Giardia causes is a major intestinal illness in the United States as well as many other countries worldwide. The life cycle is simple and direct requiring a vegetative trophozoite which attaches to the microvillus brush border of the host's intestine and a cyst which passes from host to host by the fecal-oral route. During cyst formation (encystment) induced by bile, Giardia trophozoites form a protective cyst wall filament rich in the cyst wall specific sugar N-acetylgalactosamine (GalNAC, as a novel [GalNAC beta1 yields 3 GalNAc]n homopolymer). GalNAc, undetected in non-encysting trophozoites, is synthesized from glucose during encystment by the activity of five inducible, nonsedimentable enzymes: Glucosamine 6-phosphate isomerase, GPI, glucosamine 6-phosphate N-acetylase (GlcNPA), phosphoacetylglucosamine mutase (PAG1cNM), UDP-N- acetylglucosamine pyrophosphorylase (UDP-GlcNAcPP), and UDP-N- acetylglucosamine 4' epimerase (UDP-GlcNAcE). The goal of our laboratories is an in-depth study of enzyme regulation for, the molecular biology for the control of, and the possible development of new chemotherapeutic agents that can target this pathway thus acting to prevent the formation of cysts and thus aiding in the control of giardiasis. To date GPI, UDP-GlcNAcPP and CWS have been purified (or partially purified) and characterized, and GPI and UDP-GlcNAcPP have been cloned and sequenced. We have shown also that GPI is transcriptionally regulated while UDP-GlcNAcPP is constitutive but unidirectionally activated toward GalNAc synthesis by glucosamine 6-PO4 the anabolic product of GPI. Before more in depth studies of the regulation of this pathway can be undertaken, it is essential to understand more about the three enzymes which have not yet been purified or characterized. Thus, we plan to use molecular techniques coupled with enzyme analyses to 1) determine whether GlcNPA, PAGlcNM, and UDP-GlcNAcE activities are induced at a transcriptional level (as is the case with GPI) or at a post- transcriptional level (as is the case with Giardia's UDP-N- acetylglucosamine pyrophosphorylase), and 2) determine if GlcNPA, PAG1cNM, and UDP-GlcNAcE are regulatory enzymes in the GalNAc synthetic pathway by purifying (or expressing) these enzymes and characterizing them with respect to enzyme kinetics, and possible activators and inhibitors.