Association of xanthine oxidase with the bovine milk-fat-globule membrane. Nature of the enzyme-membrane association.
1. Xanthine oxidase (EC 1.2.3.2) was found to represent more than 8% of the intrinsic protein of the bovine milk-fat-globule membranes. 2. Less than 25% of the xanthine oxidase activity of the fat-globule membrane was solubilized with 0.1 M-sodium pyrophosphate buffer or 2M-NaCl. Of the particulate activity remaining 56% was solubilized with Triton X-100. 3. The xanthine oxidase activity solubilized with buffer, 2M-NaCl or Triton X-100 was not liberated as the free enzyme. Only tryptic digestion was found to release the free enzyme from the fat-globule membrane. Tryptic digestion also liberated free xanthine oxidase from those fractions solubilized by buffer or NaCl, but not from those fractions solubilized with Triton X-100 or by sonication. 4. The effect of membrane association on the catalytic properties of the enzyme could be mimicked by low pH or by the presence in the assay mixture of certain concentrations of 2-methyl-propan-2-ol, but not 1,4-dioxan, suggesting that hydrogen-bonding rather than low dielectric constant may be involved. 5. The origin of the milk-fat-globule membrane is discussed with reference to the intrinsic nature of the associated xanthine oxidase activity.
D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D008055:Lipids; D008566:Membranes / Q000201:enzymology; D008892:Milk / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis; D011506:Proteins / Q000032:analysis; D014969:Xanthine Oxidase / Q000032:analysis*
D008055:Lipids; D011506:Proteins; D005182:Flavin-Adenine Dinucleotide; D014969:Xanthine Oxidase; D009247:NADH, NADPH Oxidoreductases