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Cytochrome oxidase histochemistry reveals regional subdivisions in the rat periaqueductal gray matter.

The identification of different anatomical regions of the periaqueductal gray matter of rats was addressed in the present study by using the histochemical staining for the mitochondrial enzyme cytochrome oxidase. At caudal and middle levels, cytochrome oxidase histochemistry clearly demonstrates the existence of four subdivisions: dorsal, dorsolateral, ventrolateral and medial, whereas in sections from the rostral periaqueductal gray matter only two concentric bands are identifiable on the basis of the degree of cytochrome oxidase activity.

Animals↗

Decreased rat brain cytochrome oxidase activity after prolonged hypoxia.

Three weeks of hypoxic exposure results in a spectrum of systemic physiological and local brain tissue adaptations. Cytochrome oxidase histochemistry was used as an indicator of changes in energy demand in response to hypoxia. We found overall cytochrome oxidase activity decreased in hypoxic adapted rats as compared to normoxic control rats. Some regions, notably layer 3 of the frontal cerebral cortex, layer 4 of the parietal sensory barrel fields, lacunosum moleculare hippocampi, and specific nuclei of the rostral ventral medulla, though exhibiting cytochrome oxidase activity decreases of 16-27%, still retain their relatively higher levels of activity. We conclude that there is a hypometabolic component of the rat brain adaptation to continued hypoxia.

Animals↗

Decreased cytochrome oxidase activity and changes in phospholipids in heart mitochondria from hypothyroid rats.

The effect of hypothyroidism on kinetic characteristics of cytochrome oxidase in rat heart mitochondria was studied. Mitochondrial preparations from control and hypothyroid rats had equivalent Km values for cytochrome c, while the maximal activity of the oxidase was significantly decreased (more than 30%) in mitochondrial preparations from hypothyroid rats. This decrease is associated to a parallel decrease in state 3 respiration. The cytochrome aa3 content was slightly decreased (by around 15%) in mitochondria from hypothyroid rats. The Arrhenius plot characteristics differ for cytochrome oxidase activity in mitochondria from hypothyroid rats as compared with control rats in that the breakpoint of the biphasic plot is shifted to a higher temperature. Cardiolipin content was markedly decreased in the mitochondrial membrane from hypothyroid rats. No alterations were found in the pattern of cardiolipin fatty acid distribution of mitochondrial membrane from control and hypothyroid rats. The effects of the hypothyroid state on the activity of cytochrome oxidase, on cytochrome aa3 levels, and on cardiolipin contents were completely reversed by following the treatment of hypothyroid rats with thyroid hormone. The results support the conclusion that the depressed mitochondrial cytochrome oxidase activity in the hypothyroid state is due, at least in part, to a decrease in the cardiolipin content of the mitochondrial inner membrane.

Animals↗

The nature of the exchange coupling between high-spin Fe(III) heme o3 and CuBII in Escherichia coli quinol oxidase, cytochrome bo3: MCD and EPR studies.

Fully oxidized cytochrome bo3 from Escherichia coli has been studied in its oxidized and several ligand-bound forms using electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectroscopies. In each form, the spin-coupled high-spin Fe(III) heme o3 and CuB(II) ion at the active site give rise to similar fast-relaxing broad features in the dual-mode X-band EPR spectra. Simulations of dual-mode spectra are presented which show that this EPR can arise only from a dinuclear site in which the metal ions are weakly coupled by an anisotropic exchange interaction of J 1 cm-1. A variable-temperature and magnetic field (VTVF) MCD study is also presented for the cytochrome bo3 fluoride and azide derivatives. New methods are used to extract the contribution to the MCD of the spin-coupled active site in the presence of strong transitions from low-spin Fe(III) heme b. Analysis of the MCD data, independent of the EPR study, also shows that the spin-coupling within the active site is weak with J approximately 1 cm-1. These conclusions overturn a long-held view that such EPR signals in bovine cytochrome c oxidase arise from an S' = 2 ground state resulting from strong exchange coupling (J > 10(2) cm-1) within the active site.

Animals↗

A novel terminal oxidase, cytochrome baa3 purified from aerobically grown Pseudomonas aeruginosa: it shows a clear difference between resting state and pulsed state.

