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PubMed2006

Gene damages of mitochondrial DNA encoding cytochrome oxidase of intestinal epithelial cells in hemorrhagic shock rats.

OBJECTIVE: To investigate the detrimental effects of hemorrhagic shock on the structure and function of mitochondria DNA (mtDNA) encoding cytochrome oxidase genes in intestinal epithelial cells. METHODS: Wistar rats were used and divided into two groups: hemorrhagic shock group and control group. Hemorrhagic shock model of rats was utilized in this experiment. The mtDNA was extracted from the intestinal epithelial cells and amplified by polymerase chain reaction (PCR) with different primers of cytochrome oxidase (COX I, COX II and COX III). The products of PCR were directly sequenced. RESULTS: Hemorrhagic shock could result in the point mutagenesis in mitochondrial genome encoding cytochrome oxidase (COX I and COX II). There were 4, 4, 22, 16, 35 point mutations in COX I from 5545 to 6838 bp in 5 shocked rats. There were five point mutations in COX II from 7191 to 7542 bp at the site of t7191c, t7212c, a7386g, a7483g, c7542g in 1 shocked rat. There was no mutation found in COX III. CONCLUSIONS: Hemorrhagic shock could significantly induce the damage of the gene of cytochrome oxidase encoded by mtDNA.

Animals↗
PubMed1988

Neuronal degeneration in the brain of the brindled mouse. Histochemical demonstration of decreased cytochrome oxidase activity in the cerebellum and brain stem.

In order to investigate the levels of cytochrome oxidase activity in neuronal mitochondria in the brain of the brindled mouse hemizygote (BM), the cerebella and brain stems from 12 pairs of brindled and normal littermates aged 13-16 days were examined. The diaminobenzidine method for light- and electronmicroscopic histochemistry was adopted. Light microscopy revealed that mitochondria in the normal cerebellum showed an intensely positive reaction to diaminobenzidine, whereas those in the BM cerebellum showed a very weak reaction indicating an evident reduction of cytochrome oxidase activity. Electron microscopy disclosed a diaminobenzidine-OsO4 product densely appearing on the inner membranes of most mitochondria in Purkinje cells in the normal cerebellum. However, it was very faint or absent in those in the BM cerebellum. The same was true in Golgi II cells, granule cells, glomeruli and brain stem nuclei, but the degree of reduction was not uniform among these structures. In conclusion, there is not only a generalized reduction of cytochrome oxidase activity but also a topographical predilection of areas showing a reduction of the enzyme in the BM cerebellum and brain stem. These facts may explain the pathogenesis of neuronal degeneration in the brain of the BM.

Animals↗
PubMed1985

Kinetic studies on formation of cytochrome oxidase of Rhodopseudomonas capsulata after a shift from phototrophic to chemotrophic growth.

Rhodopseudomonas capsulata cells were shifted from phototrophic (anaerobic, light) to chemotrophic (semiaerobic, dark, 10% air saturation) growth conditions. During the adaptation period of 4 h, the bacteriochlorophyll content of cells and membranes decreased, and a newly synthesized 65-kilodalton polypeptide of the cytochrome oxidase was incorporated into the membrane fraction. The enzymatic activity of the cytochrome oxidase increased strongly after a lag time of 2 h. The amount of cytochrome oxidase protein does not follow the same kinetics. The relative amount of a membrane-bound cytochrome c of low molecular weight, which has been proposed to be a donor for the cytochrome oxidase, increased during adaptation.

Bacterial Proteins↗
PubMed1991

Affinity chromatography isolation of human cytochrome oxidase and small-scale Western immunoblot probing of the enzyme complex in mitochondrial cytopathy patients.

Isolation of human cytochrome oxidase by a one-step affinity chromatography procedure on a Sepharose 4B-ferrocytochrome c matrix following solubilization with the nonionic detergent laurylmaltoside yields an enzyme isolate of adequate purity for producing polyclonal antisera. Such an antiserum produced a distinctive immunoreactive profile in Western immunoblot studies to that reported using the enzyme isolated with ionic detergents. A sensitive and highly reproducible Western immunoblotting method is described for probing mitochondrial fractions prepared from small frozen skeletal muscle biopsies with an antiserum against the human placenta cytochrome oxidase. Application of this method to mitochondrial cytopathy patients with partial cytochrome oxidase deficiency shows that the detected subunits are synthesized in these patients.

Blotting, Western↗
PubMed1986

Cytochrome c peroxidase activity of bovine heart cytochrome oxidase incorporated in liposomes and generation of membrane potential.

Cytochrome oxidase vesicles catalyzed the peroxidatic oxidation of ferrocytochrome c. The maximal peroxidase activity in the absence of an uncoupling agent was 9.8 mol ferrocytochrome c oxidized/(s X mol heme a), indicating a 5-fold activation compared with the soluble enzyme system. The peroxidase activity was further enhanced 1.2 to 2.1 times upon addition of an uncoupler, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. The stoichiometry of the reduction of hydrogen peroxide by ferrocytochrome c was established to be 1 : 2, indicating water formation. Potassium cyanide (0.14 mM) completely inhibited the peroxidase activity. The inhibition by 1 mM CO was 40-77% depending on the energized state of cytochrome oxidase vesicles, but in contrast, 85% inhibition was observed with the soluble enzyme. In the energized state the enzyme showed a slightly lower affinity for CO than in the deenergized state. Coupled with the peroxidase activity, a membrane potential of 72 mV was registered transiently; this may be physiologically significant in relation to the energy transduction mechanism.

Animals↗
PubMed2001

Site-directed mutations in the mitochondrially encoded subunits I and III of yeast cytochrome oxidase.

Since yeast is amenable to mitochondrial transformation, designed mutations can be introduced in the mitochondrially encoded subunits of the respiratory complexes. In the present work, six mutations have been introduced by the biolistic method into yeast (Saccharomyces cerevisiae) cytochrome oxidase subunits I and III. The effects of these mutations on respiratory growth competence, cytochrome oxidase activity and optical properties were then characterized. Firstly, the conserved glutamate Glu-243 in the D-channel of subunit I was replaced by an asparagine or an aspartate residue. The effects of the mutations showed that Glu-243, which is essential for proton movement in bacterial oxidases, is also required for the activity of the eukaryotic enzyme. Secondly, four mutations associated with human disease were introduced in yeast, allowing detailed analysis of their deleterious effects on cytochrome oxidase function: Met-273-->Thr, Ile-280-->Thr and Gly-317-->Ser, affecting residues located in or near the K-channel in subunit I, and a short in-frame deletion comprising residues Phe-102 to Phe-106 in subunit III (DeltaF102-F106). The subunit III mutation was highly deleterious and abolished enzyme assembly. The change Gly-317-->Ser had no effect on respiratory function. However, mutations Met-273-->Thr and Ile-280-->Thr were mildly deleterious, decreased cytochrome oxidase activity and slightly perturbed the properties of the binuclear centre.

Electron Transport Complex IV↗
PubMed1992

A comparison of glycogen phosphorylase a and cytochrome oxidase histochemical staining in rat brain.

The utility of metabolic markers that index functional neuronal circuits is widely appreciated. The present study asks whether patterns of the metabolic enzyme, active glycogen phosphorylase, parallel those of the neuronal marker, cytochrome oxidase. Fresh frozen rat brain sections (30 microns) were processed for either active glycogen phosphorylase or cytochrome oxidase at each of ten levels of the neuraxis. Although these metabolic markers predominate in different cellular compartments--glycogen phosphorylase in the astrocytic compartment and cytochrome oxidase in the neuronal compartment--the patterns of high, moderate, and low levels of activity for both enzymes were generally parallel. These similarities extended to detailed patterns of heterogeneous staining within structures, in particular, to laminated and modular distribution within cerebral and cerebellar cortical structures. The modular distribution was evident in barrel structures in the cerebral cortex and in parasagittal compartments in the vermis of the cerebellum. Conspicuous differences between the two patterns occurred in white matter, in subcortical grey matter regions such as the nucleus accumbens, diagonal band, amygdala, and globus pallidus, and in the superior olivary nuclei of the brainstem as well as in nonneural structures such as the choroid plexus and ependyma. Discrete patchiness was characteristic of active glycogen phosphorylase distribution in the limbic neuropil of the dentate gyrus and entorhinal cortex. The strong parallels between active glycogen phosphorylase and cytochrome oxidase distribution support the view that glycogen phosphorylase, despite its glial localization, can reflect neuronal metabolic demands.

Animals↗
PubMed1990

Quantitative cytochemical studies of cytochrome oxidase activity in rat dorsal root ganglion cells.

