The plasma-membrane Ca2+-ATPase of Leishmania donovani is an extrusion pump for Ca2+

Controlled exposure of Leishmania donovanipromastigotes to hypotonic shock results in the formation of deflagellated unsealed ghosts of original polarity that largely retain the pellicular microtubular structure associated with plasma membrane of the parasite. Gentle shearing followed by suspension of the purified membrane in appropriate isotonic buffer containing Mg²⁺ (4 mM) results in the formation of sealed everted vesicles. The presence of Mg²⁺ (4 mM) appears to be essential for efficient sealing and also to prevent leakiness. ATP-dependent Ca²⁺ accumulation can be demonstrated in these vesicles. K_m values for Ca²⁺ and ATP were 125 nM and 0.8 mM respectively. The accumulated Ca²⁺ reaches a concentration of 1.1 mM. Ca²⁺ uptake is completely inhibited by vanadate (40 ƁM) and several thiol-modifying agents. Using 5,5ƀ-dithiobis-(2-nitrobenzoic acid) as the modifying agent, an excellent correlation between loss of enzyme activity and transport capability and their parallel regeneration in the presence of 2 mM dithiothreitol was demonstrated. Using 2ƀ,7-bis(carboxyethyl)-5(6)-carboxyfluorescein as the fluorescent pH probe, it was observed that Ca²⁺ entry into the vesicles is accompanied by an outward movement of H⁺ from the vesicles. Taken together, this paper establishes that the high-affinity transmembrane Ca²⁺-ATPase [Ghosh, Ray, Sarkar and Bhaduri (1990) J. Biol. Chem. 265, 11345Ő11351; Majumdar, Mukherjee, Ray and Bhaduri (1992) J. Biol. Chem. 267, 18440Ő18446] is an extrusion pump for Ca²⁺ in this human pathogen.