Oxygen can be reduced by one and two electron transfer reactions to create the toxic species superoxide (O2) and hydrogen peroxide (H2O2), which can react further to create secondary toxic species such as hydroxyl radical (OH) or lipid hydroperoxides. An important tissue source of toxic oxygen species is the purine catabolism enzyme xanthine oxidase. Experimental and clinical studies have demonstrated that this enzyme serves as an important locus for O2 and H2O2 in ischemia-reperfusion injury (organ transplantation, stroke, heart attack), sepsis, burns and acute viral infection. In many of these situations, xanthine oxidase can also be released into the circulation to exert injury to distal tissues, such as the pulmonary vascular bed. We have recently observed that this source of cytotoxic oxidants can specifically bind to glycosaminoglycans of cell surfaces, thus we hypothesize the xanthine oxidase can be concentrated several thousand-fold at the cell membrane, with significant metabolic and toxicologic implications. Endocytosis may then be a route for the intracellular uptake of bound xanthine oxidase. Thus, the goal of this project is to learn more about cell surface glycosaminoglycan-xanthine oxidase interactions. We will examine properties of xanthine oxidase binding to immobilized glycosaminoglycans and cell surfaces, we will also examine kinetic characteristics of bound xanthine oxidase, the interaction between xanthine oxidase and other enzymes known to bind to vascular endothelium (i.e. extracellular superoxide dismutase and lipoprotein lipase) and how the ability of bound xanthine oxidase to generate O2 and H2O2 is affected. These experiments address a fundamental aspect of cell biochemistry which has important clinical implications. They will provide insight into the significant, but poorly understood role which reactive oxygen species play in disease processes and tissue pathology.
