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At least 1,243 records · Page 69Linked to original sources
PubMed1979

Definition of cytochrome c binding domains by chemical modification: kinetics of reaction with beef mitochondrial reductase and functional organization of the respiratory chain.

An assay has been developed to study the steady-state kinetics of the reduction of cytochrome c by purified beef heart mitochondrial cytochrome c reductase (cytochrome bc(1) complex, complex III). An analogue of coenzyme Q(2) (2,3-dimethoxy-5-methyl-6-decylhydroquinone) was employed as an antimycin-sensitive reductant. The kinetics of reaction of ten different mono(4-carboxy-2,6-dinitrophenyl) derivatives of horse cytochrome c were determined. The modified proteins showed higher apparent K(m) values than the native protein and greater sensitivity to ionic strength, defining an interaction domain on cytochrome c for purified cytochrome c reductase. This interaction site is located on the front surface of the molecule (which contains the exposed heme edge) and surrounds the point at which the positive end of the dipole axis crosses the surface of the protein. The site is similar to that previously determined for mitochondrial cytochrome c oxidase and yeast cytochrome c peroxidase, suggesting that the primary interaction with redox partners is directed by the dipolar charge distribution on cytochrome c. The extensive overlapping of the interaction domains for the mitochondrial cytochrome c oxidase and reductase indicates that cytochrome c must be mobile in order to transfer electrons between them, depending on their relative positions in the membrane. Whether such mobility is necessary in intact mitochondria depends on whether the interactions with the complete membrane-bound system are the same as with the purified components.

Animals↗
PubMed1985

Development and enzyme activity of protein bodies in proteinoplasts of tobacco root cells.

The development of protein bodies in proteinoplasts of tobacco (Nicotiana tabacum L. var. Wis. 38) roots was investigated with TEM, HVEM, and enzyme cytochemistry. These plastids contain a three-dimensional network of fenestrated tubules which originate from invaginations of the inner membrane of the plastid envelope. Elaboration of the network occurs in parallel with cell differentiation: slender tubules common to plastids in meristematic cells undergo dilation as protein accumulates during cell differentiation; proteinoplasts of vacuolate and root cap cells usually contain a large protein body. The contents of the peripheral tubules, originating from the inner membrane, are less electron dense than the tubules making up the central network. Localized dilations within the tubular network result in the formation of dense spheroidal structures, protein bodies, apparently as a result of continued protein accumulation via tubules connecting to the central network. Protein might be imported from segments of rough ER attached to or apposed to the outer membrane of the proteinoplast envelope. The presence of catalase (E.C. 1.11.1.6), peroxidase (E.C. 1.11.1.7), and cytochrome oxidase (E.C. 1.9.3.1) was demonstrated by cytochemistry with diaminobenzidine (DAB) as substrate. Oxidized DAB was found in protein bodies after incubation in each of the specific reaction media. While aminotriazole and sodium azide inhibited oxidation of DAB by catalase and peroxidase, respectively, only potassium cyanide completely inhibited oxidation of DAB in protein bodies. We conclude that protein bodies of proteinoplasts in tobacco roots are not sites for storage of protein, rather protein bodies contain heme protein(s) with strong oxidase activity that may convey a specific function to proteinoplasts.

Enzymes↗
PubMed2006

Duox2 exhibits potent heme peroxidase activity in human respiratory tract epithelium.

The dual oxidase isozymes Duox1 and Duox2 exhibit functional NADPH:O(2) oxidoreductase activity in thyroid and respiratory tract cells and are thought to be essential for H(2)O(2) generation in these tissues. However, it is not universally accepted that the heme peroxidase domains of the Duox isozymes are functional. To address this question, we modulated Duox2 expression in human tracheobronchial epithelial (TBE) cell culture systems and quantified peroxidase activity. We discovered that interferon-gamma (IFN-gamma) induced robust peroxidase activity in TBE cells that paralleled Duox2 expression. IFN-gamma-induced peroxidase activity was abolished in the presence of sodium azide, which implicated the activation of a heme peroxidase. IFN-gamma-induced peroxidase activity was abolished in TBE cell lines expressing anti-Duox2 short hairpin RNA transcripts. Together, these data unequivocally demonstrated that Duox2 contains a functional heme peroxidase in intact respiratory tract epithelium.

