This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Entamoeba invadens and Entamoeba histolytica are related enteric pathogens. The former is a pathogen affecting several reptile taxons and the later is a pathogen in man. Jacob proteins are abundant components of the cyst wall in E. invadens and homologues of it exist in E. histolytica. The Jacob proteins have conserved lectin-like domains and are likely important for maintenance of cyst wall integrity in these pathogens. Putative endoprotease sites are conserved in their sequence across Entamoeba species and therefore a proteolytic event may be important for proper function of the cyst wall. That most proteases in the E. histolytica genome are cysteine proteases suggests that the putative Jacob proteases are of this type. FITC label peptides containing the putative Entamoeba protease sites have been constructed for use in strategies to detect protease activity using approaches including MALDI-TOF mass spectrometric analysis. This study attempts to identify specific protease activities in E. histolytica cell extracts in strategies using known protease inhibitors. As results indicate, specific inhibitors will be used for affinity enrichment of the proteases and proteomics approaches will be used to identify the active protease(s).