The cytochrome oxidase activity of rat dorsal root ganglion cells was cytochemically and quantitatively measured. The 100 microns thick tissue slices fixed for 10 min were incubated in a DAB medium for 0, 30, 60, and 90 min at 37 degrees C, and electron-dense deposit areas within the mitochondrial intermembrane-intracristal spaces were measured with a computer-controlled image analyzer. The activity was expressed as deposit accumulation rate filling the mitochondrial space, (1 unit corresponds to the deposit filling 100% of the mitochondrial space per hour). Three different activities of the enzyme in either large pale cells or small dark cells of sensory neurons could be distinguished, based on the quantitative analysis, as large cells with intense (0.83 units), intermediate (0.38), and weak activity (0.22), and small cells with the same degrees of activity (1.04, 0.35, and 0.11, respectively). The results indicate that accumulation rate measurements of reaction product may be useful to quantitatively present the cytochrome oxidase reactivity of mitochondria, and that the degree of enzyme activity may contribute to the identification of functional differences in sensory neurons.

Animals↗
PubMed1987

Abnormal kinetic behavior of cytochrome oxidase in a case of Leigh disease.

Cultured skin fibroblasts from a child with fatal lacticacidemia displayed an abnormally high lactate:pyruvate ratio of 77:1, compared with control values of 22:1-27:1. When protease-treated isolated mitochondria were used, activity of the respiratory-chain enzymes was found to be approximately 60% of normal, and adenosine triphosphate synthesis was found to be normal with all substrates tested. In mitochondria prepared by means of digitonin treatment, adenosine triphosphate synthesis was depressed with all substrates tested, suggesting a defect in the operation of the cytochrome oxidase complex. In disrupted whole cells from the patient, cytochrome oxidase activity was 56% of the activity in the control cell line with the lowest activity. In the presence of a twofold excess of oxidized cytochrome c, patient cells showed 31% of the activity in controls. Cytochrome oxidase activity in both sonicated whole-cell preparations and in sonicated mitochondria displayed abnormal kinetics with regard to the substrate-reduced cytochrome c, which was particularly evident in the presence of excess oxidized cytochrome c. We believe that kinetically abnormal cytochrome oxidase complex is responsible for the biochemical and clinical abnormalities present in this patient.

Acidosis, Lactic↗
PubMed1983

Evidence for the sequential assembly of cytochrome oxidase subunits in rat liver mitochondria.

The assembly of cytochrome oxidase was studied in isolated rat liver mitochondria and isolated rat hepatocytes labelled in vitro with L-[35S]methionine. This was achieved by studying the temporal association of radioactive subunits which are immunoabsorbed with antibodies against subunits I, II and the holoenzyme. Antibodies against the holoenzyme were shown to be highly specific for subunit V. The results show that subunit I appears in the holoenzyme late in the assembly process. No radioactive subunit I is absorbed with antiserum against subunit II or the holoenzyme (subunit V) after a 30 min pulse in either isolated mitochondria or hepatocytes. However, both antisera absorb radioactive subunits I after a 150 min chase in isolated hepatocytes. This was confirmed using antibodies against subunit I, which absorbed only radioactive subunit I after a 30 min pulse but absorbed radioactive subunits I-III and VI after a 150 min chase. Thus, the late assembly of radioactive subunit I is explained by a temporal sequence in the assembly process and not by the presence of a large, non-radioactive pool of subunit I. Using the above approach and the three specific antisera, the following temporal sequence in the assembly of cytochrome oxidase was established. Subunits II and III assemble rapidly with each other or with cytoplasmically translated subunit VI. This complex of three peptides in turn assembles slowly with subunit I or with the other cytoplasmically translated subunits. The early association of subunit VI with the mitochondrially translated subunits II and III suggests a possible role of the former in integration of the holoenzyme.

Animals↗
PubMed1980

CO binding to mitochondrial mixed valence state cytochrome oxidase at low temperatures.

The kinetics and thermodynamics of the reaction of mixed valence state membrane-bound cytochrome oxidase with CO over the 178-203 K range has been studied by multichannel optical spectroscopy at three wavelength pairs (444-463 nm in the Soret region, and 590-630 and 608-630 nm in the alpha region) and analysed by non-linear optimization techniques. As in the case of the fully reduced membrane-bound cytochrome oxidase-CO reaction (Clore, G.M. and Chance, E.M. (1978) Biochem J. 175, 709-725), the normalized progress curves at the three wavelength pairs are significantly different indicating, on the basis of Beer's law, the presence of a minimum of three optically distinct species. The only model that satisfies the triple statistical requirement of a standard deviation within the standard error of the data, a random distribution of residuals and good determination of the optimized parameters, is a two species sequential mechanism: flash photolysis of the mixed valence state cytochrome oxidase-CO complex (species IIMC) yields unliganded mixed valence state cytochrome oxidase (species EM) and free CO which then recombine to form species IMC; species IMC is then converted into species IIMC. All the thermodynamic parameters describing the model are calculated and compared to those obtained for the fully reduced membrane-bound cytochrome oxidase-CO reaction (Clore and Chance (1978) Biochem. J. 175, 709-725). Although there are some qualitative similarities in the kinetics and thermodynamics of the reactions of mixed valence state (alpha 23+Cu+B.ALPHA 3+Cu2+A) and fully reduced (a3 2+Cu B + . a2+Cu A+) cytochrome oxidase with CO, there are large and significant quantitative differences in zero-point activation energies and frequency factors; over the temperature range studied, the mixed valence state cytochrome oxidase-CO reaction is found to proceed at a significantly slower rate than the fully reduced cytochrome oxidase-CO reaction. These differences indicate that changing the valence states of cytochrome a and CuA has a significant effect on the CO binding properties of cytochrome a 3 and possibly CuB.

Carbon Monoxide↗
PubMed1990

Effects of chronic alcohol consumption on the steady-state kinetics properties of cytochrome oxidase in rat liver.

The effect of chronic alcohol consumption on steady-state kinetic characteristics of cytochrome oxidase in rat liver was studied using submitochondrial particles prepared from ethanol-fed and control rats. Preparations from both control and alcoholic rats had equivalent apparent Km values for cytochrome c of 13 microM in the presence of phenazine methosulfate or 19 microM with N,N,N',N'-tetramethylphenylene diamine as oxidation-reduction mediators at physiological ionic strength. Both preparations showed comparable stimulation (approx. 3-fold) of oxidase activity following detergent solubilization of the membrane and similar temperature dependence for oxidase activity. Under all conditions, preparations from alcohol-fed rats displayed 30 to 50% lower rats of cytochrome oxidase activity per unit membrane protein than those from control rats. The diminution in specific activity per mg protein was accompanied by a similar decline in heme aa3 content, as has been noted in previous studies. When expressed on a turnover number basis, the molecular activity of cytochrome oxidase (natoms O/min per nmol heme a) was equivalent in both alcoholic and control preparations. The results indicate that the intrinsic kinetic characteristics of cytochrome oxidase are not changed by alcohol consumption. The data suggest that the characteristic decline in heme aa3 content and cytochrome oxidase specific activity seen in ethanol-fed rats does not arise from alterations in the accessibility of the oxidase towards cytochrome c, or from changes in bulk phase lipid composition or physical properties. The results support the conclusion that ethanol consumption decreases the membrane content of functionally active oxidase molecules, but does not change the catalytic properties of these oxidase molecules.

Alcoholism↗
PubMed1996

Quantitative histochemical study of cytochrome oxidase in the dLGN of aging rats.

We carried out a quantitative histochemical study of the enzyme cytochrome oxidase (CO) in neurons of the dorsal lateral geniculate nucleus (dLGN) of male Wistar rats aged 3, 18, 24 and 28 months. The results show that the activity of cytochrome oxidase decreases significantly between 24 and 28 months. We also checked whether a correlation existed between neuronal size and enzymatic activity. Low correlation coefficients were obtained which were between 0.4139 at 3 months 0.2092 at 28 months. Nevertheless, we observed a certain relationship between both parameters, and therefore we classified the neurons as light, moderate and dark according to their optical density, which correlates with enzyme cytochrome oxidase activity, and as small, medium and large depending on their size. We found that light neurons were scarcely represented in the dLGN. At the age of 3 months, the most frequent neurons were moderate, medium-size ones, and dark, small ones. The population of moderate neurons increased with age, reaching 74.5% at the 28th month, 52.2% of which corresponded to medium-size neurons. In the same group dark neurons decreased, falling to a total of 15.3% made up of medium and large-size ones. These results could be interpreted as reflecting a decrease in the bioenergetic competence of the neurons of this nucleus in old age.

Aging↗
PubMed1981

Electrostatic interaction of cytochrome c with cytochrome c1 and cytochrome oxidase.