Antineoplastic Agents↗
PubMed1959

Rapid cytochemical identification of phagosomes in various tissues of the rat and their differentiation from mitochondria by the peroxidase method.

1. Granules characterized by their ability to segregate foreign proteins (phagosomes) were identified in the cells of many rat organs after intravenous administration of horseradish peroxidase, by using the conventional test with benzidine for the histochemical detection of peroxidase. The largest numbers of phagosomes were identified in kidney and liver. Considerable numbers were observed cytochemically in pancreas, prostate, epididymis, thymus, spleen, bone marrow, small intestine, heart, pituitary, and mouse mammary carcinoma. 2. The variation in size of the phagosomes ranging from the limit of microscopic visibility up to 5 micro diameter, previously described for kidney, was also observed to occur in many of the other organs. The average size of the phagosomes in different organs was also different, the phagosomes of the liver, for example being on the average smaller than those of the kidney, pancreas, and prostate. 3. In squash preparations of kidney and liver, the phagosomes appeared often in curved rows following the course of the cell membranes of epithelial cells. In several other organs, they appeared aggregated in cells located in the vicinity of blood or lymphatic vessels or capillaries. 4. After injection of peroxidase directly into the brain of a rabbit, a striking concentration of peroxidase was observed in phagosomes of endothelial cells of capillaries and vessels, surrounding the site of injection. It was suggested that this localization may offer an explanation for the so called blood-brain barrier. 5. The cytochemical peroxidase method was applied to smears of isolated fractions of kidney and liver. Only the isolated phagosomes, but not the isolated nuclei, mitochondria, and microsomes, reacted with benzidine after administration of peroxidase. The contamination of conventionally prepared nuclear, mitochondrial, and microsomal fractions of kidney and liver with phagosomes of different sizes was observed. By correlating the cytochemical peroxidase test of smears of isolated fractions with the colorimetric determination of peroxidase, acid phosphatase, and cytochrome oxidase in the same fractions, the differentiation of the phagosomes from mitochondria and other cell granules was facilitated. 6. The marked difference in the osmotic properties of phagosomes and mitochondria, detectable after treatment with 70 per cent alcohol, and the difference in their affinities towards basic fuchsin, made it possible to differentiate the phagosomes from the mitochondria. It was found by this simple procedure that kidney cells of normal rats contain a large number of phagosomes ranging in size from 0.5 to 3 micro, whereas liver cells of normal rats contain relatively few phagosomes of this size but many smaller ones (0.2 to 0.5 micro diameter). These increased in size after treatment of the rats with horseradish peroxidase.

Acid Phosphatase↗
PubMed2002

Imaging of electrochemical enzyme sensor on gold electrode using surface plasmon resonance.

Three types of imaging, namely layer structure, electrochemical reaction, and enzyme sensor response, were achieved by applying surface plasmon resonance (SPR) measurement to an electrochemical biosensor. We constructed glucose oxidase based mediator type sensors on a gold electrode by spotting the mediator that contained horseradish peroxidase and spin coating the glucose oxidase film. The layer structure of the sensor was imaged by means of angle scanning SPR measurement. The single sensor spot (about 1 mm in diameter) consisted of about 100 x 100 pixels and its spatial structure was imaged. The multilayer structure of the enzyme sensor had a complex reflectance-incident angle curve and this required us to choose a suitable incident angle for mapping the redox state. We chose an incident angle that provided the most significant reflection intensity difference by using data obtained from two angle scanning SPR measurements at different electrode potentials. At this incident angle, we controlled the electrochemical states of the spotted mediator in cyclic voltammetry and imaged the degree to which the charged site density changed. Finally, we mapped the enzymatic activity around the mediator spot by the enzymatic reoxidation of pre-reduced mediator in the presence of glucose.

Biosensing Techniques↗
PubMed1992

A simple and inexpensive glucose oxidase substrate system for enzyme immunoassay.

