DEMONSTRATION OF CYANIDE-SENSITIVE, FORMALIN-RESISTANT OXIDASE-LIKE ACIVITY WITH THE N-PHENYL-P-PHENYLENEDIAMINE CYTOCHROME OXIDASE METHOD.
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Aniline Compounds↗
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Photoactivation of Chlorophyll Synthesis and Cytochrome Oxidase Activity in Anaerobically Germinated Seedlings of Echinochloa crusgalli var. Oryzicola.
When seeds of Echinochloa crusgalli var. oryzicola are germinated in dark anaerobic conditions (99.995% N(2)), the seedlings do not have detectable protochlorophyll(ide). Two hours after exposure to light aerobic conditions, they begin to synthesize chlorophyll. The lag in greening is shorter in seedlings exposed to light for 24 hours before exposure to air. Seedlings maintained in light anaerobic conditions exhibit no lag in greening upon transfer to an aerobic environment. Preillumination of anaerobically grown seedlings does not result in any chlorophyll accumulation. Phytochrome is probably the receptor for photoactivation of chlorophyll synthesis, since activation is achieved by red light alone, but not by far red light or red plus far red light. The cytochrome oxidase activity in anaerobically germinated seedlings is 30% of the normal level found in aerobically grown seedlings. Preillumination was also found to activate the ability of anaerobically germinated seedlings to increase their cytochrome oxidase activity upon exposure to air.
Journal Article↗
Kinetic evidence for the re-definition of electron transfer pathways from cytochrome c to O2 within cytochrome oxidase.
The reaction with O2 of equimolar mixtures of cytochrome c and cytochrome c oxidase in high and low ionic strength buffers has been examined by flow-flash spectrophotometry at room temperature. In low ionic strength media where cytochrome c and the oxidase are bound in an electrostatic, 1:1 complex some of the cytochrome c is oxidised at a faster rate than a metal centre of the oxidase. In contrast, when cytochrome c and cytochrome c oxidase are predominantly dissociated at high ionic strength cytochrome c oxidation occurs only slowly (t1/2 = 5 s) following the complete oxidation of the oxidase. These results demonstrate that maximal rates of electron transfer from cytochrome c to O2 occur when both substrates are present on the enzyme. The heterogeneous oxidation of cytochrome c observed in the complex implies more than one route for electron transfer within the enzyme. Possibilities for new electron transfer pathways from cytochrome c to O2 are proposed.
Animals↗
The relationship between CNS metabolism and cytoarchitecture: a review of 14C-deoxyglucose studies with correlation to cytochrome oxidase histochemistry.
Since the inception of the 14C-deoxyglucose method and its extension to in vivo imaging of regional cerebral glucose metabolism in humans by positron emission tomography, uncertainty has persisted concerning the type of work to which regional metabolism is coupled, as well as the distribution of this work within the neuron. 14C-deoxyglucose studies indicate that functionally-coupled neural metabolism is more apparent in axon terminals and perhaps dendrites than neuronal perikarya. Moreover, it appears that most of the metabolism in axon terminals is accounted for by Na+-K+-ATPase activity. Nevertheless, cytochrome oxidase histochemistry reveals the presence of intensely reactive mitochondria in soma-dendrite regions opposite presynaptic axon terminals, thereby indicating that continuous temporal and spatial summation of postsynaptic graded potentials is associated with increased metabolism. While the situation concerning the relative postsynaptic metabolic prices of EPSP's and IPSP's remains uncertain, the presence of elevated levels of cytochrome oxidase activity within certain classes of presynaptic terminals indicates that active excitation and inhibition is associated with increases in presynaptic metabolism. This observation has been confirmed in 14C-deoxyglucose studies. Nevertheless, studies of neonatal hippocampus indicate that, before metabolic activity shifts to dendritic and telodendritic regions of electrophysiological activity, metabolism is high in somal foci of biosynthesis.
Animals↗
Cytochrome oxidase activity in the enamel organ during amelogenesis in rat incisors.
