253. Cellular constituents. The chemistry of xanthine oxidase. Part III. Estimations of the co-factors and the catalytic activities of enzyme fractions from cow's milk

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

doi:10.1039/jr9560001219

Cited by 91 publications

(22 citation statements)

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“…DCPIP is frequently used as electron acceptor in enzyme-catalyzed oxidation reactions. For bovine xanthine oxidase, the rate of electron transfer from conventional substrates to DCPIP approximates that for electron transfer to the physiologic acceptor, molecular oxygen (Avis et al, 1956). However, the electron acceptors receive their electrons at fundamentally different cofactors: the electron transfer to oxygen takes place at the FAD site (Komai et al, 1969), whereas DCPIP or other artificial acceptors (e.g., phenazine methosulfate or ferricyanide) directly receive the electrons from the reduced molybdenum atom .…”

Section: Discussionmentioning

confidence: 99%

Optimization of the Expression of Human Aldehyde Oxidase for Investigations of Single-Nucleotide Polymorphisms

Foti

Hartmann

Coelho

et al. 2016

Drug Metabolism and Disposition

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Aldehyde oxidase (AOX1) is an enzyme with broad substrate specificity, catalyzing the oxidation of a wide range of endogenous and exogenous aldehydes as well as N-heterocyclic aromatic compounds. In humans, the enzyme's role in phase I drug metabolism has been established and its importance is now emerging. However, the true physiologic function of AOX1 in mammals is still unknown. Further, numerous single-nucleotide polymorphisms (SNPs) have been identified in human AOX1. SNPs are a major source of interindividual variability in the human population, and SNP-based amino acid exchanges in AOX1 reportedly modulate the catalytic function of the enzyme in either a positive or negative fashion. For the reliable analysis of the effect of amino acid exchanges in human proteins, the existence of reproducible expression systems for the production of active protein in ample amounts for kinetic, spectroscopic, and crystallographic studies is required. In our study we report an optimized expression system for hAOX1 in Escherichia coli using a codon-optimized construct. The codon-optimization resulted in an up to 15-fold increase of protein production and a simplified purification procedure. The optimized expression system was used to study three SNPs that result in amino acid changes C44W, G1269R, and S1271L. In addition, the crystal structure of the S1271L SNP was solved. We demonstrate that the recombinant enzyme can be used for future studies to exploit the role of AOX in drug metabolism, and for the identification and synthesis of new drugs targeting AOX when combined with crystallographic and modeling studies.

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Section: Discussionmentioning

confidence: 99%

Optimization of the Expression of Human Aldehyde Oxidase for Investigations of Single-Nucleotide Polymorphisms

Foti

Hartmann

Coelho

et al. 2016

Drug Metabolism and Disposition

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“…XO Activity-XO activity was monitored spectrophotometrically at 295 nm immediately before each set of experiments through its ability to oxidize xanthine to uric acid (⑀ ϭ 9,600 M Ϫ1 s Ϫ1 ) (30). In some experiments, XO activity was also monitored by superoxide radical anion production that was followed by cytochrome c Fe(III) (50 M) reduction at 550 nm (⌬⑀ ϭ 21,000 M Ϫ1 cm Ϫ1 ) (31).…”

Section: Methodsmentioning

confidence: 99%

Production of the Carbonate Radical Anion during Xanthine Oxidase Turnover in the Presence of Bicarbonate

Bonini¹,

Miyamoto²,

Mascio

et al. 2004

Journal of Biological Chemistry

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Xanthine oxidase is generally recognized as a key enzyme in purine catabolism, but its structural complexity, low substrate specificity, and specialized tissue distribution suggest other functions that remain to be fully identified. The potential of xanthine oxidase to generate superoxide radical anion, hydrogen peroxide, and peroxynitrite has been extensively explored in pathophysiological contexts. Here we demonstrate that xanthine oxidase turnover at physiological pH produces a strong one-electron oxidant, the carbonate radical anion. The radical was shown to be produced from acetaldehyde oxidation by xanthine oxidase in the presence of catalase and bicarbonate on the basis of several lines of evidence such as oxidation of both dihydrorhodamine 123 and 5,5-dimethyl-1-pyrroline-N-oxide and chemiluminescence and isotope labeling/mass spectrometry studies. In the case of xanthine oxidase acting upon xanthine and hypoxanthine as substrates, carbonate radical anion production was also evidenced by the oxidation of 5,5-dimethyl-1-pyrroline-N-oxide and of dihydrorhodamine 123 in the presence of uricase. The results indicated that Fenton chemistry occurring in the bulk solution is not necessary for carbonate radical anion production. Under the conditions employed, the radical was likely to be produced at the enzyme active site by reduction of a peroxymonocarbonate intermediate whose formation and reduction is facilitated by the many xanthine oxidase redox centers. In addition to indicating that the carbonate radical anion may be an important mediator of the pathophysiological effects of xanthine oxidase, the results emphasize the potential of the bicarbonate-carbon dioxide pair as a source of biological oxidants.