A simple and inexpensive glucose oxidase substrate system was developed for enzyme immunoassay. This system is based on the formation of a coloured complex between polyvinyl alcohol or starch and iodine produced from iodide in the presence of hydrogen peroxide generated by the glucose oxidase reaction. The rate of iodine production was enhanced by the supplementation of molybdate. In an enzyme immunoassay for anti-Salmonella antibodies using glucose oxidase as the indicator enzyme, the molybdate-enhanced polyvinyl alcohol- and starch-glucose-iodide substrate systems were as sensitive as a conventional glucose oxidase assay system employing horseradish peroxidase as a secondary enzyme and a suitable hydrogen-donating chromogen.

Animals↗
PubMed1995

The origin of the oxidative burst in plants.

A large number of publications recently have drawn strong analogies between the production of active oxygen species in plant cells and the "oxidative burst" of the phagocyte, even to the point of constructing elaborate models involving receptor mediated G-protein activation of a plasmalemma NADPH oxidase in plant cells. However there are potentially other active oxygen species generating systems at the plant cell surface. The present work examines these alternatives with particular emphasis on the rapid production of active oxygen species, in common with a number of other systems, by suspension-cultured cells of French bean on exposure to an elicitor preparation from the fungal pathogen Colletotrichum lindemuthianum. The cells show a rapid increase in oxygen uptake which is followed shortly afterwards by the appearance of a burst of these active oxygen species, as measured by a luminescence assay, which is probably all accounted for by hydrogen peroxide. An essential factor in this production of H2O2 appears to be transient alkalinization of the apoplast where the pH rises to 7.0-7.2. Dissipation of this pH change with a number of treatments, including ionophores and strong buffers, substantially inhibits the oxidative burst. Little evidence was found for enhanced activation of a membrane-bound NADPH oxidase. However the production of H2O2 under alkaline conditions can be modelled in vitro with a number of peroxidases, one of which, an M(r) 46,000 wall-bound cationic peroxidase, is able to sustain H2O2 production at neutral pH unlike the other peroxidases which only show low levels of this reaction under such conditions and have pH optima at values greater than 8.0. On the basis of such comparative pH profiles between the cells and the purified peroxidase and further inhibition studies a direct production of H2O2 from the wall peroxidase in French bean cells is proposed. These experiments may mimic some of the responses to plant pathogens, particularly the hypersensitive response, which is an important feature of resistance. A cell wall peroxidase-origin for the oxidative burst is clearly different from a model consisting of receptor activation of a plasmalemma-localised NADPH oxidase generating superoxide. An alternative simple and rapid mechanism thus exists for the generation of H2O2 which does not require such multiple proteinaceous components.

Fabaceae↗
PubMed1998

Substitution of lysine-362 in a putative proton-conducting channel in the cytochrome c oxidase from Rhodobacter sphaeroides blocks turnover with O2 but not with H2O2.

The recently reported X-ray structures of cytochrome oxidase reveal structures that are likely proton-conducting channels. One of these channels, leading from the negative aqueous surface to the heme a3/CuB bimetallic center, contains a lysine as a central element. Previous work has shown that this lysine (K362 in the oxidase from Rhodobacter sphaeroides) is essential for cytochrome c oxidase activity. The data presented demonstrate that the K362M mutant is impeded in the reduction of the heme a3/CuB bimetallic center, probably by interfering with the intramolecular movement of protons. The reduction of the heme-copper center is required prior to the reaction with dioxygen to form the so-called peroxy intermediate (compound P). This block can be by-passed to some extent by the addition of H2O2, which can react with the enzyme without prereduction of the heme-copper center and can then be reduced to water using electrons from cytochrome c. Hence, the K362M mutant, though lacking oxidase activity, exhibits cytochrome c peroxidase activity. Rapid mixing techniques have been used to determine the kinetics of this peroxidase activity at concentrations of H2O2 up to 0.5 M. The Km for peroxide is about 50 mM and the Vmax is 50 electrons s-1, which is considerably slower than the turnover that can be obtained for the oxidase activity of the wild-type enzyme (1200 s-1). The turnover of the mutant oxidase with H2O2 appears to be limited by the rate of reaction of the enzyme with peroxide to form compound P, rather than the rate of reduction of compound P to water by cytochrome c. The data require a reexamination of the proposed roles of the putative proton-conducting channels.

