Further characterization of a secreted lipase from the human pathogen Leishmania donovani by determining the effect of various metal ions on its enzymatic activity.

摘要

Leishmania donovani, a protozoan parasite, is the causative agent of the often fatal disease visceral leishmaniasis. The current treatments available are minimal and toxic to the patient. It has been shown that these organisms exhibit lipolytic activity during their growth in vitro. Lipases are enzymes that are known to aid in the development and virulence of several pathogenic organisms such as Candida albicans and Staphylococcus warneri. Little information is known, however, about the role of lipases in Leishmania species. We hypothesize that lipase may play a part in Leishmaniau27s ability to survive within the human host as well as its pathogenesis. In past studies, the L. donovani secretory lipase gene (LdLip3) was episomally expressed with an HA tag by transfected promastigotes. The purified protein was tested for enzyme activity by performing assays with McIlvaineu27s buffer pH 4-8 at 26 C, 37 C, and 42 C using 4MU-palmitate as a substrate. The results showed that there was an average thirtyfold increase in specific activity when comparing the purified protein to the unpurified supernatant samples. Furthermore, metal ions are known to be cofactors of a variety of enzymes, greatly affecting their activity, hence, in the current study a panel of metal ions was tested to determine their effect on the activity of purified LdLip3. Optimal conditions for this enzyme were established at pH 8, 42 C, with the addition of Zn+2, whereas the addition of Mn+2 consistently produced a strong inhibitory effect. We are currently in the process of validating this previously collected data via the same experimentation protocol to determine reproducibility. Chelating agent studies with EDTA, also underway, will determine if the effect of metal ions on the enzyme activity could be reversed. Thus far, EDTA has countered the addition of the cofactors. Future studies include inhibitory studies with organophosphates that have been shown to inhibit lipases, in the hope to develop new drug targets for this parasite.