• Archives of Pharmacal Research
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• v.17 no.5
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• pp.318-322
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• 1994

Upon gel filtration, the commercial bovine milk xanthine oxidase preparation was fractionated into two preparations showing enzyme activity. Native polyacrylamide gel electrophoresis showed that one was in a dimeric form and the other was a monomer having molecular weight of 150 kDa. It was also found that this commercial enzyme existed mostly in an active monomeric form without loss of enzyme activity. The rabbit antisera produced against two enzyme preparations cross-reacted well each other. In SDS-polyacrylamide gtel electro-phoresis, however, both enzyme preparations yielded two smaller protein bands below 150 kDa, which appeared to bind with both antisera with high affinity but not to retain enzyme activity. It implies that bovine milk xanthine oxidase can lose its activity when monomeric subunit is further degraded.

• Applied Biological Chemistry
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• v.21 no.2
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• pp.112-118
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• 1978

Xanthine oxidase from bovine thyroid glands was purified to apparent homogeneity when judged by analytical disc gel electrophoresis. The purification procedures include pancreatin digestion, butanol extraction, ammonium sulfate precipitation, calcium phosphate gel adsorption, ultrafiltration, calcium phosphate gel-cellulose column chromatography, gel filtration, preparative Sephadex G-25 column electrophoresis, and preparative polyacrylamide gel electrophoresis. The enzyme was enriched 1,000-fold. However, its specific activity was markedly low as compared with highly purified milk enzyme. Thyroidal xanthine oxidase exhibited a low specificity for substrates and electron acceptors. The kinetic properties of thyroid xanthine oxidase were found to be similar to those of the milk enzyme on the basis of Michaelis constants for common substrates.

• Applied Biological Chemistry
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• v.21 no.3
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• pp.137-143
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• 1978

Xanthine oxidase from bovine thyroid glands was found to contain FAD, molybdenum and iron in a ratio 1:0. 36:1. 6. The molecular weight of the thyroid enzyme was similar to that of the milk enzyme when estimated by gel filtration and polyacrylamide gel electrophoresis. The optimum pH for the enzyme activity was 7.8. The pH of the isoelectric point was determined to be 6.2 by electrofocusing. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis experiment indicated that the enzyme was dissociated into subunits and that the molecular weight for the smallest subunit was 65,000 daltons. Absorption spectra were dissimilar between milk and thyroid xanthine oxidase.

• Korean Journal of Food Science and Technology
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• v.21 no.5
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• pp.726-734
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• 1989

To analyze the components of dense coat fractions associated with fat globule membrane, The membrane was treated with various concentrations of Triton X-100, a non-ionic detergent, and the composition of lipids associated to the detergent insoluble material was analyzed. The amount of protein, phospholipid, cholesterol and ganglioside in milk fat globule membrane was reduced consistently with increasing concentrations of Triton X-100. Butyrophilin (band 12), xanthine oxidase (band 3) and band 16 as constituents of insoluble coat materials was revealed after electrophorisis on SDS-polyacrylamide gels. Phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl serine, phosphatidyl inositol and sphingomyelin were identified as the major phospholipids of the coat materials without selective concentration relative to the original membranes. Percentages of total phospholipid were not changed by any of the treatments. Fatty acids of total lipid were myristate, palmitate, stearate (major saturated acids), oleate and linoleate (major unsaturated acids). Cholesterol contents on a protein basis were slightly reduced with increasing concentrations of Triton X-100. Cholesterol adhered to protein more tightly than other constituents The contents of gangliosides was proportionally refuted with increasing concentration of Triton X-100.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.261-261
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• 1994

Bovine milk xanthine oxidase (E.C.1.1.3.22, XO) purchased from Sigma Chemical Co. had the three protein fragments below 150 kDa on 7.5％ SDS-PAGE, which did not show enzyme activity. To remove these fragments, the enzyme preparation was further purified through Sephadex G-200 column chromatography. Two peaks exhibiting enzymatic activity were separated very closely to the void volume, which were revealed as two different enzyme forms, dimeric and monomeric, confirmed by activity staining on native PAGE. Anti sera-against each of the two enzyme forms were raised by subcutaneous injection at multiple sites on the back of rabbits during 4 weeks. On the immunodiffusion test, it was found that both of the antisera of the two forms could react with each other, which implied that their epitopes were identical In the Western blot analysis of mouse liver cytosol fraction, it was found that rabbit anti-XO antibody bound well with the protein band of monomeric mouse liver XO of about 150kDa. Based on this result, mouse liver cDNA 1 ibrary was screened by in situ hybridizat ion wi th rabbi t anti -XO antibody as probe. Through the immunological screening, recombinant phages giving positive signal by the production of XO were selected and further purified. To validate these clones, purified phages were lysogenized in E. coli Y1089 and their lysates were analysed for enzyme activity and immunoreactivity, It was verified that lysates of the purified recombinant phage lysogens exhibited the enzymatic activity as well as bound wi th XO antibody, when induced by IPTG. The above results assert that selected recombinant phage carries mouse liver XO gene.