Immunological Identification and Determination of Xanthine Oxidase in Cells and Tissues

Abstract

Antibodies against purified xanthine oxidase (xanthine: O 2 oxidoreductase, EC 1.2.3.2) from bovine milk lipid globule membranes were raised in rabbits and guinea pigs, and immunoglobulins (G) were purified by immuno-adsorption. In addition, a monoclonal antibody against xanthine oxidase was isolated from a murine ‘hybridoma’ cell-line. All antibody preparations recognized the antigen both in its soluble and membrane-bound form in milk lipid globules and mammary tissue as well as the antigen present in the capillary endothelium of lung and liver. Xanthine oxidases from these different sources were purified by immunoaffinity chromatography and compared by one- and two-dimensional gel electrophoresis and by mapping the peptides obtained after complete digestion of the radioiodinated proteins with trypsin. No differences were found between the enzymes isolated from the various bovine tissue preparations. The distribution of xanthine oxidase protein was determined in a selection of bovine cell cultures and tissues using an ultrasensitive enzymic radioimmunoassay. All fractions tested, including material in which we have not detected xanthine oxidase protein by immunolocalization techniques such as brain, macrophages, and MDBK cells, were found to contain some enzyme protein. However, the levels of protein in these cells were much lower (by factors from 10 to 2,000) than the levels in lactating mammary tissue. By comparison, the concentration of the enzyme in eye lens tissue was at the limit of detection, i.e. by two orders of magnitude lower than the lowest values in other cells and tissues. These results are in accordance with the assumption that xanthine oxidase is a constitutive enzyme and that the enzyme protein present in various cell types is indistinguishable, by immunological and enzymic criteria, from milk xanthine oxidase. The very high concentrations of the protein in mammary gland epithelium and in particular in milk lipid globule membranes might reflect a specific function in the secretion of milk lipids. The possible functional significance of the high levels of xanthine oxidase in capillary endothelial cells is discussed.