The plasma-membrane Ca2+-ATPase of Leishmania donovani is an extrusion pump for Ca2+.
Controlled exposure of Leishmania donovani promastigotes 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+ (4mM) 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 Km 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 microM) and several thiol-modifying agents. Using 5,5'-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 2'.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, 11345-11351; Majumdar, Mukherjee, Ray and Bhaduri (1992) J. Biol. Chem. 267, 18440-18446] is an extrusion pump for Ca2+ in this human pathogen.
Mandal
D
D
Leishmania Group, Enzyme Division, Indian Institute of Chemical Biology, Calcutta, India.
Mukherjee
T
T
Sarkar
S
S
Majumdar
S
S
Bhaduri
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism / Q000469:parasitology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology* / Q000378:metabolism; D011522:Protons; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology; D013347:Subcellular Fractions / Q000378:metabolism / Q000648:ultrastructure
D004791:Enzyme Inhibitors; D011522:Protons; D015800:Protozoan Proteins; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium