title: Magnesium-Dependent Ecto-ATP Diphosphohydrolase Activity in Leishmania donovani.
abstract: In this work, we have described the expression of ecto-ATPDase on the external surface of Leishmania donovani. This enzyme has the ability to hydrolyze extracellular ATP. There is a low level of ATP hydrolysis in the absence of divalent cation 2.5 ± 0.51 nM Pi 107cells/h which shows the divalent cation-dependent activity of this enzyme in the intact parasite. However, MgCl2stimulated the ATP hydrolysis to a greater extent compared with CaCl2and ZnCl2. This activity was also observed when replaced by MnCl2. The Mg-dependent ecto-ATPase activity was 46.58 ± 6.248 nM Pi 107cells/h. The apparent Kmfor ATP was 5.76 mM. Since Leishmania also possesses acid phosphatase activity and to discard the possibility that the observed ATP hydrolysis was due to acid phosphatase, the effect of pH was examined. In the pH range 6.0-9.0, in which the cells were viable, the phosphatase activity decreased while ATPase activity increased. To show that the observed ATP hydrolysis was not due to phosphatase or nucleotidase activity, certain inhibitors for these enzymes were tested. Vandate and NaF inhibited the phosphatase activity; Ammonium molybdate inhibited 5'-nucleotidase activity, but these inhibitors did not inhibit the observed ATP hydrolysis. However, when ADP was used as a substrate, there was no inhibition of ATP hydrolysis showing the possibility of ATP diphosphohydrolase activity. To confirm that this Mg-dependent ATPase activity is an ecto-ATPase activity, we used an impermeable inhibitor, 4,4'-diisothiocyanostilbene 2,-2'-disulfonic acid, as well as suramin, an antagonist of P2-purinoceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-dependent ATPase activity in a dose-dependent manner. The presence of L. donovani E-NTPDase activity was demonstrated using antibodies against NTPDase by Western blotting and flow cytometry. The presence of Mg2+-dependent ATP diphosphohydrolase activity on the surface of L. donovani modulates the nucleotide concentration and protects the parasite from the lytic effects of the nucleotides mainly ATP. Ecto-ATPDase from L. donovani may be further characterized as a good antigen and as a target for immunodiagnosis and drug development, respectively.
authors: Division of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences (Indian Council of Medical Research), Patna, 800007, India.
authors: Preeti
authors: P
authors: Sinha
authors: Division of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences (Indian Council of Medical Research), Patna, 800007, India.
authors: Ranjeet Kumar
authors: RK
authors: Paswan
authors: Division of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences (Indian Council of Medical Research), Patna, 800007, India.
authors: Anjali
authors: A
authors: Kumari
authors: Division of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences (Indian Council of Medical Research), Patna, 800007, India.
authors: Sanjay
authors: S
authors: Kumar
authors: Division of Immunology, Rajendra Memorial Research Institute of Medical Sciences (Indian Council of Medical Research), Patna, India.
authors: Sanjeeva
authors: S
authors: Bimal
authors: Division of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences (Indian Council of Medical Research), Patna, India.
authors: Pradeep
authors: P
authors: Das
authors: Division of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences (Indian Council of Medical Research), Patna, 800007, India. drcslal@gmail.com.
authors: Chandra Shekhar
authors: CS
authors: Lal
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001081:Apyrase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D004795:Enzyme Stability; D007700:Kinetics; D007893:Leishmania donovani / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D008274:Magnesium / Q000378:metabolism*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D015800:Protozoan Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D001081:Apyrase; D008274:Magnesium
keywords: Adenosine; Ecto-ATPDase activity; Leishmania donovani; Magnesium