Linear correlation between acetoacetate/beta-hydroxybutyrate in arterial blood and oxidized flavoprotein/reduced pyridine nucleotide in freeze-trapped human liver tissue.

A comparison of two methods of measuring liver mitochondrial redox state demonstrated that a linear correlation exists between acetoacetate/beta-hydroxybutyrate ratio in arterial blood (arterial ketone body ratio; AKBR) and oxidized flavoprotein/reduced pyridine nucleotide in human liver tissue (FP/PN) as measured by tissue fluorescence spectroscopy, such that [FP/PN] = 0.64 + 0.49 x [AKBR] (r = 0.84, P less than 0.001). This result supports the validity of AKBR as a method of measuring the hepatic mitochondrial redox state of pyridine nucleotide using arterial blood.     

Cerebral energy state, mitochondrial function, and redox state measurements in transient ischemia.

Earlier results are reviewed suggesting that transient pronounced, incomplete cerebral ischemia could be more deleterious for the recovery of brain tissue energy state than a complete interruption of the blood flow. Measurements of respiratory function of brain mitochondria, isolated after 30 min of either complete or incomplete ischemia, demonstrated a similar inhibition of respiratory activity and maximal phosphorylation rates in both situations. This inhibition was totally normalized during recirculation after complete ischemia while a further deterioration was found after incomplete ischemia. The in vivo alterations of the cortical tissue distribution of redox states during transient, incomplete ischemia (15--60 min) were measured using a flying spot fluorometer, which gives a real-time and on-line display of the tissue distribution of NADH and oxidized flavoprotein. A reoxidation in both systems was demonstrated during the recirculation period and the distribution of redox states showed no further heterogeneity in the postischemic period as compared to the preischemic distribution. It is concluded that reoxygenation of the brain tissue is possible even after long periods of incomplete ischemia. The normal distribution of redox states during recirculation suggests that mechanisms other than an impaired or inhomogeneous oxygen delivery during the postischemic period are responsible for the failure in recovery of mitochondrial function and tissue energy state.     

The Respiratory Chain of Plant Mitochondria. II. Oxidative Phosphorylation in Skunk Cabbage Mitochondria

Mitochondria were prepared from the spadices of skunk cabbage (Symplocarpus foetidus) whose respiratory rate with succinate and malate showed 15% to 30% sensitivity to cyanide inhibition, and which showed respiratory control by added ADP. The observed respiratory control ratios ranged from 1.1 to 1.4. The change in pH of the mitochondrial suspension was recorded simultaneously with oxygen uptake: alkalinization of the medium, expected for phosphorylation of ADP, coincided with the period of acceleration in oxygen uptake caused by addition of an ADP aliquot. The ADP/O ratios obtained were 1.3 for succinate and 1.9 for malate. In the presence of 0.3 mm cyanide, the ADP/O ratio for succinate was zero, while that for malate was 0.7. These results are consistent with the existence of an alternate oxidase which interacts with the flavoprotein and pyridine nucleotide components of the respiratory chain and which, in the presence of cyanide, allows the first phosphorylation site to function with an efficiency of about 70%. In the absence of respiratory inhibitors, the efficiency of each phosphorylation site is also about 70%. This result implies that diversion of reducing equivalents through the alternate oxidase, thereby bypassing the 2 phosphorylation sites associated with the cytochrome components of these mitochondria, occurs to a negligible extent during the oxidative phosphorylation of ADP or State 3.Addition of ADP or uncoupler to skunk cabbage mitochondria respiring in the controlled state or State 4, results in reduction of cytochrome c and the oxidation of the cytochromes b, ubiquinone and pyridine nucleotide. A site of interaction of ADP with the respiratory chain between cytochromes b and cytochrome c is thereby identified by means of the crossover theorem. Flavoprotein measured by fluorescence is also oxidized upon addition of ADP or uncoupler, but flavoprotein measured by optical absorbance changes becomes more reduced under these conditions. Depletion of the mitochondria by pretreatment with ADP and uncoupler prevents reduction of most of the fluorescent flavoprotein by succinate. These results indicate that skunk cabbage mitochondria contain both high and low potential flavo-proteins characterized by different fluorescence/absorbance ratios similar to those demonstrated to be part of the respiratory chain in mitochondria from animal tissues.     

Oxidation-reduction ratio studies of mitochondria in freeze-trapped samples. NADH and flavoprotein fluorescence signals.

