Cellular constituents. The chemistry of xanthine oxidase. Part I. The preparation of a crystalline xanthine oxidase from cow's milk

Avis, P. G.; Bergel, F.; Bray, R. C.

doi:10.1039/jr9550001100

Cited by 121 publications

(59 citation statements)

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“…When stored as a solution, some XO was precipitated. Light microscopy revealed the presence of microcrystals of XO in the precipitate in a shape similar to that previously described by Avis et al (13).…”

supporting

confidence: 54%

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Solubilization and Purification of Xanthine Oxidase from Bovine Milk Fat Globule Membrane

Spitsberg

Gorewit

1998

Protein Expression and Purification

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confidence: 54%

“…Ball's method is still used for preparation of XO from milk on a commercial scale. This method, as well as others (13,14), includes a proteolytic digestion step to release the particulate XO. Waud et al (15) described a method for XO isolation and purification without the proteolytic digestion step, thus avoiding proteolytic degradation of XO.…”

mentioning

confidence: 99%

Solubilization and Purification of Xanthine Oxidase from Bovine Milk Fat Globule Membrane

Spitsberg

Gorewit

1998

Protein Expression and Purification

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“…The concentration of mutant enzyme was quantified spectrophotometrically by using a molar absorption coefficient of 37,800 M Ϫ1 ⅐cm Ϫ1 at 450 nm (25). The activity-to-flavin ratio (AFR) was obtained at 25°C as described (26). In steadystate kinetics of xanthine-NAD activity, bovine milk XDH and the DTT-treated W336A mutant were incubated at 25°C in 0.1 M pyrophosphate buffer (pH 8.5) containing 0.2 mM EDTA, 0.15 mM xanthine, and various concentrations of NAD ϩ , and the NAD-dependent activities were determined.…”

Section: Methodsmentioning

confidence: 99%

Unique amino acids cluster for switching from the dehydrogenase to oxidase form of xanthine oxidoreductase

Kuwabara

Nishino

Okamoto

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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In mammals, xanthine oxidoreductase is synthesized as a dehydrogenase (XDH) but can be readily converted to its oxidase form (XO) either by proteolysis or modification of cysteine residues. The crystal structures of bovine milk XDH and XO demonstrated that atoms in the highly charged active-site loop (Gln-423-Lys-433) around the FAD cofactor underwent large dislocations during the conversion, blocking the approach of the NAD ؉ substrate to FAD in the XO form as well as changing the electrostatic environment around FAD. Here we identify a unique cluster of amino acids that plays a dual role by forming the core of a relay system for the XDH͞XO transition and by gating a solvent channel leading toward the FAD ring. A more detailed structural comparison and sitedirected mutagenesis analysis experiments showed that Phe-549, Arg-335, Trp-336, and Arg-427 sit at the center of a relay system that transmits modifications of the linker peptide by cysteine oxidation or proteolytic cleavage to the active-site loop (Gln-423-Lys-433). The tight interactions of these residues are crucial in the stabilization of the XDH conformation and for keeping the solvent channel closed. Both oxidative and proteolytic generation of XO effectively leads to the removal of Phe-549 from the cluster causing a reorientation of the bulky side chain of Trp-336, which then in turn forces a dislocation of Arg-427, an amino acid located in the active-site loop. The conformational change also opens the gate for the solvent channel, making it easier for oxygen to reach the reduced FAD in XO.X anthine oxidoreductase (XOR) is a homodimer of molecular weight 290,000, and each subunit of the enzyme contains one molybdo-pterin (Mo-pt) cofactor, two distinct [2Fe-2S] centers, and one flavin adenine dinucleotide (FAD) cofactor (1, 2). The mammalian XORs catalyze the hydroxylation of hypoxanthine or xanthine at the Mo center, and reducing equivalents thus introduced into the enzymes are transferred via two [2Fe-2S] centers to FAD, where the reduction of NAD ϩ or molecular oxygen occurs (3). These enzymes are synthesized as the dehydrogenase form [xanthine dehydrogenase (XDH)] but can be readily converted to the oxidase form [xanthine oxidase (XO)] reversibly by oxidation of sulfhydryl residues or irreversibly by proteolysis (1, 2, 4-6). XDH shows a preference for NAD ϩ reduction at the FAD reaction site (although it still displays considerable reactivity with oxygen), whereas XO fails to react with NAD ϩ and exclusively uses dioxygen as its substrate, leading to formation of superoxide anion and hydrogen peroxide (1, 7). Previous investigations have suggested that the XDH͞XO conversion is related to milk lipid secretion (8, 9) and is implicated in diseases characterized by oxygen radical-induced tissue damage such as postischemic reperfusion injury (10-13). Thus, the XDH͞XO transition has attracted much attention from both basic and clinical researchers, not only because of the mechanistic interest in the different reactivity of FAD toward NAD ϩ or oxygen subs...

