Properties of porcine white adipose tissue and liver mitochondrial subpopulations.

Properties of porcine white adipose tissue heavy and light mitochondrial subpopulations were investigated so as to identify any functional heterogeneity. Liver mitochondrial subpopulations were concurrently evaluated since their properties have been studied in some detail. Mitochondrial subpopulations were isolated by means of differential centrifugation and the relative purity estimated using marker enzymes. Due to the greater contamination of the light mitochondrial fractions, mtDNA content, determined by PCR analysis, was used as a basis to demonstrate any mitochondrial heterogeneity. Enzymatic activity, electron microscopy, lipid analysis and Western blotting were used to characterise the different populations. With the exception of liver cytochrome c oxidase, the enzymatic capacity of adipose and liver heavy mitochondria ranged between approximately two- and threefold higher than the corresponding light fraction. The cardiolipin content and mean mitochondrial diameters paralleled these differences, suggesting an increased mitochondrial mass rather than a functional difference. However, the cytochrome c oxidase activity of the liver heavy mitochondria was 4.75-fold higher relative to the light fraction. A strong correlation between cytochrome c oxidase activity and the subunit I content was evident. Adipose tissue mitochondrial subpopulations would seem to possess a comparable oxidative capacity per gram mitochondrial protein, while liver heavy mitochondria possess an increased oxidative capacity and mass.     

The distribution of synaptosomal oxidative enzymes after exposure to deoxycholate

Abstract— The distributions of NADH₂ dehydrogenase, NADH, cytochrome c reductase and cytochrome oxidase have been determined utilizing synaptosomal isolation techniques. Deoxycholate was used to determine compartmentation and/or ‘latency’ of these activities. NADPH, dehydrogenase proved to be a soluble and mitochondrial enzyme and the activity of this enzyme was not appreciably changed by deoxycholate treatment. NADH_g cytochrome c reductase proved to be a mitochondrial enzyme with considerable activity in microsomal fractions. Deoxycholate treatment increased activity in the synaptosomal fraction 8.3-fold. A bimodal activation pattern was observed with synaptosomal and mitochondrial NADH, cyrochrome c reductase upon exposure to increasing concentrations of deoxycholate, with enhancement of activity at 0.25 % (w/v) and 0.50 % (w/v) deoxycholate. The enzyme was stable at concentrations of deoxycholate less than 0.25% (w/v) but was irreversibly inactivated at concentrations higher than 0.25% (w/v). The mechanism of this activation pattern appeared to be a combination of enzyme release and inactivation. Similar results were not observed in liver mitochondria. Cytochrome oxidase, a known mitochondrial marker, exhibited a 17-fold increase in synaptosomal activity with deoxycholate treatment. The synaptosomal cytochrome oxidase activity after deoxycholate treatment approached the activity in the free mitochondrial fraction. The percentage of mitochondrial protein in synaptosomal fractions was estimated to be about 30 per cent from a comparison of the respective total (deoxycholate-treated) activities. On the basis of these data we suggest that the synaptosomal fraction possesses a relatively sizable energy-producing potential which may be of significance in vivo.     

Coenzyme Q-pool function in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria

We have investigated the role of the Coenzyme Q pool in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria. Antimycin A and myxothiazol inhibit glycerol-3-phosphate cytochromec oxidoreductase in a sigmoidal fashion, indicating that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III. The inhibition of ubiquinol cytochromec reductase is linear at low concentrations of both inhibitors, indicating that sigmoidicity of antimycin A and myxothiazol inhibition is not a direct property of antimycin A and myxothiazol binding. Glycerol-3-phosphate cytochromec oxidoreductase is strongly stimulated by added CoQ₃, indicating that endogenous CoQ is not saturating. Application of the pool equation for nonsaturating ubiquinone allows calculation of theK _m for endogenous CoQ of glycerol-3-phosphate dehydrogenase of 3.14mM. The results of this investigations reveal that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III in brown adipose tissue mitochondria; moreover, its concentration is far below saturation for maximal electron transfer activity in comparison with other branches of the respiratory chain connected with the CoQ pool. HPLC analysis revealed a lower amount of CoQ in brown adipose mitochondria (0.752 nmol/mg protein) in comparison with mitochondria from other tissues and the presence of both CoQ₉ and CoQ₁₀.     