The recording of oxidation-reduction-related fluorescence signals of oxidized flavoprotein (Fp) and reduced pyridine nucleotide (PN) from isolated mitochondria at temperatures below -80 degrees C can be accompanished with a high degree of accuracy and a wide dynamic range. The specific low temperature enhancement of the fluorescence signals due to increased quantum yield and to multiple scattering affords increased accuracy and less interference due to screening pigments such as hemoglobin and myoglobin. Since the metabolic processes are arrested and the recording speed can be greatly diminished, the technique can operate with a much smaller concentration of mitochondria than is needed at room temperature, and the method is suitable for localized oxidation-reduction measurements. The Fp and PN signals originate from the mitochondrial matrix space in which they represent the major fluorochromes. Since Fp and PN are near oxidation-reduction equilibrium, the ratio of the two fluorescence intensities, suitably normalized, approximates the oxidation-reduction ratio of oxidized flavoprotein/reduced pyridine nucleotide. Thus, this technique affords a foundation for the resolution of oxidation-reduction states in two and three dimensions.     

Scanning fluorometer for the rapid assessment of pyridine nucleotide and flavoprotein fluorescence changes in tissues in vivo

This paper describes a scanning fluorometer which produces images in real time of the distribution of pyridine nucleotide or flavoprotein fluorescence at the surface of tissues in vivo. The basic difference between this device and others reported in the literature is that fluorescence changes at any selected point within the image can be quantified as they occur. We suggest that the apparatus has potential application in those areas of surgery where vascular replacement or repair is required and where it would be advantageous to have an immediate measure of the cellular response to a return of blood flow.     

Functional Imaging of Mitochondria in Saponin-permeabilized Mice Muscle Fibers

Confocal laser-scanning and digital fluorescence imaging microscopy were used to quantify the mitochondrial autofluorescence changes of NAD(P)H and flavoproteins in unfixed saponin-permeabilized myofibers from mice quadriceps muscle tissue. Addition of mitochondrial substrates, ADP, or cyanide led to redox state changes of the mitochondrial NAD system. These changes were detected by ratio imaging of the autofluorescence intensities of fluorescent flavoproteins and NAD(P)H, showing inverse fluorescence behavior. The flavoprotein signal was colocalized with the potentiometric mitochondria-specific dye dimethylaminostyryl pyridyl methyl iodide (DASPMI), or with MitoTracker™ Green FM, a constitutive marker for mitochondria. Within individual myofibers we detected topological mitochondrial subsets with distinct flavoprotein autofluorescence levels, equally responding to induced rate changes of the oxidative phosphorylation. The flavoprotein autofluorescence levels of these subsets differed by a factor of four. This heterogeneity was substantiated by flow-cytometric analysis of flavoprotein and DASPMI fluorescence changes of individual mitochondria isolated from mice skeletal muscle. Our data provide direct evidence that mitochondria in single myofibers are distinct subsets at the level of an intrinsic fluorescent marker of the mitochondrial NAD–redox system. Under the present experimental conditions these subsets show similar functional responses.     

吡啶核苷酸转氢酶的结构及功能

吡啶核苷酸转氢酶(pyridine nucleotide transhydrogenases)是能直接催化NADP(H)与NAD(H)之间氢负离子可逆转移的氧化还原酶, 主要调控分解代谢和合成代谢过程中NADH与NADPH之间的动态平衡. 膜结合吡啶核苷酸转氢酶(TH)是ATP依赖性跨膜蛋白, 由2个亚基构成, 每个亚基包含dⅠ、dⅡ和dⅢ三个结构域. 在TH结合可变催化机制中, 氢负离子的转移总是与质子转移相偶联. 可溶性吡啶核苷酸转氢酶(STH)是非能量依赖性的黄素蛋白, 以可溶性多聚体形式存在. 目前认为,很多因活性氧自由基异常增多而引起的线粒体疾病都与TH的活性有关, 包括糖尿病、癌症、神经退行性疾病及心血管疾病等. TH分子机制的研究将有助于揭示这些线粒体疾病的致病机理以及为其诊断和基因治疗提供分子依据. STH作用机理的研究及其在辅酶再生系统中的应用, 将会推动代谢工程和工业生物催化过程的进一步发展.     

