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At least 955 records · Page 53Linked to original sources
PubMed1999

Electrostatic immobilization of glucose oxidase in a weak acid, polyelectrolyte hyperbranched ultrathin film on gold: fabrication, characterization, and enzymatic activity.

In this paper we show that hyperbranched polymers can be used as a host matrix for electrostatic entrapment of enzymes. Specifically, amine-functionalized glucose oxidase (GOx+) and horseradish peroxidase, as well as poly(amidoamine) dendrimer-modified horseradish peroxidase, reversibly sorb into polyanionic, hyperbranched poly(sodium acrylate) (PAA-) films that are on the order of a few hundred angstroms thick. The quantity of GOx+ entrapped within the PAA- films depends on the nature of film preparation but is typically on the order of 0.06 unit/cm2. The extent to which entrapped GOx+ retains its activity depends on the film history, but for PAA-/GOx+ composites not exposed to glucose and stored at 4 degrees C, the original activity is retained for up to 68 days and perhaps longer.

Acrylates↗
PubMed2002

Indirect evidence of direct electron communication between the active site of galactose oxidase and a graphite electrode.

Bi-enzymatic biosensor based on galactose oxidase (GalOD) and horseradish peroxidase (HRP) using ferrocene as an efficient mediator was constructed. When a dependence of a working potential on the sensor performance was examined, an unusual behaviour was observed. With increasing of an applied working potential a lower concentration of substrate to attain full linear range was needed. A fully linear dependence from the first substrate addition was observed at and above the working potential of 150 mV. This activation of the biosensor response by an applied working potential very well corresponds with a formal potential of GalOD (156 mV). When a membrane prevented GalOD access to the electrode surface was applied, no activation effect of a working potential on the sensor performance was observed. Thus, it can be assumed that direct electron communication between GalOD and the electrode occurred.

Binding Sites↗
PubMed1984

Action of free radical generating systems upon the biological and immunological properties of caeruloplasmin.

Exposure of human caeruloplasmin, an acute phase protein with antioxidant properties, to a mixture of xanthine/hypoxanthine and xanthine oxidase as a source of reactive oxygen intermediates decreased its ferroxidase and ascorbate oxidase activities and its ability to inhibit lipid peroxidation. Immunological reactivity was also altered. Exposure to hydrogen peroxide mimicked these effects. Exposure to low-intensity u.v. irradiation depressed caeruloplasmin's ability to inhibit iron-catalysed hyaluronic acid degradation. The results may explain the mechanism of the observed inactivation of caeruloplasmin within human rheumatoid synovial fluid.

Ascorbate Oxidase↗
PubMed1989

The peroxidase/oxidase activity of soybean lipoxygenase--I. Triplet excited carbonyls from the reaction with isobutanal and the effect of glutathione.

Soybean lipoxygenase shows a secondary peroxidase/oxidase activity: The aerobic reaction with isobutanal, enhanced by hydrogen peroxide as a cosubstrate, yields acetone, exhibits chemiluminescence and consumes oxygen (phi cl = 1.3 x 10(-9) photons/O2 molecule consumed). 9,10-Dibromoanthracene-2-sulfonate increases the photoemission (kET tau 0 = 2 x 10(4) M-1; phi cl = 0.9 x 10(-7) photons/O2), whereas it is diminished by sorbate, tryptophan, 2-methyl-1,4-naphthoquinone, glutathione, and superoxide dismutase. In the presence of hydrogen peroxide the lipoxygenase reaction with glutathione yields yet another excited state. From the well-known reactions promoted by horseradish-peroxidase, these features are concluded to indicate the novel activity of soybean lipoxygenase. With isobutanal as a substrate lipoxygenase acts as an oxidase and as a peroxidase. The mechanism suggested leads to photoemissive triplet excited acetone as expected from the cleavage of an intermediate dioxetane.

Aldehydes↗
PubMed2006

Expression of TPO and ThOXs in human thyrocytes is downregulated by IL-1alpha/IFN-gamma, an effect partially mediated by nitric oxide.

