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PubMed1986

Resonance Raman evidence for an exchangeable protein hydrogen associated with the heme a group of cytochrome oxidase.

When cytochrome-c oxidase is soaked in D2O, downshifts of the cytochrome a formyl C = O stretching mode are seen in the resonance Raman (RR) spectra (413.1 nm excitation) of both the resting and reduced forms. Other changes observed in the reduced protein RR spectra are consistent with involvement of the cytochrome a formyl group in the deuterium effect. The D2O-induced RR changes are fully developed during 3-5 days incubation, but are incomplete after 1 h. Extraction of the heme a chromophore in deuterated solvents eliminates these changes, implying that the exchangeable proton is on a protein group in the cytochrome a pocket which H-bonds to the heme formyl. The rate of the D2O exchange process is unaffected by enzyme turnover, thus reducing the likelihood that the cytochrome a formyl H-bond is directly involved in the redox-linked mechanism of proton pumping.

Animals↗
PubMed1989

Double labeling of cytochrome oxidase and gamma-aminobutyric acid in central nervous system neurons of adult cats.

The relationship between the levels of cytochrome oxidase and gamma-aminobutyric acid (GABA) was investigated within single neurons by double labeling the 2 markers in the same section. Double staining was equally effective when immunogold-silver staining of GABA was followed by indirect immunoperoxidase labeling of cytochrome oxidase, or when cytochrome oxidase histochemistry was followed by immunogold-silver staining of GABA. Neurons in the perigeniculate nucleus (PGN) and basket cell terminals in the cerebellum were GABA positive and rich in cytochrome oxidase. Interneurons of the lateral geniculate nucleus (LGN) as well as stellate and Golgi cells of the cerebellum were GABA-rich but poor in cytochrome oxidase. These results demonstrate that there is no consistent relationship between the levels of cytochrome oxidase and GABA in neurons.

Animals↗
PubMed1984

Influence of vesicle size and oxidase content on respiratory control in reconstituted cytochrome oxidase vesicles.

Previous work has shown that the respiratory control or coupling exhibited by reconstituted cytochrome oxidase systems can be markedly sensitive to the lipid composition, the lipid to protein ratio, and the vesicle size. In this work we have attempted to ascertain which, if any, of these factors plays a definitive role in determining the observed coupling. Vesicles prepared from dioleoylphosphatidylcholine-dioleoylphosphatidylethanolamine (1:4) were fractionated by DEAE chromatography to obtain a population homogeneous with respect to their lipid to protein ratio. This subpopulation was then applied to a Sepharose 4B-CL column to separate the vesicles by size. Fractions eluted from the column were assayed for enzyme activity in the presence and absence of carbonyl cyanide ( trifluoromethoxy )phenylhydrazone plus valinomycin. The coupling ratio was found to be dependent upon vesicle size; the smaller the vesicles, the higher the ratio. This suggests that lipid composition per se does not determine coupling characteristics. Reducing vesicle size for a given lipid to protein ratio has two effects. First, the radius of curvature is increased and, second, the average number of oxidase molecules per vesicle is reduced. In order to identify which of these factors was responsible for the observed tighter coupling, the oxidase was reconstituted with dioleoylphosphatidylcholine into vesicles of a defined size and the number of oxidase molecules per vesicle varied. The highest coupling ratios were observed for vesicles containing on average only one oxidase dimer. As the fraction of vesicles containing more than one protein was increased, the coupling ratio rapidly declined.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-Free System↗
PubMed1990

Development of synaptic transmission at autonomic synapses in vitro revealed by cytochrome oxidase histochemistry.

