Enzymatic Reducing System Research Articles

Effect of Reduction of Redox Modifications of Cys-Residues in the Na,K-ATPase α1-Subunit on Its Activity.

Sodium-potassium adenosine triphosphatase (Na,K-ATPase) creates a gradient of sodium and potassium ions necessary for the viability of animal cells, and it is extremely sensitive to intracellular redox status. Earlier we found that regulatory glutathionylation determines Na,K-ATPase redox sensitivity but the role of basal glutathionylation and other redox modifications of cysteine residues is not clear. The purpose of this study was to detect oxidized, nitrosylated, or glutathionylated cysteine residues in Na,K-ATPase, evaluate the possibility of removing these modifications and assess their influence on the enzyme activity. To this aim, we have detected such modifications in the Na,K-ATPase α1-subunit purified from duck salt glands and tried to eliminate them by chemical reducing agents and the glutaredoxin1/glutathione reductase enzyme system. Detection of cysteine modifications was performed using mass spectrometry and Western blot analysis. We have found that purified Na,K-ATPase α1-subunit contains glutathionylated, nitrosylated, and oxidized cysteines. Chemical reducing agents partially eliminate these modifications that leads to the slight increase of the enzyme activity. Enzyme system glutaredoxin/glutathione reductase, unlike chemical reducing agents, produces significant increase of the enzyme activity. At the same time, the enzyme system deglutathionylates native Na,K-ATPase to a lesser degree than chemical reducing agents. This suggests that the enzymatic reducing system glutaredoxin/glutathione reductase specifically affects glutathionylation of the regulatory cysteine residues of Na,K-ATPase α1-subunit.

Mechanisms of Action and Reduced Cardiotoxicity of Pixantrone; a Topoisomerase II Targeting Agent with Cellular Selectivity for the Topoisomerase IIα Isoform.

Pixantrone is a new noncardiotoxic aza-anthracenedione anticancer drug structurally related to anthracyclines and anthracenediones, such as doxorubicin and mitoxantrone. Pixantrone is approved in the European Union for the treatment of relapsed or refractory aggressive B cell non-Hodgkin lymphoma. This study was undertaken to investigate both the mechanism(s) of its anticancer activity and its relative lack of cardiotoxicity. Pixantrone targeted DNA topoisomerase IIα as evidenced by its ability to inhibit kinetoplast DNA decatenation; to produce linear double-strand DNA in a pBR322 DNA cleavage assay; to produce DNA double-strand breaks in a cellular phospho-histone γH2AX assay; to form covalent topoisomerase II-DNA complexes in a cellular immunodetection of complex of enzyme-to-DNA assay; and to display cross-resistance in etoposide-resistant K562 cells. Pixantrone produced semiquinone free radicals in an enzymatic reducing system, although not in a cellular system, most likely due to low cellular uptake. Pixantrone was 10- to 12-fold less damaging to neonatal rat myocytes than doxorubicin or mitoxantrone, as measured by lactate dehydrogenase release. Three factors potentially contribute to the reduced cardiotoxicity of pixantrone. First, its lack of binding to iron(III) makes it unable to induce iron-based oxidative stress. Second, its low cellular uptake may limit its ability to produce semiquinone free radicals and redox cycle. Finally, because the β isoform of topoisomerase II predominates in postmitotic cardiomyocytes, and pixantrone is demonstrated in this study to be selective for topoisomerase IIα in stabilizing enzyme-DNA covalent complexes, the attenuated cardiotoxicity of this agent may also be due to its selectivity for targeting topoisomerase IIα over topoisomerase IIβ.

Autoxidation and Oxygen Binding Properties of Recombinant Hemoglobins with Substitutions at the αVal-62 or βVal-67 Position of the Distal Heme Pocket

The E11 valine in the distal heme pocket of either the α- or β-subunit of human adult hemoglobin (Hb A) was replaced by leucine, isoleucine, or phenylalanine. Recombinant proteins were expressed in Escherichia coli and purified for structural and functional studies. (1)H NMR spectra were obtained for the CO and deoxy forms of Hb A and the mutants. The mutations did not disturb the α1β2 interface in either form, whereas the H-bond between αHis-103 and βGln-131 in the α1β1 interfaces of the deoxy α-subunit mutants was weakened. Localized structural changes in the mutated heme pocket were detected for the CO form of recombinant Hb (rHb) (αV62F), rHb (βV67I), and rHb (βV67F) compared with Hb A. In the deoxy form the proximal histidyl residue in the β-subunit of rHb (βV67F) has been altered. Furthermore, the interactions between the porphyrin ring and heme pocket residues have been perturbed in rHb (αV62I), rHb (αV62F), and rHb (βV67F). Functionally, the oxygen binding affinity (P50), cooperativity (n50), and the alkaline Bohr Effect of the three α-subunit mutants and rHb (βV67L) are similar to those of Hb A. rHb (βV67I) and rHb (βV67F) exhibit low and high oxygen affinity, respectively. rHb (βV67F) has P50 values lower that those reported for rHb (αL29F), a B10 mutant studied previously in our laboratory (Wiltrout, M. E., Giovannelli, J. L., Simplaceanu, V., Lukin, J. A., Ho, N. T., and Ho, C. (2005) Biochemistry 44, 7207-7217). These E11 mutations do not slow down the autoxidation and azide-induced oxidation rates of the recombinant proteins. Results from this study provide new insights into the roles of E11 mutants in the structure-function relationship in hemoglobin.