Morphological and functional alterations in Hashimoto's thyroiditis (HT) are predominantly mediated by Th1 cytokines through apoptotic cell death. This ultimate step could be preceded by functional injuries in thyroid hormone synthesis. The action of two Th1 cytokines (IL-1alpha/IFN-gamma) on thyroperoxidase (TPO) and thyroid oxidase (ThOXs) expression was tested in human thyrocytes isolated from normal tissues, Graves' disease (GD) tissues, and autonomous toxic nodules. There was no evidence of cell death. Nitric oxide (NO) release was induced by cytokines but was absent when NG-nitro-L-arginine methyl ester (L-NAME) was coincubated. When thyrotropin (TSH)-incubated normal and GD thyrocytes were treated with IL-1alpha/IFN-gamma, TPO and ThOXs protein and mRNA expression dropped, a decrease partially prevented by L-NAME, suggesting that NO acts as a mediator of Th1 effects. In thyrocytes from autonomous toxic nodules, the high level of TPO and ThOXs protein expression was not influenced by TSH or by cytokines, a finding partially reproduced when normal thyrocytes were treated with increasing concentrations of TSH. In conclusion, incubation of normal or GD thyrocytes with Th1 cytokines induces a significant reduction in TSH-increased expression of both TPO and ThOXs, an effect partially mediated by NO. The thyroid cell function can therefore be severely affected in HT, even when cells remain viable. In autonomous toxic nodules, cells become partially insensitive to exogenous Th1 cytokines.

Cells, Cultured↗
PubMed1989

A new cytochemical method for ultrastructural detection of liposomes in tissues in vivo.

Multilamellar vesicles (MLVs) have been used as drug carriers to increase efficacy or decrease toxicity of a variety of therapeutic agents, including antineoplastics, antibiotics, and immunomodulators. Although analysis of the disposition of encapsulated materials is relatively simple using radiolabels or single enzymes, determining the cellular and subcellular disposition of intact MLVs, i.e., those that still retain their encapsulated materials, is much less straightforward. We have developed a technique that allows demonstration of the uptake of intact MLVs by Kupffer cells. The method is based on co-localization of paired enzymes, glucose oxidase (GO), and horseradish peroxidase (HRP). The rationale for the localization is that H2O2 generated from glucose and oxygen by GO acts as the substrate for the HRP-mediated oxidative polymerization of diaminobenzidine. Therefore, only sites of co-localization of GO and HRP should stain. Mice were injected IV with phosphatidyl choline MLVs encapsulating HRP and GO. Encapsulated enzymes were separated from non-encapsulated by passing the MLVs over a Sepharose 2B column. Control mice were injected with equivalent amounts of free GO. Mice were sacrificed 30 min after injection and liver tissue was fixed in 3% cacodylate-buffered glutaraldehyde for at least 18 hr. Tissues were washed in buffer, then stained in medium containing glucose, diaminobenzidine HCl, and dimethylsulfoxide in 0.1 M cacodylate buffer. In animals injected with MLV-encapsulated GO and HRP, vacuoles in Kupffer cells and some endothelial cells contained electron-dense reaction product. No other cell type, including polymorphonuclear leukocytes, was stained. In control animals no staining was seen. Our results indicate that encapsulation of paired enzymes may be a feasible method to demonstrate the cellular and subcellular disposition of intact liposomes.

3,3'-Diaminobenzidine↗
PubMedDate not supplied

Multiple structures and functions of cytochrome oxidase.

X-ray absorption studies have been used to investigate the structure of the four redox centers (2Fe, 2Cu) of the terminal enzyme in the respiratory chain, cytochrome c oxidase in the resting oxidized form as well as in the functional intermediates that are freeze-trapped. Methods of x-ray fluorescence detection for these low-concentration samples together with low-temperature cryostats and simultaneous optical monitoring were developed to ensure good signal-to-noise data and sample integrity. The resting oxidized form contains a sulfur bridge between the copper and iron of the active site which are separated by approximately 3.8 A. This separation of the active site metal atoms was uniquely identified by comparison of both the iron and copper EXAFS data and iron EXAFS of the copper-depleted enzyme. In the reduced state, the CO or O2 is bound to the active site iron having a structure identical to CO or oxy hemoglobin while the sulfur remains with the active site copper. Little change in structure is observed for the other iron and copper. It is the sulfur bridged active site form that is isolated by the Yonetani and Caughy methods with greater than or equal to 85% homogeneity but not the Hartzell-Beinert or similar methods. Another form observed in the redox cycle is also fully oxidized but lacks the sulfur bridged active site with the iron of the active site having a structure identical to that of the peroxidases. This form exhibits peroxidase as well as oxidase activity, and a stable intermediate is formed with hydrogen and ethylhydrogen peroxide in which the iron of the active site is structurally similar to that of the peroxidase intermediate. The active site copper, however, does not participate in the peroxidatic role and the structures of the other iron and copper are identical to those of the sulfur bridged resting oxidized form. Thus this unique enzyme has peroxidase activity which may serve to safeguard its main oxidase function.

