Characterization of plasma membrane bound inorganic pyrophosphatase from Leishmania donovani promastigotes and amastigotes

Background: Currently, a major problem in the management of visceral leishmaniasis or kala-azar, especially in the Indian subcontinent, is the growing unresponsiveness to conventional antimonial therapy. Membrane bound pyrophophatase (PPases) do not exist in plasma membrane from mammals. Thus, H+-PPases from Leishmania plasma membrane might be potential target in rational chemotherapy of the disease caused by Leishmania parasites. Objective: To characterize the activities of inorganic pyrophophatase (PPase) in the plasma membrane of Leishmania donovani promastigote and amastigote. Methods: Culture method of promastigote and amastigote were developed. We assayed PPase activity in isolated plasma membrane of L. donovani. Results: We characterized K+-PPase present in the plasma membrane of Leishmania donovani and investigated its possible role in the survival of promastigote and amastigote. PPase activity was stimulated by K+ ions and sodium orthovanadate, inhibited by pyrophosphate analogs imidodiphosphate and alendronate, KF, DCCD, thiol reagent parachloromercuribenzenesulfonate (PCMBS), N-ethylmaliemide (NEM), phenylarsineoxide, ABC superfamily transport modulator verapamil and was also by F1 F0-ATPase inhibitor quercetin. Conclusion: We conclude that there are significant differences within promastigote, amastigote and mammalian host in cytosolic pH homeostasis to merit the inclusion of PPase transporter as putative targets for rational drug design. Abbreviations used: PPase-Pyrophosphatase, NEM-N-ethylmaliemide, PAO-Phenylarsineoxide, KF-Potassium fluoride, DCCD-N, N -dicclohexylcarbodiimide, PCMBS-Parachloromer curibenzenesulfonate, SA-Sodium azide, SOV- Sodium orthovanadate, IDP-Imidodiphosphate, QTN-Quercetin, LDC-Leishmania donovani cell, DPC-Digitonin permeabilized cell, PM- Plasma membrane, PPi-Pyrophosphate, gp-Glycoprotein, Pro-Promastigote, Am-Amastigote