Effect of disulfiram (DS) on mitochondria from rat hippocampus: Metabolic compartmentation of DS neurotoxicity

This experiment was designed to study the acute effects of disulfiram on mitochondrial enzymes in nonsynaptic and synaptic mitochondria from rat hippocampus. Cytochromec oxidase, monoamine oxidase-B, glycerolphosphate acyltransferase and betahydroxybutyrate dehydrogenase were studied. Differences in enzyme activity were seen in controls. Cytochromec oxidase activity was higher in synaptic mitochondria whereas glycerolphosphate acyltransferase activity was higher in nonsynaptic mitochondria. Mitochondria from disulfiram treated rats, particularly synaptic mitochondria, exhibited lower specific activities of cytochromec oxidase and monoamine oxidase-B. These alterations were not limited to either the inner or outer mitochondrial membrane. Transmission electron microscopy revealed that mitochondria from disulfiram treated rats were severely altered in isolated preparations as well as in those from whole tissue. This study shows that disulfiram exerts a differential effect on mitochondrial subpopulations.     

The localisation of succinic dehydrogenase in the muscles of Nereis virens and Homarus gammarus

Summary The localisation of succinic dehydrogenase and cytochrome oxidase in body muscles of Nereis virens and in tail muscles of Homarus gammarus was studied. Pig heart muscle was used for some comparisons.Electron microscopic studies on tissue sections generally showed well developed and independent mitochondria in Homarus gammarus. A lower degree of independence characterised the less developed mitochondria of Nereis virens.Sections were stained with nitro-BT. Light microscopic studies showed a distinct and selective staining of the mitochondria in sections of Homarus gammarus. In addition to the few mitochondria of Nereis virens strings within the cytoplasm became distinctly blue. Electron microscopic studies on Nereis virens showed a higher electron density along the membranes of the vesicular sarcotubular system in incubated than in non-incubated sections.The fractions obtained on centrifugation of the homogenised tissues were used for combined enzyme studies and electron microscopic investigation. Similarly prepared fractions from the two invertebrates showed a similar electron microscopic appearance. The supernatants obtained by centrifugation at 12,000 g for 10 minutes contained vesicles different from the majority of those in the mitochondrial fractions. These supernatants had rather considerable activities of succinate-cytochrome c reductase and of cytochrome c oxidase. The activity of succinate-cytochrome c reductase was most pronounced in the supernatants of Nereis virens and much greater than the cytochrome c oxidase activity in these fractions. The ratio between succinate-cytochrome c reductase activity and cytochrome c oxidase activity in the supernatants of Nereis virens was about three times that in the corresponding fractions of Homarus gammarus.Manometric studies were performed to get the effect of added succinate on the O₂ uptake of the supernatants obtained by centrifugation at 12,000 g for 10 minutes. A distinctly larger increase in oxygen consumption characterised the supernatants of Nereis virens.The results presented indicate the occurrence of an extra-mitochondrial succinic dehydrogenase in Nereis virens. This conclusion was related to the occurrence of alternative oxidative systems in the muscles of this invertebrate.The literature dealing with an extra-mitochondrial localisation of succinic dehydrogenase is briefly reviewed as well as the electron microscopic studies concerning transformations between the membrane structures of cells.     

Effect of Ageing and Ischemia on Enzymatic Activities Linked to Krebs’ Cycle,Electron Transfer Chain,Glutamate and Aminoacids Metabolism of Free and Intrasynaptic Mitochondria of Cerebral Cortex