Animals↗
PubMed2006

Identification of ethylene-mediated protein changes during nodulation in Medicago truncatula using proteome analysis.

Ethylene has been hypothesised to be a regulator of root nodule development in legumes, but its molecular mechanisms of action remain unclear. The skl mutant is an ethylene-insensitive legume mutant showing a hypernodulation phenotype when inoculated with its symbiont Sinorhizobium meliloti. We used the skl mutant to study the ethylene-mediated protein changes during nodule development in Medicago truncatula. We compared the root proteome of the skl mutant to its wild-type in response to the ethylene precursor aminocyclopropane carboxylic acid (ACC) to study ethylene-mediated protein expression in root tissues. We then compared the proteome of skl roots to its wild-type after Sinorhizobium inoculation to identify differentially displayed proteins during nodule development at 1 and 3 days post inoculation (dpi). Six proteins (pprg-2, Kunitz proteinase inhibitor, and ACC oxidase isoforms) were down-regulated in skl roots, while three protein spots were up-regulated (trypsin inhibitor, albumin 2, and CPRD49). ACC induced stress-related proteins in wild-type roots, such as pprg-2, ACC oxidase, proteinase inhibitor, ascorbate peroxidase, and heat-shock proteins. However, the expression of stress-related proteins such as pprg-2, Kunitz proteinase inhibitor, and ACC oxidase, was down-regulated in inoculated skl roots. We hypothesize that during early nodule development, the plant induces ethylene-mediated stress responses to limit nodule numbers. When a mutant defective in ethylene signaling, such as skl, is inoculated with rhizobia, the plant stress response is reduced, resulting in increased nodule numbers.

Amino Acid Sequence↗
PubMed2000

Development and comparison of biosensors for in-vivo applications.

Electrochemical biosensors have been of increasing interest, especially those developed to be directly applied in diagnostic areas, such as neuroscience. We have been interested in developing a range of biosensors for monitoring glucose, lactate, pyruvate, and glutamate in order to study on-line both brain function in the laboratory and to monitor brain health in neurointensive care. For a biosensor to function effectively in these situations, it has to combine the following characteristics: quick response and high sensitivity, good reproducibility and adequate stability. In this study we compared the performance of a number of different amperometric biosensors strategies. These included ferrocene mediation of immobilised enzymes (system A), a redox hydrogel based system (system B), and a conducting polymer approach using polyaniline (system C). All assays were operated as flow-injection systems with upstream immobilised enzyme beds if necessary. When calibrated for H2O2 systems A and B reacted quickly enough to give quantitative conversion up to 0.2 mM. Above this concentration the response was limited by horseradish peroxidase enzyme kinetics and eventually enzyme loading. System C showed a restricted H2O2 response. When calibrated for glucose (by use of immobilised glucose oxidase) system B exhibited the highest sensitivity but its analytical range was restricted because the system became limited by H2O2 response. System A had low sensitivity for analyte compared to H2O2 and system B, but a greater useful range. Problems of mediator cycling between the immobilised enzymes are discussed. System C gave an excellent linear range but sensitivity was limited by background noise. Stability and reproducibility of the systems are also described. In conclusion, from this study the ferrocene system proved to be overall most useful and has now been used in the first dual on-line monitoring of glucose and lactate in patients in neurointensive care.

Biosensing Techniques↗
PubMed1996

Mechanism of simultaneous iodination and coupling catalyzed by thyroid peroxidase.

