Regional alterations in neuronal activity in dystonic hamster brain determined by quantitative cytochrome oxidase histochemistry

The neural mechanisms underlying idiopathic dystonia are currently unknown. Genetic animal models, such as the dt(sz) hamster, a model of idiopathic paroxysmal dystonia, may be helpful to providing insights into the pathophysiology of this common movement disorder. Recent metabolic mapping studies in the hamster model, using 2-deoxyglucose autoradiography, demonstrated altered 2-deoxyglucose uptake in motor areas such as the striatum, ventral thalamic nuclei, red nucleus, and deep cerebellar nuclei, during dystonic attacks. Whereas the 2-deoxyglucose method is thought to reflect mainly acute alterations of synaptic activity, determination of cytochrome oxidase activity has been suggested as a method of choice to examine sustained baseline changes in neuronal activity. Therefore, in the present study quantitative cytochrome oxidase histochemistry was used to identify chronic regional alterations in the absence of dystonic attacks in mutant hamsters. For comparison with recent 2-deoxyglucose studies, cytochrome oxidase activity was also determined during a dystonic attack, which was induced by mild stress. Cytochrome oxidase was determined in 109 brain regions of dystonic hamsters and non-dystonic, age-matched control hamsters. In the absence of a dystonic attack, a tendency to decreased cytochrome oxidase activity was found in most brain regions, possibly due to retarded brain development in mutant hamsters. Significant decreases in cytochrome oxidase activity were found in motor areas and limbic structures, such as hippocampus, piriform cortex, fundus striatum, globus pallidus, substantia nigra pars reticulata, mediodorsal nucleus of the thalamus, ventral pallidum, and interpositus nucleus of the cerebellum. After induction of a dystonic attack, the trend of decreased cytochrome oxidase activity disappeared, except in globus pallidus and interpositus nucleus of the cerebellum. Although the significant alterations in cytochrome oxidase activity in the absence of a dystonic attack were moderate, the data are in line with previous findings in the mutant hamsters, indicating that dysfunctions of the basal ganglia and their output nuclei are involved in the dystonic condition. Altered neural activity in limbic structures, found in the absence of dystonic attacks in mutant hamsters, may contribute to the stress-susceptibility of the animals.     

Metabolic responses of the neonatal rabbit brain to hydrocephalus and shunting

The metabolic changes that occur in the neonatal brain as a result of hydrocephalus, and the response to ventriculoperitoneal shunting, vary with the maturational stage of the brain. In this study, local glucose utilization (LCMRglu) and oxidative metabolic capacity were estimated using 2-deoxyglucose autoradiography and cytochrome oxidase histochemistry, respectively. Hydrocephalus was induced in rabbit pups via intracisternal kaolin injections at 4-6 days of age. Shunting occurred at 19-26 days of age and the animals were sacrificed at ages ranging from 33 to 331 days. In normal animals there was a high glucose demand early in life which showed a decrease at about 60 days of age. In rabbits sacrificed prior to 60 days of age the controls showed the highest LCMRglu with significant decreases in both the hydrocephalic and shunted animals. After 60 days of age the shunted animals had higher LCMRglu than both the hydrocephalic and control subjects. Oxidative metabolic capacity peaked before 50 days of age in normal animals. At the youngest age, both the hydrocephalic and shunted animals showed higher cytochrome oxidase density rates than the control rabbits. In the older group, the hydrocephalic animals remained high while the shunted animals approximated the control densities. Neither the changes seen in the LCMRglu nor the oxidative metabolic capacity were correlated with changes in cell packing density or increased intracranial pressure. These data suggest that when the brain is compromised by hydrocephalus, there is an initial compensatory increase in oxidative metabolic capacity. The development of the glycolytic pathway appears to be retarded by hydrocephalus, but with shunting and the passage of time, the LCMRglu rebounds to levels above that of controls.     