The effect of ageing and the relationships between the catalytic properties of enzymes linked to Krebs’ cycle, electron transfer chain, glutamate and aminoacid metabolism of cerebral cortex, a functional area very sensitive to both age and ischemia, were studied on mitochondria of adult and aged rats, after complete ischemia of 15 minutes duration. The maximum rate (V _max) of the following enzyme activities: citrate synthase, malate dehydrogenase, succinate dehydrogenase for Krebs’ cycle; NADH-cytochrome c reductase as total (integrated activity of Complex I–III), rotenone sensitive (Complex I) and cytochrome oxidase (Complex IV) for electron transfer chain; glutamate dehydrogenase, glutamate–oxaloacetate- and glutamate–pyruvate transaminases for glutamate metabolism were assayed in non-synaptic, perikaryal mitochondria and in two populations of intra-synaptic mitochondria, i.e., the light and heavy mitochondrial fraction. The results indicate that in normal, steady-state cerebral cortex, the value of the same enzyme activity markedly differs according (a) to the different populations of mitochondria, i.e., non-synaptic or intra-synaptic light and heavy, (b) and respect to ageing. After 15 min of complete ischemia, the enzyme activities of mitochondria located near the nucleus (perikaryal mitochondria) and in synaptic structures (intra-synaptic mitochondria) of the cerebral tissue were substantially modified by ischemia. Non-synaptic mitochondria seem to be more affected by ischemia in adult and particularly in aged animals than the intra-synaptic light and heavy mitochondria. The observed modifications in enzyme activities reflect the metabolic state of the tissue at each specific experimental condition, as shown by comparative evaluation with respect to the content of energy-linked metabolites and substrates. The derangements in enzyme activities due to ischemia is greater in aged than in adult animals and especially the non-synaptic and the intra-synaptic light mitochondria seems to be more affected in aged animals. These data allow the hypothesis that the observed modifications of catalytic activities in non-synaptic and intra-synaptic mitochondrial enzyme systems linked to energy metabolism, amino acids and glutamate metabolism are primary responsible for the physiopathological responses of cerebral tissue to complete cerebral ischemia for 15 min duration during ageing.     

Cytochrome c oxidase activity in T/t 6 (balanced lethal) mutant mice

R-1 (1450g) and R-2 (25,000g) liver fractions from T/t ⁶ and B6CBAF1 hybrid mice were analyzed for their protein content, mitochondria concentrations, and activities of three respiratory-chain enzymes of the mitochondrial inner membrane: cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase, E.C. 1.9.3.1), -glycerophosphate dehydrogenase [l-glycerol-3-phosphate: (acceptor) oxidoreductase, E.C. 1.1.99.5], and succinate-cytochrome c reductase. Only cytochrome c oxidase activity, calculated as units per 1010 mitochondria, was significantly lower in both R-1 and R-2 fractions of T/t ⁶ mice. Cytochrome c oxidase activity varied greatly among T/t ⁶ mice, as did their liver mitochondria concentrations and body weights. Cytochrome c oxidase activity in the R-1 fraction of T/t ⁶ mice, calculated as units per 1010 mitochondria per gram of body weight, averaged about 40% lower than in B6CBAF1 mice. -Glycerophosphate dehydrogenase activity was often elevated in T/t ⁶ mice, particularly in the R-2 fraction. The T/t locus, a complex genetic locus on chromosome 17, may contain genes important to the function and biogenesis of mitochondria.This investigation was supported by institutional funds from the Jackson Laboratory and by an allocation from NIH Biomedical Research Support Grant (RR-05545). The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Mechanism of electron transport during thiosulfate oxidation in an obligately mixotrophic bacterium Thiomonas bhubaneswarensis strain S10 (DSM 18181T)

This study describes the thiosulfate-supported respiratory electron transport activity of Thiomonas bhubaneswarensis strain S10 (DSM 18181^T). Whole-genome sequence analysis revealed the presence of complete sox (sulfur oxidation) gene cluster (soxCDYZAXB) including the sulfur oxygenase reductase (SOR), sulfide quinone reductase (SQR), sulfide dehydrogenase (flavocytochrome c (fcc)), thiosulfate dehydrogenase (Tsd), sulfite dehydrogenase (SorAB), and intracellular sulfur oxidation protein (DsrE/DsrF). In addition, genes encoding respiratory electron transport chain components viz. complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (ubiquinone-cytochrome c reductase), and various types of terminal oxidases (cytochrome c and quinol oxidase) were identified in the genome. Using site-specific electron donors and inhibitors and by analyzing the cytochrome spectra, we identified the shortest thiosulfate-dependent electron transport chain in T. bhubaneswarensis DSM 18181^T. Our results showed that thiosulfate supports the electron transport activity in a bifurcated manner, donating electrons to quinol (bd) and cytochrome c (Caa ₃ ) oxidase; these two sites (quinol oxidase and cytochrome c oxidase) also showed differences in their phosphate esterification potential (oxidative phosphorylation efficiency (P/O)). Further, it was evidenced that the substrate-level phosphorylation is the major contributor to the total energy budget in this bacterium.

