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

Abstract

Controlled exposure of Leishmania dono ani 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 Mg# + (4 mM) results in the formation of sealed everted vesicles. The presence of Mg# + (4 mM) appears to be essential for efficient sealing and also to prevent leakiness. ATP-dependent Ca# + accumulation can be demonstrated in these vesicles. K m values for Ca# + and ATP were 125 nM and 0n8 mM respectively. The accumulated Ca# + reaches a concentration of 1n1 mM. Ca# + uptake is completely inhibited by vanadate (40 µM) and several

A novel ATP‐binding cassette transporter from Leishmania is involved in transport of phosphatidylcholine analogues and resistance to alkyl‐phospholipids

“…For localization of the LiABCG4–GFP chimeras, live parasites were pelleted, washed three times in PBS, attached to poly l ‐lysine‐coated coverslips, and images were acquired with a Zeiss Axiophot microscope (Oberkochen, Germany) equipped with a SPOT camera (Diagnostic Instruments). In order to differentiate the PM from intracellular organelles, parasites were incubated in hypotonic buffer (5 mM Tris‐HCl, 100 μM PMSF, pH 7.4) for 30 min on ice and gently homogenized resulting in the formation of deflagellated ghosts that preserve the microtubular structure associated with the parasite PM (Mandal et al ., 1997). Expression of tagged LiABCG4 was verified by Western blot analysis using polyclonal anti‐GFP antibodies (1:7500) (Molecular Probes), and a HRP‐conjugated secondary goat anti‐rabbit (1:5000).…”

Camptothecin-induced Imbalance in Intracellular Cation Homeostasis Regulates Programmed Cell Death in Unicellular Hemoflagellate Leishmania donovani

“…Cell suspensions were kept at 4°C and were mixed by mild vortexing to detach flagella from the cell body. Formation of unsealed ghosts was confirmed by total leakage of the marker cytoplasmic enzymes (26).…”

Detection and Localization of a Ca2+-ATPase Activity in Toxoplasma gondii.

“…as well as in some protozoans and protozoan parasites (Paramecium tetraurelia, Hauser et al, 1998;Leishmania donovani, Ghosh et al, 1990, Mandal et al, 1997Plasmodium falciparum, Dyer et al, 1996: Dictyostelium discoideum, Rooney et al, 1994and Trypanosoma cruzi, Lu et al, 1998). Dyer et al (1996) cloned a P-type ATPase gene from Plasmodium falciparum that encodes an enzyme analogous to the Ca-ATPase of the endoplasmic reticulum (SERCA).…”