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“…Different concentrations of tested compounds were added to the samples before the enzyme was added and their effects on the generation of uric acid was used to calculate regression lines and IC 50 values. 18 Allopurinol was used as a positive standard.…”

Section: Xanthine Oxidase Activity Assaymentioning

confidence: 99%

In vitro and in vivo Antioxidant, Antihemolytic and Anti-inflammatory Activities of Santolina chamaecyparissus Extracts

Djarmouni¹,

Baghiani²,

Adjadj³

et al. 2018

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Introduction: Santolina chamaecyparissus L. is a small medicinal herb, cultivated in Europe, Asia and Africa due to the antihelmintic, antiseptic, anti spasmodic, bactericidal, fungicidal, digestive and vulnerary properties. Despite this, S. chamaecyparissus aerial part extractions have not been examined for antioxidant, antihemolytic and anti-inflammatory properties. Methods: S. chamaecyparissus aerial parts were extracted with solvents of varying polarity: methanol (crude) extract (CrE) chloroform extract (CHE), ethyl acetate extract (EAE), and aqueous extract (AE). The content of total phenolics, and flavonoids in all the extracts were determined with spectrophotometric methods. Both enzymatic and non-enzymatic methods were used to evaluate the antioxidant activity of the extracts. Antioxidant activity of all the extracts were investigated using free radical scavenging activity (1,1-diphenyl-2-picrylhydrazyl, DPPH• and 2,2'-azino-bis-3-ethyl benzthiazoline-6-sulfonic acid (ABTS) assay), capacity of the inhibition of linoleic acid peroxidation (β-carotene assay), chelation of metals (iron chelating assay) and inhibition of xanthine oxidase (XO) activity. To investigate antihemolytic activity of the extracts, the 2, 2,-azobis (2-amidinopropane) dihydrochloride (AAPH) assay was used to induce erythrocyte oxidative hemolysis. An in vivo approach was carried out on mice treated with the methanol extract at a dose of 100 mg/kg/day for 21 consecutive days, and one group was treated with vitamin C (vitamin C 50 mg/kg) as a standard drug. To determine the improvement of antioxidant potential, basic biochemical parameters were used in tissue (liver), plasma and whole blood. Phorbol 12-myristate 13-acetate (PMA)-induced ear edema was used to investigate the antiinflammatory activity of methanolic extract in mice. Results: Among all the extracts analyzed, the EAE exhibited a higher phenolic and flavonoids content (373.83 ± 0.23 mg gallic acid/g, and 61.51±7.86 mg quercetin/g respectively), followed by CE and CHE. DPPH and ABTS scavenging assays showed that EAE exhibited the highest effect in the both assays, with an IC 50 of 0.01 mg/ml and 0.0085 mg/ml respectively. All extracts moderately inhibited linoleic acid oxidation, with 57 % inhibition. The CE had a considerable chelating activity on ferrous iron (IC 50 = 0.32 mg/ml). In the enzymatic method by xanthine oxidase, results demonstrated that EAE had the highest XO inhibitory effect on both XO activity and Cyt-c reduction with IC50= 0.052±0.0003 mg/ml and 0.057±0.0006 mg/ml, respectively followed by CHE and CE. In the cellular system, CrE and CHE showed a hemolysis effect with % hemolysis (62.81% ± 1.43), (62.71% ± 1.01). However, EAE provided protection against AAPH-induced hemolysis with 53.67% ± 0.97. The in vivo assay exhibited a significant decrease (79.56%) of the content of malondialdehyde (MDA) in the liver and increased glutathione (GSH) and catalase (71.95% and 59.16% respectively). The methanol extract clearly demonstrated anti-inflammatory effects...

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253. Cellular constituents. The chemistry of xanthine oxidase. Part III. Estimations of the co-factors and the catalytic activities of enzyme fractions from cow's milk

Cited by 91 publications

References 0 publications

Optimization of the Expression of Human Aldehyde Oxidase for Investigations of Single-Nucleotide Polymorphisms

Optimization of the Expression of Human Aldehyde Oxidase for Investigations of Single-Nucleotide Polymorphisms

Production of the Carbonate Radical Anion during Xanthine Oxidase Turnover in the Presence of Bicarbonate

In vitro and in vivo Antioxidant, Antihemolytic and Anti-inflammatory Activities of Santolina chamaecyparissus Extracts

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