     

A new method for flat-embedding large native cryostat sections for targeting small preneoplastic lesions in comparative ultrastructural and ultracytochemical investigations

 Ultrastructural studies of rare and small cellular lesions in pathologically altered tissue are difficult to perform by applying conventional electron microscopic preparation. The search for lesions, often consisting of only a few cells in randomly obtained small specimen blocks, is time consuming and often without success. The methodological requirements for comparative enzyme cytochemical and morphological studies, i.e., preservation of both enzyme activity and ultrastructure, are divergent. By processing large native cryostat sections for electron microscopy, small preneoplastic focal lesions were successfully targeted in liver and kidney. Glucose-6-phosphatase, alkaline phosphatase, acid phosphatase, catalase, and cytochrome c oxidase activities were distinctly localized to endoplasmic reticulum, canalicular membrane, lysosomes, peroxisomes, and mitochondria, respectively, in the morphologically altered cells. Fixation of serial cryostat sections and enzyme reactions were both carried out through a semipermeable membrane except those for cytochrome c oxidase, which was demonstrated after fixation through the membrane by floating the section in incubation medium containing cytochrome c. Thereafter, the sections were flat embedded and polymerized between epoxy resin disks and aluminum dishes fitting exactly together. The objects of interest were identified in the light microscope, cut out, and reembedded in reversed gelatine capsules. By using this technique an ultrastructural preservation was achieved similar to that seen after immersion fixation. The enzyme activities were clearly localized without diffusion of the reaction product or unspecific deposits. The procedure permits precise targeting and complex studies of rare and small lesions, and opens new perspectives for the use of cryo-preserved tissue. Accepted: 10 March 1998     

Modification of respiratory-chain enzyme activities in brown adipose tissue mitochondria by idebenone (hydroxydecyl-ubiquinone)

Idebenone (IDE), a synthetic analog of coenzyme Q, strongly activates glycerol phosphate (GP) oxidation in brown adipose tissue mitochondria. GP oxidase, GP cytochrome c oxidoreductase and GP dehydrogenase activities were all significantly stimulated by 13 μM IDE. Substituted derivatives of IDE acetyl- and methoxyidebenone had similar activating effects. When succinate was used as substrate, no activation by IDE could be observed. The activation effect of IDE could be explained as release of the inhibition of glycerol phosphate dehydrogenase by endogenous free fatty acids. NADH oxidoreductase activity and oxidation of NADH-dependent substrates were inhibited by IDE. The extent of the inhibition and IDE concentration dependence varied when various substrates were tested, being highest for pyruvate and lowest for 2-oxoglutarate. This study thus showed that the effect of IDE on various mitochondrial enzymes is very different and thus its therapeutic use should take into account its specific effect on various mitochondrial dehydrogenases in relation to particular defects of mitochondrial respiratory chain.     

Multiphasic oxidation-reduction of cytochrome b in the succinate-cytochrome c reductase

The triphasic course previously reported for the reduction of cytochrome b in the succinate-cytochrome c reductase by either succinate or duroquinol has been shown to be dependent on the redox state of the enzyme preparation. Prior reduction with increasing concentrations of ascorbate leads to partial reduction of cytochrome c₁, and a gradual decrease in the magnitude of the oxidation phase of cytochrome b. At an ascorbate concentration sufficient to reduce cytochrome c₁ almost completely, the reduction of cytochrome b by either succinate or duroquinol becomes monophasic. Owing to the presence of a trace amount of cytochrome oxidase in the reductase preparation employed, the addition of cytochrome c makes electron flow from substrate to oxygen possible. Under such circumstances, the addition of a limited amount of either succinate or duroquinol leads to a multiphasic reduction and oxidation of cytochrome b. After the initial three phases as described previously, cytochrome b becomes oxidized before cytochrome c₁ when the limited amount of added substrate is being used up. However, at the end of the reaction when cytochrome c_a is being rapidly oxidized, cytochrome b becomes again reduced. The above observations support a cyclic scheme of electron flow in which the reduction of cytochrome b proceeds by two different routes and its oxidation controlled by the redox state of a component of the respiratory chain.     