We have studied the development of synaptic transmission by innervating sympathetic neurons in vitro and monitoring synaptic activity with both physiological recording and cytochrome oxidase histochemistry. The onset of synaptic transmission was reflected in increased cytochrome oxidase reaction product within individual neurons. Within 24 hours of co-culture, relatively low frequency suprathreshold potentials were recorded in approximately 20% of the innervated neurons. At this stage the cytochrome oxidase activity of innervated neurons, as assessed by optical density of the histochemical reaction product, was increased twofold compared with uninnervated neurons. Over the next 2-4 days of innervation, changes in the pattern and extent of synaptic activity and superthreshold events were accompanied by a net fourfold increase in cytochrome oxidase activity levels compared with noninnervated neurons. The increase in density of cytochrome oxidase reaction product observed after innervation was reversed completely by blockade of synaptic transmission. Differences in the efficacy of synaptic input provided to the sympathetic neurons by appropriate versus inappropriate presynaptic sources was determined by co-culturing sympathetic neurons with explants that contained either preganglionic neurons or somatic motor neurons. Although sympathetic neurons innervated by motor neuron explants had increased levels of cytochrome oxidase activity compared with noninnervated controls, the density of cytochrome oxidase reaction product was even greater in sympathetic neurons innervated by preganglionic explants. We conclude that both the onset of innervation of sympathetic neurons as well as the subsequent maturation of synaptic function is directly reflected in graded increases in cytochrome oxidase reaction product.

Adrenergic Fibers↗
PubMed1991

Control of cytochrome oxidase activity. A transient spectroscopy study.

The kinetics of cytochrome oxidase reconstituted into small phospholipid vesicles (COV) has been followed by transient optical spectroscopy under steady-state and pre-steady-state conditions, in the presence and absence of ionophores. The effect of valinomycin on the activity of reconstituted cytochrome oxidase is shown to depend on the absolute concentration of the ionophore and on the number of turnovers elapsed by the enzyme; this novel observation, which escaped previous investigations, may account for important differences in results and therefore in interpretation of the mechanism of control of the enzyme activity as between Brunori et al. (Brunori, M., Sarti, P., Colosimo, A., Antonini, G., Malatesta, F., Jones, M.G., and Wilson, M.T. (1985) EMBO J. 4, 2365-2368), Gregory and Ferguson-Miller (Gregory, L., and Ferguson-Miller, S. (1989) Biochemistry 28, 2655-2662) and Capitanio et al. (Capitanio, N., De Nitto, E., Villani, G., Capitanio, G., and Papa, S. (1990) Biochemistry 29, 2939-2944). Quantitative analysis of the optical spectra acquired within 10 ms over a large wavelength and time range (500-650 nm and 5 ms to 60 s) under different experimental conditions, indicates that the electrical component of the transmembrane electrochemical gradient controls the rate of the internal electron transfer from cytochrome a-CuA to cytochrome a3-CuB as well as the cytochrome c to cytochrome a electron transfer. The slow down of cytochrome oxidase activity observed in the presence of valinomycin after several (greater than 10) turnovers is attributed to alkalinization of the vesicle interior, which affects the internal electron transfer rate. These two mechanisms of control act most likely independently. A "cubic scheme," which illustrates the effect of the electrochemical gradient on two states of cytochrome oxidase characterized by different redox and proton pumping activities is presented and discussed.

Animals↗
PubMed1986

Immunochemical evidence for an inactive form of cytochrome oxidase in mitochondrial membranes of ethanol-fed rats.

Previous studies have established that rats fed ethanol chronically exhibit a 50% decrease in hepatic mitochondrial cytochrome oxidase compared to pair-fed controls, based on both heme aa3 content and specific activity. To determine whether the 'missing' 50% of cytochrome oxidase is present in the membrane but catalytically inactive, or entirely absent, we used immunochemical techniques to determine the content of cytochrome oxidase protein in hepatic submitochondrial particles. Rabbit antiserum against purified rat liver cytochrome oxidase precipitated cytochrome oxidase from detergent-solubilized submitochondrial particles. Immunoinhibition titrations of a fixed amount of anti-oxidase serum with increasing amounts of submitochondrial particle protein showed that similar percentages of added oxidase activity were recovered in supernatants after immunoprecipitation with preparations from both alcoholic and control rats. Similarly, titrations of a fixed amount of submitochondrial particle protein with increasing amounts of antiserum showed comparable decreases in oxidase activity. Equivalent amounts of protein were obtained in immunoprecipitates from both preparations. Immunoprecipitates demonstrated comparable oxidase subunit profiles by electrophoresis, except that one additional band, migrating in the region of oxidase subunit IV, was present in samples from alcoholic rats. The data indicate that cytochrome oxidase immunologic reactivity is quantitatively similar in both types of membranes. The results suggest that the 'missing' cytochrome oxidase is actually present within the membranes of alcoholic animals in an inactive form, apparently devoid of heme aa3.

