The regulation of xanthine oxidase. Inhibition by reduced nicotinamide–adenine dinucleotide of rat liver xanthine oxidase type D and of chick liver xanthine dehydrogenase

1. Rat liver xanthine oxidase type D (NAD(+)-dependent) and chick liver xanthine oxidase are inhibited by NADH, which competes with NAD(+). 2. The addition of a NADH-reoxidizing system in the assay of these enzyme activities is proposed. 3. Rat liver xanthine oxidase type O (oxygen-dependent) is not affected by NADH.     

Inhibition of xanthine oxidase — xanthine — iron mediated lipid peroxidation by eugenol in liposomes

The effect of eugenol on xanthine oxidase (XO) xanthine(X)-Fe⁺³-ADP mediated lipid peroxidation was studied in liver microsomal lipid liposomes. Eugenol inhibited the lipid peroxidation in a dose dependent manner as assessed by formation of thiobarbituric acid reactive substances. When tested for its effect on XO activity per se, (by measuring uric acid formation) eugenol inhibited the enzyme to an extent of 85% at 10 µm concentration and hence formation of O₂ also However, the concentration of eugenol required for XO inhibition was more in presence of metal chelators such as EDTA, EGTA and DETAPAC, but not in presence of deferoxamine, ADP and citrate. The antiperoxidative effect of eugenol was about 35 times more and inhibition of XO was about 5 times higher as compared to the effect of allopurinol. Eugenol did not scavenge O₂ generated by phenazine methosulfate and NAD but inhibited propagation of peroxidation catalyzed by Fe²⁺ EDTA and lipid hydroperoxide containing liposomes. Eugenol inhibits XO-X-Fe⁺³ ADP mediated peroxidation by inhibiting the XO activity per se in addition to quenching various radical species. (Mol Cell Biochem 166: 65-71, 1997)     

NADH oxidase activity of rat liver xanthine dehydrogenase and xanthine oxidase—contribution for damage mechanisms

The involvement of xanthine oxidase (XO) in some reactive oxygen species (ROS) -mediated diseases has been proposed as a result of the generation of and H₂O₂ during hypoxanthine and xanthine oxidation. In this study, it was shown that purified rat liver XO and xanthine dehydrogenase (XD) catalyse the NADH oxidation, generating and inducing the peroxidation of liposomes, in a NADH and enzyme concentration-dependent manner. Comparatively to equimolar concentrations of xanthine, a higher peroxidation extent is observed in the presence of NADH. In addition, the peroxidation extent induced by XD is higher than that observed with XO. The in vivo-predominant dehydrogenase is, therefore, intrinsically efficient at generating ROS, without requiring the conversion to XO. Our results suggest that, in those pathological conditions where an increase on NADH concentration occurs, the NADH oxidation catalysed by XD may constitute an important pathway for ROS-mediated tissue injuries.     

The nonenzymic hydrolysis of Δ2-thiazoline-2-carboxylate: The product of the suspected physiological reaction catalyzed by d-amino acid oxidase

Δ²-Thiazoline-2-carboxylate, the product of the suspected physiological reaction catalyzed by d-amino acid oxidase, is stable to hydrolysis at 37°C and pH 7 or above, but it hydrolyzes readily at pH 5 or below to give a mixture of N- and S-oxalylcysteamines; the N-oxalyl derivative predominates at pH's above 1 while the S-oxyalyl compound is the major product at high acidities. The pH-rate profile looks like the superposition of two bell-shaped curves. The initial increase in the rate as the pH is lowered is controlled by a pK_a of 3.95 and from pH 1 to 3 the rate is relatively constant (k = 6.7 × 10⁻⁴s⁻¹ at 37°C and ionic strength 0.5 m). Below pH 1 the rate increases again to a maximum in 1 m HCl and then decreases in more highly acidic solutions. The rate of conversion of S-oxalylcysteamine to N-oxalylcysteamine is inversely proportional to the hydrogen ion concentration from pH 3 to 5 but becomes largely independent of pH from pH 1 to 2. In the pH-independent region the rate is comparable with that observed by others for S-acetylcysteamine but in the pH-dependent region the rate is 20 to 25 times faster for the oxalyl derivative than for the acetyl compound. At pH 1, N-oxalylcysteamine is partially converted to the S-oxalyl derivative but the rate of hydrolysis (k = 1.0 × 10⁻⁵s⁻¹ at 37°C) to cysteamine and oxalate of this partially equilibrated system occurs at a comparable rate. The results of this investigation are rationalized in terms of what is known about other thiazoline hydrolyses and intramolecular S to N acyl migrations. The main differences in the present case are presumably due to the fact that thiazoline-2-carboxylate can undergo hydrolysis by two reaction manifolds, one with the carboxyl unprotonated and the other with it protonated. The relevance of these results to possible reactions of thiazoline-2-carboxylate in vivo is briefly considered.     