Catalysis↗
PubMed2005

NAD(P)H oxidase contributes to the progression of remote hepatic parenchymal injury and endothelial dysfunction, but not microvascular perfusion deficits.

Oxidative stress occurs in remote liver injury, but the origin of the oxidant generation has yet to be thoroughly delineated. Some reports suggest that the source of the distant oxidative stress originates from the site of initial insult [i.e., xanthine oxidase (XO)]; however, it could also be derived from sources such as phagocytic and/or vascular NAD(P)H oxidase (Nox) enzymes. With a murine model of bilateral hindlimb ischemia-reperfusion, we describe here a mechanism for Nox-dependent oxidant production that contributes, at least in part, to remote hepatic parenchymal injury and sinusoidal endothelial cell (SEC) dysfunction. To determine whether Nox enzymes were the source of oxidants, mice were treated immediately after the onset of hindlimb ischemia with specific inhibitors to XO (50 mg/kg ip allopurinol) or Nox (10 mg/kg ip gp91ds-tat and 3 mg/kg ip apocynin). After 1 h of ischemia, hindlimbs were reperfused for either 3 or 6 h. Inhibition of XO failed to provide any improvement in parenchymal injury, SEC dysfunction, neutrophil accumulation, or microvascular dysfunction. In contrast, the inhibition of Nox enzymes prevented the progression (6 h) of parenchymal injury, significantly protected against SEC dysfunction, and completely prevented signs of neutrophil-derived oxidant stress. At the same time, however, inhibition of Nox failed to protect against the early parenchymal injury and microvascular dysfunction at 3 h of reperfusion. These data confirm that microvascular perfusion deficits are not essential for the pathogenesis of remote hepatic parenchymal injury. The data also suggest that Nox enzymes, not XO, are involved in the progression of compromised hepatic parenchymal and endothelial integrity during a systemic inflammatory response.

Animals↗
PubMed1997

Comparison of DNA adduct formation by aristolochic acids in various in vitro activation systems by 32P-post-labelling: evidence for reductive activation by peroxidases.

Aristolochic acid I (AAI) and aristolochic acid II (AAII), the two major components of the carcinogenic plant extract aristolochic acid (AA), are known to be mutagenic and to form DNA adducts in vivo. According to the structures of the major DNA adducts identified in animals and humans, nitroreduction is the crucial pathway in the metabolic activation of these naturally occurring nitroarenes to their ultimate carcinogenic species. Using the nuclease P1-enhanced version of the 32P-post-labelling assay we investigated the formation of DNA adducts by AAI and AAII in different in vitro activation systems in order to determine the most suitable in vitro system mimicking target tissue activation. Although DNA adducts resulting from oxidative activation of AAs have not yet been identified both reductive and oxidative in vitro systems were employed. In vitro incubations were conducted under standardized conditions (0.3 mM AAs; 4 mM dNp as calf thymus DNA) using rat liver microsomes, xanthine oxidase (a mammalian nitroreductase), horseradish peroxidase, lactoperoxidase and chemical reduction by zinc. Enzymatic incubations were performed under aerobic and anaerobic conditions. A combination of two independent chromatographic systems (ion-exchange chromatography and reversed-phase HPLC) with reference compounds was used for the identification of DNA adducts detected by the 32P-post-labelling assay. The two known major adducts of AAI or AAII found in vivo were generated by all in vitro systems except for incubations with AAII and horseradish peroxidase where two unknown adducts predominated. Irrespective of the in vitro activation system used, the majority of adduct spots obtained were identified as the previously characterized four AA-DNA adducts: dA-AAI, dA-AAII, dG-AAI and dG-AAII. This indicates that both reductive and peroxidative activation of AAI or AAII resulted in chromatographically indistinguishable DNA adducts. Thus, peroxidase mediated activation of AAs led to the formation of the same adducts that had been observed in vivo and upon reductive activation in several in vitro systems. Quantitative analyses of individual adducts formed in the various in vitro systems revealed relative adduct labelling (RAL) values over a 100,000-fold range from 4 in 10(3) for activation of AAII to deoxyadenosine adducts by zinc to only 3 in 10(8) for activation of AAII by lactoperoxidase. The extent of DNA modification by AAI was higher than by AAII in all enzymatic in vitro systems. Only activation by zinc resulted in higher total binding to exogenous DNA by AAII than by AAI. Aerobic incubations with rat liver microsomes generated AAI- and AAII-DNA adduct profiles reproducing profiles in target tissue (forestomach) of rats, thus providing the most appropriate activation among the in vitro systems tested.