Infectious disease complications of simultaneous pancreas kidney transplantation

Gram-positive thermophilic Bacillus species contain cytochrome caa3-type cytochrome c oxidase as their main terminal oxidase in the respiratory chain. To identify alternative oxidases, we isolated several mutants from B. stearothermophilus defective in the caa3-type oxidase activity [Sakamoto, J. et al (1996) FEMS Microbiol. Lett. 143, 151-158]. A novel oxidase was isolated from membrane preparations of one of the mutants, K17. The oxidase was composed of two subunits with molecular masses of 56 and 19 kDa, and contained protoheme IX, heme O, heme A, and Cu in a ratio of 1:0.7:0.2:3. CO difference spectra indicate that the high-spin heme is mainly heme O. These results suggest that the enzyme belongs to the heme-copper oxidase family and is a cytochrome b(o/a)3-type oxidase, whose high-spin heme is mainly heme O and partly heme A. The enzyme oxidized cytochrome c-551, which is a membrane-bound lipoprotein of thermophilic Bacillus. The turnover rate of the activity (Vmax = 190 s[-1]) and its affinity for cytochrome c-551 (Km = 0.15 microM) were much higher than those for yeast and equine heart cytochromes c. The oxidase activity was enhanced by the presence of salts and inhibited by sodium cyanide with a Ki value of 19 microM. The enzyme kinetics suggests that cytochrome c-551 is the natural substrate to this oxidase. Furthermore, the oxidase had similarity to cytochrome ba3-type oxidase from Thermus thermophilus in the subunit composition, partial amino acid sequence, and prosthetic groups, and therefore is suggested to belong to a unique subgroup of the heme-copper oxidase family together with the Thermus enzyme and archaeal oxidases such as Sulfolobus SoxABCD.     

Intra-BLA or Intra-NAc Infusions of the Dopamine D? Receptor Partial Agonist, BP 897, Block Intra-NAc Amphetamine Conditioned Activity.

Recent studies have shown that both systemic and intra-nucleus accumbens (NAc) or intra-amygdala administration of dopamine D? receptor ligands modulate reward-related learning. A previous study (H. Aujla, H. Sokoloff, & R. J. Beninger. 2002) showed that systemic administration of the partial dopamine D? receptor agonist BP 897 selectively blocked the expression, but not the acquisition, of amphetamine-conditioned activity. This suggested the hypothesis that intra-NAc or intra-basolateral amygdala (BLA) BP 897 would attenuate the expression, but not the acquisition, of amphetamine-conditioned activity. Rats were habituated to activity-monitoring chambers for 5 days, for 1 hr each day. Conditioning occurred on the next 3 days, followed by a single 1-hr test session. Intra-NAc or intra-BLA infusions of BP 897 during test, but not during conditioning, attenuated intra-NAc amphetamine conditioned activity. Results indicate that the ability of BP 897 to attenuate the expression of conditioned activity is mediated in part by the NAc and BLA. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Classical conditioning modifies cytochrome oxidase activity in the auditory system

The effects of excitatory classical conditioning on cytochrome oxidase activity in the central auditory system were investigated using quantitative histochemistry. Rats in the conditioned group were trained with consistent pairings of a compound conditional stimulus (a tone and a light) with a mild footshock, to elicit conditioned suppression of drinking. Rats in the pseudorandom group were exposed to pseudorandom presentations of the same tone, light and shock stimuli without consistent pairings. Untrained rats in a naive group did not receive presentations of the experimental stimuli. The findings demonstrated that auditory fear conditioning modifies the metabolic neuronal responses of the auditory system, supporting the hypothesis that sensory neurons are responsive to behavioural stimulus properties acquired by learning. There was a clear distinction between thalamocortical and lower divisions of the auditory system based on the differences in metabolic activity evoked by classical conditioning, which lead to an overt learned behavioural response versus pseudorandom stimulus presentations, which lead to behavioural habituation. Increases in cytochrome oxidase activity indicated that tone processing is enhanced during associative conditioning at upper auditory structures (medial geniculate nucleus and secondary auditory cortices). In contrast, metabolic activation of lower auditory structures (cochlear nuclei and inferior colliculus) in response to the pseudorandom presentation of the experimental stimuli suggest that these areas may be activated during habituation to tone stimuli. Together these findings show that mapping the metabolic activity of cytochrome oxidase with quantitative histochemistry can be successfully used to map regional long-lasting effects of learning on brain systems.     