Synthesis and evaluation of β-carboline derivatives as potential monoamine oxidase inhibitors

Previous studies have shown that harmine is a reversible inhibitor of human monoamine oxidase A (MAO-A). Moreover, the crystal structure of human MAO-A in complex with harmine has been recently solved. This crystal structure shows that close to the methoxy group of the harmine moiety, a lipophilic pocket is left vacant within the binding site of human MAO-A. Our objective was to optimize the ??-carboline series against human MAO-A in order to explore this pocket. Therefore, a series of ??-carboline derivatives has been synthesized. The compounds were evaluated for their human monoamine oxidase A and B inhibitory potency and their K_i values were estimated. The results show that O-alkylated compounds with lipophilic groups like cyclohexyl, phenyl and aliphatic chains increase the inhibition of MAO-A compared to harmine. Compound 3e, with the trifluorobutyloxy group, was the most active of this series, with a K_i against MAO-A of 3.6 nM. Molecular docking studies show that the trifluorobutyloxy chain occupies the hydrophobic pocket vacant with harmine. The O-alkylated compounds are less active on MAO-B than on MAO-A. However, several compounds show a better inhibition on MAO-B compared to harmine. Compound 3f, with the cyclohexylmethoxy chain, displayed the best inhibitory activity against MAO-B with a K_i value of 221.6 nM. This cyclohexyl bearing analogue is also a potent MAO-A inhibitor with a K_i value of 4.3 nM. Molecular docking studies show that the cyclohexyl chain also occupies a hydrophobic pocket but in different ways in MAO-A or MAO-B.     

Theoretical study of an anti-Markovnikov addition reaction catalyzed by β-cyclodextrin

Journal of Molecular Modeling - Human sodium-dependent glucose co-transporter 2 (hSGLT2) is a crucial therapeutic target in the treatment of type 2 diabetes. In this study, both comparative...     

Structure–activity relationships of fraxamoside as an unusual xanthine oxidase inhibitor

Fraxamoside, a macrocyclic secoiridoid glucoside featuring a hydroxytyrosol group, was recently identified as a xanthine oxidase inhibitor (XOI) comparable in potency in vitro to the standard antigout drug allopurinol. However, this activity and its considerably higher value than its derivatives oleuropein, oleoside 11-methyl ester, and hydroxytyrosol are not explained by structure–activity relationships (SARs) of known XOIs. To exclude allosteric mechanisms, we first determined the inhibition kinetic of fraxamoside. The resulting competitive mechanism prompted a computational SAR characterization, combining molecular docking and dynamics, which fully explained the behavior of fraxamoside and its derivatives, attributed the higher activity of the former to conformational properties of its macrocycle, and showed a substantial contribution of the glycosidic moiety to binding, in striking contrast with glycoside derivatives of most other XOIs. Overall, fraxamoside emerged as a lead compound for a new class of XOIs potentially characterized by reduced interference with purine metabolism.     

HZE ⁵⁶Fe-ion irradiation induces endothelial dysfunction in rat aorta: role of xanthine oxidase

Ionizing radiation has been implicated in the development of significant cardiovascular complications. Since radiation exposure is associated with space exploration, astronauts are potentially at increased risk of accelerated cardiovascular disease. This study investigated the effect of high atomic number, high-energy (HZE) iron-ion radiation on vascular and endothelial function as a model of space radiation. Rats were exposed to a single whole-body dose of iron-ion radiation at doses of 0, 0.5 or 1 Gy. In vivo aortic stiffness and ex vivo aortic tension responses were measured 6 and 8 months after exposure as indicators of chronic vascular injury. Rats exposed to 1 Gy iron ions demonstrated significantly increased aortic stiffness, as measured by pulse wave velocity. Aortic rings from irradiated rats exhibited impaired endothelial-dependent relaxation consistent with endothelial dysfunction. Acute xanthine oxidase (XO) inhibition or reactive oxygen species (ROS) scavenging restored endothelial-dependent responses to normal. In addition, XO activity was significantly elevated in rat aorta 4 months after whole-body irradiation. Furthermore, XO inhibition, initiated immediately after radiation exposure and continued until euthanasia, completely inhibited radiation-dependent XO activation. ROS production was elevated after 1 Gy irradiation while production of nitric oxide (NO) was significantly impaired. XO inhibition restored NO and ROS production. Finally, dietary XO inhibition preserved normal endothelial function and vascular stiffness after radiation exposure. These results demonstrate that radiation induced XO-dependent ROS production and nitroso-redox imbalance, leading to chronic vascular dysfunction. As a result, XO is a potential target for radioprotection. Enhancing the understanding of vascular radiation injury could lead to the development of effective methods to ameliorate radiation-induced vascular damage.     