Cytochrome oxidase (CO), one of the membrane-bound mitochondrial enzymes involved in oxidative phosphorylation, reflects the functional activity of mitochondria. Mitochondria in the enamel organ show drastic changes in localization during amelogenesis (Smith. INSERM, 1984;125:273-282). In understanding the functional aspects of the enamel organ, it is essential that one knows the exact CO activity in the respective mitochondria. The present study examines the CO activity of mitochondria in the enamel organ of rat incisors throughout the various stages of amelogenesis using light and transmission electron microscopy. CO activity was examined histochemically according to Seligman et al. (J. Cell. Biol., 1968;38:1-14) in decalcified sections of the upper and lower incisors of the rat. In the secretory stage, half of the mitochondria in the ameloblasts accumulated in the infranuclear region were reactive for CO. Both the population and CO activity of the infranuclear mitochondria of ameloblasts decreased significantly in the later stage where the enamel matrix secretion was almost complete. The CO-reactive mitochondria in the cells of the stratum intermedium (SI) gradually increased in number throughout the secretory stage. In the maturation stage, the ameloblasts contained intensively CO-reactive giant mitochondria in the proximal region and regular sized ones in the distal cytoplasm that were mostly devoid of detectable CO reactivity. The proportion of CO-reactive mitochondria in the supranuclear region and the population of mitochondria in the infranuclear regions of the smooth-ended ameloblasts were significantly higher as compared with the respective values in the ruffle-ended ameloblasts. In the late stages of enamel maturation, ameloblasts containing a large number of ferritin-filled pigment vesicles possessed numerous CO-reactive mitochondria between those vesicles in the supranuclear region, implicating an active role of the ameloblasts in iron transfer into the maturing enamel. The papillary layer cells possessed numerous intensively CO-reactive mitochondria throughout the maturation stage. A stage-related variation in the localization of CO-reactive mitochondria in the enamel organ of rat incisors was quantitatively demonstrated. It is conceivable that maturation stage ameloblasts form a functional unit with the papillary layer cells, and operate in energy-requiring events such as active ion transport to, and water and matrix protein removal from the maturating enamel. A sign of such functional integrity among the types of the enamel organ cells (ameloblasts, cells of SI, cells of stellate reticulum, and outer enamel epithelial cells) cannot be seen in the secretory stage. The secretory ameloblasts may function in matrix formation and calcium regulation in a less cooperative manner with the other cells of the enamel organ as compared to the maturation stage ameloblasts.
Amelogenesis↗
Synthesis and degradation of cytochrome oxidase subunit mRNAs in neurons: differential bigenomic regulation by neuronal activity.
Cytochrome oxidase (CO) plays a key role in oxidative capacity of neurons and serves as a sensitive indicator of neuronal activity. The mechanism(s) involved in the regulation of this bigenomic-encoded mitochondrial enzyme is still not clearly understood. Previous studies have shown changes in the level of its subunit mRNAs encoded by the nuclear or mitochondrial genome in response to changing neuronal activity. Our goals in the present study were to determine whether such changes were due to RNA synthesis rate or stability or both. The level of CO activity of neurons in primary cultures assayed histochemically was increased after depolarizing KCl treatment. The steady-state levels of CO subunit II (CO II; mitochondrial-encoded) and IV (CO IV; nuclear-encoded) mRNAs were up-regulated in response to 5 hr of 20 mM KCl treatment. By using gene-specific probes, the relative rates of synthesis of CO II and IV mRNA were elevated significantly after KCl treatment (P < 0.05). The degradation of CO II and IV mRNAs was monitored by (3)H-uridine pulse-chase labeling, which revealed half-lives of 84 min for CO II mRNA and 50 min for CO IV mRNA. Under KCl treatment, the half-life of CO IV was increased to 102 min, but there was no statistically significant change in the half-life of CO II mRNA. These results indicate that mitochondrial subunit CO II mRNA is regulated mainly at the transcriptional level, whereas the nuclear subunit CO IV mRNA is regulated at both the synthetic and the degradative levels. Both subunits, however, are tightly governed by neuronal activity.
Animals↗
Organization of the nucleus of the solitary tract in the hamster: acetylcholinesterase, NADH dehydrogenase, and cytochrome oxidase histochemistry.
The distribution of acetylcholinesterase (AChE), NADH dehydrogenase (NADHd), and cytochrome oxidase (CO) was determined in the nucleus of the solitary tract (NST) in the golden hamster. Histochemical staining was compared to cytoarchitectonic subdivisions of the NST (Whitehead: J. Comp. Neurol. 276:547-572, 1988) and to terminal fields of primary afferents of the nerves that innervate the tongue. These three histochemical methods resulted in differential staining patterns within the NST that were related to certain subdivisions. Transganglionic transport of horseradish peroxidase (HRP) was used to determine the central projections of the chorda tympani (CT), the lingual branch of the trigeminal (L-V), and the lingual-tonsilar branch of the glossopharyngeal nerves (L-IX). Alternate or the same brain sections were processed to reveal transported HRP, and NADHd or AChE levels. Increased staining of the neuropil with NADHd and AChE was coincident with the dense part of the afferent terminal fields of all three nerves in the NST and the laterally adjacent dorsomedial part of the spinal trigeminal nucleus. CO showed this pattern only for the most rostral part of the CT field. The densest AChE staining coincided with gustatory afferent terminal fields. The histochemical staining facilitated the interpretation of the organization of the NST. For example, at caudal levels of the gustatory NST, it is suggested that taste processing is localized predominantly in the medial part of the rostral central, and somatosensory processing in the rostral lateral subdivision. AChE or NADHd staining should facilitate studies of connections, topography, and neuroplastic changes of the gustatory NST.