Expression of the Escherichia coli bo-type ubiquinol oxidase with a chimeric subunit II having the CuA-cytochrome c domain from the thermophilic Bacillus caa3-type cytochrome c oxidase

The C-terminal periplasmic domain of subunit II of the Escherichia coli bo-type ubiquinol oxidase was replaced with the counterpart of the thermophilic Bacillus caa3-type cytochrome c oxidase containing the CuA-cytochrome c domain by means of gene engineering techniques. The chimeric terminal oxidase was expressed by a pBR322 derivative in a terminal oxidase deficient mutant of E. coli, although the amount of the chimeric enzyme was smaller than that of the Escherichia coli bo-type ubiquinol oxidase expressed by the original cytochrome bo-expressing plasmid. The chimeric enzyme showed much higher TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity than the wild-type cytochrome bo, but lower activity than the thermophilic Bacillus caa3-type cytochrome c oxidase. The chimeric subunit II was confirmed to bind to heme C. These results suggest that the CuA-cytochrome c domain grafted to this membrane anchor can facilitate electron transfer from reduced TMPD to low-spin protoheme b in subunit I.     

Regulation of cytochrome c oxidase by interaction of ATP at two binding sites, one on subunit VIa

Cytochrome c oxidase isolated from a wild-type yeast strain and a mutant in which the gene for subunit VIa had been disrupted were used to study the interaction of adenine nucleotides with the enzyme complex. At low ionic strength (25 mM potassium phosphate), in the absence of nucleotides, the cytochrome c oxidase activity of the mutant enzyme lacking subunit VIa was higher than that of the wild-type enzyme. Increasing concentrations of ATP, in the physiological range, enhanced the cytochrome c oxidase activity of the mutant much more than the activity of the wild-type strain, whereas ADP, in the same concentration range, had no significant effect on the activity of the cytochrome c oxidase of either strain. These results indicate an interaction of ATP with subunit VIa in the wild-type enzyme that prevents the stimulation of the activity observed in the mutant enzyme. The stimulation of the mutant enzyme implies the presence of a second ATP binding site on the enzyme. Quantitative titrations with the fluorescent adenine nucleotide analogues 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) and 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) confirmed the presence of two binding sites for adenine nucleotides per monomer of wild-type cytochrome c oxidase and one binding site per monomer of mutant enzyme. Covalent photolabeling of yeast cytochrome c oxidase with radioactive 2-azido-ATP further confirmed the presence of an ATP binding site on subunit VIa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome c oxidase from eucaryotes but not from procaryotes is allosterically inhibited by ATP

The activity of reconstituted cytochrome c oxidase from bovine heart but not from Rhodobacter sphaeroides is allosterically inhibited by intraliposomal ATP, which binds to subunit IV. The activity of cytochrome c oxidase of wild-type yeast and of a subunit VIa-deleted yeast mutant, measured with Tween 20-solubilized mitochondria in the presence of an ATP-regenerating system, was also allosterically inhibited by ATP, indicating the general validity of this mechanism of "respiratory control" in eucaryotic cytochrome c oxidases (Arnold and Kadenbach, Eur. J. Biochem. (1997) 249, 350-354). Deletion of subunit VIa changes the biphysic into monophysic kinetics of the yeast enzyme in the presence of ADP. A tenfold higher amount of horse heart cytochrome c, as compared to yeast cytochrome c, was required to relieve the ATP inhibition of the yeast enzyme.     

Histochemical characterization of NADPH-diaphorase activity in area 17 of diurnal and nocturnal primates and rodents

NADPH-diaphorase (NADPH-d) activity was studied comparatively in area 17 of four mammalian species, two primates and two rodents. Three brain hemispheres each from adult capuchin-monkeys, owl-monkeys, agoutis and guinea pigs were fixed with aldehyde fixatives by perfusion and 200 microns sections were submitted to NADPH-d histochemistry, using the indirect malic enzyme method. In all species studied the neuropil pattern of enzymes activity presented a clear layered appearance. In primates, histochemical staining was most intense in layer IVc, while in rodents the highest intensity of the neuropil reaction was in supragranular layers (II and III). Comparison of cell density in grey and white matter showed that the majority of NADPH-d-positive neurones were located in the white matter of primates but not of rodents. Since NADPH-d is a nitric oxide synthase the results are very important for comparative functional studies of neuromediators and their correlations with laminar and modular organization of area 17 of the mammalian brain.     

