Mechanism for high stability of liposomal glucose oxidase to inhibitor hydrogen peroxide produced in prolonged glucose oxidation

Glucose oxidase (GO) was encapsulated in the liposomes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) to increase the enzyme stability through its decreased inhibition because of hydrogen peroxide (H(2)O(2)) produced in the glucose oxidation. The GO-containing liposomes (GOLs) were completely free of the inhibition even in the complete conversion of 10 mM glucose at 25 degrees C because the H(2)O(2) concentration was kept negligibly low both outside and inside liposomes throughout the reaction. It was interestingly revealed that the H(2)O(2) produced was decomposed not only by a slight amount of catalase originally contained in the commercially available GO but also by the lipid membranes of GOL. As compared to the GOL-catalyzed reaction, the free GO-catalyzed reaction more highly accumulated H(2)O(2) because of the more rapid glucose conversion despite containing free catalase, leading to the completely inhibited GO before reaching a sufficient glucose conversion. This suggested that only the liposomal catalase could continue to catalyze the H(2)O(2) decomposition. The effect of the glucose oxidation rate, i.e., the H(2)O(2) production rate on the liposomal GO inhibition, was also examined employing the various GOLs with different permeabilities to glucose present in their external phase. It was concluded that the liposomal GO free of the inhibition could be obtained when the GOL-catalyzed H(2)O(2) formation rate was limited by such a suitable lipid bilayer as POPC membrane so that the rate was well-balanced with the sum of the above two H(2)O(2) decomposition rates. The highly stable GOL obtained in the present paper was shown to be a useful biocatalyst for the prolonged glucose oxidation.     

Optimal covalent immobilization of glucose oxidase-containing liposomes for highly stable biocatalyst in bioreactor

The glucose oxidase-containing liposomes (GOL) were prepared by entrapping glucose oxidase (GO) in the liposomes composed of phosphatidylcholine (PC), dimyristoyl L-alpha-phosphatidylethanolamine (DMPE), and cholesterol (Chol) and then covalently immobilized in the glutaraldehyde-activated chitosan gel beads. The immobilized GOL gel beads (IGOL) were characterized to obtain a highly stable biocatalyst applicable to bioreactor. At first, the glutaraldehyde concentration used in the gel beads activation as well as the immobilizing temperature and time were optimized to enhance the immobilization yield of the GOL to the highest extent. The liposome membrane composition and liposome size were then optimized to obtain the greatest possible immobilization yield of the GOL, the highest possible activity efficiency of the IGOL, and the lowest possible leakage of the entrapped GO during the GOL immobilization. As a result, the optimal immobilization conditions were found to be as follows: the liposome composition, PC/DMPE/Chol = 65/5/30 (molar percentage); the liposome size, 100 nm; the glutaraldehyde concentration, 2% (w/v); the immobilizing temperature, 4 degrees C; and the immobilizing time, 10 h. Furthermore, the optimal IGOL prepared were characterized by its rapidly increasing effective GO activity to the externally added substrate (glucose) with increasing temperature from 20 to 40 degrees C, and also by its high stability at 40 degrees C against not only the thermal denaturation in a long-term (7 days) incubation but also the bubbling stress in a bubble column. Finally, compared to the conventionally immobilized glucose oxidase (IGO), the higher operational stability of the optimal IGOL was verified by using it either repeatedly (4 times) or for a long time (7 days) to catalyze the glucose oxidation in a small-scale airlift bioreactor.     

Novel immobilized liposomal glucose oxidase system using the channel protein OmpF and catalase