High spatial resolution readout of 3-D metabolic organ structure: an automated, low-temperature redox ratio-scanning instrument

Intraorgan compartmentation of metabolic processes plays an important role in the understanding of the physiological function of the integrated organ under normal as well as under pathological conditions. We describe here a technique by which 3-D information on tissue redox state may be obtained by means of automated scanning of surface fluorescence. The instrument allows for serial scanning of frozen tissue. A typical scan of a tissue volume of 3 X 3 X 2 mm at a linear resolution of 50 micron and a spatial resolution of ca. 3 X 10(-7) ml includes 144,000 single-point measurements of pyridine nucleotide and flavoprotein fluorescence within the tissue block. The scanning process is fully computerized and programs have been developed which allow 2- or 3-D reconstruction of the data in terms of "redox ratio models," exemplified here by a 3-D model of a spreading depression wave in the cerebral cortex of a gerbil.     

The Respiratory Chain of Plant Mitochondria: XVII. Flavoprotein-Cytochrome b(562) Interaction in Antimycin-treated Skunk Cabbage Mitochondria

During the transition from the aerobic steady state with succinate as substrate to anaerobiosis, in suspensions of skunk cabbage (Symplocarpus foetidus) mitochondria treated with antimycin A, cytochrome b(562) becomes reoxidized to the extent of about 20%, synchronously with the reduction of cytochrome c(549). This reoxidation occurs in both the absence and presence of m-chlorobenzhydroxamic acid, a specific inhibitor for the alternate terminal oxidase of plant mitochondria. A flavoprotein component, amounting to 13% to 15% of the total nonfluorescent mitochondrial flavoprotein, undergoes reduction synchronously with the oxidation of cytochrome b(562) during the aerobic to anaerobic transition with succinate as substrate in the presence of both antimycin A and m-chlorobenzhydroxamic acid. This flavoprotein component remains reduced in the presence of cyanide. The half-time for reduction of the flavoprotein component and cytochrome c(549) and for oxidation of cytochrome b(562) during the aerobic to anaerobic transition with succinate as substrate in the presence of both antimycin A and m-chlorobenzhydroxamic acid is 2 seconds. The half-times for oxidation of cytochrome c(549) and the flavoprotein component are 2.1 and 170 milliseconds, respectively, during the anaerobic to aerobic transition induced by addition of 14 mum O(2) to the mitochondrial suspensions. The half-time for reduction of cytochrome b(562) under these conditions is 150 milliseconds, synchronous with the flavoprotein component. The synchrony of the flavoprotein oxidation and of the cytochrome b(562) reduction at a rate much slower than that of cytochrome c(549) oxidation implies that, in antimycin-treated plant mitochondria, the state of the cytochrome b(562)/antimycin complex is regulated by the redox state of this flavoprotein component, rather than by cytochrome c(549). It is tentatively suggested that these two components are not part of the main sequence of the respiratory chain, but may be part of a multienzyme complex active in the hydroxylation reactions required for ubiquinone biosynthesis in the inner mitochondrial membrane.     

Calcium-dependent activation of mitochondrial metabolism in mammalian cells

Endogenous fluorophores provide a simple, but elegant means to investigate the relationship between agonist-evoked Ca2+ signals and the activation of mitochondrial metabolism. In this article, we discuss the methods and strategies to measure cellular pyridine nucleotide and flavoprotein fluorescence alone or in combination with Ca2+-sensitive indicators. These methods were developed using primary cultured hepatocytes and neurons, which contain relatively high levels of endogenous fluorophores and robust metabolic responses. Nevertheless, these methods are amendable to a wide variety of primary cell types and cell lines that maintain active mitochondrial metabolism.     

Effects of glucagon on the redox states of cytochromes in mitochondria in situ in perfused rat liver

The effects of glucagon on the respiratory function of mitochondria in situ were investigated in isolated perfused rat liver. Glucagon at the concentrations higher than 20 pM and cyclic AMP (75 microM) stimulated hepatic respiration, and shifted the redox state of pyridine nucleotide (NADH/NAD) in mitochondria in situ to a more reduced state as judged by organ fluorometry and beta-hydroxybutyrate/acetoacetate ratio. The organ spectrophotometric study revealed that glucagon and cyclic AMP induced the reduction of redox states of cytochromes a(a3), b and c+c1. Atractyloside (4 micrograms/ml) abolished the effects of glucagon on these parameters and gluconeogenesis from lactate. These observations suggest that glucagon increases the availability of substrates for mitochondrial respiration, and this alteration in mitochondrial function is crucial in enhancing gluconeogenesis.     

