Purification of an active glucosylceramide synthase in T.cruzi

T A. PIÑERO; M LANDONI; V G. DUSCHAK; A S. COUTO

Córdoba- Argentina

Congreso; XLIV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2008

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

            The genome determination of three human-infective protozoa, including Trypanosoma cruzi, the agent of Chagas disease, has prompt the research to the identification and validation of novel drug targets. Glycosphingolipids are important regulators of many different cellular processes such as signaling control, cell cycle progression and differentiation. Thus, attention has been focus on them as potential drug targets to treat a wide variety of diseases. Glucosylceramides are membrane lipids that contribute to the physical properties and physiological functions of membranes; they serve as basic precursors for over 300 species of glycosphingolipids and its synthesis and degradation seem to contribute to the control of the level of ceramide, which is regarded as a second messenger involved in many biological processes. Previously, we have shown the presence of an active Glucosylceramide synthase (GCS) in T.cruzi which is the key enzyme in this biosynthetic pathway. In the current work we present a method for the isolation of GCS from epimastigotes forms of T. cruzi.  Purification of GCS was achieved via a 5 step protocol involving a Green-dye agarose affinity column. This procedure led to a 70 kDa protein whose specific activity was proven using NBD-Ceramide and UDP-Glucose as susbstrates. On going studies are being carried out to make the biochemical characterization of the enzyme.