Animals↗
PubMed1998

Anaerobic bacterial metabolism in the ancient eukaryote Giardia duodenalis.

The protozoan parasite, Giardia duodenalis, shares many metabolic and genetic attributes of the bacteria, including fermentative energy metabolism which relies heavily on pyrophosphate rather than adenosine triphosphate and as a result contains two typically bacterial glycolytic enzymes which are pyrophosphate dependent. Pyruvate decarboxylation and subsequent electron transport to as yet unidentified anaerobic electron acceptors relies on a eubacterial-like pyruvate:ferredoxin oxidoreductase and an archaebacterial/eubacterial-like ferredoxin. The presence of another 2-ketoacid oxidoreductase (with a preference for alpha-ketobutyrate) and multiple ferredoxins in Giardia is also a trait shared with the anaerobic bacteria. Giardia pyruvate:ferredoxin oxidoreductase is distinct from the pyruvate dehydrogenase multienzyme complex invariably found in mitochondria. This is consistent with a lack of mitochondria, citric acid cycle, oxidative phosphorylation and glutathione in Giardia. Giardia duodenalis actively consumes oxygen and yet lacks the conventional mechanisms of oxidative stress management, including superoxide dismutase, catalase, peroxidase, and glutathione cycling, which are present in most eukaryotes. In their place Giardia contains a prokaryotic H2O-producing NADH oxidase, a membrane-associated NADH peroxidase, a broad-range prokaryotic thioredoxin reductase-like disulphide reductase and the low molecular weight thiols, cysteine, thioglycolate, sulphite and coenzyme A. NADH oxidase is a major component of the electron transport pathway of Giardia which, in conjunction with disulphide reductase, protects oxygen-labile proteins such as ferredoxin and pyruvate:ferredoxin oxidoreductase against oxidative stress by maintaining a reduced intracellular environment. As the terminal oxidase, NADH oxidase provides a means of removing excess H+, thereby enabling continued pyruvate decarboxylation and the resultant production of acetate and adenosine triphosphate. A further example of the bacterial-like metabolism of Giardia is the utilisation of the amino acid arginine as an energy source. Giardia contain the arginine dihydrolase pathway, which occurs in a number of anaerobic prokaryotes, but not in other eukaryotes apart from trichomonads and Chlamydomonas reinhardtii. The pathway includes substrate level phosphorylation and is sufficiently active to make a major contribution to adenosine triphosphate production. Two enzymes of the pathway, arginine deiminase and carbamate kinase, are rare in eukaryotes and do not occur in higher animals. Arginine is transported into the trophozoite via a bacterial-like arginine:ornithine antiport. Together these metabolic pathways in Giardia provide a wide range of potential drug targets for future consideration.

Amino Acids↗
PubMedDate not supplied

[Observations about the use of different types of enzymes in immunohistological diagnosis of autoimmune diseases. Comparative study with indirect immunofluorescence technique (author's transl)].

The Authors have evaluated the use of anti-human immunoglobulins conjugated with peroxidase, alkaline-phosphatase, glucose-oxidase as a diagnostic tool for assaying autoantibodies. A comparison study was done using the classical indirect immunofluorescence technique. Immunoenzymatic techniques, of minor cost compared to immunofluorescence, have revealed a better appreciation of global and particular structure of the tissue in examination. From the three enzymes examined peroxidase revealed itself as first choice, because, other than having on overlapping sensitivity to immunofluorescence, it can allow a further ultrastructural study.

Alkaline Phosphatase↗