Animals↗
PubMed1985

Specific labeling and partial inactivation of cytochrome oxidase by fluorescein mercuric acetate.

Addition of 1 eq of fluorescein mercuric acetate (FMA) to beef heart cytochrome oxidase was found to inhibit the steady-state electron transfer activity by 50%, but further additions up to 10 eq had no additional effect on activity. The partial inhibition caused by FMA is thus similar to that observed with other mercury compounds (Mann, A. J., and Auer, H. E. (1980) J. Biol. Chem. 255, 454-458). The fluorescence of FMA was quenched by a factor of 10 upon binding to cytochrome oxidase, consistent with the involvement of a sulfhydryl group. However, addition of mercuric chloride to FMA-cytochrome oxidase resulted in an increase in fluorescence, suggesting that FMA was displaced from the high affinity binding site. Cytochrome c binding to FMA-cytochrome oxidase resulted in a 10% decrease in the fluorescence, possibly caused by Forster energy transfer from FMA to the cytochrome c heme. The binding site for FMA in cytochrome oxidase was investigated by carrying out sodium dodecyl sulfate gel electrophoresis under progressively milder dissociation conditions. When FMA-cytochrome oxidase was dissociated with 3% sodium dodecyl sulfate and 6 M urea, FMA was predominantly bound to subunit II following electrophoresis. However, when the dissociation was carried out at 4 degrees C in the absence of urea with progressively smaller amounts of lithium dodecyl sulfate, the labeling of subunit II decreased and that of subunit I increased. These experiments demonstrate that mercury compounds bind to a high affinity site on cytochrome oxidase, possibly located in subunit I, but then migrate to subunit II under the normal sodium dodecyl sulfate gel electrophoresis conditions. A definitive assignment of the high affinity binding site in the native enzyme cannot be made, however, because it is possible that mercury compounds can migrate from one sulfhydryl to another under even the mildest electrophoresis conditions.

Animals↗
PubMed1982

Protein-protein interactions of cytochrome oxidase in inner mitochondrial membranes. The effect of liposome fusion on protein rotational mobility.

Rotational diffusion of cytochrome oxidase in the inner membrane of rat liver mitochondria was measured by detecting the decay of absorption anisotropy after photolysis of the heme a3.CO complex by a vertically polarized laser flash. As in previous experiments with beef heart mitochondria (Kawato, S., Sigel, E., Carafoli, E., and Cherry, R. J. (1980) J. Biol. Chem. 255, 5508-5510), co-existence of rotating cytochrome oxidase (mean rotational relaxation time, phi, of 700 to 1400 microseconds) and immobilized cytochrome oxidase (phi greater than 20 ms) was observed in mitochondria and mitoplasts. The effect of lipid/protein ratio by weight (L/P) on the relative proportions of mobile and immobile cytochrome oxidase was investigated following the fusion of soybean phospholipid vesicles with mitoplasts. The fusion procedure yielded four separate fractions upon sucrose density gradient centrifugation with L/P as follows: 0.3 in Pellet, 0.7 in Band 3, 1.5 in Band 2, and 3.0 in Band 1. The percentage of rotationally mobile cytochrome oxidase (phi = 700 to 1000 microseconds) in each of the different bands was found to be 16% in Pellet, 25% in Band 3, 47% in Band 2, and 76% in Band 1 at 37 degrees C. The dependence of the amount of mobile cytochrome oxidase on L/P indicates that the fraction of aggregated protein progressively decreases with decreasing concentration of proteins in the membrane. Thus, the large immobile fraction of cytochrome oxidase in mitochondrial inner membranes can be explained by nonspecific protein aggregation which is a consequence of the low L/P. The decrease in the mobile fraction in Pellet compared with mitoplasts was shown to be due to the pH 6.5 incubation used for fusion.