The reactivity of immobilized glucose oxidase-containing liposomes (IGOL) prepared in our previous work (Wang et al. [2003] Biotechnol Bioeng 83:444-453) was considerably improved here by incorporating the channel protein OmpF from Escherichia coli into the liposome membrane as well as by entrapping inside the liposome's aqueous interior not only glucose oxidase (GO), but also catalase (CA), both from Aspergillus niger. CA was used for decomposing the hydrogen peroxide produced in the glucose oxidation reaction inside the liposomes. The presence of OmpF enhanced the transport of glucose molecules from the exterior of the liposomes to the interior. In a first step of the work, liposomes containing GO and CA (GOCAL) were prepared and characterized. A remarkable protection effect of the liposome membrane on CA inside the liposomes at 40 degrees C was found; the remaining CA activity at 72 h incubation was more than 60% for GOCAL, while less than 20% for free CA. In a second step, OmpF was incorporated into GOCAL membranes, leading to the formation of OmpF-embedded GOCAL (abbreviated GOCAL-OmpF). The activity of GO inside GOCAL-OmpF increased up to 17 times in comparison with that inside GOCAL due to an increased glucose permeation across the liposome bilayer, without any leakage of GO or CA from the liposomes. The optimal system was estimated to contain on average five OmpF molecules per liposome. Finally, GOCAL-OmpF were covalently immobilized into chitosan gel beads. The performance of this novel biocatalyst (IGOCAL-OmpF) was examined by following the change in glucose conversion, as well as by following the remaining GO activity in successive 15-h air oxidations for repeated use at 40 degrees C in an airlift bioreactor. IGOCAL-OmpF showed higher reactivity and reusability than IGOL, as well as IGOL containing OmpF (IGOL-OmpF). The IGOCAL-OmpF gave about 80% of glucose conversion even when the catalyst was used repeatedly four times, while the corresponding conversions were about 60% and 20% for the IGOL and IGOL-OmpF, respectively. Due to the absence of CA, IGOL-OmpF was less stable and resulted in drastically inhibited GO.     

Preparation and characterization of reactive and stable glucose oxidase-containing liposomes modulated with detergent

Glucose oxidase-containing liposomes (GOL) as well as detergent-modulated glucose oxidase-containing liposomes were prepared and characterized, focusing not only on the reactivity of the liposomes upon external addition of glucose but also on the leakage of the entrapped glucose oxidase (GO) from the liposomes with the aim of developing a reactive and stable liposomal GO system. The membranes of the GOL prepared were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and modulated with either Triton X-100 or cholate. In the absence of added detergent, no GO leakage from the GOL was observed while its enzymatic activity was very low (low glucose permeability). As detergent-modulated liposomes, mixed POPC/Triton X-100 and mixed POPC/cholate liposomes (abbreviated as TL and CL, respectively) were prepared at different effective detergent/POPC molar ratios (R(e)) ranging from R(e) = 0 to R(e) = R(e) (sat) (R(e) (sat) is the critical value of R(e) at which the liposome membrane is saturated with detergent). The reactivity of GO-loaded TL (abbreviated as GOTL) or GO-loaded CL (GOCL) increased drastically with increase in the respective detergent content in the liposomes. In the case of GOTL, at R(e) (sat) = 0.40, a high reactivity was measured with a simultaneous high extent of GO leakage, suggesting that the observed enzymatic reaction was catalyzed mainly by leaked GO, caused by the interaction of Triton X-100 with the POPC membrane. On the other hand, GOCL prepared at R(e) (sat) = 0.43 showed relatively high reactivity with only a small extent of GO leakage, suggesting that most of the enzyme reaction was limited by the glucose permeation across the bilayers of GOCL. The GO leakage from GOCL was found to occur mostly during the rearrangement of the liposomal membrane during the preparation of the GOCL (mixing the GOL and cholate). Fluorescence polarization measurements of membrane-associated DPH (1,6-diphenyl-1,3,5-hexatriene) indicated that CL prepared by modifying POPC with cholate did not lead to a drastic change in membrane fluidity, indicating that the interacting cholate molecules did not penetrate deeply into the POPC bilayers. In summary, it was clearly shown that the membrane permeability of GOL can be quite simply modulated by mixing it with a certain amount of cholate to form highly reactive and stable GOCL with minimal enzyme leakage.     

Free radicals and their biological significance: present and future]

Free radicals are usually active species which have unpair electron (S) in molecules or Atomic groups. Of the free radicals, O2- and .OH could easily be produced in mammalian cells, by oxidation and reduction cycle catalyzed by fravoproteins and by iron + H2O2 reaction, respectively. Other free radicals would also be produced in mammalian cell, such as amino acid radicals, semiquinone radicals and flavine radicals etc. In general, free radicals cause cell injury though membrane lipid peroxidation and DNA strand cleavage and some other mechanisms.     