The reactions of horse heart cytochrome c with succinate-cytochrome c reductase and cytochrome oxidase were studied as a function of ionic strength using both spectrophotometric and oxygen electrode assay techniques. The kinetic parameter Vmax/Km for both reactions decreased very rapidly as the ionic strength was increased, indicating that electrostatic interactions were important to the reactions. A new semiempirical relationship for the electrostatic energy of interaction between cytochrome c and its oxidation-reduction partners was developed, in which specific complementary charge-pair interactions between lysine amino groups on cytochrome c and negatively charged carboxylate groups on the other protein are assumed to dominate the interaction. The contribution of individual cytochrome c lysine amino groups to the electrostatic interaction was estimated from the decrease in reaction rate caused by specific modification of the lysine amino groups by reagents that change the charge to 0 or -1. These estimates range from -0.9 kcal/mol for lysines immediately surrounding the heme crevice of cytochrome c to 0 kcal/mol for lysines well removed from the heme crevice region. The semiempirical relationship for the total electrostatic energy of interaction was in quantitative agreement with the experimental ionic strength dependence of the reaction rates when the parameters were based on the specific lysine modification results. The electrostatic energies of interaction between cytochrome c and its reductase and oxidase were nearly the same, providing additional evidence that the two reactions take place at similar sites on cytochrome c.

Animals↗
PubMed1992

[Cytochrome oxidase induction following oxidative stress produced by adriamycin in the heart of rats fed olive oil].

Cytochrome oxidase is extremely susceptible to modifications that could occur at membrane level such as, for example, the deep influence that cardiolipin has on its maximal activity. Moreover, even the different ratio of fatty acid species in cardiolipin molecule could affect cytochrome oxidase properties. The influence of dietary fat and of endogenous lipid peroxidation with regard to cytochrome c oxidase activity and cytochrome a+a3 concentrations in mitochondria from rat heart has been investigated. Whilst the endogenous oxidative stress led to increase the peroxidation products, detected by means of hydroperoxides and conjugated dienes determinations, the features of the lipid environment were not affected as shown by fluorescence polarization technique. However, the oxidative stress was able to induce a significant increase of cytochrome a+a3 level even if the phenomenon did not reach the same extent as in the case of previous investigations on rat liver. Moreover, the results vary with the different fat source used and this might indicate a possible involvement of different dietary fats in peroxidation mechanisms. Furthermore the data we have obtained enable us to confirm once more that an induction of the synthesis of cytochrome a+a3 might be related to an enhanced production of peroxides at membrane level.

Animals↗
PubMed1980

Ultrastructural demonstration of succinic dehydrogenase and cytochrome oxidase activity in sporozoites of Babesia ovis and Theileria annulata (Apicomplexa: Piroplasmea) in salivary glands of tick vectors (Rhipicephalus bursa, Hyalomma anatolicum excavatum).

Salivary gland stages ("sporozoites") of Babesia ovis and Theileria annulata (Apicomplexa: Piroplasmea) in female ixodid ticks were studied for ultracytochemical activity of the respiratory enzymes, succinic dehydrogenase (SDH), and cytochrome oxidase. Both SDH and cytochrome oxidase were demonstrated in the sporozoites and the mitochondria in these stages. Identified in this way the final reaction product of SDH was located mainly at the inner side of the mitochondrial boundary, though it was also visible in the internal space of the organelle. Cytochrome oxidase activity always was confined to the wall of mitochondria. This enzyme was demonstrated also in the erythrocyte stage of B. ovis. The cytochemical results indicate respiratory potential of the piroplasmean stages studied. Cristate or typical protozoan mitochondria have not been observed in sporozoites of Babesia or Theileria. This report is the first demonstration of mitochondria, or mitochondrialike activity in Babesia.

Animals↗
PubMed1986

Distribution of cytochrome oxidase in rat brain: studies with diaminobenzidine histochemistry in vitro and [14C]cyanide tissue labeling in vivo.

Studies in experimental animals and post-mortem studies in humans have indicated that the level of the mitochondrial enzyme cytochrome oxidase within brain anatomical pathways is regulated by the long-term functional use of those pathways. To study this relationship, we have measured cytochrome oxidase spectrophotometrically in punch biopsies from different brain regions of rat. We compared these assays against results from the diaminobenzidine histochemical technique. We found a high degree of correlation (r = 0.90) between the density of diaminobenzidine reaction product and enzyme activity. This validates the usefulness of the diaminobenzidine technique for anatomical localization and measurement of this enzyme. To study the feasibility of using radioactive cyanide as an in vivo ligand of cytochrome oxidase, we performed quantitative autoradiographic analysis of rat brains of animals given an intravenous bolus injection of [14C]cyanide. Analysis of the arterial blood curve indicated a complex redistribution of cyanide between red blood cells, plasma, and tissues. Brain labeling reached peak levels at 1 min and then fell despite rising concentrations of free plasma cyanide. Analysis of autoradiographic images revealed good anatomical resolution. The density of labeling in individual structures over time failed to show a strong correlation with cytochrome oxidase activity or diaminobenzidine reaction product.

3,3'-Diaminobenzidine↗