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. 1974 Oct;143(1):149–157. doi: 10.1042/bj1430149

Association of xanthine oxidase with the bovine milk-fat-globule membrane. Catalytic properties of the free and membrane-bound enzyme

Michael S Briley 1,*, Robert Eisenthal 1
PMCID: PMC1168363  PMID: 4156954

Abstract

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 Km 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, BM1, 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, BM2, and free xanthine oxidase. Fractions BM1 and BM2 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 BM1, 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 BM1. The binding constant and stoicheiometry were determined. 4. Proteolytic digestion of fraction BM1 liberated free xanthine oxidase from the fat-globule membrane with a corresponding alteration in X/N value.

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Selected References

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