Permeabilization of phospholipid bilayer membranes induced by gas-liquid flow in an airlift bubble column

The permeability of 5(6)-carboxyfluorescein (CF) through the phospholipid bilayer membranes was measured by using the system in which the CF-containing phospholipid vesicles (liposomes) were suspended in the gas-liquid flow in an external loop airlift bubble column. The airlift was operated at various superficial gas velocities UG up to 2.4 cm/s at 25 and 40 degrees C. The CF-containing liposomes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) had the nominal diameters of 50, 100, and 200 nm. The 50- and 100-nm liposomes were stable at 40 degrees C for 5 h even at a high UG value of 2.4 cm/s based on the observed turbidity of the liposome suspension in the airlift. On the other hand, the 200-nm liposomes were stable at a low UG value of 1.4 cm/s, although a progressive decrease in size of the liposomes was implied at the high UG value of 2.4 cm/s. The permeability coefficient PCF of CF through the lipid membrane of the 100-nm liposomes was significantly increased with increasing UG at a high temperature of 40 degrees C, while at a low temperature of 25 degrees C the PCF value was little dependent on UG. As a typical result on the above liposomes, the PCF value (9.2 x 10(-11) cm/s) at 40 degrees C and UG = 2.4 cm/s in the airlift was more than 15 times larger than that at 25 degrees C in the static liquid corresponding to UG = 0. In addition, the dependence of the PCF value on UG at 40 degrees C became more significant with increasing the size of liposomes suspended. The results obtained revealed that the permeability of the liposome membranes could be regulated by suspending the liposomes in the gas-liquid flow in the airlift without modulating the membrane composition of liposomes.     

RNA oxidation catalyzed by cytochrome c leads to its depurination and cross-linking, which may facilitate cytochrome c release from mitochondria

Growing evidence indicates that RNA oxidation is correlated with a number of age-related neurodegenerative diseases, and RNA oxidation has also been shown to induce dysfunction in protein synthesis. Here we study in vitro RNA oxidation catalyzed by cytochrome c (cyt c)/H(2)O(2) or by the Fe(II)/ascorbate/H(2)O(2) system. Our results reveal that the products of RNA oxidation vary with the oxidant used. Guanosine residues are preferentially oxidized by cyt c/H(2)O(2) relative to the Fe(II)/ascorbate/H(2)O(2) system. GC/MS and LC/MS analyses demonstrated that the guanine base was not only oxidized but also depurinated to form an abasic sugar moiety. Results from gel electrophoresis and HPLC analyses show that RNA formed a cross-linked complex with cyt c in an H(2)O(2) concentration-dependent manner. Furthermore, when cyt c was associated with liposomes composed of cardiolipin/phosphatidylcholine, and incubated with RNA and H(2)O(2), it was found cross-linked with the oxidized RNA and dissociated from the liposome. Results of the quantitative analysis indicate that the release of the cyt c from the liposome is facilitated by the formation of an RNA-cyt c cross-linked complex. Thus, RNA oxidation may facilitate the release of cyt c from the mitochondrial membrane to induce apoptosis in response to oxidative stress.     

Carboxymethylated glucan inhibits lipid peroxidation in liposomes

Protective capabilities were studied of carboxymethylated (1-->3)-beta-D-glucan from Saccharomyces cerevisiae cell wall against lipid peroxidation in phosphatidylcholine liposomes induced by OH radicals produced with Fenton's reagent (H2O2/Fe2+) and also by microwave radiation using absorption UV-VIS spectrophotometry. A significant decrease in the conjugated diene production, quantified as Klein oxidation index, was observed in the presence of a moderate amount of added glucan. Increase of the oxidation index was accompanied with enhanced carboxyfluorescein leakage as a result of liposome membrane destabilization. This process was markedly suppressed with glucan present in the liposome suspension. Therefore, glucan may be considered as a potent protector against microwave radiation-induced cell damage.     