Respiratory oscillations and heat evolution in synchronous cultures of Candida utilis

Synchronous cultures of the budding yeast Candida utilis prepared by continuous-flow size selection showed respiratory oscillations when the energy source was either glucose, acetate or glycerol. The period of the oscillations was about one-third of the cell cycle time (i.e. about 0.5 h). No fluctuations in heat evolution could be detected. In organisms growing with acetate or glycerol, the effects of cyanide, N,N'-dicyclohexylcarbodi-imide and carbonyl cyanide m-chlorophenylhydrazone (maximum inhibition of respiration at respiratory maxima, maximum uncoupling of energy conservation at respiratory minima) suggest that the control mechanism responsible for the oscillations is mitochondrial respiratory control in vivo. The effects of cyanide and N,N'-dicyclohexylcarbodi-imide on the respiration of cultures growing synchronously with glucose were different from those for cultures growing with the non-fermentable substrates; this suggests that the mitochondrial respiratory system interacts with the early reactions of glucose utilization.     

The Respiratory Chain of Plant Mitochondria: XII. Some Aspects of the Energy-linked Reverse Electron Transport from the Cytochromes c to the Cytochromes b in Mung Bean Mitochondria

The cytochromes c of mung bean (Phaseolus aureus) mitochondria become reduced when sulfide, a cytochrome oxidase inhibitor free from uncoupling side effects, is added to the aerobic mitochondrial suspension in the absence of added substrate. The cytochromes b remain largely oxidized. Subsequent addition of ATP results in partial oxidation of the cytochromes c and partial reduction of the cytochromes b due to ATP-driven reverse electron transport through the second site of energy conservation, or coupling site, of the respiratory chain. Cytochrome a is also oxidized under these conditions, but there is no concomitant reduction of the flavoprotein components, of ubiquinone, or of endogenous pyridine nucleotide. The reaction is abolished by oligomycin. The reducing equivalents transported from the cytochromes c and a in ATP-driven reverse electron transport are about 2-fold greater than those which appear in the cytochromes b. It is suggested that the equivalents not accounted for are present in a coupling site enzyme at the second site of energy conservation which interacts with the respiratory chain carriers by means of the dithiol-disulfide couple; this couple would not show absorbance changes with redox state over the wavelength range examined. With succinate present, reverse electron transport can be demonstrated at both coupling sites in both the aerobic steady state and in anaerobiosis. ATP-driven reverse electron transport in anaerobiosis maintains cytochrome a 30% oxidized while endogenous pyridine nucleotide is 50% reduced.     

Spectral properties of fluorescent flavoproteins of isolated rat liver mitochondria

The different flavoproteins contributing to flavin fluorescence of isolated rat liver mitochondria have distinct excitation and emission spectra. The NAD-linked flavin component was identified as alpha-lipoamide dehydrogenase, while the non-NAD-linked component was found to be electron transfer flavoprotein. The differences in excitation and emission properties of the mitochondrial flavoproteins permit selective recording of their redox state changes in isolated mitochondria.     

UNDAMPED OSCILLATIONS OF PYRIDINE NUCLEOTIDE AND OXYGEN TENSION IN CHEMOSTAT CULTURES OF KLEBSIELLA AEROGENES

Klebsiella aerogenes was grown in chemostat culture with the pH controlled to ±0.01 and temperature to ±0.1°C. The oxygen tension of the culture was regulated by changing the partial pressure of oxygen in the gas phase and recorded by means of an oxygen electrode. Reduced pyridine nucleotide was monitored continuously in the culture by means of direct fluorimetry. On applying an anaerobic shock to the culture, damped oscillations in pyridine nucleotide fluorescence were obtained. Further anaerobic shocks decreased the damping and eventually gave rise to undamped oscillations of a 2–3 min period which continued for several days. These oscillations were paralleled by oscillations of the same frequency in respiration rate. The amplitude of the oscillations in the respiration rate was equivalent to only 1% of the total steady-state respiration, whereas that of pyridine nucleotide oscillations was equivalent to 10% of the total aerobic/anaerobic fluorescence response. The oscillations ceased on interrupting the glucose feed but restarted on adding excess glucose to the culture. Addition of succinate also restarted the oscillations so that they appear not to be of glycolytic origin. The frequency of oscillations varied with growth rate and conditions. Oscillations of much lower frequency were obtained under limited-oxygen and anaerobic conditions than under fully aerobic conditions. Under glucose-limited conditions, fluctuations were found in adenosine triphosphate (ATP) content which were in phase with the pyridine nucleotide oscillations, but under nitrogen-limited growth conditions no such fluctuations in ATP were observed. The primary oscillating pathway could not be identified but the mechanism would appear to be quite different from that involved in oscillations observed in yeast cells. The synchronization of oscillations and observations of negative damping could be explained by a syntalysis effect.     

