Studies on mitochondrial ATPase of Leishmania donovani using digitonin-permeabilized promastigotes.
Mitochondrial ATPase of Leishmania donovani was characterized using digitonin-permeabilized promastigotes and the results were compared with those from isolated mitochondria. Maximum mitochondrial ATPase activity was obtained in promastigotes permeabilized with digitonin at a final concentration of 20 microM and the specific activity of the enzyme was 46% and 57% higher than that of homogenized and sonicated promastigotes, respectively. At concentrations above 20 microM digitonin inhibited ATPase activity and the degree of inhibition increased with increasing concentrations of the detergent. The ATPase activity of promastigotes remained DCCD-sensitive when permeabilized with digitonin at concentrations up to 120 microM but the enzyme became increasingly resistant to this inhibitor as digitonin concentrations were increased to 140 microM and more, indicating the loss of functional activity of the enzyme. The pH and temperature optima for mitochondrial ATPase were determined to be 7.5 and 30 degrees C, respectively. Mg2+ ions were essential for ATPase activity but free Mg2+ ions were found to be inhibitory. A Mg2+/ATP ratio of 1:3 supported the optimum ATPase activity. Sulfite and hexanol activated the enzyme but failed to prevent the inhibition by free Mg2+ ions. The results indicate that digitonin-permeabilized promastigotes provide an ideal system for studying the mitochondrial ATPase of L. donovani.
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Division of Membrane Biology, Central Drug Research Institute, Lucknow, India.
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000438:Alcohols / Q000494:pharmacology; D000818:Animals; D002463:Cell Membrane Permeability / Q000187:drug effects; D004072:Digitonin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007893:Leishmania donovani / Q000187:drug effects / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology; D009840:Oligomycins / Q000494:pharmacology
D000438:Alcohols; D007477:Ions; D009840:Oligomycins; D000251:Adenosine Triphosphatases; D008274:Magnesium; D004072:Digitonin