Long-term alcohol consumption and brown adipose tissue in man

The purpose of the present work was to study whether long-term alcohol consumption in man affects the development of brown adipose tissue. The adipose tissue around the thoracic aorta and common carotid arteries was collected at medicolegal autopsies on adults with a positive record of heavy alcohol consumption. Adults without any evident history of alcohol consumption served as controls. Histochemical reactions of the oxidative mitochondrial enzymes, cytochrome oxidase and succinate dehydrogenase were studied in samples of this adipose tissue and the activities of the enzymes were measured biochemically. There was histological evidence of some multilocular adipose tissue around the thoracic aorta and common carotid tissue from the non-drinkers was mostly unilocular resembling white adipose tissue. Histochemical evidence of brown adipose tissue was found in all alcohol consumers, but also in some of the controls. Biochemical cytochrome oxidase (CYO) and succinate dehydrogenase measurements in isolated mitochondria showed activity in 70% of the cases of drinkers and in one of the eight controls. Activity of CYO was measurable in the mitochondria from two other controls. The protein content of the samples from the alcoholics was twice that of the controls. The results suggest that chronic alcohol intake may induce a change in the white adipose tissue around the thoracic aorta and common carotid arteries of human adults into brown fat.     

Bioflavonoid effects on the mitochondrial respiratory electron transport chain and cytochrome c redox state

Abstract

The polyphenolic structure common to flavonoids enables them to donate electrons and exert anti-oxidant activity. Since the mitochondrial electron transport chain consists of a series of redox inter-mediates, the effect of flavonoids in a complex mixture of polyphenols, as well as related pure flavonoids, was evaluated on the rat liver mitochondrial electron transport chain. A French maritime pine bark extract (PBE), a complex mixture of polyphenols and related pure flavonoids, was able to reduce cytochrome c reversibly, possibly by donation of electrons to the iron of the heme group; the donated electrons can be utilized by cytochrome c oxidase. Among single flavonoids tested, (-)-epicatechin gallate had the greatest ability to reduce cytochrome c. In addition, PBE competitively inhibited electron chain activity in both whole mitochondria and submitochondrial particles. A 3.5-fold increase in the apparent K_m value for succinate was calculated from reciprocal plots. Among the flavonoids tested, taxifolin and (-)-epicatechin gallate showed minor inhibitory effects, while (±)-catechin and (+)-epicatechin were ineffective. Activities of NADH-ubiquinone, succinate-ubiquinone, and ubiquinol-cytochrome c reductases were inhibited by low concentrations of PBE to a similar extent. However, inhibition of cytochrome c oxidase activity required 4-fold higher PBE concen-trations. These results suggest that flavonoids reduce cytochrome c and that PBE inhibits electron transport chain activity mainly through NADH-ubiquinone, succinate-ubiquinone, and ubiquinol-cytochrome c reductases.     

Behaviour of some mitochondrial enzymes in ripening tomato fruit

The activities of four mitochondrial enzymes were studied in four stages of ripening tomato fruit. The highest enzyme activity was recorded for malate dehydrogenase followed by cytochrome c oxidase. Succinate dehydrogenase and NADH oxidase levels were low and could only be determined in the green stage of the fruit. However, peaks of various enzyme activities coincided in identical mitochondrial fractions on the sucrose density gradient. Moreover, the levels of malate dehydrogenase and cytochrome c oxidase were constant during the ripening process while the other two enzymes, succinate dehydrogenase and NADH oxidase, declined. This might indicate that mitochondria retain some of their essential functions through the ripening process.     

