brucei, a close relative of T. cruzi. In this study, we show that LAPTc mediates selleck catalog the major leucyl aminopepti dase activity in T. cruzi extracts and, thus, it likely has important functions in physiological processes involving protein and peptide processing, degradation of proteins and amino acid recycling. T. cruzi, Leishmania spp. and T. brucei lack the biosynthetic pathways to synthesize the essential amino acids of humans, including leucine. In spite of the metabolic relevance of amino acids for these parasites, their transport and recycling are poorly known. Although many putative amino acid transporter genes have been identified in silico, only arginine and proline transporters have been biochemically character ized in T. cruzi. Considering that a biosynthetic pathway is missing, T.

cruzi must acquire leucine through specific transport and or recycling. Since amastigotes live and divide within host cells where the concentration of free amino acids is low, leucine aminopeptidases would play a major role in leucine supply to the parasite through hydrolysis of exogenous and endogenous pro teins and peptides. Inactivation of LAPTc activity by spe cific inhibitors or through gene disruption may help reveal its functional properties and thus its importance to the host T. cruzi interface. Conclusions LAPTc is a 330 kDa homohexameric enzyme that med iates the major leucyl aminopeptidase activity in T. cruzi. Inter monomer disulfide bonds do not take part in the assembly of the active oligomer. LAPTc is a member of the metallopeptidase M17 family or leucyl aminopeptidase family.

It retains its oligomeric structure after losing activity and is expressed by all T. cruzi forms. Methods Parasites and preparation of enzyme extract T. cruzi epimastigote, amastigote and trypomastigote forms from Berenice stock were cultured and purified as described previously. Cell free extracts were pre pared from 100 ml of epimastigote culture in the log phase. Parasites were harvested by centrifugation and washed four times in PBS. Cells were resuspended in 1. 0 ml of Milli Q water in the presence of 10 uM of the protease inhibitors trans epoxysuccinyl L leucylamido butane and tosyl lysylchloro methane and disrupted by three cycles of freezing at 20 C and thawing. The insoluble material was removed by centrifugation and the supernatant, referred to hereafter as enzyme extract, was immediately used for the assays or stored at 80 C.

Protein content was determined by the Bradford method. Assay of peptidase activity T. cruzi aminopeptidase activity was assayed on the fluorogenic substrates L Leu 7 amido 4 methylcoumarin, N carbobenzoxy Leu AMC, L Pro AMC and Asp AMC, which were purchased from Sigma Aldrich. Enzyme activity was determined by measuring the fluorescence of AMC released by hydrolysis Carfilzomib of the substrates as described pre viously. Assays were performed by incubating 1.