Thyroid peroxidase (TPO) simultaneously catalyzes two very different types of reaction in the thyroid gland- iodination and coupling. The present study addresses the mechanism of this simultaneous dual activity. Compound I, the two-electron oxidation product of TPO, exists in two different forms--an oxoferryl porphyrin pi-cation radical and an oxoferryl protein radical. It has been proposed that iodination is mediated by the porphyrin pi-cation radical form of TPO compound I, while coupling is mediated by the protein radical form. However, results obtained in the present study favor the view that both iodination and coupling are mediated by the porphyrin pi-cation radical form of compound I. In the first part of the study, we compared coupling and iodination activities of two peroxidases with very similar crystal structures--cytochrome c peroxidase (CcP) and lignin peroxidase (LiP). Although these two peroxidases have very similar three-dimensional structures, CcP forms a compound I only of the protein radical type, whereas compound I of LiP exists only as a porphyrin pi-cation radical. Comparison of the catalytic activities of the two enzymes showed that diiodotyrosine (DIT)-stimulated coupling activity of LiP was significantly greater than that of CcP. Moreover, lignin peroxidase displayed very significant iodinating activity at acid pHs, whereas iodination with CcP was negligible at all pHs tested. Our findings with these two structurally similar peroxidases suggested that TPO-catalyzed iodination and coupling could both be mediated by the porphyrin pi-cation radical form of compound I. More direct evidence in support of this view was obtained in the second part of this study, employing TPO and lactoperoxidase (LPO) model systems in which iodination and coupling occurred simultaneously. Heme spectral analysis was used to correlate formation of the protein radical form of compound I with the kinetics of the iodination and coupling reactions. Formation of the compound I protein radical was not observed until the iodination and coupling reactions had almost been completed. In separate experiments it was shown that the spontaneous conversion of the porphyrin pi-cation radical form of TPO or LPO compound I to the protein radical form was markedly inhibited by a low concentration of iodide, especially in the presence of an iodide acceptor. These studies provide compelling evidence that both iodination and coupling are mediated by the porphyrin pi-cation radical form of compound I. This was further substantiated by the finding that coupling was inhibited in the presence of excess iodide, an observation readily explained by competition between iodide and DIT residues in thyroglobulin for oxidation by the porphyrin pi-cation radical.

Animals↗
PubMed1999

In vivo and in vitro evidence for hydrogen peroxide (H2O2) accumulation in the epidermis of patients with vitiligo and its successful removal by a UVB-activated pseudocatalase.

To date there is compelling in vitro and in vivo evidence for epidermal H2O2 accumulation in vitiligo. This paper reviews the literature and presents new data on oxidative stress in the epidermal compartment of this disorder. Elevated H2O2 levels can be demonstrated in vivo in patients compared with healthy controls by utilizing Fourier-Transform Raman spectroscopy. H2O2 accumulation is associated with low epidermal catalase levels. So far, four potential sources for epidermal H2O2 generation in vitiligo have been identified: (i) perturbed (6R)-L-erythro 5,6,7,8 tetrahydrobiopterin (6BH4) de novo synthesis/recycling/regulation; (ii) impaired catecholamine synthesis with increased monoamine oxidase A activities; (iii) low glutathione peroxidase activities; and (iv) "oxygen burst" via NADPH oxidase from a cellular infiltrate. H2O2 overproduction can cause inactivation of catalase as well as vacuolation in epidermal melanocytes and keratinocytes. Vacuolation was also observed in vitro in melanocytes established from lesional and nonlesional epidermis of patients (n = 10) but was reversible upon addition of catalase. H2O2 can directly oxidize 6BH4 to 6-biopterin, which is cytotoxic to melanocytes in vitro. Therefore, we substituted the impaired catalase with a "pseudocatalase". Pseudocatalase is a bis-manganese III-EDTA-(HCO3-)2 complex activated by UVB or natural sun. This complex has been used in a pilot study on 33 patients, showing remarkable repigmentation even in long lasting disease. Currently this approach is under worldwide clinical investigation in an open trial. In conclusion, there are several lines of evidence that the entire epidermis of patients with vitiligo is involved in the disease process and that correction of the epidermal redox status is mandatory for repigmentation.

Animals↗
PubMed2000

Response of putative indices of copper status to copper supplementation in human subjects.

No sensitive functional index is currently available to assess Cu status in healthy human populations. This study evaluated the effect of Cu supplementation on putative indices of Cu status in twelve women and twelve men, aged between 22 and 45 years, who participated in a double-blind placebo controlled crossover study. The study consisted of three 6-week supplementation regimens of 3 mg CuSO4, 3 mg Cu-glycine chelate and 6 mg Cu-glycine chelate, each separated by placebo periods of equal length. Women had significantly higher caeruloplasmin oxidase activity (P < 0.001), caeruloplasmin protein concentration (P < 0.05), and serum diamine oxidase activity (P < 0.01) at baseline than men. Erythrocyte and leucocyte superoxide dismutase activity, leucocyte cytochrome c oxidase activity, and erythrocyte glutathione peroxidase activity did not respond to Cu supplementation. Platelet cytochrome c oxidase activity was significantly higher (P < 0.01), after supplementation with 6 mg Cu-glycine chelate in the total group and in women but did not change in men. Caeruloplasmin oxidase activity was significantly higher (P < 0.05), in men after supplementation with 3 mg Cu-glycine chelate, while caeruloplasmin protein concentration was significantly lower in men after supplementation with 6 mg Cu-glycine chelate (P < 0.05). Serum diamine oxidase activity was significantly higher after all supplementation regimens in the total group and in both men and women (P < 0.01). These results indicate that serum diamine oxidase activity is sensitive to changes in dietary Cu intakes and may also have the potential to evaluate changes in Cu status in healthy adult human subjects.

