An intracellular calcium store is present in Leishmania donovani glycosomes.
A fourth intracellular Ca2+ pool in Leishmania donovani was identified by permeabilizing plasma membrane with digitonin. In Fura 2 loaded cells Ca2+ was released synergistically when mitochondrial function was blocked by antimycin and oligomycin. Vanadate did not have any effect if applied before incorporation of these mitochondrial poisons. However, the same inhibitor which inhibits Ca2+-ATPase activity of endoplasmic reticulum was able to release Ca2+ at a slow rate when added after antimycin and oligomycin. Alkalization of cytoplasmic pH allowed further release of Ca2+ essentially from the acidocalcisome. Purified glycosomes could mediate Ca2+ uptake mechanism in presence of vanadate whereas bafilomycin, a specific and potent inhibitor of vacuolar proton pump did not have any effect. Glycosomal Ca2+-ATPase activity was optimum at pH 7.5. The apparent Km for calciumin presence of vanadate was 12 nM. Taken together, it may be suggested that a vanadate-insensitive Ca2+-ATPase is present in the membrane of this microbody. Presence of glycosomal Ca2+ was further confirmed by imaging of Ca2+ activity in the Fura 2 loaded purified organelle using confocal laser. Results reveal that newly localized glycosomal calcium may essentially be an effective candidate to play a significant role in cellular function.
Gupta
Shreedhara
S
Department of Biological Chemistry, Infectious Diseases Group, Indian Institute of Chemical Biology, Kolkata 700032, India.
Raychaudhury
Bikramjit
B
Banerjee
Shouvik
S
Das
Banasri
B
Datta
Salil C
SC
D000818:Animals; D000968:Antimycin A / Q000031:analogs & derivatives / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry; D016257:Fura-2 / Q000031:analogs & derivatives / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007476:Ionophores / Q000494:pharmacology; D007893:Leishmania donovani / Q000187:drug effects / Q000378:metabolism*; D018942:Macrolides / Q000494:pharmacology; D008830:Microbodies / Q000187:drug effects / Q000378:metabolism*; D018613:Microscopy, Confocal; D009840:Oligomycins / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014475:Uncoupling Agents / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007476:Ionophores; D018942:Macrolides; D009840:Oligomycins; D014475:Uncoupling Agents; C049925:fura-2-am; C032456:antimycin; C057620:bafilomycin A; D000001:Calcimycin; D014638:Vanadates; D000968:Antimycin A; D000252:Calcium-Transporting ATPases; D004072:Digitonin; D002118:Calcium; D016257:Fura-2