The mitochondrial adenosine triphosphatase of Acanthamoeba castellanii. Oscillatory accumulation of enzyme activity, enzyme protein and F1-inhibitor during the cell cycle.

1. The mitochondrial ATPase of Acanthamoeba castellanii accumulated discontinuously in synchronous cultures prepared by a minimally perturbing size-selection technique. 2. Enzyme activity per ml of culture doubled overall during one cell cycle time of 8 h, but oscillated to give seven maxima during this period. Similar oscillations were observed in the specific activities of ATPase and of the naturally occurring inhibitor protein. 3. These variations in enzyme activity reflected changes in amount of enzyme protein as assayed by an immunological technique. 4. Large variations in I50 values (micrograms of inhibitor/mg of protein necessary for 50% inhibition of inhibitor-sensitive activity) for inhibition of ATPase activity by seven different inhibitors of energy conservation were observed. Activity was more sensitive to inhibition by oligomycin, efrapeptin, citreoviridin and quercetin when values were highest. 5. The results are discussed in relation to the phased organization of biosynthesis and degradation of cellular components known to occur during the cell cycle of this organization.     

Evaluation of electron-transfer flavoprotein and alpha-lipoamide dehydrogenase redox states by two-channel fluorimetry and its application to the investigation of beta-oxidation

A new method permitting the simultaneous evaluation of the redox states of alpha-lipoamide dehydrogenase and electron-transfer flavoprotein in intact rat liver mitochondria by two-channel fluorimetry is described. It is shown that correction for the partial overlap of emission spectra can readily be introduced after a calibration procedure is performed. This method was applied to the investigation into regulation of palmitoylcarnitine oxidation. It was found that in the presence of rotenone, malonate and a redox buffer for the mitochondrial NAD-system, the beta-oxidation flux was sensitive to variations in redox state of respiratory chain electron carriers at low states of NAD reduction. Therefore, the concept of beta-oxidation control caused solely by the NAD redox state can no longer be sustained.     

Involvement of fibrinogen specific binding in erythrocyte aggregation

Respiratory oscillations in continuous yeast cultures can be accounted for by cyclic energization of mitochondria, dictated by the demands of a temperature-compensated ultradian clock with a period of 50 min. Inner mitochondrial membranes show both ultrastructural modifications and electrochemical potential changes. Electron transport components (NADH and cytochromes c and c oxidase) show redox state changes as the organisms cycle between their energized and de-energized phases. These regular cycles are transiently perturbed by uncouplers of energy conservation, with amplitudes more affected than period; that the characteristic period is restored after only one prolonged cycle, indicates that mitochondrial energy generation is not part of the clock mechanism itself, but is responding to energetic requirement.     

Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators

Current methods for determining ambient redox potential in cells are labor-intensive and generally require destruction of tissue. This precludes single cell or real time studies of changes in redox poise that result from metabolic processes or environmental influences. By substitution of surface-exposed residues on the Aequorea victoria green fluorescent protein (GFP) with cysteines in appropriate positions to form disulfide bonds, reduction-oxidation-sensitive GFPs (roGFPs) have been created. roGFPs have two fluorescence excitation maxima at about 400 and 490 nm and display rapid and reversible ratiometric changes in fluorescence in response to changes in ambient redox potential in vitro and in vivo. Crystal structure analyses of reduced and oxidized crystals of roGFP2 at 2.0- and 1.9-A resolution, respectively, reveal in the oxidized state a highly strained disulfide and localized main chain structural changes that presumably account for the state-dependent spectral changes. roGFP1 has been targeted to the mitochondria in HeLa cells. Fluorometric measurements on these cells using a fluorescence microscope or in cell suspension using a fluorometer reveal that the roGFP1 probe is in dynamic equilibrium with the mitochondrial redox status and responds to membrane-permeable reductants and oxidants. The roGFP1 probe reports that the matrix space in HeLa cell mitochondria is highly reducing, with a midpoint potential near -360 mV (assuming mitochondrial pH approximately 8.0 at 37 degrees C). In other work (C. T. Dooley, T. M. Dore, G. Hanson, W. C. Jackson, S. J. Remington, and R. Y. Tsien, submitted for publication), it is shown that the cytosol of HeLa cells is also unusually reducing but somewhat less so than the mitochondrial matrix.