Modified oscillation behavior and decreased D-3-hydroxybutyrate dehydrogenase activity in diabetic rat liver mitochondria

One month after induction of diabetes in adult white rats with streptozotocin or 4–10 months after its induction by pancreatectomy (in every case glycemia was over 3 g/liter), the following alterations were observed in liver mitochondria: (a) a decrease of amplitude and an increase of the damping factor of volume oscillations induced by potassium ions and valinomycin; (b) a 50% decrease of d-3-hydroxybutyrate dehydrogenase (HBD) activity in mitochondria disrupted by repeated freeze-thawing; (c) a similar decrease in the rate of d-3-hydroxybutyrate oxidation by intact mitochondria; (d) a significant increase of cytochrome oxidase activity and cytochrome aa₃ content. Measurement of succinate dehydrogenase and NADH dehydrogenase activity, the cytochrome b, c₁, and c content, and the P:O ratio for mitochondria oxidizing d-3-hydroxybutyrate did not reveal significant differences between control and diabetic rat mitochondria. In the streptozotocin-injected rats, the variation of HBD activity and the modification of the mitochondrial oscillation pattern were time-dependent phenomena, both effects reaching their maximal expression about 1 month after the onset of diabetes. The variation of HBD activity followed a biphasic course, since it rose to above the control level during the first 2 weeks of diabetes, then fell progressively to about half the control value after the third week. Treatment of diabetic rats with NPH insulin (5 IU twice daily, for 3 days, reinforced by the same dose 45 min before sacrifice) restored the mitochondrial oscillation pattern, HBD activity, and rate of d-3-hydroxybutyrate oxidation by intact mitochondria to their normal values.     

Long photophase is not a sufficient stimulus to reduce thermogenic capacity in winter-acclimatized short-tailed field voles (Microtus agrestis) during long-term cold acclimation

The thermogenic capacity of brown adipose tissue in winter- and summer-acclimatized short-tailed field voles (Microtus agrestis) was investigated by examining changes in mass of brown adipose tissue, the ratio of white adipose tissue to brown adipose tissue, the concentration of the uncoupling protein (thermogenin) in whole depots (μg) and in mitochondrial mass (μg·mg^-1) and the activity of cytochrome c oxidase in the depots (mmol·min^-1). The concentration of thermogenin in winter-acclimatized voles (n=8), per brown adipose tissue depot and per mitochondrial mass, was significantly higher than in summer-acclimatized voles (n=6). There was no significant difference in the level of cytochrome c oxidase activity between these two groups. Four groups of winter-acclimatized voles (n=6 in each group) were exposed to 5°C for 10, 20, 50 and 100 days in a 14L:10D photoperiod. Body mass, brown adipose tissue mass, white adipose tissue mass and basal metabolic rate were significantly positively related to the length of time cold exposed up to 100 days. There was a significant inverse relationship between the ratio of white to brown adipose tissue mass and the duration of cold exposure. There was no significant relationship between thermogenin concentration, either per depot or in mitochondrial mass of brown adipose tissue, with the length of time cold exposed. The level of cytochrome c oxidase activity increased significantly from control levels to a maximum after 10 days in the cold but decreased from 10 days onwards. In winter-acclimatized M. agrestis, a 14L:10D photoperiod is not a sufficient stimulus to reduce thermogenic capacity during cold acclimation. Indeed, some changes in the indirect parameters reflecting thermogenesis, notably the increase in basal metabolic rate and the decrease in the ratio of white to brown adipose tissue mass, indicated that despite the long photophase the thermogenic capacity was slightly further enhanced during the cold acclimation.     

Enzymatic Activity in Mitochondria from Orobanche

Mitochondria from Orobanche were analysed for the activities of aconitate hydratase, isocitrate dehydrogenase, succinate dehydro-genase, fumarate hydratase, malate dehydrogenase, NADH oxidase, substrate-cytochrome c oxidoreductases, glutamate dehydrogenase, aminotransferases, ATPase and “malic” enzyme. The specific activities of isocitrate dehydrogenase, NADH oxidase, substrate-cytochrome c oxidoreductases and glutamate dehydrogenase in the mitochondria) fraction from parasite tissue compared favourably with those reported for most of the mitochondria from growing and storage tissues. Succinate dehydrogenase, fumarate hydratase and aspartate aminotransferase were of intermediate activity, while aconitate hydratase and malate dehydrogenase had rather low activity, and “malic” enzyme had very low activity in comparison with other preparations. The relevance of these findings in relation to mitochondrial metabolism in the parasite is discussed. No evidence was obtained to suggest any basic abnormality in the biochemical properties of the mitochondria from Orobanche centua which may be correlated with its obligatorily parasitic existence.     