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“…A small portion of the nonfunctional population is the demolybdo species which is free of molybdenum but retains molybdopterin as documented by the phosphate analyses (see below). Xanthine oxidase with a spectral ratio of 4.8-5.0 can be obtained by subjecting enzyme that has been purified through the DEAE-Sephadex tThe functionality of a preparation of xanthine oxidase is determined by measuring its activity-to-flavin ratio (AFR) (14). Activity is defined as the change in absorbance at 295 nm per min when enzyme is incubated with 0.1 mM xanthine in 0.1 M pyrophosphate buffer (pH 8.5) at 250C.…”

mentioning

confidence: 99%

Covalently bound phosphate residues in bovine milk xanthine oxidase and in glucose oxidase from Aspergillus niger: a reevaluation.

Johnson

London

Rajagopalan

1989

Proc. Natl. Acad. Sci. U.S.A.

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The reported presence of covalently bound phosphate residues in flavoproteins has significant implications with regard to the catalytic mechanisms and structural stability of the specific enzymes themselves and in terms of general cellular metabolic regulation. These considerations have led to a reevaluation of the presence of covalently bound phosphorus in the flavoproteins xanthine oxidase (xanthine: oxygen oxidoreductase, EC 1.1.3.22) and glucose oxidase (13-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4). Milk xanthine oxidase purified by a procedure that includes anion-exchange chromatography is shown to contain three phosphate residues. All three are noncovalently associated with the protein, two with the FAD cofactor, and one with the molybdenum cofactor.Results of chemical analysis and 31p NMR spectroscopy indicate that enzyme purified by this method contains no phosphoserine residues. Xanthine oxidase preparations purified by chromatography on calcium phosphate gel in place of DEAESephadex yielded higher phosphate-to-protein ratios, which could be reduced to the expected values by additional purification on a folate affinity column. Highly active, highly purified preparations of glucose oxidase are shown to contain only the two phosphate residues of the FAD cofactor. The covalently bound bridging phosphate reported by others may arise in aged or degraded preparations of the enzyme but appears not to be a constituent of functional glucose oxidase. These results suggest that the presence of covalent phosphate residues in other flavoproteins should be rigorously reevaluated as well.Most molybdenum-containing enzymes bind the metal as a complex with a reduced, phosphorylated pterin termed molybdopterin (1, 2) that is tightly, but not covalently, bound to the protein. Because of its instability when released, molybdopterin is difficult to extract quantitatively as a single species (1). The presence of the phosphate ester in molybdopterin and in various inactive derivatives thereof has been extremely useful in attempts to evaluate the pterin content of various enzyme preparations. For example, the phosphate content of chicken liver sulfite oxidase is one per subunit (1) and of chicken liver xanthine dehydrogenase and milk xanthine oxidase is three per subunit (two in the FAD cofactor and one in molybdopterin) (3). In contrast to the results obtained in our laboratory, Davis et al. (4) have described a preparation of milk xanthine oxidase that apparently contained a fourth phosphate residue in the form of a covalently bound phosphoserine. In view of this contradiction, we have reinvestigated the phosphate content of xanthine oxidase purified from bovine milk by several different procedures. We find, as documented below, three phosphate residues and no phosphoserine in enzyme purified by anion exchange and/or affinity chromatography. In contrast, enzyme preparations containing more than three phosphate residues are obtained if purification is limited to chromatography on calcium phosphate gel. However,...

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Cellular constituents. The chemistry of xanthine oxidase. Part I. The preparation of a crystalline xanthine oxidase from cow's milk

Cited by 121 publications

References 0 publications

Solubilization and Purification of Xanthine Oxidase from Bovine Milk Fat Globule Membrane

Solubilization and Purification of Xanthine Oxidase from Bovine Milk Fat Globule Membrane

Unique amino acids cluster for switching from the dehydrogenase to oxidase form of xanthine oxidoreductase

Covalently bound phosphate residues in bovine milk xanthine oxidase and in glucose oxidase from Aspergillus niger: a reevaluation.

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