A RESEARCH NOTE: ANTIOXIDANT EFFECTS ON COLOR OF AN IRRADIATED BOVINE MYOGLOBIN MODEL SYSTEM

ABSTRACT To determine whether antioxidants could protect meat color during irradiation, we extracted myoglobin (Mb) from beef hearts, treated it with one of four antioxidants (control, citric acid [CA], ascorbic acid [AA], ethylenediamine tetraacetic acid [EDTA] or propyl gallate [PG]) and irradiated it at 0.00, 1.25, or 2.50 kGy. The myoglobin model system was then evaluated for pH; Commission Internationale de L'Eclairage (CIE) L*, a* and b* values; hue angle; chroma; and thiobarbituric acid reactive substances (TBARS). Adding CA and AA to the beef myoglobin model system resulted in higher L*, a* and b* values than did EDTA and PG, indicating that these samples were lighter, redder and more yellow. EDTA‐ and PG‐containing samples were more similar to controls containing no antioxidants. Hue angle and chroma supported these findings. Irradiation had no effect on pH, color or TBARS. These results indicate that antioxidants have more influence on beef color than irradiation and that the acidic antioxidants have the potential for preserving color.PRACTICAL APPLICATIONSIrradiation has the potential to reduce the microbial load on meat products, producing safer foods for consumers. However, a number of reports have indicated that irradiation can compromise the bright, cherry red color of fresh meat. Off‐color will result in consumer rejection of products that may be safe. Antioxidants have the potential to preserve meat color by affecting the state of myoglobin; however, studying these effects in vivo is challenging because of the complexity of the system. In this model system, which was devoid of enzymatic reducing systems and equivalents, the acidic antioxidants, ascorbic acid (AA) and citric acid (CA), were most effective in preserving the color of the pigment. These antioxidants appear to have the greatest potential for preserving fresh meat color during irradiation.

A RESEARCH NOTE: EFFECT OF NATURAL ANTIOXIDANTS ON COLOR OF AN IRRADIATED BEEF MYOGLOBIN MODEL SYSTEM

ABSTRACT To determine the effect of antioxidants and irradiation on meat color, myoglobin (Mb) was extracted from beef hearts. Mb was treated with one of three antioxidants (control, grape seed extract, rosemary extract [RE] or oregano extract) and irradiated at 0.00, 1.25 or 2.50 kGy. The samples were then evaluated for pH, Commission Internationale de l'Eclairage L*, a* and b* values, hue angle, chroma and thiobarbituric reactive substances (TBARS). Antioxidants influenced pH. RE‐containing beef Mb samples had lower pH values before and after irradiation. Antioxidants also had significant effects on the color of the beef Mb model system. RE‐containing samples had the best pre‐ and postirradiated color in terms of a* value (redness) and chroma (color intensity). TBARS values were very low and did not differ regardless of antioxidant or irradiation dose.PRACTICAL APPLICATIONSIrradiation can reduce the microbial load on meat products and improve product safety for consumers. However, irradiation, because of its potential to cause oxidative damage, can compromise the red color of fresh meat. Off‐color will result in consumer rejection of products which may be safe. Antioxidants have the potential to preserve meat color by affecting the state of myoglobin and maintaining the iron in the reduced state such that it can oxygenate to the bright cherry red color, which consumers expect. However, studying these effects in vivo is challenging because of the complexity of the system. In this model system, devoid of enzymatic reducing systems and equivalents, natural antioxidants, including grape seed extract, rosemary extract (RE) and oregano extract, were evaluated for their potential to preserve color during irradiation. RE appeared to be the most effective in preserving red color intensity. Extracts of this herb appear to have potential for ameliorating some of the color damage that often accompanies the irradiation of fresh red meat.