Acetylcholinesterase↗
The mechanism of antibody inhibition of proton pumping by cytochrome oxidase vesicles.
Antibodies previously shown to inhibit vectorial proton translocation through cytochrome oxidase vesicles were converted to F(ab')2 and Fab'. Neither fragment inhibited proton pumping, although binding capacity was present. However, when a surrogate Fc was added to F(ab')2, inhibition of proton translocation was restored; indicating that the inhibition is due to steric hindrance. These results provide insight into mechanisms of energy transduction by oxidase.
Animals↗
Improved contrast in histochemical detection of cytochrome oxidase: metallic ions protocol.
The standard current technique for demonstration of cytochrome oxidase (CyOx) provides low-contrast diaminobenzidine (DAB) polymer. In order to enhance the contrast with divalent metalic ions, we have screened a number of buffers and found that Hepes, Mops and cacodylate neither precipitate these ions nor inactivate CyOx in a concentration of 0.1 M. Staining thus obtained shows a broad range of gradations between black and white. With fresh tissue the resulting image is superior to that obtained with the brown DAB product, even if a recommended blue filter or printing on very hard paper are used. The technique is as simple as the one which is currently standard. Fixed tissue, cut either in a cryostat or vibratome, can be stained well when floating but not when mounted on slides. The stained floating tissue can be used for electron microscopy, but has no advantage over the standard method.
Animals↗
Entorhinal lesions result in shrinkage of the outer molecular layer of rat dentate gyrus leading subsequently to an apparent increase of glutamate decarboxylase and cytochrome oxidase activities.
In intact dentate gyrus, glutamate decarboxylase immunoreactivity (GAD) and cytochrome oxidase activity (CyO) showed different distributions patterns. Entorhinal lesions caused increases of GAD and CyO in the outer molecular layer (OML) of the ipsilateral side. Submicroscopical localization of these enzymes did not change, except for CyO labeling more astrocytic mitochondria. The increase in numerical density of GAD puncta correlated quantitatively with shrinkage of OML, whereas in the whole molecular layer the number of GAD puncta remained unchanged. Hence, the localized increase of enzyme activities and lysosomes is apparently related to shrinkage of OML, but does not indicate plasticity of GABAergic neurons.
Afferent Pathways↗
Effect of vestibular nerve section on cytochrome oxidase activity in the vestibular ganglion cells of the squirrel monkey.
Cytochrome oxidase (CO) activity of the vestibular ganglion cells of the squirrel monkey was demonstrated histochemically under normal and experimental conditions. Under general anesthesia, right vestibular nerve section was performed on adult squirrel monkeys between the vestibular ganglion and brain stem. The left side was left intact and was used as a within-animal normal control. One squirrel monkey that did not undergo vestibular nerve section was also included in the normal group. Following a survival period of seven months, neurons in the vestibular ganglion of both sides were examined. In the normal control sides, a significant negative correlation between the size of the neuron and its optical density for CO stain was observed. Many neurons in the vestibular ganglion survived after vestibular nerve section, but their cell sizes and optical densities of CO stain decreased compared with those of the control side.
Animals↗
Effect of redox state on the folding free energy of a thermostable electron-transfer metalloprotein: the CuA domain of cytochrome oxidase from Thermus thermophilus.
The unfolding of the CuA domain of cytochrome oxidase from the thermophilic bacterium Thermus thermophilus, induced by guanidine hydrochloride (GuHCl)1 at different temperatures, has been monitored by CD as well by electronic absorption (with the oxidized protein) and by fluorescence (with the reduced protein). The same unfolding curves were obtained with the different methods, providing evidence for a two-state model for the unfolding equilibrium. This was also supported by the shape of the unfolding equilibrium curves and by the observed refolding of the unfolded, oxidized protein on dilution of the denaturant. The oxidized protein cannot be unfolded by GuHCl at room temperature, and it was found to be thermally very stable as well, since, even in the presence of 7 M GuHCl, it is not fully unfolded until above 80 degrees C. For the reduced protein at room temperature, the unfolding equilibrium curve yielded a folding free energy of -65 kJ/mol. The corresponding value for the oxidized protein (-85 kJ/mol) could be estimated indirectly from a thermodynamic cycle connecting the folded and unfolded forms in both oxidation states and the known reduction potentials of the metal site in the folded and unfolded states; the potential is increased on unfolding, consistent with the higher folding stability of the oxidized form. The difference in folding stability between the oxidized and reduced proteins (20 kJ/mol) is exceptionally high, and this is ascribed to the unique structure of the dinuclear CuA site. The unfolded, reduced protein was found to refold partially on oxidation with ferricyanide.
