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. 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 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.
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February 1997
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Research Article|
February 15 1997
The plasma-membrane Ca2+-ATPase of Leishmania donovani is an extrusion pump for Ca2+
Debjani MANDAL;
Debjani MANDAL
*Leishmania Group, Enzyme Division, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Calcutta-700 032, India
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Tanmoy MUKHERJEE;
Tanmoy MUKHERJEE
*Leishmania Group, Enzyme Division, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Calcutta-700 032, India
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Sibaji SARKAR;
Sibaji SARKAR
†241 Lineberger Comprehensive Cancer Center CB no. 7295, University of North Carolina, Chapel Hill, NC 27599, U.S.A.
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Sabita MAJUMDAR;
Sabita MAJUMDAR
‡Biophysics Division, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Calcutta-700 032, India
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Amar BHADURI
Amar BHADURI
§
*Leishmania Group, Enzyme Division, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Calcutta-700 032, India
§To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
June 26 1996
Revision Received:
October 02 1996
Accepted:
October 09 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1997
1997
Biochem J (1997) 322 (1): 251–257.
Article history
Received:
June 26 1996
Revision Received:
October 02 1996
Accepted:
October 09 1996
Citation
Debjani MANDAL, Tanmoy MUKHERJEE, Sibaji SARKAR, Sabita MAJUMDAR, Amar BHADURI; The plasma-membrane Ca2+-ATPase of Leishmania donovani is an extrusion pump for Ca2+. Biochem J 15 February 1997; 322 (1): 251–257. doi: https://doi.org/10.1042/bj3220251
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