Lifelong severe verrucosis associated with human papillomavirus type 2: report of a case with a 38-year follow-up

Sulfite ion (HSO3-) is one of the products when elemental sulfur is oxidized by the hydrogen sulfide:ferric ion oxidoreductase of Thiobacillus ferrooxidans AP19-3. Under the conditions in which HSO3- is accumulated in the cells, the iron oxidase of this bacterium was strongly inhibited by HSO3-. Since cytochrome c oxidase is one of the most important components of the iron oxidase enzyme system in T. ferrooxidans, effects of HSO3- on cytochrome c oxidase activity were studied with the plasma membranes of HSO3(-)-resistant and -sensitive strains of T. ferrooxidans, OK1-50 and AP19-3. The enzyme activity of AP19-3 compared with OK1-50 was strongly inhibited by HSO3-. To investigate the inhibition mechanism of HSO3- in T. ferrooxidans, cytochrome c oxidases were purified from both strains to an electrophoretically homogeneous state. Cytochrome c oxidase activity of a purified OK1-50 enzyme was not inhibited by 5 mM HSO3-. In contrast, the same concentration of HSO3- inhibited the enzyme activity of AP19-3 50%, indicating that the cytochrome c oxidase of OK1-50 was more resistant to HSO3- than that of AP19-3. Cytochrome c oxidases purified from both strains were composed of three subunits. However, the molecular weight of the largest subunit differed between OK1-50 and AP19-3. Apparent molecular weights of the three subunits of cytochrome c oxidases were 53,000, 24,000, and 19,000 for strain AP19-3 and 55,000, 24,000, and 19,000 for strain OK1-50, respectively.     

Multiplex PCR/LDR for detection of K-ras mutations in primary colon tumors

The use of near-infrared spectroscopy to measure noninvasively changes in the redox state of cerebral cytochrome oxidase in vivo is controversial. We therefore tested these measurements using a multiwavelength detector in the neonatal pig brain. Exchange transfusion with perfluorocarbons revealed that the spectrum of cytochrome oxidase in the near-infrared was identical in the neonatal pig, the adult rat, and in the purified enzyme. Under normoxic conditions, the neonatal pig brain contained 15 micromol/L deoxyhemoglobin, 29 micromol/L oxyhemoglobin, and 1.2 micromol/L oxidized cytochrome oxidase. The mitochondrial inhibitor cyanide was used to determine whether redox changes in cytochrome oxidase could be detected in the presence of the larger cerebral hemoglobin concentration. Addition of cyanide induced full reduction of cytochrome oxidase in both blooded and bloodless animals. In the blooded animals, subsequent anoxia caused large changes in hemoglobin oxygenation and concentration but did not affect the cytochrome oxidase near-infrared signal. Simultaneous blood oxygenation level-dependent magnetic resonance imaging measurements showed a good correlation with near-infrared measurements of deoxyhemoglobin concentration. Possible interference in the near-infrared measurements from light scattering changes was discounted by simultaneous measurements of the optical pathlength using the cerebral water absorbance as a standard chromophore. We conclude that, under these conditions, near-infrared spectroscopy can accurately measure changes in the cerebral cytochrome oxidase redox state.     

Preferential localization of monoamine oxidase type A activity in neurons of the locus coeruleus and type B activity in neurons of the dorsal raphe nucleus of the rat: a detailed enzyme histochemical study

Using enzyme histochemistry for monoamine oxidase (MAO) activity, we have examined whether MAO type A or type B or both are localized in neurons of the locus coeruleus (LC) and dorsal raphe nucleus (DR) of the rat. After pretreatment with various concentrations of the MAO type A inhibitor clorgyline or the type B inhibitor deprenyl, non-fixed frozen sections of the brain were histochemically stained for MAO activity with tyramine as a common substrate for the two types. MAO activity of the stained neuron was determined by measuring optical density of the staining. Percentage inhibition of the control MAO activity was plotted against increasing concentrations of the inhibitors. MAO activity of LC neurons was inhibited by low concentrations of clorgyline with a monophasic dose-response curve but not with a biphasic curve. Higher concentrations of deprenyl were needed to inhibit of LC neurons. MAO activity of DR neurons was inhibited by low concentrations of deprenyl with a monophasic dose-response curve. Clorgyline inhibited the MAO activity of DR neurons at only higher concentrations. When the sections without inhibitor pretreatment were incubated with the type A preferential substrate serotonin, the MAO activity was strongly stained in LC neurons but very weakly in DR neurons. With the type B preferential substrate beta-phenylethylamine, the staining was intense in DR neurons while very faint in LC neurons. These findings suggest that (i) almost all the MAO activity in LC neurons is of type A, and (ii) the MAO activity in DR neurons is predominantly of type B.     

