THE PLASMA-MEMBRANE CA2-ATPASE OF LEISHMANIA-DONOVANI IS AN EXTRUSIONPUMP FOR CA2+()

Controlled exposure of Leishmania donovani promastigotes to hypotonic shock results in the formation of deflagellated unsealed ghosts of ori ginal polarity that largely retain the pellicular microtubular structu re associated with plasma membrane of the parasite. Gentle shearing fo llowed by suspension of the purified membrane in appropriate isotonic buffer containing Mg2+ (4 mM) results in the formation of sealed evert ed vesicles. The presence of Mg2+ (4 mM) appears to be essential for e fficient sealing and also to prevent leakiness. ATP-dependent Ca2+ acc umulation can be demonstrated in these vesicles. K-m values for Ca2+ a nd ATP were 125 nM and 0.8 mM respectively. The accumulated Ca2+ reach es a concentration of 1.1 mM. Ca2+ uptake is completely inhibited by v anadate (40 mu M) and several thiol-modifying agents. Using 5,5'-dithi obis-(2-nitrobenzoic acid) as the modifying agent, an excellent correl ation between loss of enzyme activity and transport capability and the ir parallel regeneration in the presence of 2 mM dithiothreitol was de monstrated. Using 2',7-bis(carboxyethyl)-5(6)-carboxyfluorescein as th e fluorescent pH probe, it was observed that Ca2+ entry into the vesic les is accompanied by an outward movement of H+ from the vesicles. Tak en together, this paper establishes that the high-affinity transmembra ne Ca2+-ATPase [Ghosh, Ray, Sarkar and Bhaduri (1990) J. Biol. Chem. 2 65, 11345-11351; Majumdar, Mukherjee, Ray and Bhaduri (1992) J. Biol. Chem. 267, 18440-18446] is an extrusion pump for Ca2+ in this human pa thogen.