RESUMEN

In Leishmania spp. ATP utilizing enzymes serves as a key role in preserving integrity of host cells for survival of parasite. Earlier reports suggested that Adenylate kinase (AK) a phosphotransferase enzyme released by Leishmania donovani secretome, involved in modulating levels of NTPs. In the present study, we cloned, expressed and characterized recombinant putative AK. Based on a sequence and phylogeny analysis, we identified the prominent features of the seven AK isoforms of Leishmania donovani and assigned our putative AK as LdAK2a. The Km value of LdAK2a for ATP and AMP substrate were 204 µM and 184 µM, respectively and Vmax was calculated as 1.6 µmol min-1 mg-1 protein. Ap5A, a known inhibitor of AK inhibited LdAK2a with estimated Ki values of 280 nM and 230 nM for ATP and AMP respectively. CD spectral studies were carried out to estimate its structural stability. Recombinant LdAK2a was found to prevent ATP mediated cell cytolysis of Raw 264.7 macrophages in vitro, which was determined by LDH assay and MMP assay. This is the first report which validates that Leishmanial AK2a can prevent ATP mediated cytolysis of macrophage cells and thereby probably play a role in preserving integrity of host cells for survival of parasite.

Asunto(s)

Adenosina Trifosfato/metabolismo , Adenilato Quinasa/metabolismo , Interacciones Huésped-Parásitos , Leishmania donovani/enzimología , Macrófagos/patología , Macrófagos/parasitología , Adenilato Quinasa/genética , Animales , Muerte Celular , Cinética , Leishmaniasis Visceral , Ratones , Células RAW 264.7