Studies on Milk Xanthine Oxidase

Massey, Vincent; Brumby, P. E.; Komai, Hirochika; Palmer, Graham

doi:10.1016/s0021-9258(18)91738-2

Cited by 368 publications

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“…There has been a tendency to assume (cf. Massey, Brumby, Komai & Palmer, 1969) that all of these constituents would not be likely to function as a single catalytic unit in the molecule and that there must be two presumably equivalent active centres (Bray, 1963). We now present evidence based on FAD loss (Bray, Chisholm, Hart, Meriwether & Watts, 1966) during inactivation of the reduced enzyme by iodoacetamide (Bray & Watts, 1965) indicating that xanthine oxidase operates, during aerobic xanthine oxidation at least, with a single active centre.The interpretation of earlier quantitative studies on reaction ofxanthine oxidase with iodoacetamide, by measuring iodide liberation (Bray & Watts, 1965) and following FAD loss (A. J. Chisholm, A. M. Scott & R. C. Bray, unpublished work), was complicated by the use as starting material of preparations with non-stoicheiometric FAD/Mo ratios.…”

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Reaction with iodoacetamide and the number of active centres in xanthine oxidase

McGartoll

Bray

1969

Biochemical Journal

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Active milk xanthine oxidase has 2 moles of FAD, 2g.atoms of Mo and 8g.atoms of Fe/mole of protein of mol.wt. 275000 (Hart, McGartoll, Chapman & Bray, 1969). There has been a tendency to assume (cf. Massey, Brumby, Komai & Palmer, 1969) that all of these constituents would not be likely to function as a single catalytic unit in the molecule and that there must be two presumably equivalent active centres (Bray, 1963). We now present evidence based on FAD loss (Bray, Chisholm, Hart, Meriwether & Watts, 1966) during inactivation of the reduced enzyme by iodoacetamide (Bray & Watts, 1965) indicating that xanthine oxidase operates, during aerobic xanthine oxidation at least, with a single active centre.The interpretation of earlier quantitative studies on reaction ofxanthine oxidase with iodoacetamide, by measuring iodide liberation (Bray & Watts, 1965) and following FAD loss (A. J. Chisholm, A. M. Scott & R. C. Bray, unpublished work), was complicated by the use as starting material of preparations with non-stoicheiometric FAD/Mo ratios. These problems were overcome for the present work by the use of the salicylate method for preparing the enzyme, this giving samples free from demolybdoxanthine oxidase (Hart et al. 1969). The enzyme used was also free from detectable impurities in the ultracentrifuge and in recycling gel filtration on Sephadex G-200. Techniques and analytical methods were as described by Hart et al. (1969). Reaction of enzyme, reduced with xanthine, with iodo[14C]acetamide was carried out at pH5-5 to minimize side reactions (Bray & Watts, 1965) and was terminated by the addition of cysteine. The product from the reaction was dialysed, then purified by recycling gel filtration on Sephadex G-200. After three cycles it showed a single symmetrical peak, radioactivity (detected with a flow scintillation counter from Packard Instruments Ltd., Wembley, Middx.), residual xanthine oxidase

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Reaction with iodoacetamide and the number of active centres in xanthine oxidase

McGartoll

Bray

1969

Biochemical Journal

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“…), stained with saturated ethanolic uranylacetate and lead citrate, and examined in a Philips EM 300. Protein concentrations were obtained by the method of Lowry et al ( 9), 5'-nueleotidase, thiamine pyrophosphatase, glucose-6-phosphatase, and succinic dehydrogenase ( 14), xanthine oxidase (12), and galactosyltransferase (18,21) as described in the literature. UDPase assays were performed as for thiamine pyrophosphatase, using 2 mM UDP.…”

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Enzymic characteristics of fat globule membranes from bovine colostrum and bovine milk.

Powell¹,

Järlfors²,

Brew³

1977

The Journal of Cell Biology

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Fat globule membranes have been isolated from bovine colostrum and bovine milk by the dispersion of the fat in sucrose solutions at 4~ and fractionation by centrifugation through discontinuous sucrose gradients. The morphology and enzymic characteristics of the separated fractions were examined.Fractions comprising a large proportion of the total extracted membrane were thus obtained having high levels of the Golgi marker enzymes UDP-galactose Nacetylglucosamine fl-4-galactosyltransferase and thiamine pyrophosphatase. A membrane-derived form of the galactosyltransferase has been solubilized from fat and purified to homogeneity. This enzyme is larger in molecular weight than previously studied soluble galactosyltransferases, but resembles in size the galactosyltransferase of lactating mammary Golgi membranes.In contrast, when fat globule membranes were prepared by traditional procedures, which involved washing the fat at higher temperatures, before extraction, galactosyltransferase was not present in the membranes, having been released into supernatant fractions. When the enzyme released by this procedure was partially purified and examined by gel filtration, it was found to be of a degraded form resembling in size the soluble galactosyltransferase of milk. The release is therefore attributed to the action of proteolytic enzymes.Our observations contrast with previous biochemical studies which suggested that Golgi membranes do not contribute to the milk fat globule membrane. They are, however, consistent with electron microscope studies of the fat secretion process, which indicate that secretory vesicle membranes, derived from the Golgi apparatus, may provide a large proportion of the fat globule membrane.While the composition and origin of the membranes surrounding the fat globules of milk have been the subject of many studies (1,8,16,23,24), there are serious differences between the conclusions reached on the basis of electron microscope and biochemical analyses.The elegant microscope studies of Wooding (23, 24) suggest that, within the lactating mammary epithelial cell, fat droplets originate near the basal portion with no surrounding membrane, but that during passage to the apex of the cell before secretion they become associated with membranebounded vesicles derived from the Golgi apparatus. Under some conditions, fat droplets are observed within the cell included in vesicles which also contain aqueous constituents and casein micelles (25). Fat globule membranes can therefore originate without any contribution from the plasmalemma. Normally the acquisition of the membrane occurs at the plasmalemma during secre-

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“…XO was isolated and purified by the method of Massey et a1. 6 The identity and purity of the enzyme was ascertained by optical spectra, AFR (=120; activity to flavin ratio) and PFR (=5.8; protein to flavin ratio) values. Analytical grade PdC12 and NaH2P04 were used.…”

Section: Chemical Physics Group Tata Institute Of Fundamental Researc...mentioning

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Mixed inhibition of the oxidoreductase activity of xanthine oxidase by Pd2+ ion

Sau¹,

Mondal²,

Mitra³

1994

J. Chem. Soc., Chem. Commun.

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Studies on Milk Xanthine Oxidase

Cited by 368 publications

References 31 publications

Reaction with iodoacetamide and the number of active centres in xanthine oxidase

Reaction with iodoacetamide and the number of active centres in xanthine oxidase

Enzymic characteristics of fat globule membranes from bovine colostrum and bovine milk.

Mixed inhibition of the oxidoreductase activity of xanthine oxidase by Pd2+ ion

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