A novel type of cytochrome c oxidase was purified to homogeneity from Pseudomonas aeruginosa which was grown aerobically. The purified oxidase contained two molecules of heme a, two atoms of copper, and one molecule of protoheme per molecule. One of the two heme a molecules in the oxidase reacted with carbon monoxide, so that the enzyme was of baa3-type. The oxidase molecule was composed of three subunits with molecular weights of 38,000, 57,000, and 82,000. Although the oxidase oxidized ferrocytochrome c-550 obtained from the bacterial cells grown aerobically, the oxidizing activity was not high. The "resting form" and the "pulsed form" of the oxidase were observed clearly with this enzyme, and the transition from the resting form to the pulsed form was accompanied by a distinct change of the enzymatic activity. The difference in the kinetics of the catalytic reactions between the two forms is discussed.

Carbon Monoxide↗

Protein-lipid interactions in cytochrome oxidase from Saccharomyces cerevisiae. Effects of detergents and reconstitution of enzyme activity by phospholipids by using cholate-mediated exchange.

Cytochrome oxidase, purified from the yeast Saccharomyces cerevisiae, was shown to have associated phospholipid, cholate or detergent, which could be varied by dialysis or (NH4)2SO4 precipitation of the protein. Cholate and the detergents Triton X-100 and Tween 80 were shown to differ in their ability to support enzyme activity. Changes in the Vmax, but not the Km, for ferrocytochrome c as the cholate concentration was varied indicate that cholate increases the number of exposed active sites of the enzyme. Cholate was used to introduce chosen phospholipids into the lipid environment of yeast cytochrome oxidase. Kinetic studies clearly showed that cholate can mediate exchange of exogenous for endogenous phospholipid. All phospholipids screened supported activity up to the basal value for the unsubstituted enzyme, whereas mitochondrial phosphatidylethanolamine and various phosphatidlycholines (except 1,2-dipalmitoyl-sn-glycero-3-phosphocholine) produced enhanced activity. A detailed kinetic examination revealed that the major effect of phosphatidylethanolamine is to increase k+1, whereas the major effect of phosphatidylcholine is to increase K+2 in the minimal kinetic scheme E + S k+1 in equilibrium k-1 ES k+2 leads to E + P Cardiolipin, although supporting activity, does not give any enhancement of k+1 or k+2 over the values for the cholate control. The relevance of these observations to protein-lipid interactions in cytochrome oxidase is discussed.

Cholic Acids↗

Resolution of two compound C-type intermediates in the reaction with oxygen of mixed-valence state membrane-bound cytochrome oxidase.

The reaction of mixed-valence state membrane-bound cytochrome oxidase with oxygen has been studied by difference spectroscopy with reference to the unliganded state and by the low temperature technique of Chance and coworkers. Three intermediates, compound A2 and two compound C-type components denoted C606 and C610, have been resolved in time and wavelength in the alpha region. Their optical properties are defined in the visible range. Compound A2 disappearance and compound C606 formation exhibit first-order kinetics with identical rate constants: 2.4 . 10(-3) s-1 at -94 degrees C. Compound A2 has its alpha band maximum at 590 nm and shares an isosbestic point at 595 nm with the C606 species. The alpha band of this intermediate peaks at 606 nm. Compound C610 is the real end point of the reaction and its alpha band maximum appears at 610 nm. Compound C606 is interpreted as resulting from the transfer of one electron from heme alpha 3 copper to oxygen and compound C610 as expressing a molecular reorganization due to the effect of the temperature. Structural requirements for the location of CuB in the active site are discussed. It is concluded that the three observed compounds are the only intermediates formed in the reaction between oxygen and mixed-valence state membrane-bound cytochrome oxidase.

Animals↗

The conformational states of cytochrome oxidase in the mitochondrion.

THE Soret spectrum of "resting" cytochrome oxidase in cytochrome-c depleted mitochondria has been determined. The spectrum obtained is dependent on the rate at which the oxidase is turning over. In the least active preparations, the spectrum is almost pure "oxidized" oxidase. With increasing activity the spectrum is converted to a mixture of "oxidized" and "oxygenated" oxidases. It is concluded that the same conformational differences between the two non-reduced forms that are found in the purified enzyme also occur in these cytochrome-c depleted mitochondria.

Animals↗

Cytochrome oxidase, acetylcholinesterase, and NADPH-diaphorase staining in human supratemporal and insular cortex: evidence for multiple auditory areas.