Adult↗
PubMed1986

Glucose oxidase as label in histological immunoassays with enzyme-amplification in a two-step technique: coimmobilized horseradish peroxidase as secondary system enzyme for chromogen oxidation.

A sensitive staining procedure for glucose oxidase (GOD) as marker in immunohistology is described. The cytochemical procedure involves a two-step enzyme method in which GOD and horseradish peroxidase (HRP) are coimmobilized onto the same cellular sites by immunological bridging or by the principle of avidin-biotin interaction. In this coupled enzyme technique, H2O2 generated during GOD reaction is the substrate for HRP and is utilized for the oxidation of chromogens such as 3,3'-diaminobenzidine or 3-amino-9-ethylcarbazole. Due to the immobilization of the capture enzyme HRP in close proximity to the marker enzyme (GOD), more intense and specific staining is produced than can be obtained with soluble HRP as coupling enzyme in the substrate medium. Indirect antibody labelled and antibody bridge techniques including the avidin (streptavidin)-biotin principle have proven the usefulness of this GOD labelling procedure for antigen localization in paraffin sections. Antigens such as IgA in tonsil, alpha-fetoprotein in liver and tissue polypeptide antigen in mammary gland served as models. The immobilized two-step enzyme procedures have the same order of sensitivity and specificity as comparable immunoperoxidase methods. The coupled GOD-HRP principle can be superior to conventional immunoperoxidase labelling for the localization of biomolecules in tissue preparations rich in endogenous peroxidase activities.

Antigens↗
PubMed2001

Oxidative responses of resistant and susceptible cereal leaves to symptomatic and nonsymptomatic cereal aphid (Hemiptera: Aphididae) feeding.

The impact of the leaf-chlorosis-eliciting Russian wheat aphid, Diuraphis noxia (Mordvilko), and the nonchlorosis-eliciting bird cherry-oat aphid, Rhopalosiphum padi (L.), feeding on D. noxia-susceptible and -resistant cereals was examined during the period (i.e., 3, 6, and 9 d after aphid infestation) that leaf chlorosis developed. After aphid number, leaf rolling and chlorosis ratings, and fresh leaf weight were recorded on each sampling date, total protein content, peroxidase, catalase, and polyphenol oxidase activities of each plant sample were determined spectrophotometrically. Although R. padi and D. noxia feeding caused significant increase of total protein content in comparison with the control cereal leaves, the difference in total protein content between R. padi and D. noxia-infested leaves was not significant. Although R. padi-feeding did not elicit any changes of peroxidase specific activity in any of the four cereals in comparison with the control leaves, D. noxia feeding elicited greater increases of peroxidase specific activity only on resistant 'Halt' wheat (Triticum aestivum L.) and susceptible 'Morex' barley (Hordeum vulgare L.), but not on susceptible 'Arapahoe' and resistant 'Border' oat (Avena sativa L.). D. noxia-feeding elicited a ninefold increase in peroxidase specific activity on Morex barley and a threefold on Halt wheat 9 d after the initial infestation in comparison with control leaves. Furthermore, D. noxia feeding did not elicit any differential changes of catalase and polyphenol oxidase activities in comparison with either R. padi feeding or control leaves. The findings suggest that D. noxia feeding probably results in oxidative stress in plants. Moderate increase of peroxidase activity (approximately threefold) in resistant Halt compared with susceptible Arapahoe wheat might have contributed to its resistance to D. noxia, whereas the ninefold peroxidase activity increase may have possibly contributed to barley's susceptibility. Different enzymatic responses in wheat, barley, and oat to D. noxia and R. padi feeding indicate the cereals have different mechanisms of aphid resistance.

Animals↗