvale, CA).

The intracellular concentration of ATP under each condition was determined by using a luciferase assay according to the manufacturer’s instructions (Sigma). ATP concentrations were estimated by using a calibration curve (ATP disodium salt; Sigma). Luminescence (λ at 570 nm) was detected using a Synergy H1 multimode microplate reader (BioTek, USA) as described previously (86 (link)).

[γ-³²P]ATP and ⁴⁵CaCl₂ were purchased from Perkin-Elmer, USA. [³H]Glucose and ⁴⁵CaCl₂ were products of DuPont NEN. The Ca²⁺ ionophore A23187 (calcimycin), EGTA (ethylene glycol-bis (β-aminoethyl ether)-N,N,N’,N’-tetra acetic acid), Mops (4-morpholinepropanesulfonic acid), liquid scintillation cocktail (Sigma-Fluor S-4023), ATP disodium salt, LaCl₃, K-oxalate, benzene, 1,2-DCB was obtained from Sigma, Co. (St. Louis, MO). CaCl₂ and other chemicals ware purchased from J.T. Baker (México). The Ca²⁺ ionophore A23187 was dissolved in ethanol. Benzene and 1,2-DCB were dissolved in methanol. Ethanol and methanol never reached a concentration higher than 1 % (v/v) in the reaction media after the addition of 1,2-DCB or A23187.

Fetal bovine serum (FBS) and RPMI 1640 media, sodium pyruvate, penicillin-streptomycin and L-glutamine, were purchased from Invitrogen Gibco, Incl. LIT, RPMI 1640-modified medium and HMI-9 media were prepared at Tissue culture facility in Instituto de Parasitología y Biomedicina “López-Neyra”. Surfact-Amps NP40 was supplied by Thermo Scientific and Tween 20 by Merck. Sodium dodecyl sulfate (SDS) was purchased from Affymetrix. Chlorophenol red-β-D-galactopyranoside (CPRG), resazurin sodium salt, amphotericin B, p-formaldehyde, pentamidine isethionate salt, benznidazole, nifurtimox, posaconazole, ATP disodium salt, propidium iodide, podophyllotoxin, ribonuclease A (RNase A) from bovine pancreas, phorbol 12-myristate 13-acetate (PMA),3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were obtained from Sigma Aldrich. CellTiter-Glo Luminescent Cell Viability Assay was purchased from Promega Corporation and 4′, 6-diamidino-2-phenylindole (DAPI) Prolong from Molecular Probes.