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Roger Harrison - One of the best experts on this subject based on the ideXlab platform.
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Physicochemical and Kinetic Properties of Purified Sheep's Milk Xanthine Oxidoreductase
Journal of Dairy Science, 2004Co-Authors: Mustapha Benboubetra, Abderahmene Baghiani, Djebbar Atmani, Roger HarrisonAbstract:Abstract Xanthine oxidoreductase (XOR) was purified for the first time from sheep's Milk. The ultraviolet-visible absorption spectrum was essentially identical to those of the corresponding bovine, human, and goats' Milk Enzymes and showed an A 280 / A 450 ratio of 5.35±0.24, indicating a high degree of purity. Like Milk XOR from other species, sheep's Milk Enzyme showed a single band on SDS-PAGE corresponding to a subunit with approximate M r 150,000. Xanthine oxidase activity of purified sheep's Milk XOR (0.69±0.04μmole urate min −1 mg −1 ) was low relative to that of the bovine Milk Enzyme (1.83±0.02μmole urate min −1 mg −1 ), but higher than those of human or goats' Milk XOR. As in the latter 2 cases, the low activity of sheep's Milk XOR can be attributed to its relatively low molybdenum content (0.18 atoms per subunit), compared with that of the bovine Milk Enzyme (0.56 atoms Mo per subunit). Consistent with this, NADH oxidase activity of sheep's Milk XOR was similar to that of Enzymes purified from bovine, human, or goats' Milk. The presence of desulpho-Enzyme in sheep's Milk XOR was demonstrated by resulfuration experiments, whereby xanthine oxidase activity was increased by approximately 75%.
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Goats' Milk xanthine oxidoreductase is grossly deficient in molybdenum.
The Journal of dairy research, 2004Co-Authors: Djebbar Atmani, Mustapha Benboubetra, Roger HarrisonAbstract:Xanthine oxidoreductase (XOR) was purified from goats' Milk. The u.v.-visible absorption spectrum was essentially identical to those of the corresponding bovine and human Milk Enzymes and showed an A280/A450 ratio of 5.20+/-0.12, indicating a high degree of purity. Like bovine and human Milk XORs, Enzyme purified from goats' Milk showed a single band on SDS-PAGE corresponding to a subunit with approximate Mr 150,000. On Western blotting, mouse monoclonal anti-human XOR antibody cross-reacted with purified caprine and bovine XORs. The specific xanthine oxidase activity of goats' Milk XOR, however, was very much lower than that of bovine XOR, although NADH oxidase activities of XOR from the two sources were similar. In these respects, the caprine Milk XOR mirrors the human Milk Enzyme, in which case the kinetic effects have previously been attributed to relatively low molybdenum content. The molybdenum content of goats' Milk XOR also was shown to be relatively low, with 0.09 atoms Mo per subunit, compared with 055 atoms Mo per subunit for the bovine Enzyme. A parallel purification of human Milk XOR showed 0.03 atoms Mo per subunit. The possible physiological significance of the low molybdenum content of the caprine Milk Enzyme and of its correspondingly low enzymic activity is discussed.
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Xanthine oxidoreductase in human mammary epithelial cells: activation in response to inflammatory cytokines.
Biochimica et biophysica acta, 1998Co-Authors: Susanna Page, Mustapha Benboubetra, Debbie Powell, C R Stevens, David R. Blake, Fishaye Selase, Adrian J. Wolstenholme, Roger HarrisonAbstract:Xanthine oxidoreductase (XOR) in human mammary epithelial cells was shown to have low true specific activity, similar to that in breast Milk. Enzymic activity was increased in response to inflammatory cytokines; increases of 2–2.5-fold being seen with TNF-α and IL-1β and of approximately 8-fold with IFN-γ. No significant increase was seen with IL-6. A combination of IFN-γ and TNF-α, or of these two cytokines plus IL-1β, led to responses representing the sum of those obtained by using the individual cytokines. The 8-fold increase in enzymic activity, stimulated by IFN-γ, corresponded to only a 2–3-fold increase in specific mRNA, suggesting the possibility of post-translational activation; a possibility strongly supported by the corresponding 2–3-fold rise in XOR protein, as determined by ELISA. In no case was cytokine-induced activation accompanied by changes in the oxidase–dehydrogenase ratio of XOR. These data strongly support a role for XOR in the inflammatory response of the human mammary epithelial cell, and provide further evidence of post-translational activation of a low activity form of human XOR, similar to that previously observed in vivo for the breast Milk Enzyme.
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Purification and partial characterization of xanthine oxidase from human Milk.
