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

1974 ◽  
Vol 143 (1) ◽  
pp. 149-157 ◽  
Author(s):  
Michael S. Briley ◽  
Robert Eisenthal
Keyword(s):  
Xanthine Oxidase ◽  
Milk Fat ◽  
Oxidase Activity ◽  
Nadh Oxidase ◽  
Catalytic Properties ◽  
Bovine Milk ◽  
N Value ◽  
Xanthine Oxidase Activity ◽  
Membrane Bound ◽  
Fat Globule

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.

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

1975 ◽  
Vol 147 (3) ◽  
pp. 417-423 ◽  
Author(s):  
M S Briley ◽  
R Eisenthal
Keyword(s):  
Xanthine Oxidase ◽  
Milk Fat ◽  
Oxidase Activity ◽  
Bovine Milk ◽  
Membrane Association ◽  
Milk Fat Globule Membrane ◽  
Xanthine Oxidase Activity ◽  
Milk Fat Globule ◽  
Fat Globule ◽  
Triton X

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.

The oxidation of lipids by components of bovine milk-fat globule membrane

1977 ◽  
Vol 44 (3) ◽  
pp. 495-507 ◽  
Author(s):  
J. C. Allen ◽  
Catherine Humphries
Keyword(s):  
Heat Treatment ◽  
Xanthine Oxidase ◽  
Isoelectric Focusing ◽  
Milk Fat ◽  
Oxidase Activity ◽  
Bovine Milk ◽  
Zwitterionic Surfactant ◽  
Milk Fat Globule Membrane ◽  
Milk Fat Globule ◽  
Fat Globule

SummaryBovine milk-fat globule membrane was solubilized with a zwitterionic surfactant and subjected to chromatography on agarose, with the surfactant in the eluant. Fractions were tested for their effects on the oxidation of buffered linoleate. The maximum oxidative capability was greatly enhanced by the addition of Cu, and became associated with the phospholipids.Further chromatography of the retarded protein peak from agarose on Sephadex G-200, again in the presence of surfactant, gave 2 protein peaks. Oxidative effectiveness resided almost entirely in the first peak, which was devoid of phospholipid, but high in xanthine oxidase activity. This fraction was subjected to isoelectric focusing, and the xanthine oxidase from this was highly pro-oxidative. Furthermore, its oxidative capability was almost doubled on heat treatment.

scholarly journals Preparation of monoclonal antibodies to xanthine oxidase and other proteins of bovine milk-fat-globule membrane

1980 ◽  
Vol 188 (3) ◽  
pp. 925-928 ◽  
Author(s):  
I H Mather ◽  
C S Nace ◽  
V G Johnson ◽  
R A Goldsby
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Xanthine Oxidase ◽  
Cell Lines ◽  
Milk Fat ◽  
Bovine Milk ◽  
Serial Transfer ◽  
Milk Fat Globule Membrane ◽  
Milk Fat Globule ◽  
Fat Globule

Nine hybridomas secreting monoclonal antibody to proteins of bovine milk-fat-globule membrane were isolated. All nine cell lines continued to secrete monoclonal antibody after serial transfer in culture and after passage as solid tumours in Balb/cJ mice. Four of the cell lines secreted monoclonal antibody specific for xanthine oxidase, one of the major proteins of milk-fat-globule membrane.

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