Neuroglobin and cytoglobin as potential enzyme or substrate

The possible enzymatic activities of neuro- and cytoglobin as well as their potential function as substrates in enzymatic reactions were studied. Neuro- and cytoglobin are found to show no appreciable superoxide dismutase, catalase, and peroxidase activities. However, the internal disulfide bond (CD7–D5) of human neuroglobin can be reduced by thioredoxin reductase. Furthermore, our in vivo and in vitro studies show that Escherichia coli cells contain an enzymatic reducing system that keeps the heme iron atom of neuroglobin in the Fe 2+ form in the presence of dioxygen despite the high autoxidation rate of the molecule. This reducing system needs a low-molecular-weight compound as co-factor. In vitro tests show that both NADH and NADPH can play this role. Furthermore, the reducing system is not specific for neuroglobin but allows the reduction of the ferric forms of other globins such as cytoglobin and myoglobin. A similar reducing system is present in eukaryotic tissue protein extracts.

Effects of Chilling and Ethanol Treatments on the Antioxidant Systems of Cucumber Seedling Roots

Previous experiments have shown that soaking 24-hr-old cucumber seedlings in ethanol leads to an increase in chilling tolerance. Seedlings were immersed in a 500-mM ethanol solution for 2 hr prior to chilling, rinsed, and placed in chilling immediately after treatment. All seedlings (except the control group) were treated for 2 hr after a 24-hr germination period at 25°C and a second group was held for 3 hr to allow the ethanol-induced chilling tolerance to dissipate. At this time, the seedlings were retreated with a second ethanol treatment identical to the first. The induction of chilling tolerance seen in both treatment groups was measured as an increase in root growth and decreases in lipid peroxidation and electrolyte leakage after exposure to chilling temperatures (2°C). Although ethanol treatment does clearly enhance chilling tolerance, the mechanisms by which it does so are unclear. Some hypotheses have suggested that ethanol acts as an anaesthetic, preventing a membrane phase change from a liquid to a gel-like state. Other evidence indicates that ethanol is a scavenger of the hydroxyl radical, working in concert with enzymatic-reducing systems and antioxidants to directly prevent damage caused by active oxygen species. Because enzymatic reducing systems and antioxidants have been shown to combat stress, the activities of three H2O2-scavenging enzymes (catalase, glutathione reductase, and ascorbate-specific peroxidase), along with the relative concentrations of reduced and oxidized forms of glutathione and ascorbate as affected by both chilling and ethanol treatments will be discussed in relation to chilling susceptibility.

24] Bezafibrate derivatives as potent effectors of hemoglobin

Publisher Summary This chapter discusses various methods concerned with the studies of new potent allosteric effectors in erythrocyte suspensions and describes examples of solution studies in the presence of these effectors, using equilibrium and kinetic methods to investigate new aspects of the allosteric equilibrium. X-Ray analyses of the hemoglobin(Hb)-compound complexes have revealed that all these compounds bind at the interface of α and β subunits deep in the water-filled central cavity at a distance from the hemes and the 2,3-diphosphoglycerate(DPG)-binding site. The availability of allosteric effectors that could be used in erythrocyte suspensions would be advantageous in studying dilute Hb solutions for the following reasons: (1) inside the erythrocytes the high concentration of Hb reduces the presence of dimers to negligible amounts and (2) the presence of the cellular enzymatic reducing system continuously keeps the formation of methemoglobin to insignificant levels.

Metabolism-based transformation of myoglobin to an oxidase by BrCCl3 and molecular modeling of the oxidase form.

The stoichiometric reductive debromination of BrCCl3 to a trichloromethyl radical by myoglobin caused the prosthetic heme to become covalently cross-linked to the protein moiety and transformed myoglobin from an oxygen storage protein to an oxidase. This was shown in experiments in which oxygen consumption was measured during redox cycling of the altered myoglobin in the presence of ascorbate or an enzymatic reducing system containing diaphorase and NADH. Redox cycling eventually led to loss of the protein-bound heme adduct and oxidase activity of myoglobin. We have used molecular modeling and the known structure of the protein-bound heme adduct to identify probable mechanisms for transformation of myoglobin to an oxidase. Based on these modeling studies, the most likely structure of the experimentally observed adduct involves ligation to the heme iron of the epsilon-nitrogen atom of histidine 97 and/or that of histidine 64. The model structures revealed access of solvent to the heme active site, which could facilitate oxygen reduction. The transformation of myoglobins and perhaps other hemoproteins to oxidases may have toxicological importance in causing the tissue damage resulting from exposure to various xenobiotics and endogenous chemicals as well as explaining how hemoproteins are inactivated during catalysis.

