Energy charge,phosphorylation potential and proton motive force in chloroplasts

Adenylate concentrations were measured in intact chloroplasts under a variety of conditions. Energy charge was significant in the dark and increased in the light, but remained far below values expected from observed phosphorylation potentials in broken chloroplasts, which were 80 000 M^?1 or more in the light. With nitrite as electron acceptor, phosphorylation potentials in intact chloroplasts were about 80 M^?1 in the dark and only 300 M^?1 in the light. Similar phosphorylation potentials were observed, when oxaloacetate, phosphoglycerate or bicarbonate were used as substrates. ΔG′_ATP was ?42 kJ/mol in darkened intact chloroplasts, ?46 kJ/mol in illuminated intact chloroplasts and ?60 kJ/mol in illuminated broken chloroplasts. Uncoupling by NH₄Cl, which stimulated electron transport to nitrite or oxaloacetate and decreased the proton gradient, failed to decrease the phosphorylation potential of intact chloroplasts. Also, it did not increase the quantum requirement of CO₂ reduction. It is concluded that the proton motive force as conventionally measured and phosphorylation potentials are far from equilibrium in intact chloroplasts. The insensitivity of CO₂ reduction and of the phosphorylation potential to a decrease in the proton motive force suggests that intact chloroplasts are over-energized even under low intensity illumination. However, such a conclusion is at variance with available data on the magnitude of the proton motive force.     

The relation between phosphate potential and growth rate of Streptococcus cremoris

During energy limited growth the phosphate potential of the adenine nucleotide pool of Streptococcus cremoris was independent of growth rate. The ratio of the phosphate potential and proton motive force under these conditions was 2.6 to 2.7. Under carbon-limiting conditions the phosphate potential increased with the growth rate which is the result of a decrease of the organic phosphate content of the cells at higher growth rates. The ATP/ADP ratio of energy- and carbon-limited cultures had the same value and it is proposed that this ratio is kept constant by a near equilibrium reaction in the glycolysis.     

Formation of fermentation products and extracellular protease during anaerobic growth of Bacillus licheniformis in chemostat and batch-culture

For a relaxed (rel-), protease producing (A-type) and a stringent (rel+), not-protease producing (B-type) variant of Bacillus licheniformis we determined fermentation patterns and products, growth parameters and alkaline protease-production (if any) in anaerobic, glucose-grown chemostats and batch-cultures. Glucose is dissimilated via glycolysis and oxidative pentose phosphate pathway simultaneously; the relative share of these two routes depends on growth phase (in batch) and specific growth rate (in chemostat). Predominant products are lactate, glycerol and acetaldehyde for A-type batches and acetaldehyde, ethanol, acetate and lactate for B-type batches. Both types show a considerable acetaldehyde production. In chemostat cultures, the fermentation products resemble those in batch-culture. From the anaerobic batches and chemostats, we conclude that the A-type (with low ATP-yield) will have a YATPmax of probably 12.9 g/mol and the B-type (with high ATP-yield) a YATPmax of about 10.1 g/mol. For batch-cultures, both types have about the same, high Yglucose (12 g/mol). So, the slow-growing A-type has a relatively high efficiency of anaerobic growth (i.e. an efficient use of ATP) and the fast-growing B-type a relatively low efficiency of anaerobic growth. In aerobic batch-cultures, we found 48, respectively 41% glucose-carbon conversion into mainly glycerol and pyruvate, respectively acetate as overflow metabolites in the A- and B-type. In both aerobic and anaerobic batch-cultures of the A-type, protease is produced predominantly in the logarithmic and early stationary phase, while a low but steady production is maintained in the stationary phase. Protease production occurs via de novo synthesis; up to 10% of the total protease in a culture is present in a cell-associated form. Although anaerobic protease production (expressed as protease per amount of biomass) is much higher than for aerobic conditions, specific rates of production are in the same range as for aerobic conditions while, most important, the substrate costs of anaerobic production are very much higher than for aerobic conditions.     

The ability of acidic pH,growth inhibitors,and glucose to increase the proton motive force and energy spilling of amino acid-fermenting <Emphasis Type="Italic">Clostridium sporogenes</Emphasis> MD1 cultures

Clostridium sporogenes MD1 grew rapidly with peptides and amino acids as an energy source at pH 6.7. However, the proton motive force (p) was only –25 mV, and protonophores did not inhibit growth. When extracellular pH was decreased with HCl, the chemical gradient of protons (ZpH) and the electrical membrane potential () increased. The p was –125 mV at pH 4.7, even though growth was not observed. At pH 6.7, glucose addition did not cause an increase in growth rate, but increased to –70 mV. Protein synthesis inhibitors also significantly increased . Non-growing, arginine-energized cells had a of –80 mV at pH 6.7 or pH 4.7, but was not detected if the F₁F₀ ATPase was inhibited. Arginine-energized cells initiated growth if other amino acids were added at pH 6.7, and and ATP declined. At pH 4.7, ATP production remained high. However, growth could not be initiated, and neither nor the intracellular ATP concentration declined. Based on these results, it appears that C. sporogenes MD1 does not need a large p to grow, and p appears to serve as a mechanism of ATP dissipation or energy spilling.Mandatory disclaimer: Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.     

