Association of xanthine oxidase with the bovine milk fat globule membrane. Nature of the enzyme membrane association

Abstract

Xanthine oxidase (EC 1.2.3.2) represented more than 8% of the intrinsic protein of the bovine milk fat globule membrane. 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. 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. 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.