Evidence for a radical mechanism of halogenation of monochlorodimedone catalyzed by chloroperoxidase

A radical species of monochlorodimedone has been characterized by its high reactivity with molecular O2. Horseradish peroxidase greatly accelerated O2 uptake by acidic solutions of this substrate; the enzymatic reaction required exogenous H2O2 only with freshly prepared substrate solutions, and the total substrate oxidized was equal to the sum of H2O2 added and O2 consumed. However, with excess Br- and horseradish peroxidase, or high Br- or Cl- and chloroperoxidase, a 1:1 stoichiometry between H2O2 and substrate was observed. In the absence of halide, the stoichiometry of the chloroperoxidase-catalyzed oxidation of monochlorodimedone changed to two molecules of the organic donor per H2O2. Moreover, in the absence of halide, at substrate:H2O2 ratios greater than 2.0, chloroperoxidase catalyzed significant O2 uptake; this enzyme-dependent autoxidation of monochlorodimedone also occurred in the presence of Cl- or Br-, when H2O2 was limiting. These data, and recent evidence from this laboratory for free hypohalous acid as the first product of chloroperoxidase-catalyzed halide oxidation [B. W. Griffin (1983) Biochem. Biophys. Res. Commun. 116, 873-879], strongly support a mixed enzymatic/nonenzymatic radical chain process as the mechanism for halogenation of monochlorodimedone by chloroperoxidase. Both horseradish peroxidase and chloroperoxidase can catalyze either bromination or oxidation of this substrate, depending on the experimental conditions. Implications of these results for the mechanism of HOCl formation catalyzed by chloroperoxidase are considered.     

Stability and reactivity of liposome‐encapsulated formate dehydrogenase and cofactor system in carbon dioxide gas‐liquid flow

Formate dehydrogenase from Candida boidinii (CbFDH) is potentially applicable in reduction of CO₂ through oxidation of cofactor NADH into NAD⁺. For this, the CbFDH activity needs to be maintained under practical reaction conditions, such as CO₂ gas‐liquid flow. In this work, CbFDH and cofactor were encapsulated in liposomes and the liposomal enzymes were characterized in an external loop airlift bubble column. The airlift was operated at 45°C with N₂ or CO₂ as gas phase at the superficial gas velocity U_G of 2.0 or 3.0 cm/s. The activities of liposomal CbFDH/cofactor systems were highly stable in the airlift regardless of the type of gas phase because liposome membranes prevented interactions of the encapsulated enzyme and cofactor molecules with the gas‐liquid interface of bubbles. On the other hand, free CbFDH was deactivated in the airlift especially at high U_G with CO₂ bubbles. The liposomal CbFDH/NADH could catalyze reduction of CO₂ in the airlift giving the fractional oxidation of the liposomal NADH of 23% at the reaction time of 360 min. The cofactor was kept inside liposomes during the reaction operation with less than 10% of leakage. All of the results obtained demonstrate that the liposomal CbFDH/NADH functions as a stable catalyst for reduction of CO₂ in the airlift. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

A method for measuring H2O2 based on the potentiation of peroxidative NADPH oxidation by superoxide dismutase and scopoletin.

NADPH oxidation catalyzed by horseradish peroxidase is considerably increased by scopoletin and superoxide dismutase. These effects were used to develop a method for measuring H2O2 in a horseradish peroxidase, superoxide dismutase, and scopoletin system by measuring the NADPH oxidation rate. The optimal concentration of each reactant was determined. H2O2 could be detected and measured when it was present free in the medium or when it was produced by an H2O2-generating system, such as glucose-glucose oxidase or NADPH oxidase from thyroid plasma membranes. H2O2 was measured either by taking aliquots of the incubation medium or by placing NADPH directly in the medium and following the kinetics of NADPH oxidation. This latter approach required smaller amounts of biological material. In contrast to other methods, the H2O2 which is measured is regenerated. This method is 10 times more sensitive than the standard scopoletin method for H2O2 measurement and will detect a H2O2 production rate as low as 0.2 nmol per hour. The method is particularly suitable for biological systems in which small quantities of biological material are available.     

Activation by ATP of calcium-dependent NADPH-oxidase generating hydrogen peroxide in thyroid plasma membranes