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

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

Cytochrome oxidase and NADPH-diaphorase on the afferent relay branch of the optokinetic reflex in the opossum

In the present study, histochemical techniques combined with more conventional anatomical methods were used to refine the identification of the nucleus of the optic tract and the nuclei of the accessory optic system in the opossum. The distribution of the enzyme cytochrome oxidase (CO) was examined in the cells and the neuropil of the opossum's mesodiencephalic region. Strong CO labeling was present in the nucleus of the optic tract (NOT)-dorsal terminal nucleus (DTN). Alternate sections, taken from animals that had received bilateral injections of horseradish peroxidase centered in the region of the inferior olive, were subjected to assays for CO and horseradish peroxidase. The region occupied by CO-labeled cells in the NOT-DTN superimposed with the one defined by retrogradely labeled cells. Cell counts along the NOT-DTN anteroposterior axis revealed that although the olivary and CO-positive cells were confined within similar boundaries, the latter are up to twofold more numerous than the former. As revealed by cytochrome oxidase histochemistry, the outlines of the NOT-DTN, the other pretectal nuclei and the nuclei belonging to the accessory optic system coincided with those revealed by the histochemistry for nicotinamide dinucleotide phosphate diaphorase (NADPH-d). After an intraocular injection of cholera toxin beta subunit and alternate sections processing for NADPH-d and CO, the distribution of labeled retinal terminal fields in the mesodiencephalic region was shown to be coincident with regions of high levels of histochemical labeling. These results are discussed in the light of previous anatomofunctional assessments of the pretectum and accessory optic system.     

Action of L-acetylcarnitine on different cerebral mitochondrial populations from cerebral cortex

The maximum rate (Vmax) of some mitochondrial enzymatic activities related to the energy transduction (citrate synthase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, cytochrome oxidase) and amino acid metabolism (glutamate dehydrogenase, glutamate-pyruvate-transaminase, glutamate-oxaloacetate-transaminase) was evaluated in non-synaptic (free) and intra-synaptic mitochondria from rat brain cerebral cortex. Three types of mitochondria were isolated from rats subjected to i.p. treatment with L-acetylcarnitine at two different doses (30 and 60 mg.kg-1, 28 days, 5 days/week). In control (vehicle-treated) animals, enzyme activities are differently expressed in non-synaptic mitochondria respect to intra-synaptic "light" and "heavy" ones. In fact, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, glutamate-pyruvate-transaminase and glutamate-oxaloacetate-transaminase are lower, while citrate synthase, cytochrome oxidase and glutamate dehydrogenase are higher in intra-synaptic mitochondria than in non-synaptic ones. This confirms that in various types of brain mitochondria a different metabolic machinery exists, due to their location in vivo. Treatment with L-acetylcarnitine decreased citrate synthase and glutamate dehydrogenase activities, while increased cytochrome oxidase and alpha-ketoglutarate dehydrogenase activities only in intra-synaptic mitochondria. Therefore in vivo administration of L-acetylcarnitine mainly affects some specific enzyme activities, suggesting a specific molecular trigger mode of action and only of the intra-synaptic mitochondria, suggesting a specific subcellular trigger site of action.     

Electron transfer kinetics during the reduction and turnover of the cytochrome caa3 complex from Bacillus subtilis

