IMMUNOAFFINITY LOCALIZATION OF THE ENZYME XANTHINE-OXIDASE ON THE OUTSIDE SURFACE OF THE ENDOTHELIAL-CELL PLASMA-MEMBRANE

Background. Reactive oxygen metabolites generated from endothelial xan thine oxidase (XO) trigger reperfusion injury in many organs. We evalu ated the possibility that endothelial XO was localized on the endothel ial cell surface, as well as within the cytoplasm. Methods. Primary cu ltures of bovine (BAECs) and porcine (PAECs) aortic endothelial cells were grown in media documented to be free of XO. Polyclonal and monocl onal antibodies were developed against XO. These antibodies were used to evaluate BAEC and PAEC for cell surface XO through immunofluorescen ce staining, hybridoma cell surface labeling, and endothelial cell sur face binding Results. These antibodies bound specifically to the surfa ce of these cells when the membrane was shown to be intact and imperme able land the cytoplasm inaccessible) to immunoglobulins. Moreover hyb ridoma cells expressing monoclonal antibody to XO bound specifically t o the endothelial cell surface. Finally, intact endothelial cells boun d specifically to the anti-XO polyclonal antibodies immobilized to the surface of a Petri dish. The integrity of these endothelial cell plas ma membranes was demonstrated by the subsequent growth and replication of these cells in culture. Conclusions. These findings indicate that XO is present on the outside surface of the endothelial cell plasma me mbrane. This would not only explain the known in vivo efficacy of intr avascularly administered Large molecular weight antioxidants (such as superoxide dismutase) but could have important implications for inflam matory signaling.