Xanthine oxidase inactivation by reagents that modify thiol groups.
Ontology highlight
ABSTRACT:
SUBMITTER: Green RC
PROVIDER: S-EPMC1198348 | biostudies-other | 1967 Nov
REPOSITORIES: biostudies-other
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Project description:Milk is a source of antimicrobial systems such as xanthine oxidoreductase, which has been proposed to modulate the oral and gut microbiota of infants. Heat treatments are applied to milk to ensure its microbial safety, however, the effects of heat on this antimicrobial enzyme are not known. The effects of batch pasteurization (BP), high-temperature short time (HTST), and ultra high temperature (UHT) on kinetics of inactivation of xanthine oxidase and its antimicrobial properties were determined. Xanthine oxidase activity was preserved by HTST (100%). Partial (8%) and nearly complete (95%) enzyme inactivation were observed for BP and UHT milks, respectively. K m values of 100 μM and V max values of 6.85, 5.12, 6.31, and 0.40 μmol/min/mg were determined for xanthine oxidase in raw, BP, HTST, and UHT milks, respectively. These results demonstrate that xanthine oxidase maintains apparent affinity and activity for its substrate when milk is treated by BP and HTST and yet the enzyme is inactivated with UHT. To investigate heat treatment-induced alterations in the biological activity of xanthine oxidase, heat treated milks were compared to raw milk for their ability to inhibit the growth of S. aureus. Raw, BP, and HTST milk xanthine oxidase efficiently inhibited S. aureus growth. However, these antibacterial properties were lost when milk was subjected to UHT. These results demonstrate that HTST and BP preserves bovine milk xanthine oxidase activity compared with UHT and that, the judicious selection of thermal treatments could be exploited to preserve the antimicrobial properties of bovine milk.
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Project description:Xanthine oxidase activation occurs in sepsis and results in the generation of uric acid (UrAc) and reactive oxygen species (ROS). We aimed to evaluate the effect of xanthine oxidase inhibitors (XOis) in rats stimulated with lipopolysaccharide (LPS). LPS (10 mg/kg) was administered intraperitoneally (i.p.) immediately after allopurinol (Alo, 2 mg/kg) or febuxostat (Feb, 1 mg/kg) every 24 h for 3 days. To increase UrAc levels, oxonic acid (Oxo) was administered by gavage (750 mg/kg per day) for 5 days. Animals were divided into the following 10 groups (n = 6 each): (1) Control, (2) Alo, (3) Feb, (4) LPS, (5) LPSAlo, (6) LPSFeb, (7) Oxo, (8) OxoLPS, (9) OxoLPSAlo, and (10) OxoLPSFeb. Feb with or without Oxo did not aggravate sepsis. LPS administration (with or without Oxo) significantly decreased the creatinine clearance (ClCr) in LPSAlo (60%, P < 0.01) versus LPS (44%, P < 0.05) and LPSFeb (35%, P < 0.05). Furthermore, a significant increase in mortality was observed with LPSAlo (28/34, 82%) compared to LPS treatment alone (10/16, 63%) and LPSFeb (11/17, 65%, P < 0.05). In addition, increased levels of thiobarbituric acid reactive substances (TBARS), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 were observed at 72 h compared to the groups that received LPS and LPSFeb with or without Oxo. In this study, coadministration of Alo in LPS-induced experimental sepsis aggravated septic shock, leading to mortality, renal function impairment, and high ROS and proinflammatory IL levels. In contrast, administration of Feb did not potentiate sepsis, probably because it did not interfere with other metabolic events.
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Project description:Glyceryl trinitrate (GTN) has found widespread use for the treatment of angina pectoris, a pathological condition manifested by chest pain resulting from insufficient blood supply to the heart. Metabolic conversion of GTN, a nitric oxide (NO) pro-drug, into NO induces vasodilation and improves blood flow. Patients develop tolerance to GTN after several weeks of continuous use, limiting the potential for long-term therapy. The mechanistic cause of nitrate tolerance is relatively unknown. We developed a cell culture model of nitrate tolerance that utilizes stable isotopes to measure metabolism of 15N3-GTN into 15N-nitrite. We performed global metabolomics to identify the mechanism of GTN-induced nitrate tolerance and to elucidate the protective role of vitamin C (ascorbic acid). Metabolomics analyses revealed that GTN impaired purine metabolism and depleted intracellular ATP and GTP. GTN inactivated xanthine oxidase (XO), an enzyme that is critical for the metabolic bioactivation of GTN into NO. Ascorbic acid prevented inactivation of XO, resulting in increased NO production from GTN. Our studies suggest that ascorbic acid has the ability to prevent nitrate tolerance by protecting XO, but not aldehyde dehydrogenase (another GTN bioactivating enzyme), from GTN-induced inactivation. Our findings provide a mechanistic explanation for the previously observed beneficial effects of ascorbic acid in nitrate therapy.
