Characteristics of membrane-bound and soluble forms of xanthine oxidase from milk and endothelial cells of capillaries.
Xanthine oxidase (xanthine:O2 oxidoreductase, EC 1.2.3.2) was purified from bovine milk lipid globules to electrophoretic homogeneity (Mr 155,000) and antibodies were raised against it in rabbits. By immunolocalization techniques, the xanthine oxidase antigen was detected in milk lipid globules and mammary gland epithelium, but also in capillary endothelium from various tissues, including liver, lung and intestine. These findings were paralleled by measurements of xanthine oxidase activities in the tissues, both in a membrane-associated and a soluble form. Addition of hypoxanthine to fractions containing native xanthine oxidase did not promote lipid peroxidation, in contrast to the widely used in vitro system for lipid peroxidation which involves addition of xanthine oxidase preparations. Extraction with buffers of high ionic strength and with nonionic detergents removed only part of the enzyme from the membranes. Immunoprecipitates from the soluble supernatant fractions, using anti-xanthine oxidase IgG, were enriched in the Mr 155,000 polypeptide. Patterns of proteolytic cleavage products of the xanthine oxidase monomer from capillaries and milk lipid globules were similar but not identical. Immunoprecipitates from soluble fractions of milk lipid globules and tissues were enriched in both xanthine oxidase and NADH-cytochrome c reductase activities. Electrophoretic separation of proteins from milk lipid globule membranes under non-denaturing conditions revealed a close correlation of xanthine oxidase and part of the NADH-cytochrome c reductase activity, but showed different activity profiles of NADH-ferricyanide reductase and xanthine oxidase.
D000273:Adipose Tissue / Q000201:enzymology; D000818:Animals; D002196:Capillaries / Q000201:enzymology*; D002417:Cattle; D004727:Endothelium / Q000201:enzymology; D005260:Female; D007158:Immunologic Techniques; D007774:Lactation; D008099:Liver / Q000201:enzymology; D008168:Lung / Q000201:enzymology; D008321:Mammary Glands, Animal / Q000201:enzymology; D008565:Membrane Proteins / Q000378:metabolism; D008892:Milk / Q000201:enzymology*; D009245:NADH Dehydrogenase / Q000378:metabolism; D011247:Pregnancy; D012995:Solubility; D014969:Xanthine Oxidase / Q000378:metabolism*
D008565:Membrane Proteins; D014969:Xanthine Oxidase; D009245:NADH Dehydrogenase