The pattern of cytochrome oxidase, acetylcholinesterase, and NADPH-diaphorase activity was studied in the supratemporal plane, the posterior part of the superior temporal gyrus, and the insula of normal human brains. Five dark cytochrome oxidase regions were found: (i) on Heschl's gyrus (area TC of von Economo and Koskinas); (ii) on the planum polare (area TC/TG); (iii) posterior to Heschl's gyrus (within area TA); (iv) on the posterior convexity of the superior temporal gyrus (within area TA); and (v) on the posterosuperior insula (area IB). More lightly stained cortex separated these regions (areas IA, TD, and part of TB). The laminar distribution of cytochrome oxidase activity varied in different areas. Acetylcholinesterase-positive fibers predominated in area TC and pyramidal neurons in areas TA and IA and in parts of TB; a mixture of fiber and neuronal staining was found in TC/TG, TD, and IB. NADPH-diaphorase positive profiles included large darkly stained nonpyramidal neurons, mostly in infragranular layers and in subcortical white matter, small faintly stained cells, and a dense array of fibers. The NADPH-diaphorase staining pattern did not vary between areas. The present results suggest that the supratemporal plane, the posterior part of the superior temporal gyrus, and the insula contain at least eight putative cortical areas. Comparison with activation studies by others suggest that, apart from the primary auditory area, six other putative areas may be auditory whereas one putative area, on posterior insula, may be vestibular.

Acetylcholinesterase↗

Regulation of cytochrome oxidase activity in the rat forebrain throughout adulthood.

Measures of metabolic activity can provide useful indices of the effects of aging on neural function, since sustained changes in neural activity alter metabolic demand and the activity of metabolic enzymes. Previous reports of effects of aging on key enzymes for oxidative metabolism are mixed, however, with some reports that activity declines in the aging brain and others that activity remains stable or increases. We used high-resolution, quantitative histochemistry to test whether cytochrome oxidase (CO) activity changes in the forebrain during adulthood and senescence, measuring activity in each layer of the hippocampus and several cerebral cortical areas. In most forebrain regions, average cytochrome oxidase activity was slightly higher in middle-aged than in young adult rats but did not differ between middle-aged and old rats. Thus, there was no significant change in cytochrome oxidase activity with senescence. Additional analyses indicated that cytochrome oxidase activity is regulated regionally in the brain, as well as focally, and that differences in regional regulation may contribute to variation in CO activity among individuals, which was greater in young and old rats than in middle-aged animals.

Age Factors↗

Preparation of reconstituted cytochrome oxidase vesicles with defined trans-membrane protein orientations employing a cytochrome c affinity column.

Reconstituted cytochrome oxidase systems in which the majority of the vesicles contain a single oxidase dimer can be prepared. It is shown that, when these are passed through a cytochrome c affinity column, only those vesicles oriented outwards (such that the active site is available to external cytochrome c) are bound to the support matrix. Protein-free vesicles and vesicles containing an inwardly oriented enzyme are eluted in the void volume. Subsequently, vesicles containing an outwardly oriented enzyme can be eluted from the column at high salt concentrations. This protocol has been used successfully to resolve vesicles of either oxidase orientation when the enzyme is reconstituted with a variety of lipid mixtures. The recovery of oxidase activity from the column ranged between 75 and 94%.

Animals↗

Neuronal uptake and laminar distribution of tritiated aspartate, glutamate, gamma-aminobutyrate and glycine in the prestriate cortex of squirrel monkeys: correlation with levels of cytochrome oxidase activity and their uptake in area 17.

The neuronal uptake and laminar distribution of cortically injected tritium-labeled gamma-aminobutyrate (GABA), aspartic acid, glutamate and glycine was examined in the prestriate cortex of squirrel monkeys. The intent of this investigation was not to examine the role of these amino acids as neurotransmitters, but to correlate the distribution of tritium-labeled neurons with their levels of cytochrome oxidase activity. A comparison of the number of these labeled neurons was made between the metabolically active "puff" and the less active "nonpuff" regions. In addition, these results were contrasted with the findings in area 17. With each tritiated amino acid tested, labeled neurons that had either high or low levels of cytochrome oxidase activity were present in all laminae. However, the density of labeled neurons varied between lamina for a given amino acid as well as between different amino acids. While many neurons that were cytochrome oxidase-reactive were also tritium-labeled, cytochrome oxidase activity was not a prerequisite for the sequestering of tritium label. In fact, many of the labeled neurons exhibited relatively low levels of cytochrome oxidase activity. Similar to area 17, few aspartate- or glutamate-labeled neurons were present in laminae II-III. The number of labeled neurons for both amino acids increased in laminae IV-VI, with the greatest increase observed in laminae V-VI. Gamma-aminobutyrate-labeled neurons were more prevalent in laminae I and upper II than in the other laminae, whereas in area 17, a greater proportion of the labeled neurons were found in laminae V-VI. With the exception of the uppermost laminae, where GABA-labeled neurons were more abundant, the number of glycine-labeled neurons was significantly greater throughout most laminae than with the other amino acids examined. The density of glycine-labeled neurons in lamina IV, however, was significantly less than the number observed in lamina III even though lamina III was farther away from the injection site which was at the boundary between laminae V-VI. Glycine-labeled neurons were, on average, larger than those labeled with any other amino acid. Similar to area 17, more GABA- and glycine-labeled neurons were observed within the puff regions than in nonpuff regions. No puff/nonpuff differences were observed in the distribution of leucine-injected controls. Labeled neurons for each amino acid included stellate-, fusiform- and pyramidal-shaped cells, each of varying sizes. However, outside the intensely labeled injection sites, no GABA-labeled pyramidal cells were observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗

Histochemical assessment of cytochrome oxidase activity for monitoring ischemic muscle injury.

Electromyographic detection of increased neuromuscular conduction latency that follows electrostimulation of spinal nerves has been used widely as a clinical tool for detection of ischemic muscle injury. We hypothesized that a biochemical marker for stagnant hypoxia provides more direct evidence for evaluating severity and monitoring resolution of ischemic muscle injury than the EMG. To detect intracellular changes in oxygen use during experimental ischemia, ultrathin sections of rat gastrocnemius muscle were treated with reducing agent, 3,3'-diaminobenzidine (DAB), a histochemical marker for intramitochondrial cytochrome oxidase activity. The observed decrease in mitochondrial uptake of DAB suggested that a decrease in cytochrome oxidase activity was associated with experimental ischemia. Neuromuscular conduction latency in rat gastrocnemius muscle was also quantitated after electrostimulation of the sciatic nerve. Ligation of the femoral artery produced ischemic tissue injury, during which recordings of the EMG showed that conduction latency increased [from a mean +/- SEM control value of 3.09 +/- 0.13 to 3.92 +/- 0.22 ms (N = 11, P less than 0.001).] The changes in both histochemically detectable cytochrome oxidase activity and neuromuscular conduction latency were reversed by reperfusion. Response of the rat tissue to arterial occlusion was thereby shown to be a physiologic model for skeletal muscle response to ischemia. In addition, histochemical detection of cytochrome oxidase activity was shown to be a sensitive intracellular marker for decreased oxygen use during ischemic muscle injury.

Animals↗

Photoreactivation of the cytochrome oxidase complex with cyanide: the reaction of heme a3 photoreduction.

Electron transfer activity of isolated cytochrome oxidase inhibited by low concentrations of cyanide by 93-95% was shown to rise no less than three times under exposure to visible light. Irradiation with visible light was found to increase the rate of reduction of cytochrome oxidase heme groups in the presence of sodium dithionite. Based on these results, it is suggested that the modification of the catalytic and spectral characteristic of the cytochrome oxidase-cyanide complex is due to the photostimulation of the intramolecular electron transport at the interheme (heme a heme a3) transfer stage, i.e., is caused by photoreduction of the enzyme's heme a3-CN complex.

Animals↗

EPR study of heme x NO complexes of ascorbic acid-reduced Pseudomonas cytochrome oxidase and corresponding model complexes.

The EPR spectra of the NO complexes of frozen solutions of ascorbic acid-reduced cytochrome oxidase (nitrite reductase) purified from Pseudomonas aeruginosa, of its heme d1-depleted form, and of heme d1 in solutions containing various nitrogenous bases are quite similar to each other as well as to several heme (iron protoporphyrin IX)-containing proteins. The NO complexes of heme d1 (an iron-chlorin) in the presence of nitrogenous bases belong to spectral type C according to Kon's classification and, thus, the energy levels of the iron are closely related to thorse of heme complexes recorded under similar conditions. Comparison of these spectra with those of complexes of known structure suggests that both heme c and heme d1 are linked with Pseudomonas cytochrome oxidase by means of a nitrogenous ligand. The EPR spectrum of the NO complex of the native enzyme exhibits a lack of resolution of the high field (gy) resonance which can be characterized in terms of a spectral contribution from both the heme c and heme d1 moieties. The similarity between the EPR spectra of the NO complexes of horse heart cytochrome c and the heme d1-depleted Pseudomonas cytochrome oxidase before and after interaction with urea suggests structural similarities involving the heme irons. The changes caused by urea are likely to be a breaking or distortion of the bond between the iron and the protein-donated nitrogenous ligand and are similar to alterations seen with NO complexes of hemoglobin under a variety of conditions.

Ascorbic Acid↗