Association of xanthine oxidase with the bovine milk-fat-globule membrane.
1. The catalytic properties of xanthine oxidase in bovine milk (EC 1.2.3.2) are dependent on the state of the enzyme, i.e. whether free or bound to the fat-globule membrane. Oxidase activity of the membrane-bound enzyme towards NADH is enhanced relative to that towards xanthine. This reflects a change in the relative K(m) values and enables the ratio of xanthine to NADH oxidase activities (X/N) to be used as a parameter for the relative amounts of free and membrane-bound xanthine oxidase in milk fractions. 2. Chromatography of buttermilk on Sepharose 2B yielded an excluded fraction, BM(1), with xanthine oxidase activity. The remaining xanthine oxidase activity was eluted as a single broad peak. This was further resolved on Sephadex G-200 into an excluded fraction, BM(2), and free xanthine oxidase. Fractions BM(1) and BM(2) had X/N values in the range 45-65, which is characteristic of membrane-bound xanthine oxidase. Purified xanthine oxidase has a mean X/N value of 110.3. Addition of fraction BM(1), heated to remove associated enzyme activities, to purified xanthine oxidase progressively enhanced its NADH oxidase activity to a value where its X/N value was characteristic of membrane-bound xanthine oxidase. This was shown to be due to binding of free enzyme to heated fraction BM(1). The binding constant and stoicheiometry were determined. 4. Proteolytic digestion of fraction BM(1) liberated free xanthine oxidase from the fat-globule membrane with a corresponding alteration in X/N value.
D000818:Animals; D002384:Catalysis; D002417:Cattle; D007042:Hypoxanthines; D066298:In Vitro Techniques; D007700:Kinetics; D008055:Lipids / Q000032:analysis; D008566:Membranes; D008892:Milk / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases; D010084:Oxidation-Reduction; D010743:Phospholipids / Q000032:analysis; D011485:Protein Binding; D011489:Protein Denaturation; D014969:Xanthine Oxidase / Q000032:analysis* / Q000378:metabolism
D007042:Hypoxanthines; D008055:Lipids; D010743:Phospholipids; D014969:Xanthine Oxidase; D009247:NADH, NADPH Oxidoreductases