An H2O2-generating fraction was prepared from porcine thyroid homogenate by differential and Percoll-density gradient centrifugations. The fraction consisted of mainly fragmented plasma membranes as judged by marker enzyme analysis and electron microscopy. The fraction produced H2O2 by reaction with NADPH only in the presence of Ca2+. The Ca2+ concentration for half-maximal activation (KCa) was about 0.1 microM and the Hill coefficient was 2. Sr2+ also activated the reaction whereas Mn2+, Zn2+, and Cd2+ inhibited it. The reaction was enhanced about twice by addition of ATP but not ADP, and inhibited by addition of hexokinase together with glucose to remove ATP. The Km value for NADPH was 35 microM and was less than 1/12 that for NADH. The NADPH oxidation rate was measured and the KCa and the Km were similar to those for the H2O2 production. The stoichiometry between the oxidation and the H2O2 formation was essentially 1. Superoxide dismutase (SOD) and KCN did not affect H2O2 production. The fraction catalyzed NADPH-cytochrome c reduction but the activity was SOD-insensitive. These results suggest that H2O2 was not generated through superoxide anion formation. NADPH-dichloroindophenol (DCIP) reductase activity was also observed and DCIP inhibited the production of H2O2. The cytochrome c and DCIP reductase activities were not influenced by Ca2+ or ATP. A unique electron transport system regulated by Ca2+ and ATP exists in the thyroid plasma membrane that produces H2O2. The concentrations of Ca2+ and ATP in thyroid cells may regulate hormone synthesis through activation of the production of H2O2, a substrate for peroxidase.     

Human neutrophils produce free radicals from the cell-zymosan interface during phagocytosis and from the whole plasma membrane when stimulated with calcium ionophore A23187.

The production of free radicals, superoxide anions (O2-), and hydrogen peroxide (H2O2) was histochemically investigated in human neutrophils that were stimulated by either phagocytosis or the calcium ionophore A23187. To demonstrate O2-, peripheral neutrophils from healthy donors were incubated at 37 degrees C in a medium containing nitroblue tetrazolium and glucose in the presence of either opsonized zymosan A and/or A23187. To demonstrate H2O2, neutrophils pretreated with a stimulant for 10 min were washed and incubated in a cerium medium containing CeCl3 and glucose in a Tris-maleate buffer. In cells engaged in phagocytosis, diformazan (for O2-) and cerium perhydroxide deposits (for H2O2) were restricted to the neutrophil-particle interface and on the inner surface of phagosomes. The remaining free surface of the plasma membrane was devoid of reaction products. In the case of neutrophils stimulated with A23187, the production of O2- and H2O2 was visualized over the whole surface of the plasma membrane. These histochemical reactions were inhibited by p-benzoquinone, superoxide dismutase, ferricytochrome c or catalase, and p-diazobenzenesulfonate (a membrane-impermeable protein denaturant). The results showed that human neutrophils produce free radicals exocellularly and that the site of production varies with different stimuli.     

Chemiluminescence flow-through biosensor for glucose with eggshell membrane as enzyme immobilization platform

In this study, a new chemiluminescence (CL) flow-through biosensor for glucose was developed by immobilizing glucose oxidase (GOD) and horseradish peroxidase (HRP) on the eggshell membrane with glutaraldehyde as a cross-linker. The CL detection involved enzymatic oxidation of glucose to D-gluconic acid and hydrogen peroxide (H2O2) and then H2O2 oxidizing luminol to produce CL emission in the presence of HRP. The immobilization condition (e.g., immobilization time, GOD/HRP ratio, glutaraldehyde concentration) was studied in detail. It showed good storage stability at 4 degrees C over a 5-month period. The proposed biosensor exhibited short response time, high sensitivity, easy operation, and simple sensor assembly, and the proposed biosensor was successfully applied to the determination of glucose in human serum.     

The oxidation of apocynin catalyzed by myeloperoxidase: proposal for NADPH oxidase inhibition

Apocynin has been used as an efficient inhibitor of the NADPH oxidase complex and its mechanism of inhibition is linked to prior activation through the action of peroxidases. Here we studied the oxidation of apocynin catalyzed by myeloperoxidase (MPO) and activated neutrophils. We found that apocynin is easily oxidized by MPO/H2O2 or activated neutrophils and has as products dimer and trimer derivatives. Since apocynin impedes the migration of the cytosolic component p47phox to the membrane and this effect could be related to its conjugation with essential thiol groups, we studied the reactivity of apocynin and its MPO-catalyzed oxidation products with glutathione (GSH). We found that apocynin and its oxidation products do not react with GSH. However, this thiol compound was efficiently oxidized by the apocynin radical during the MPO-catalyzed oxidation. We suggest that the reactivity of apocynin radical with thiol compounds could be involved in the inhibitory effect of this methoxy-catechol on NADPH oxidase complex.     