Effect of crosslinking cytochrome c oxidase

Bovine heart cytochrome c oxidase and rat liver mitochondria were crosslinked in the presence and absence of cytochrome c. Biimidate treatment of purified cytochrome oxidase, which results in the crosslinkage of all of the oxidase protomers except subunit I when ? 20% of the free amines are modified, inhibits ascorbate-N,N,N′,N′-tetramethyl-p-phenylene diamine oxidase activity. Intermolecular crosslinking of cytochrome oxidase molecules, which results in the formation of large enzyme aggregates displaying rotational correlation times ? 1 ms, does not affect oxidase activity. Crosslinking of mitochondria covalently binds the cytochrome bc₁ and aa₃ complexes to cytochrome c, and inhibits steady-state oxidase activity. Addition of cytochrome c to purified cytochrome oxidase or to cytochrome c-depleted mitoplasts increases this inhibition slightly. Cytochrome c oligomers act as competitive inhibitors of native cytochrome c; however, crosslinking of cytochrome c to cytochrome c-depleted mitoplasts or purified cytochrome oxidase results in a catalytically inactive complex. These experiments indicate that cytochrome c oxidase subunit interactions are required for activity, and that cytochrome c mobility may be essential for electron transport between cytochrome c reductase and oxidase.     

Functional activity and ultrastructure of mitochondria isolated from myocardial apoptotic tissue

Apoptosis in myocardial tissue slices was induced by extended incubation under anoxic conditions. Mitochondria were isolated from the studied tissue. A new method of isolation of mitochondria in special conditions by differential centrifugation at 1700, 10,000, and 17,000g resulted in three fractions of mitochondria. According to the data of electron microscopy the heavy mitochondrial fraction (1700g) consisted of mitochondrial clusters only, the middle mitochondrial fraction (10,000g) consisted of mitochondria with typical for isolated mitochondria ultrastructure, and the light fraction consisted of small mitochondria (2 or 3 cristae) of various preservation. The heavy fraction contained unusual structural elements that we detected earlier in apoptotic myocardial tissue—small electron-dense mitochondria incorporated in bigger mitochondria. The structure of small mitochondria from the light fraction corresponded to that of the small mitochondria from these unusual elements—mitochondrion in mitochondrion. The most important functions of isolated mitochondria are strongly inhibited when apoptosis is induced in our model. The detailed study of the activities of the two fractions of the apoptotic mitochondria showed that the system of malate oxidation is completely altered, the activity of cytochrome c as electron carrier is partly inhibited, while succinate oxidase activity is completely preserved (complexes II, III, and IV of the respiration chain). Succinate oxidase activity was accompanied by high permeability of the internal membrane for protons: the addition of uncoupler did not stimulate respiration. ATP synthesis in mitochondria was inhibited. We demonstrated that in our model of apoptosis cytochrome c remains in the intermembrane space, and, consequently, is not involved in the cascade of activation of effector caspases. The possible mechanisms of induction of apoptosis during anoxia are discussed.     

Copper deficiency in the developing rat brain: evidence for abnormal mitochondria.

Abstract— Copper deficiency was produced in developing rats by feeding a low copper diet to rats during gestation and lactation and providing the offspring the same diet. The progeny developed similar to those of an earlier model based on preconception depletion followed by marginal supplementation during gestation. Copper levels were greatly reduced in the brain, iron levels were slightly depressed, and no differences in zinc content were found. Electron microscopic examination of brain tissue revealed the presence of enlarge mitochondria from copper-deficient animals. Isolated mitochondria from copper-deficient rats showed a 30% reduction in the rate of both succinate and glutamate oxidation, and for glutamate, the respiratory control ratio (RCR) was decreased by 60%. Difference spectra displayed a four-fold reduction in cytochrome a+a₃ and slight increases in cytochrome b, c₁ and c. Enzyme analysis of isolated mitochondria revealed a five-fold decrease in cytochrome oxidase, slight increases in succinic dehydrogenase and fumarase, and small decreases in hexokinase and monoamine oxidase. No difference in peroxidation of brain lipids was evident. Determination of metabolites from fast frozen tissue suggested that the copper-deficient brain was in a more reduced state based on a doubling of both the lactate/pyruvate and α-glycerol-P/dihydroxyacetone-P ratios. Creatine-P, ATP, and ADP levels were not different.