Animals↗
PubMed1975

Embryonic development and mitochondrial function. 1. Effects of chloramphenicol infusion on the synthesis of cytochrome oxidase and DNA in rat embryos during late organogenesis.

Cytochrome oxidase, which is partially synthesized by the mitochondrion, was used as a measure for the development of mitochondrial function in rat embryos during the late stage of organogenesis. For this purpose the specific inhibitor of mitochondrial protein synthesis, chloramphenicol (CAP), served as a tool. Due to the rapid elimination rate of CAP from rats, a method for continuous infusion which would not cause immobilization to the animals was devised. 1. Pharmacokinetic studies proved that CAP reaches the embryo before placentation. Concentrations of CAP in the embryo are as high as they are in the maternal serum (about 20 mug/ml serum or g embryo) and thuse are sufficiently in supply for the inhibition of mitochondrial proteins synthesis, if 1000 mg/kg CAP are infused intravenously per 24 hrs. CAP is partially excluded from the embryonic compartment after the placental barrier has fully developed: whereas CAP concentration in the maternal serum remains at about 20 mug/ml, the concentration in the embryonic compartment drops to about 10 mug/g embryonic tissue during day 13 of gestation. 2. The average cytochrome oxidase activity per cell is very low (about 1 nmole O2/min X mug DNA-1) in embryonic tissue as it is in many other rapidly proliferating tissues. It is 15-60 times higher in slowly proliferating tissues, as, for example, the adult rat liver or brain (greater than 14 nmoles O2/min X mug DNA-1). 3. When the infusion technique is applied on day 12 of gestation, a sufficiently high concentration of CAP in embryonic tissue can be obtained to inhibit the synthesis of cytochrome oxidase. In constrast to tissues of an adult organism-as in the case of liver after partial hepatectomy- in embryonic tissues this limitation in the availablity of cytochrome oxidase appearently results in a critical reduction of energy production, which subsequently affects the DNA synthesis and embryonic growth. 4. The possible relevance and applicability of these experimental findings to man is discussed.

Animals↗
PubMed1989

Quantitative histochemical analysis of cytochrome oxidase in rat dorsal root ganglia and its co-localization with carbonic anhydrase.

A quantitative histochemical method was developed and standardized and then used to characterize the heterogeneity of cytochrome oxidase activity among primary afferent neuronal cell bodies in dorsal root ganglia of rat. In addition, the relationship between cytochrome oxidase and carbonic anhydrase activities in these neurons was determine. In tests of the procedure, the density of cytochrome oxidase reaction product evaluated repeatedly in individual neurons within sections of ganglia was found to increase linearly over incubation periods of up to 6 h. The heterogeneity in cytochrome oxidase activity in ganglia was not simply a reflection of the heterogeneity in ganglion cell sizes. On the whole, each class of ganglion cell exhibited the full range of staining densities encountered but intense staining was observed in many more large type A cells than small type B cells. The latter, together with their termination fields within the substantia gelatinosa of the spinal cord, were lightly stained. A significant positive correlation was found between neuronal size and staining density (r = 0.43). However, the large scatter in the plot of these two variables suggests that the expression of cytochrome oxidase in sensory neurons is governed to a considerable extent by properties of these neurons that are unrelated to their size. Analysis of cytochrome oxidase and carbonic anhydrase activities in the same ganglion cells revealed that all neurons with dense staining for the oxidase were anhydrase positive. Conversely, however, some intensely anhydrase-positive cells exhibited only light staining for cytochrome oxidase. The heterogeneity of cytochrome oxidase activity among neurons in dorsal root ganglia may be related to the steady state electrophysiological activity of distinct populations of sensory neurons which in turn may be related to the specific sensory modalities these populations transmit. The observation that some neurons with the greatest abundance of carbonic anhydrase do not contain high or even moderate levels of cytochrome oxidase suggests some degree of dissociation between the functional requirement for carbonic anhydrase in sensory neurons and the rate of energy expenditure in these cells.