NADPH-dependent H2O2 generation catalyzed by thyroid plasma membranes. Studies with electron scavengers

Hog thyroid plasma membrane preparations containing a Ca2+-regulated NADPH-dependent H2O2-generating system were studied. The Ca2+-dependent reductase activities of ferricytochrome c, 2,6-dichloroindophenol, nitroblue tetrazolium, and potassium ferricyanide were tested and the effect of these scavengers on H2O2 formation, NADPH oxidation and O2 consumption were measured, with the following results. 1. Thyroid plasma membrane Ca2+-independent cytochrome c reduction was not catalyzed by the NADPH-dependent H2O2-generating system. This activity was superoxide-dismutase-insensitive. 2.Of the three other electron scavengers tested, only K3Fe(CN)6 was clearly, but partially reduced in a Ca2+-dependent manner. 3. Though the NADPH-dependent reduction of nitroblue tetrazolium was very low and superoxide-dismutase-insensitive, nitroblue tetrazolium inhibited O2 consumption, H2O2 formation and NADPH oxidation, indicating that nitroblue tetrazolium inhibits the H2O2-generating system. We conclude that the thyroid plasma membrane H2O2-generating system does not or liberate O2- and that Ca2+ controls the first step (NADPH oxidation) of the H2O2-generating system.     

Oxidant-mediated inhibition of ligand uptake by the macrophage mannose receptor

We have investigated the effect of oxidants on ligand recognition and internalization by the macrophage mannose receptor. Rat bone marrow macrophages were treated with increasing concentrations of H2O2 for 30 min at 37 degrees C. Fifty percent inhibition of ligand uptake was observed at 250 microM, with only 10% of control uptake remaining following exposure to 1 mM H2O2 for 30 min. Electron micrographic analysis of macrophages following H2O2 treatment showed no morphological alterations compared to untreated cells. Ligand uptake was also inhibited by the following H2O2 generating systems: menadione, xanthine/xanthine oxidase, glucose/glucose oxidase, and phorbol 12-myristate 13-acetate-stimulated polymorphonuclear leukocytes. Inhibition could be blocked by catalase plus or minus superoxide dismutase. Treatment of macrophages at 4 degrees C with H2O2 had no effect on ligand binding, whereas treatment with H2O2 at 37 degrees C reduced binding to 15% of control levels and decreased the number of surface receptors to one-third of control cells. H2O2 treatment inhibited ligand degradation by macrophages, but did not prevent ligand movement from the surface to the interior of the cell. In addition, ligand delivery to lysosomes was blocked by oxidant treatment. These results suggest that treatment of macrophages with reagent H2O2 or H2O2-generating systems inhibits the normal ligand delivery and receptor recycling process involving the mannose receptor. Potential mechanisms might include receptor oxidation, alterations in ATP levels, or membrane lipid peroxidation.     

Glucose metabolism and NADH recycling by Treponema hyodysenteriae, the agent of swine dysentery

Glucose metabolism and the mechanisms of NADH oxidation by Treponema hyodysenteriae were studied. Under an N2 atmosphere, washed cell suspensions of the spirochete consumed glucose and produced acetate, butyrate, H2, and CO2. Approximately twice as much H2 as CO2 was produced. Determinations of radioactivity in products of [14C]glucose and [14C]pyruvate metabolism and analyses of enzyme activities in cell lysates revealed that glucose was catabolized to pyruvate via the Embden-Meyerhof-Parnas pathway. The results of pyruvate exchange reactions with NaH14CO3 and Na14COOH demonstrated that pyruvate was converted to acetyl coenzyme A (acetyl-CoA), H2, and CO2 by a clostridium-type phosphoroclastic mechanism. NADH:ferredoxin oxidoreductase and hydrogenase activities were present in cell lysates and produced H2 from NADH oxidation. Phosphotransacetylase and acetate kinase catalyzed the formation of acetate from acetyl-CoA. Butyrate was formed from acetyl-CoA via a pathway that involved 3-hydroxybutyryl-coenzyme A (CoA) dehydrogenase, butyryl-CoA dehydrogenase, and butyryl-CoA transferase. T. hyodysenteriae cell suspensions generated less H2 and butyrate under 10% O2-90% N2 than under 100% N2. Cell lysates contained NADH oxidase, NADH peroxidase, and superoxide dismutase activities. These findings indicated there are three major mechanisms that T. hyodysenteriae cells use to recycle NADH generated from the Embden-Meyerhof-Parnas pathway--enzymes in the pathway from acetyl-CoA to butyrate, NADH:ferredoxin oxidoreductase, and NADH oxidase. Versatility in methods of NADH oxidation and an ability to metabolize oxygen could benefit T. hyodysenteriae cells in the colonization of tissues of the swine large bowel.     