Biochimica et biophysica acta, 1992Co-Authors: Shahla Abadeh, Joanne Killacky, Murtapha Benboubetra, Roger HarrisonAbstract:Abstract Xanthine oxidase was purified from human Milk in yields comparable with those obtained from bovine Milk. The freshly purified Enzyme appeared homogenous in gel permeation FPLC and SDS-PAGE, consistent with its being a homodimer with total Mr290 000 ± 6000. The ultraviolet/visible absorption spectrum differed only by slightly from that of bovine Milk Enzyme and showed an A280/A450 ratio of 5.13 ± 0.29, indicating a high degree of purity. Xanthine oxidase activities of purified Enzyme varied with batches of Milk, ranging between 3 and 46 mU/mg protein; values that are some two to three orders of magnitude smaller than those shown by the most highly purified samples of bovine Milk Enzyme. Direct comparison with commercially-available bovine Milk Enzyme showed that activities involving xanthine as reducing were 1–6% that of the bovine Enzyme, whereas those involving NADH, in contrast, were of the same order for the two Enzymes. Anerobic bleaching experiments indicated that less than 2% of the human Enzyme was present as a form active with xanthine. These findings, together with the activity data, are consistent with a very high content, possibly greater than 98%, of demolybdo- and/or desulpho-forms of human Enzyme, both of which occur, to a lesser extent, in bovine xanthine oxidase. Molybdenum assay indicated that demolybdo-Enzyme could only account for some 26% of this indicated component, suggesting that desulpho-Enzyme may account for the remainder.
Lars Bläckberg - One of the best experts on this subject based on the ideXlab platform.
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Purification of carboxyl ester lipase (bile salt-stimulated lipase) from human Milk and pancreas.
Methods in enzymology, 1997Co-Authors: Lars Bläckberg, Rui-dong Duan, Berit SternbyAbstract:Publisher Summary This chapter summarizes the physiological motivation for the interest in the purification of the human Enzyme. Purification of carboxyl ester lipase is a story of purification of several Enzymes using different substrates. Thus, several strategies and methods have been used to obtain a pure Enzyme. It has been determined that all these Enzymes are the same and are found in both human Milk and pancreas. Purification of the Enzyme from pancreatic gland is more cumbersome, which may explain why there is just one published study from 1988 available for the human Enzyme. This purification scheme includes homogenization of the pancreatic tissue in the presence of trypsin inhibitor and bile salts, centrifugations, ammonium sulfate precipitation, filtration, cholate-Sepharose chromatography, and Bio-Rex and heparin-Sepharose chromatography. Purification of the Milk Enzyme is easier, owing to the presence of fewer proteases. The chapter presents different strategies for purification of the human Enzyme from Milk and pancreas, owing to the significant difference in the source of the Enzyme—Milk or pancreatic juice/pancreas.
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Recombinant human Milk bile salt-stimulated lipase. Catalytic activity is retained in the absence of glycosylation and the unique proline-rich repeats.
Journal of Biological Chemistry, 1993Co-Authors: Lennart Hansson, Lars Bläckberg, Michael Edlund, Lennart Gustav Lundberg, Mats Strömqvist, Olle HernellAbstract:Human Milk bile salt-stimulated lipase ensures efficient utilization of triacylglycerol by breast-fed infants. Cloning and sequencing of cDNA have revealed that the peptide chain consists of 722 amino acid residues showing only little homology to typical lipases. The sequence is identical to that of pancreatic carboxylic-ester hydrolase. The COOH-terminal part contains 16 proline-rich repeats of 11 residues with O-linked carbohydrate. The only N-linked sugar chain is situated close to the active-site serine. Using C127 cells and a bovine papilloma virus vector, high and stable expression of full-length lipase and of several variants, obtained by site-directed mutagenesis, was achieved. The produced proteins were purified and further characterized. Variants lacking all, or all but two, repeats were active with similar specific activity and the same bile salt dependence as the native Milk Enzyme. Changing the asparagine necessary for N-glycosylation gave the same principal results. Active recombinant full-length lipase was also produced in a bacterial system. We conclude that neither glycosylation (N- or O-linked) nor the proline-rich repeats are essential for catalytic activity or bile salt activation of human Milk bile salt-stimulated lipase.
Takeshi Nishino - One of the best experts on this subject based on the ideXlab platform.
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Crystal Structures of Urate Bound Form of Xanthine Oxidoreductase: Substrate Orientation and Structure of the Key Reaction Intermediate
Journal of the American Chemical Society, 2010Co-Authors: Ken Okamoto, Bryan T. Eger, Emil F. Pai, Yuko Kawaguchi, Takeshi NishinoAbstract:Two contradictory models have been proposed for the binding mode of the substrate xanthine to and its activation mechanism by xanthine oxidoreductase. In an effort to distinguish between the two models, we determined the crystal structures of the urate complexes of the demolybdo-form of the D428A mutant of rat xanthine oxidoreductase at 1.7 A and of the reduced bovine Milk Enzyme at 2.1 A, the latter representing a reaction intermediate. The results clearly indicate the catalytically relevant binding mode of the substrate xanthine.