Animals↗
PubMed1979

Effects of temperature on cytochrome oxidase activity in solubilized form and in lipid vesicle systems.

Isolated mammalian cytochrome oxidase gave an Arrhenius plot with a break (Tb) at about 20 degrees C when assayed in a medium containing Emasol. The activation energies above and below 20 degrees C were 9.3 (EH) and 18.9 kcal/mol (EL), respectively. Isolated cytochrome oxidase was also incorporated into vesicles of dipalmitoyl phosphatidylcholine (DPPC, phase transition temperature Tt = 40 degrees C), dimyristoyl phosphatidylcholine (DMPC, Tt = 23 degrees C) and dioleoyl phosphatidylcholine (DOPC, Tt = -22 degrees C). The DPPC system showed a nearly linear Arrhenius plot between 9 and 36 degrees C with E = 22.8 kcal/mol. When cytochrome oxidase was resolubilized from the DPPC vesicles and assayed in solution a biphasic plot was obtained again. Cytochrome oxidase-DOPC was more active than the solubilized enzyme and exhibited a biphasic Arrhenius plot with Tb = 23 degrees C. EH and EL were 6.6 and 15.8 kcal/mol, respectively. The plot for the oxidase-DMPC also showed a break (Tb = 26 degrees C) with EH = 6.6 and EL = 26.6 kcal/mol. These results indicate that the break in the Arrhenius plot reflects primarily a structural transition in the cytochrome oxidase molecule between the "hot" and "cold" conformations, as proposed previously. This transition, as well as the molecular state of cytochrome oxidase, is affected by the physical state of the membrane lipids as reflected by changes in the kinetic properties.

Animals↗
PubMed1992

Cytochrome oxidase in rat ocular tissues with special reference to copper.

The localization of cytochrome oxidase, one of the copper metalloenzymes, was determined histochemically and compared with that of copper. Copper and cytochrome oxidase coexisted in the corneal epithelium and endothelium, iris, ciliary body, lens epithelium, and retinal photoreceptor inner segment. In spite of the presence of copper, no cytochrome oxidase was demonstrated histochemically in the retinal pigment epithelium, choroid, sclera, or optic nerve. The coexistence of copper and cytochrome oxidase suggests that copper plays a role in this copper metalloenzyme, while the non-coincidence of localizations of copper and cytochrome oxidase may be attributed to histochemical problems or to some unknown function of copper.

Animals↗
PubMed1986

Functional heterogeneity in the myenteric plexus: demonstration using cytochrome oxidase as a verified cytochemical probe of the activity of individual enteric neurons.

The cytochemical technique for the demonstration of cytochrome oxidase has been used to locate the sites of chronically active neurons in the CNS. The current experiments were undertaken to validate the use of this technique for the identification of active neurons and ganglia in the myenteric plexus of the gut. The excitotoxin veratridine was used to cause repetitive firing of action potentials and to depolarize myenteric neurons over an 8-hour test period in vitro. The effects of veratridine were monitored electrophysiologically and correlated with the ability of the drug to affect the density of the cytochrome oxidase reaction product, as well as the proportion of reactive myenteric neurons. The action of veratridine on cytochrome oxidase activity was evaluated by means of video microdensitometry and computer-assisted morphometry. Effects of veratridine were considered to be specific if they were blocked by the inclusion of tetrodotoxin (TTX), which antagonizes the action of veratridine on voltage-dependent Na+ channels. Veratridine (1.0 microM) depolarized the membranes of myenteric type II neurons and greatly increased both the average density of the cytochrome oxidase reaction product in individual neuronal cell bodies and the proportion that were reactive. The concentration-effect relationship for veratridine was biphasic. As the concentration of veratridine was raised, neurons became sufficiently depolarized such that they ceased to fire action potentials. This absence of spiking was associated with a decline in the stimulation of cytochrome oxidase activity by veratridine. At still higher concentrations of veratridine the degree of neuronal depolarization continued to increase. This further depolarization was associated with a secondary increase in cytochrome oxidase activity. All effects of veratridine were blocked by TTX. It is concluded that the cytochemical demonstration of cytochrome oxidase reflects the recent metabolic history and thus the physiological activity of myenteric neurons. In control preparations, fixed without exposure to veratridine, considerable variation in cytochrome oxidase was observed between ganglia and between individual neurons within ganglia. This suggests that there are significant differences in the activity of individual ganglia and neurons of the myenteric plexus. These observations support the hypothesis that there is a functional heterogeneity that corresponds to an anatomical heterogeneity (previously demonstrated) of the myenteric plexus.