Mechanism of hydrogen peroxide formation catalyzed by NADPH oxidase in thyroid plasma membrane

The thyroid plasma membrane contains a Ca2(+)-regulated NADPH-dependent H2O2 generating system which provides H2O2 for the thyroid peroxidase-catalyzed biosynthesis of thyroid hormones. The plasma membrane fraction contains a Ca2(+)-independent cytochrome c reductase activity which is not inhibited by superoxide dismutase. But it is not known whether H2O2 is produced directly from molecular oxygen (O2) or formed via dismutation of super-oxide anion (O2-). Indirect evidence from electron scavenger studies indicate that the H2O2 generating system does not liberate O2-, but studies using the modified peroxidase, diacetyldeuteroheme horseradish peroxidase, to detect O2- indicate that H2O2 is provided via the dismutation of O2-. The present results provide indirect evidence that the cytochrome c reductase activity is not a component of the NADPH-dependent H2O2 generator, since it was removed by washing the plasma membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid without affecting H2O2 generation. Spectral studies with diacetyldeuteroheme-substituted horseradish peroxidase showed that the thyroid NADPH-dependent H2O2 generator does not catalyze superoxide anion formation. The O2- adduct compound (compound III) was formed but was completely inhibited by catalase, indicating that the initial product was H2O2. The rate of NADPH oxidation also increased in the presence of diacetylheme peroxidase. This increase was blocked by catalase and was greatly enhanced by superoxide dismutase. The O2- adduct compound (compound III) was produced in the presence of NADPH when glucose-glucose oxidase (which does not produce O2-) was used as the H2O2 generator. NADPH oxidation occurred simultaneously and was enhanced by superoxide dismutase. We conclude that O2- formation occurs in the presence of an H2O2 generator, diacetylheme peroxidase and NADPH, but that it is not the primary product of the H2O2 generator. We suggest that O2- formation results from oxidation of NADPH, catalyzed by the diacetylheme peroxidase compound I, producing NADP degree, which in turn reacts with O2 to give O2-.     

Glucose metabolism and NADH recycling by Treponema hyodysenteriae, the agent of swine dysentery.

Glucose metabolism and the mechanisms of NADH oxidation by Treponema hyodysenteriae were studied. Under an N2 atmosphere, washed cell suspensions of the spirochete consumed glucose and produced acetate, butyrate, H2, and CO2. Approximately twice as much H2 as CO2 was produced. Determinations of radioactivity in products of [14C]glucose and [14C]pyruvate metabolism and analyses of enzyme activities in cell lysates revealed that glucose was catabolized to pyruvate via the Embden-Meyerhof-Parnas pathway. The results of pyruvate exchange reactions with NaH14CO3 and Na14COOH demonstrated that pyruvate was converted to acetyl coenzyme A (acetyl-CoA), H2, and CO2 by a clostridium-type phosphoroclastic mechanism. NADH:ferredoxin oxidoreductase and hydrogenase activities were present in cell lysates and produced H2 from NADH oxidation. Phosphotransacetylase and acetate kinase catalyzed the formation of acetate from acetyl-CoA. Butyrate was formed from acetyl-CoA via a pathway that involved 3-hydroxybutyryl-coenzyme A (CoA) dehydrogenase, butyryl-CoA dehydrogenase, and butyryl-CoA transferase. T. hyodysenteriae cell suspensions generated less H2 and butyrate under 10% O2-90% N2 than under 100% N2. Cell lysates contained NADH oxidase, NADH peroxidase, and superoxide dismutase activities. These findings indicated there are three major mechanisms that T. hyodysenteriae cells use to recycle NADH generated from the Embden-Meyerhof-Parnas pathway--enzymes in the pathway from acetyl-CoA to butyrate, NADH:ferredoxin oxidoreductase, and NADH oxidase. Versatility in methods of NADH oxidation and an ability to metabolize oxygen could benefit T. hyodysenteriae cells in the colonization of tissues of the swine large bowel.