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Y-700 (1-(3-Cyano-4-(2,2-dimethylpropoxy)phenyl)-1H- pyrazole-4-carboxylic Acid): A Potent Xanthine Oxidoreductase Inhibitor with Hepatic Excretion
Journal of Pharmacology and Experimental Therapeutics, 2004Co-Authors: Atsushi Fukunari, Ken Okamoto, Takeshi Nishino, Bryan T. Eger, Emil F. Pai, Miho Kamezawa, Ichimaro Yamada, Norihisa KatoAbstract:Y-700 (1-[3-Cyano-4-(2,2-dimethylpropoxy)phenyl]-1 H -pyrazole-4-carboxylic acid) is a newly synthesized inhibitor of xanthine oxidoreductase (XOR). Steady-state kinetics with the bovine Milk Enzyme indicated a mixed type inhibition with K i and K i ′ values of 0.6 and 3.2 nM, respectively. Titration experiments showed that Y-700 bound tightly both to the active sulfo-form and to the inactive desulfo-form of the Enzyme with K d values of 0.9 and 2.8 nM, respectively. X-ray crystallographic analysis of the Enzyme-inhibitor complex revealed that Y-700 closely interacts with the channel leading to the molybdenum-pterin active site but does not directly coordinate to the molybdenum ion. In oxonate-treated rats, orally administered Y-700 (1–10 mg/kg) dose dependently lowered plasma urate levels. At a dose of 10 mg/kg, the hypouricemic action of Y-700 was more potent and of longer duration than that of 4-hydroxypyrazolo(3,4- d )pyrimidine, whereas its action was approximately equivalent to that of 2-(3-cyano-4-isobutoxyphenyl)-4-methyl-5-thiazolecarboxylic acid, a nonpurine inhibitor of XOR. In normal rats, orally administered Y-700 (0.3–3 mg/kg) dose dependently reduced the urinary excretion of urate and allantoin, accompanied by an increase in the excretion of hypoxanthine and xanthine. Y-700 (1 mg/kg) was absorbed rapidly by the oral route with high bioavailability (84.1%). Y-700 was hardly excreted via the kidneys but was mainly cleared via the liver. These results suggest that Y-700 will be a promising candidate for the treatment of hyperuricemia and other diseases in which XOR may be involved.
J L Poechmann - One of the best experts on this subject based on the ideXlab platform.
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Two forms of human Milk bile-salt-stimulated lipase
Biochemical Journal, 1992Co-Authors: J.s. Swan, M M Hoffman, M K Lord, J L PoechmannAbstract:This study provides the first direct evidence for the existence of two active forms of the human Milk Enzyme bile-salt-stimulated lipase. Heparin-based affinity chromatography and size-exclusion chromatography were used to obtain two active forms of this Enzyme with molecular masses of 97 and 120 kDa.
Olle Hernell - One of the best experts on this subject based on the ideXlab platform.
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Recombinant human Milk bile salt-stimulated lipase. Catalytic activity is retained in the absence of glycosylation and the unique proline-rich repeats.
Journal of Biological Chemistry, 1993Co-Authors: Lennart Hansson, Lars Bläckberg, Michael Edlund, Lennart Gustav Lundberg, Mats Strömqvist, Olle HernellAbstract:Human Milk bile salt-stimulated lipase ensures efficient utilization of triacylglycerol by breast-fed infants. Cloning and sequencing of cDNA have revealed that the peptide chain consists of 722 amino acid residues showing only little homology to typical lipases. The sequence is identical to that of pancreatic carboxylic-ester hydrolase. The COOH-terminal part contains 16 proline-rich repeats of 11 residues with O-linked carbohydrate. The only N-linked sugar chain is situated close to the active-site serine. Using C127 cells and a bovine papilloma virus vector, high and stable expression of full-length lipase and of several variants, obtained by site-directed mutagenesis, was achieved. The produced proteins were purified and further characterized. Variants lacking all, or all but two, repeats were active with similar specific activity and the same bile salt dependence as the native Milk Enzyme. Changing the asparagine necessary for N-glycosylation gave the same principal results. Active recombinant full-length lipase was also produced in a bacterial system. We conclude that neither glycosylation (N- or O-linked) nor the proline-rich repeats are essential for catalytic activity or bile salt activation of human Milk bile salt-stimulated lipase.