A possible hydrolysis mechanism of β—naphthyl acetate catalyzed by antibodies

The mechanism of ester hydrolysis has been extensively studied;however,the precise function of active-site residues in promoting catalysis is unclear.We describe here the structural models for the complex of a catalytic antibody Fv fragment with a phosphonate transition-state analogue,constructed by using gene cloning,sequencing and molecular modeling,mainly based on a known X-ray structure of a catalytic antibody.Hydrophobic and electrostatic analyses of the Fv/analog and Fv/substrate interaction suggest the hydrolysis mechanism:Tyr L91 and Tyr H97 play important roles to stabilize the β-naphthyl group of hapten through π-stack;His H35 donates a pair of free electrons at the atom NE2 to an active water and let it to be a partial hydroxide,which attacks the carbon atom of the carbonyl group of the substrate.Both His H35 and Arg L96 can form hydrogen bonds and stabilize the anionic tetrahedral intermediate formed during turnover.This mechanism emphasizes that an active water bridge may be formed during hydrolysis process.     

The role of conjunctival epithelial cell xanthine oxidoreductase/xanthine oxidase in oxidative reactions on the ocular surface of dry eye patients with Sjögren's syndrome

Previous papers examined lipid peroxidase levels and myeloperoxidase activity as products of oxidative and inflammatory reactions in the tear fluid of patients suffering from dry eye. The aim of the present paper was to investigate whether the enzymes xanthine oxidoreductase/xanthine oxidase known to generate reactive oxygen species contribute to oxidative reactions on the ocular surface. Xanthine oxidoreductase/xanthine oxidase were examined immunohistochemically as well as histochemically in conjunctival epithelial cells of patients suffering from dry eye. Patients with verified autoimmune dry eye (Sj?gren's syndrome) participated in our study; normal eyes served as controls. Conjunctival epithelial cells were obtained by the method of impression cytology using Millicell membranes. The results revealed a pronounced expression, as well as activity of xanthine oxidoreductase/xanthine oxidase in the conjunctival epithelium of dry eye. It is suggested that reactive oxygen species which are generated by this enzymatic system, contribute to oxidative reactions on the eye surface of patients with ocular manifestations of autoimmune disease (Sj?gren's syndrome).     

The utilization of glycollate by Micrococcus denitrificans: the β-hydroxyaspartate pathway

1. Micrococcus denitrificans utilized glycollate as sole carbon source for aerobic growth. Glyoxylate was utilized less well, and though glycine alone did not support growth it enhanced growth on glyoxylate. 2. During growth on glycollate, ¹⁴C was incorporated from [2-¹⁴C]glycollate into glycine and thence into aspartate, malate and glutamate. No phosphoglycerate was labelled at the earliest times. 3. Glyoxylate was the first product of glycollate utilization, and glycollate oxidase was inducibly formed on transfer of the organism to glycollate-containing media. 4. Extracts of glycollate-grown M. denitrificans contained negligible glyoxylate-carboligase activity and only low tartronate semialdehyde-reductase activity. 5. erythro-β-Hydroxyaspartate is a key intermediate in glyoxylate utilization by this organism. Enzymes catalysing (a) the synthesis of erythro-β-hydroxyaspartate from glyoxylate and glycine, and (b) the conversion of erythro-β-hydroxyaspartate into oxaloacetate, were inducibly formed during growth on glycollate and on other substrates yielding glyoxylate. Methods for the assay of these enzymes were developed. 6. It is concluded that in M. denitrificans the biosynthesis of cell materials from glycollate is accomplished by the `β-hydroxyaspartate pathway', a novel metabolic route that may also perform a catabolic role in glyoxylate oxidation.     

n-alkane oxidation by apseudomonas formation and β-oxidation of intermediate fatty acids

Summary From a culture broth ofPseudomonas aeruginosa (KSLA strain 473) grown on heptane as the sole source of carbon, fatty acids could be isolated after a period of decreased oxygen supply. The corresponding methyl esters—obtained by treatment with diazomethane—were separated by gas-liquid chromatography and identified by mass spectrometry. Heptylic, valeric and propionic acids were shown to be present in the original culture broth. Using the same techniques the formation of caproic acid from hexane was shown to occur, whereas the amount of butyric acid formed was extremely small and inconsistent. These results show conclusively that this microbiological oxidation of heptane and hexane proceeds by way of the corresponding fatty acids, which are further degraded by β-oxidation. The absence of caproic and valeric acids in heptane and hexane oxidation, respectively, shows that decarboxylation of fatty acids does not occur.     