Copper↗
Identification of negative-strand complements to cytochrome oxidase subunit III RNA in Trypanosoma brucei.
A substantial amount of cytochrome oxidase subunit III (COIII) mRNA continues to be synthesized de novo in Trypanosoma brucei in the presence of actinomycin D, presumably by a DNA-independent transcription process. We describe the identification of negative-strand COIII RNA molecules, characterization of their termini, and the detection of RNA-dependent RNA polymerase activity. Three lines of evidence for the existence of negative-strand COIII RNA are presented: (i) hybridization with oligonucleotide probes with the same polarity as mRNA after preliminary enrichment for putative negative-strand RNA by affinity purification; (ii) cloning and sequencing of negative-strand complements for the unedited, edited, and partially edited COIII RNA; and (iii) exact correspondence of the terminal sequences of the putative negative-strand RNA molecules to the ends of COIII RNA. The presence of negative-strand complements of COIII RNA is consistent with the notion that a significant amount of mRNA in T. brucei is synthesized by an RNA-dependent RNA polymerase with negative-strand RNA as an intermediate template.
Animals↗
The respiration and cytochrome oxidase activity of rat aorta in experimental hypertension.
Oxygen consumption and cytochrome oxidase activity of aortas of rats with experimental hypertension were found to be higher than the corresponding values for aortas of normotensive animals. The higher metabolic activity of aortas of hypertensive animals appeared to be due both to an increase in the proportion of muscle cells to connective tissue fibers and to a higher activity of the intracellular portion of the tissue.
Animals↗
Development of cytochrome oxidase blobs in visual cortex of normal and visually deprived cats.
Cytochrome oxidase (CO) blobs are central to our understanding of the columnar organization and parallel pathways in primate and cat visual cortex. In primates, development of blobs and their relationship with other columnar features of the visual cortex begins pre-natally, before visual experience. In kittens, the supragranular layers differentiate post-natally, after eye opening, raising the possibility that visual experience may influence the development of blobs in cat V1. We have examined the development of blobs in unfolded and flattened sections through the visual cortex of normally reared, dark-reared, monocularly deprived and binocularly deprived kittens. Blobs were found in superficial layers of V1 of normally reared kittens as early as 2 weeks of age, although at this age the overall CO staining in V1 was lighter than in V2. By 6 weeks of age the blobs were adult-like. A patchy pattern of CO staining was also found in V2 of young kittens but not in adults. Visual experience was not necessary for expression of the blobs and monocularly deprived kittens had well developed blobs, indicating that strong Y cell drive is not necessary for the development of blobs in cat V1. CO blobs appear in kitten V1 very early in post-natal development and their expression is independent of visual experience, suggesting that they may be an intrinsic feature of V1 organization.
Age Factors↗
Cytochrome oxidase activity reveals parcellations of the pigeon's ectostriatum.
The endogenous cytochrome oxidase activity of the pigeon's ectostriatum, the primary telencephalic structure of the tectofugal visual pathway, was histochemically demonstrated and a heterogeneous distribution of the reaction product was observed. In cross-sections the medial, central and ventrolateral parts of the ectostriatum showed high levels of activity while the centroventral and dorsolateral ectostriatum remained weakly labelled. Only slight left-right and interindividual variations were found in the pattern of labelling. These data demonstrate for the first time anatomical subdivisions within the ectostriatal core and open the possibility of functional parcellations within this structure.
Animals↗
Cytochrome oxidase activity in the gastric mucosa of patients with iron deficiency.
Cytochrome oxidase activity was assessed histochemically in gastric biopsies taken from 22 iron-deficient patients. Activity was strong only in parietal cells, and the total amount of activity appeared to be proportional to the number of parietal cells present in the sections. No evidence was found of diminished activity within parietal cells.
Adult↗
Reconstitution of the apoenzyme of cytochrome oxidase from Pseudomonas aeruginosa with heme d1 and other heme groups.
Cytochrome oxidase (EC 1.9.3.2) from Pseudomonas aeruginosa contains heme d1 and heme c in an equimolar ratio. The heme d1 can be removed from the enzyme with acidified acetone leaving an apoenzyme that contains heme c but has no oxidase activity. Reconstitution of the apoenzyme in neutral 6 M urea with heme d1 yields a reconstituted product which, after removal of the urea, has 90 to 100% of the oxidase activity of the native enzyme, a 1:1 molar ratio of the heme groups, and is indistinguishable from the native on the basis of its absorption spectral properties and its EPR spectrum. The apoenzyme can also be reconstituted with heme a, deuteroheme, hematoheme, mesoheme, and protoheme but only the heme a yields a product with any oxidase activity. The properties of these reconstituted products are compared.
Apoenzymes↗