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Project description:DFT calculations support a concerted mechanism for xanthine oxidase and aldehyde oxidase hydride displacement from the sp(2) carbon of 6-substituted 4-quinazolinones. The variations in transition state structure show that C-O bond formation is nearly complete in the transition state and the transition state changes are anti-Hammond with the C-H and C-O bond lengths being more product-like for the faster reactions. The C-O bond length in the transition state is around 90% formed. However, the C-H bond is only about 80% broken. This leads to a very tetrahedral transition state with an O-C-N angle of 109 degrees. Thus, while the mechanism is concerted, the antibonding orbital of the C-H bond that is broken is not directly attacked by the nucleophile and instead hydride displacement occurs after almost complete tetrahedral transition state formation. In support of this the C=N bond is lengthened in the transition state indicating that attack on the electrophilic carbon occurs by addition to the C=N bond with negative charge increasing on the nitrogen. Differences in experimental reaction rates are accurately reproduced by these calculations and tend to support this mechanism.
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Project description:1. The ;xanthine oxidase' activity of rat liver supernatant, most of which behaves as an NAD(+)-dependent dehydrogenase (type D) can be rapidly converted into an oxidase (type O) by thiol reagents such as tetraethylthiuram disulphide, copper sulphate, 5,5'-dithiobis-(2-nitrobenzoic acid), N-ethylmaleimide and p-hydroxymercuribenzoate. Treatment with copper sulphate, if prolonged, leads to almost complete inactivation of the enzyme. The effect of these reagents is prevented by dithioerythritol, and in all cases but that of N-ethylmaleimide is reversed by the same thiol. 2. Dithioerythritol prevents and reverses the conversion of xanthine oxidase from type D into type O brought about by storage of rat liver supernatant at -20 degrees C, preincubation under anaerobic conditions, treatment with carbon or with diethyl ether, and reverses, but does not prevent, the conversion obtained by preincubation of the whole liver homogenate. 3. Conversion of the enzyme from type D into type O is effected by preincubation of rat liver supernatant with the sedimentable fraction from rat liver but not from chick or pigeon liver. The xanthine dehydrogenase activity of chick liver supernatant is not changed into an oxidase by preincubation with the sedimentable fraction from rat liver. 4. The enzyme activity of rat liver supernatant is converted from type D into type O during purification of the enzyme: the purified enzyme can be reconverted into type D by dithioerythritol. 5. The enzyme appears as an oxidase in the supernatant of rat heart, intestine, spleen, pancreas, lung and kidney. The enzyme of all organs but intestine can be converted into a dehydrogenase by dithioerythritol.
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Project description:Mammalian xanthine oxidoreductases, which catalyze the last two steps in the formation of urate, are synthesized as the dehydrogenase form xanthine dehydrogenase (XDH) but can be readily converted to the oxidase form xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. Here, we present the crystal structure of the dimeric (M(r), 290,000) bovine milk XDH at 2.1-A resolution and XO at 2.5-A resolution and describe the major changes that occur on the proteolytic transformation of XDH to the XO form. Each molecule is composed of an N-terminal 20-kDa domain containing two iron sulfur centers, a central 40-kDa flavin adenine dinucleotide domain, and a C-terminal 85-kDa molybdopterin-binding domain with the four redox centers aligned in an almost linear fashion. Cleavage of surface-exposed loops of XDH causes major structural rearrangement of another loop close to the flavin ring (Gln 423Lys 433). This movement partially blocks access of the NAD substrate to the flavin adenine dinucleotide cofactor and changes the electrostatic environment of the active site, reflecting the switch of substrate specificity observed for the two forms of this enzyme.
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Project description:Malaria is a highly inflammatory disease caused by Plasmodium infection of host erythrocytes. However, the parasite does not induce inflammatory cytokine responses in macrophages in vitro and the source of inflammation in patients remains unclear. Here, we identify oxidative stress, which is common in malaria, as an effective trigger of the inflammatory activation of macrophages. We observed that extracellular reactive oxygen species (ROS) produced by xanthine oxidase (XO), an enzyme upregulated during malaria, induce a strong inflammatory cytokine response in primary human monocyte-derived macrophages. In malaria patients, elevated plasma XO activity correlates with high levels of inflammatory cytokines and with the development of cerebral malaria. We found that incubation of macrophages with plasma from these patients can induce a XO-dependent inflammatory cytokine response, identifying a host factor as a trigger for inflammation in malaria. XO-produced ROS also increase the synthesis of pro-IL-1β, while the parasite activates caspase-1, providing the two necessary signals for the activation of the NLRP3 inflammasome. We propose that XO-produced ROS are a key factor for the trigger of inflammation during malaria.
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Project description:The thiol reagent dithiothreitol inhibits the activity of a core GDP-fucose-N-acetylglucosaminide alpha-6-fucosyltransferase in plasma and blood-cell homogenates, while promoting the activity of alpha-2- and alpha-3-fucosyltransferases. The latter enzymes catalyse transfer of fucose on to terminal galactose and subterminal N-acetylglucosamine residues respectively. A thiol-blocking reagent N-ethylmaleimide does not affect the activity of the alpha-6-fucosyltransferase, but inhibits the other two enzymes. These results indicate the presence of a critical disulphide linkage in the alpha-6-fucosyltransferase, and provide a means of delineation of different fucosyltransferases.
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