Reduction of fluoromisonidazole, a new imaging agent for hypoxia

[ 18F]Fluoromisonidazole (1-(3-[ 18F]fluoro-2-hydroxypropyl)-2-nitroimidazole, [ 18F]FMISO) is a nitroimidazole compound that is being used as a new imaging agent for hypoxia. Because its uptake in hypoxic tissue is dependent on reduction of the nitro group on the imidazole ring, it is necessary to verify the availability of nitroreductase enzymes in a variety of tissues. FMISO reduction was studied using chemical and enzymatic reducing systems and mammalian cells. FMISO reduction by iron/HCl eliminated the absorbance peak at 325 nm caused by the nitro group. FMISO reduction by xanthine oxidase, as measured by a decrease in absorbance at 325 nm, occurred at a rate of 2.4 ± 0.3 nmol/min/ unit enzyme (mean ± SEM, N = 15). This reaction was inhibited by allopurinol. Separation of the parent drug from its reduction product following chemical and enzymatic reductions indicated that iron/ HC1 reduced the majority of the FMISO molecules present, while xanthine oxidase did not. Reduction of FMISO by NADH dehydrogenase could not be demonstrated spectrophotometrically. Measurement of the reduction of FMISO in V79 cells based on the binding of [ 3H]FMISO to cellular macromolecules was performed using a cell suspension in a three-neck flask. Hypoxic V79 cells bound [ 3H]FMISO at the rate of 0.26 ± 0.07 pmol/10 6 cells/min (N = 8). When specific inhibitors of two nitroreductase enzymes and a general inhibitor of electron transport were added to the cell suspension, no consistent, statistically significant inhibition of FMISO binding could be shown. We conclude that while inhibition of FMISO reduction by a purified nitroreductase can be shown, nitroreductase activity in cells is not inhibited so easily. This supports the hypothesis that nitroreductases are plentiful and will not limit the rate of FMISO reduction and uptake in hypoxic tumors or nonmalignant tissues.

Nitrate and nitrite uptake and reduction by intact sunflower plants

Nitrogen-starved sunflower plants (Helianthus annuus L. cv. Peredovic) cannot absorb NO 3 (-) or NO 2 (-) upon initial exposure to these anions. Ability of the plants to take up NO 3 (-) and NO 2 (-) at high rates from the beginning was induced by a pretreatment with NO 3 (-) . Nitrite also acted as inducer of the NO 2 (-) -uptake system. The presence of cycloheximide during NO 3 (-) -pretreatment prevented the subsequent uptake of NO 3 (-) and NO 2 (-) , indicating that both uptake systems are synthesized de novo when plants are exposed to NO 3 (-) . Cycloheximide also suppressed nitrate-reductase (EC 1.6.6.1) and nitrite-reductase (EC 1.7.7.1) activities in the roots. The sulfhydryl-group reagent N-ethylmaleimide greatly inhibited the uptake of NO 3 (-) and NO 2 (-) . Likewise, N-ethylmaleimide promoted in vivo the inactivation of nitrate reductase without affecting nitrite-reductase activity. Rates of NO 3 (-) and NO 2 (-) uptake as a function of external anion concentration exhibited saturation kinetics. The calculated Km values for NO 3 (-) and NO 2 (-) uptake were 45 and 23 μM, respectively. Rates of NO 3 (-) uptake were four to six times higher than NO 3 (-) -reduction rates in roots. In contrast, NO 2 (-) -uptake rates, found to be very similar to NO 3 (-) -uptake rates, were much lower (about 30 times) than NO 2 (-) -reduction rates. Removal of oxygen from the external solution drastically suppressed NO 3 (-) and NO 2 (-) uptake without affecting their reduction. Uptake and reduction were also differentially affected by pH. The results demonstrate that uptake of NO 3 (-) and NO 2 (-) into sunflower plants is mediated by energy-dependent inducible-transport systems distinguishable from the respective enzymatic reducing systems.

Oxidation properties of two hemoglobin variants with their mutation localized in the heme pocket: Hb castilla β32 (B14) [formula omitted] and Hb toulouse β66 (E10) [formula omitted], and abnormal functional properties of Hb castilla