The cytochrome caa3 complex from Bacillus subtilis is a member of the cytochrome oxidase superfamily of respiratory enzyme complexes. The key difference in the cytochrome caa3 complex lies in the addition of a domain, homologous with mitochondrial cytochrome c, that is fused to the C-terminal end of its subunit II. Measurements of steady-state and transient reduction kinetics have been carried out on the cytochrome caa3 complex. Reduction of the cyanide-bound enzyme with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) supports a sequence of electron transfer in which cytochromec is reduced initially, and this is followed by rapid internal electron transfer from cytochrome c to CuA and from CuA to cytochrome a. Steady-state kinetics with exogenous cytochrome c as the substrate demonstrates the capability of the cytochrome caa3 complex to act as a cytochrome c oxidase. The cytochrome c from B. subtilis is the most efficient cytochrome c of those tested. Steady-state kinetics with ascorbate-TMPD as the reductant, in the absence of exogenous cytochrome c, reveals a biphasic pattern even though only a single, covalent cytochrome c interaction site is present. The two-phase kinetics are characterized by a low activity phase associated with a high apparent affinity for TMPD and a high activity phase with a low affinity for TMPD. This pattern is observed over a wide range of ionic strengths and enzyme concentrations, and with both purified and membrane extract forms of cytochrome caa3. It is proposed that the biphasic steady-state kinetics of this oxidase, and other members of the cytochrome oxidase superfamily, do not result directly from different interactions with cytochrome c but are due to a change in the redox kinetics within the centers of the conventional oxidase unit itself. Our results will be related to models that account for the biphasic steady-state kinetics exhibited by cytochrome oxidase.     

Dibucaine interacts differently with membrane and protein in cytochrome c oxidase systems

Dibucaine acts as a weak protonophore in cytochrome c oxidase proteoliposomes. At low concentrations in the presence of permeant anions, it stimulates turnover and collapses enzyme-generated pH gradients. At higher concentrations, dibucaine inhibits activity of cytochrome c oxidase in proteoliposomes and the isolated enzyme. It also induces a red shift in the resting spectrum, indicating a change at the binuclear centre. This spectroscopic effect is kinetically biphasic. Dibucaine inhibits steady-state oxidase activity, but not the rate of the red shift in the cytochrome a3 Soret band during turnover. It reacts faster with the partially reduced state than with resting enzyme. The inhibition is kinetically biphasic with a noncompetitive Ki approximately 0.5 mM. Excess dibucaine effects a maximal turnover decline of 80%. At low ionic strength only the total Vmax is affected; tight binding of cytochrome c and turnover at the "tight" site are unaffected. Dibucaine may bind to an anionic site in a hydrophobic pocket, modifying electron transfer from cytochrome a and CuA to cytochrome a3 - CuB and the oxidized spectrum of the latter centre. Stimulation of turnover in cytochrome c oxidase in proteoliposomes is due to a separate membrane-dependent proton translocation catalysed by dibucaine in the presence of permeant anions.     

L-phenylalanine effect on rat brain acetylcholinesterase and Na+,K(+)-ATPase

The effect of different L-phenylalanine (Phe) concentrations (0.1-12.1 mM), on acetylcholinesterase (AChE) and Na+,K(+)-ATPase activities of brain homogenate and pure enzymes, was investigated at 37 degrees C. AChE and Na+,K(+)-ATPase activities were determined according to Ellman G. L., Courtney D., Andres V. and Featherstone R. M. (1961), Biochem. Pharmacol. 7, 88-95 and Bowler K. and Tirri R. (1974), J. Neurochem. 23, 611-613) respectively, after preincubation with Phe. AChE activity in brain homogenate or in pure eel E.electricus enzyme showed a decrease, which reached up to 18% with concentrations of 0.9-12.1 mM. Brain homogenate Na+,K(+)-ATPase activity showed an increase 16-65% with 0.24-0.9 mM of Phe, while an activity increase of 60-65% appeared with 0.9-12.1 mM of Phe. Pure enzyme activity (from porcine cerebral cortex) was not affected by high Phe concentrations, while it was increased by low concentrations. The above results suggest: a) A direct effect of Phe on AChE, b) A direct effect of low Phe concentrations and an indirect effect of high ones on Na+,K(+)-ATPase.     

Subunit II of Bacillus subtilis cytochrome c oxidase is a lipoprotein

The sequence of the N-terminal end of the deduced ctaC gene product of Bacillus species has the features of a bacterial lipoprotein. CtaC is the subunit II of cytochrome caa3, which is a cytochrome c oxidase. Using Bacillus subtilis mutants blocked in lipoprotein synthesis, we show that CtaC is a lipoprotein and that synthesis of the membrane-bound protein and covalent binding of heme to the cytochrome c domain is not dependent on processing at the N-terminal part of the protein. Mutants blocked in prolipoprotein diacylglyceryl transferase (Lgt) or signal peptidase type II (Lsp) are, however, deficient in cytochrome caa3 enzyme activity. Removal of the signal peptide from the CtaC polypeptide, but not lipid modification, is seemingly required for formation of functional enzyme.