The oxidation of α-olefins by aPseudomonas reactions involving the double bond

The preponderant pathway of octene-1 degradation by octane- and octene-1-grownPseudomonas aeruginosa cells (strain 473) starts with oxidation of the methyl group.In addition, with both types of cells minor reactions occur that involve the double bond. The formation of a 1,2-epoxide was reported earlier. In addition, the identification of the saturated C₈ fatty acid is a strong indication that the terminal methylene group is partially converted into an aldehydic group. The aldehyde seems to be formed beside the epoxide and the latter is not an intermediate. Enzymatic dihydroxylation of the double bond, if at all occurring, is masked by non-enzymatic hydrolysis of the epoxide.The formation of a saturated methyl ketone could not be detected. Nor is the olefinic group converted into a primary or secondary alcohol group under conditions which result in accumulation of octanol-1 from octane and of 7-octenol-1 from octene-1.The absence of a saturated alcohol among the intermediates in octene-1 degradation excludes hydration of the double bond as well as other mechanisms leading to saturated alcohols.Accumulation of C₈ fatty acids was effected by inhibition of -oxidation with acrylate, whereas addition of a competing alcohol substrate (octanediol-1,8) yielded detectable amounts of the alcoholic intermediates.     

Batch production of high-content fructo-oligosaccharides from sucrose by the mixed-enzyme system of β-fructofuranosidase and glucose oxidase

The production of high-content fructo-oligosaccharides from sucrose by the mixed-enzyme system of β-fructofuranosidase and glucose oxidase was investigated. The mixed-enzyme reaction was carried out in a stirred tank reactor containing 0.7 l of sucrose solution with coupled β-fructofuranosidase and glucose oxidase for 25 h. The optimum conditions for the mixed-enzyme reaction were as follows: pH, 5.5; temperature, 40°C; sucrose concentration, 400 g/l; agitation speed, 550 rpm; oxygen flow rate, 0.7 l/min; enzyme dosage, 10 units of β-fructofuranosidase with the combination of 15 units of glucose oxidase per gram sucrose. Under optimum conditions, high-content fructo-oligosaccharides up to 98% were obtained with complete consumption of sucrose and glucose by the mixed-enzyme system. Compared with the fructo-oligosaccharides produced by the β-fructofuranosidase, the high-content fructo-oligosaccharides produced by the mixed-enzyme system showed a significant difference with respect to sugar composition; i.e., a higher content of nystose was accumulated and only a trace amount of fructofuranosyl nystose was detected.     

The production of β-glucosidase in shake-flasks by Aspergillus wentii

Studies in shake-flasks showed that Aspergillus wentii produces the maximum activity of β-glucosidase among the cultures tested. The activity against cellobiose was about 2–3 fold that against 4NPG. Aspergillus wentii produced a maximum activity of 16.5 U/ml in 14 days on malt extract. It also produced a comparable amount on other simple soluble sugars, which indicates that it is constitutive and does not require an inducer. Peptone was found to the best nitrogen source for β-glucosidase production. Optimum C/N ratio was found to be 7.3. Phosphate, magnesium and trace metals did not play significant roles in the production of β-glucosidase when they were used with malt extract as a carbon source. An inoculum of 6% (v/v) of 20-h-old culture grown on malt extract produced the maxium β-glucosidase activity.     

Activation of circularly permutated β-lactamase tethered to antibody domains by specific small molecules

Regulation of enzyme activity either by its substrates or by effectors is generally known as allostery. However, it has been considered hard to alter its effector specificity, despite its potential utility as a sensitive molecular sensor. To this end, we made fusion proteins consisting of an antibody variable region Fv and a circularly permutated TEM-1 β-lactamase cpBLA. Two expression vectors encoding Fv-cpBLA with different antigen specificities were made, in which cpBLA was inserted into the linker region of the single chain Fv that specifically binds either bone-related disease marker osteocalcin (BGP) C-terminal peptide or neonicotinoid insecticide imidacloprid (ICP). The cpBLA having new termini near the active site was activated upon binding with its cognate antigen, owing to the stabilization of tethered Fv by bound antigen. As a result, both Fv-cpBLA showed specific antigen binding as well as antigen-induced enhancement in catalytic activity. Moreover, E. coli cells expressing Fv-cpBLA for ICP showed ICP concentration dependent growth in the medium containing ampicillin. The system was also applied to select for Fv-cpBLA linker mutants that confer faster growth. This will be the first of an antibody-based small molecule indicator enzyme.