We have studied the oxidation properties of two unstable hemoglobin variants: Hb Castilla ( β32 ( B14) Leu → Arg ) and Hb Toulouse ( β66 ( E10) Lys → Glu ). Hb Castilla, a very unstable variant, exhibited a rapid autoxidation rate with a concomitant appearance of hemichrome and precipitation. Inositol hexaphosphate increases the percentage of hemichrome in the ferriform of Hb Castilla. This could be interpreted either as a direct action on the oxidation step leading to hemichrome or as a displacement of the equilibrium towards a more readily oxidized conformation. Hb Toulouse also autoxidized very rapidly but did not form hemichrome. The ionization pK of the ferric form was increased (8.5 instead of 8.1 for normal) due to the more acidic environment of the iron. The reduction rates of these hemoglobins by enzymatic systems differed; thus Hb Castilla was completely reduced while Hb Toulouse remained 50% in the ferriform. Although each Hb displays an amino acid substitution which probably explains their instability, the perturbations are different in the two cases. These results are discussed in view of the differences in the stereochemical modifications of the two hemoglobins and also with regard to the general mechanism of denaturation of hemoglobins proposed by Winterbourn and Carrell ((1974) J. Clin. Invest. 54, 678). For the study of the functional properties of Hb Castilla, we have developed a method which minimized precipitation and oxidation: the use of an enzymatic reducing system prevented oxidation and temperature of experiment was decreased to 10°C in order to diminish the precipitation. Under these conditions, Hb Castilla exhibited a decreased oxygen affinity, a low degree of subunit interaction and a decreased Bohr effect. Variations in P 50 were also studied as a function of temperature.

ENZYMATIC REDUCTION OF METMYOGLOBIN IN FISH

ABSTRACTAn enzymatic reducing system for metmyoglobin was studied in two species of fish, bluefin tuna (Thunnus thynnus) and jack mackerel (Trachurus symmetricus). In both species, the enzyme was primarily located in the midsection of the fish muscle; it had an optimum pH of 7 and an optimum temperature of 25°C. The enzymatic reducing activity of both species was enhanced by anaerobic conditions and partially inhibited by the enzyme catalase, while sodium chloride was not an effective inhibitor. Ascorbic acid as an electron donor and menadione as a mediator were found to be much less efficient than nicotinamide adenine dinucleotide and methylene blue, respectively.

Catalysts of lipid peroxidation in meats

AbstractHemoprotein and non‐heme iron components are active catalysts of lipid peroxidation. The behavior of these two catalysts under a number of conditions was compared as a basis for a study of their activities in meats. In model systems, MetMb accelerated linoleic acid peroxidation in a pH range from 5.6 to 7.8; it catalyzed especially rapidly at higher pH. A complex of ferrous ion [Fe(II)] and EDTA, a non‐heme iron model, in a 1:1 ratio accelerated peroxidation at lower pH; no catalysis took place above pH 6.4. Most chelating agents eliminated Fe(II)‐EDTA catalysis, but had no effect on MetMb catalysis. Reducing agents, on the other hand, accelerated Fe(II)‐EDTA catalysis but inhibited MetMb catalysis. In model systems in which fresh dilute (1.2%, w/v) meat homogenate was the catalyst, the effect of the heme predominated. An exception was ascorbic acid; it accelerated oxidation at pH 5.6. The pattern of linoleate peroxidation catalyzed by heme‐free (H2O2‐treated) beef homogenate and shrimp homogenate was similar to that in the Fe(II)‐EDTA model system. Again, ascorbic acid accelerated the catalysis and the acceleration could be eliminated by adding chelating agents. The presence of a non‐heme iron catalyst in meat is thus indicated. Evidence is presented for both types of catalytic activity in meats. In cooked meats, heme was the dominant catalyst, but significant lipid oxidation, apparently catalyzed by a non‐heme iron‐type catalyst, occurred in cooked meats in which the heme had been destroyed by H2O2. In raw meats, lipid oxidation was inhibited at high pH because of removal of oxygen by enzymatic reducing systems. Both heme and non‐heme iron were active at lower pH values. EDTA inhibited lipid oxidation during storage, presumably by its demonstrated effect on non‐heme iron catalysis. Ascorbic acid also inhibited lipid oxidation, probably indirectly by keeping the heme pigment in the catalytic inactive ferrous state.

Reduction of triphenyltetrazolium chloride by plant tissues and its relation to molybdenum status.

TRIPHENYLTETRAZOLIUM chloride has been used recently for indicating enzymatic reducing systems in several tissues1–5 under aerobic as well as anaerobic conditions, and many other similar applications have been reviewed by Smith6. We have found that sections of petioles or stems of tomato, cauliflower, swede, kale, clover and other plants are characteristically stained by reduction of solutions of triphenyltetrazolium chloride to the insoluble red formozan pigment. Staining is intense in phloem and strong in cambium and vascular parenchyma, and often in endodermis. It is weak in cortex and epidermis, and negligible or absent in xylem and semi-lignified tissues, for example, pericycle. In leaves the mesophyll stains less than the phloem and vascular parenchyma. Our results confirm closely those of Gall7 (with bean stems) and others6 unknown to us until this work was well advanced.