Immunoaffinity localization of the enzyme xanthine oxidase on the outside surface of the endothelial cell plasma membrane

S. Vickers, H. J. Schiller, James Hildreth, G. B. Bulkley

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

Background. Reactive oxygen metabolites generated from endothelial xanthine oxidase (XO) trigger reperfusion injury in many organs. We evaluated the possibility that endothelial XO was localized on the endothelial cell surface, as well as within the cytoplasm. Methods. Primary cultures of bovine (BAECs) and porcine (PAECs) aortic endothelial cells were grown in media documented to be free of XO. Polyclonal and monoclonal antibodies were developed against XO. These antibodies were used to evaluate BAEC and PAEC for cell surface XO through immunofluorescence staining, hybridoma cell surface labeling, and endothelial cell surface binding. Results. These antibodies bound specifically to the surface of these cells when the membrane was shown to be intact and impermeable (and the cytoplasm inaccessible) to immunoglobulins. Moreover, hybridoma cells expressing monoclonal antibody to XO bound specifically to the endothelial cell surface. Finally, intact endothelial cells bound specifically to the anti-XO polyclonal antibodies immobilized to the surface of a Petri dish. The integrity of these endothelial cell plasma 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 membrane. This would not only explain the known in vivo efficacy of intravascularly administered large molecular weight antioxidants (such as superoxide dismutase) but could have important implications for inflammatory signaling.

Original languageEnglish (US)
Pages (from-to)551-560
Number of pages10
JournalSurgery
Volume124
Issue number3
DOIs
StatePublished - 1998
Externally publishedYes

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Xanthine Oxidase
Endothelial Cells
Cell Membrane
Enzymes
Hybridomas
Cytoplasm
Monoclonal Antibodies
Immobilized Antibodies
Antibodies
Reperfusion Injury
Superoxide Dismutase
Fluorescent Antibody Technique
Immunoglobulins
Swine
Cell Culture Techniques
Antioxidants
Molecular Weight
Staining and Labeling
Oxygen
Growth

ASJC Scopus subject areas

  • Surgery

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Immunoaffinity localization of the enzyme xanthine oxidase on the outside surface of the endothelial cell plasma membrane. / Vickers, S.; Schiller, H. J.; Hildreth, James; Bulkley, G. B.

In: Surgery, Vol. 124, No. 3, 1998, p. 551-560.

Research output: Contribution to journalArticle

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