Animals↗
PubMed1995

Cloning and characterization of COX14, whose product is required for assembly of yeast cytochrome oxidase.

Nuclear respiration-deficient mutants of Saccharomyces cerevisiae previously assigned to complementation group G93 lack cytochromes a and a3 and detectable cytochrome oxidase activity. Other respiratory chain carriers and the ATPase complex are present at near wild-type levels, indicating that the mutations specifically affect cytochrome oxidase. Since synthesis of the mitochondrially derived subunits 1, 2, and 3 of cytochrome oxidase is normal, the defect cannot be related to transcription of the endogenous genes or processing and translation of the corresponding RNAs. The results of Western analysis of the cytochrome oxidase subunits encoded in nuclear DNA also argues against an effect of the mutations on expression of these constituents. The G93 mutants are complemented by a nuclear gene, designated COX14. The product of this gene is a low molecular mass protein of 7,960 Da. A gene fusion expressing a biotinylated form of Cox14p complements cox14 mutants, indicating partial functional equivalence. The biotinylated derivative has been helpful in localizing Cox14p to the mitochondrial membrane and demonstrating that it is not a hitherto unrecognized subunit of cytochrome oxidase, although it does appear to be associated with a high molecular weight complex. This evidence, combined with the assembly-arrested phenotype of cox14 mutants, indicates that Cox14p, like several other recently described mitochondrial constituents, provides an important function at some late stage of the cytochrome oxidase assembly pathway.

Amino Acid Sequence↗
PubMed1999

Hepatic cytochrome oxidase in rats with microsurgical cholestasis or portocaval shunt.

AIMS: portocaval shunt and extrahepatic cholestasis are experimental models of chronic hepatic insufficiency of different etiology and histological characteristics, and which probably also differ in the mechanism of impairment of oxidative metabolism. To test this hypothesis we measured hepatic cytochrome oxidase. METHODS: cytochrome oxidase was assayed with a histochemical technique in three groups of Wistar rats: A (n = 8) control; B (n = 8) microsurgical extrahepatic cholestasis; and C (n = 8) end-to-side portocaval shunt. RESULTS: cytochrome oxidase activity was lowest in group B, both in the left middle (p = 0.00019) and in the inferior caudate (p = 0.00014) hepatic lobes, and was highest in group C in both hepatic lobes, especially in the left middle lobe (p = 0.0029). CONCLUSION: the decrease in cytochrome oxidase activity in the liver of rats with extrahepatic cholestasis and the increase in animals subjected to portal flow deprivation demonstrate the different nature of the impairment in hepatic oxidative metabolism in these two pathological conditions.

Animals↗
PubMed1986

A gene in Paracoccus for subunit III of cytochrome oxidase.

The region of Paracoccus denitrificans chromosome where the genes coding for cytochrome oxidase (cytochrome aa3) subunits are located has been cloned. DNA sequencing revealed an open reading frame that codes for a protein homologous to the subunit III of the eukaryotic, mitochondrial enzyme. This subunit is absent from the isolated Paracoccus oxidase. It now seems that it is part of the native enzyme in the bacterial cytoplasmic membrane. This may explain the observed discrepancies in the function of the isolated enzyme.

Base Sequence↗