Iron transport in Francisella in the absence of a recognizable TonB protein still requires energy generated by the proton motive force

The mechanism of iron transport in Francisella is still a puzzle since none of the sequenced Francisella strains appears to encode a TonB protein, the energy transducer of the proton motive force necessary to act on the bacterial outer membrane siderophore receptor to allow the internalization of iron. In this work we demonstrate using kinetic experiments of radioactive Fe³⁺ utilization, that iron uptake in Francisella novicida, although with no recognizable TonB protein, is indeed dependent on energy generated by the proton motive force. Moreover, mutants of a predicted outer membrane receptor still transport iron and are sensitive to the iron dependent antimicrobial compound streptonigrin. Our studies suggest that alternative pathways to internalize iron might exist in Francisella.     

Reconsideration of the efficiency of energy transduction in Paracoccus denitrificans during growth under a variety of culture conditions

1. Theoretical overall P/2e^- ratios were calculated for growth of Paracoccus denitrificans under a variety of culture conditions on the basis of a growth model and recently published schemes of electron transport and associated proton translocation. 2. Experimental overall P/2e^- ratios were calculated, using the specific rate of ATP synthesis determined in cultures grown under aerobic carbon source-limited conditions. 3. The experimental P/2e^- was equal to the theoretical P/2e^- for aerobic sulphate-limited growth with gluconate or succinate as carbon source, on the assumption that site 1 phosphorylation was completely absent. 4. The experimental and the theoretical P/2e^- were similar for growth on nitrate as terminal electron acceptor and gluconate, mannitol or succinate as carbon source, on the assumption that nitrate enters the cell via the electroneutral nitrate-nitrite antiport system. 5. The experimental and theoretical P/2e^- were similar for growth on nitrite as terminal electron acceptor and mannitol or succinate as carbon source. 6. The experimental P/2e^- was substantially lower than the theoretical P/2e^- for aerobic growth with nitrate as nitrogen source and gluconate or mannitol as carbon source. The amount of energy needed for assimilative reduction of nitrate to ammonia was calculated from this difference.Dedicated to Prof. H. G. Schlegel on the occasion of his 60th birthday     

Chronic environmental pollution alters adenylate levels in needles and fine roots of Scots pine

Contents of ATP, ADP, AMP, inorganic phosphate, and values of ATP/ADP ratio, adenylate energy charge (AEC), phosphorylation potential (PP) and adenylate kinase activity were analysed in needles and fine roots of Scots pine trees grown at the polluted and control (free of acute air pollution) site. Also chemical properties of the soil and mineral elements in needles from both sites were analysed. In comparison with the control, developing needles from the polluted site contained less ATP, the same amount of ADP and more AMP, and had lower values of ATP/ADP, AEC and PP. In one-year-old needles from the polluted site no change or a decrease in ATP was recorded, while ADP decreased, AMP increased, AEC did not change, and ATP/ADP ratio and PP were higher. In fine roots from the polluted site AMP level was higher, while ATP, ADP, ATP/ADP ratio, PP and AEC were lower than in the control.     

Corn phosphoglycolate phosphatase: Modulation of activity by pyridine nucleotides and adenylate energy charge

The activity of corn phosphoglycolate phosphatase (EC 3.1.3.18), a bundle sheath chloroplastic enzyme, is modulated, in vitro, both by NADP(H) and adenylate energy charge. The Vmax of the enzyme is increased by NADP (25%) and NADPH (16%) whatever the pH used, 7.0 or 7.9 respective pH of the stroma in the dark and in the light. At both pH, the adenylate energy charge alone has a positive effect with two peaks of activation, characteristics for this enzyme, at 0.2 and a maximum at 0.8 accentuated under nonsaturating concentration of phosphoglycolate. At low energy charge, NADP(H) increased the activation with an additive effect most particularly observed at pH 7.9 under saturating phosphoglycolate concentration; at high energy charge, NADP(H) had a positive or negative effect on the activation, depending on the pH value and the concentrations of substrate and NADP(H).The ferredoxin-thioredoxin system does not regulate the activity since i) DTT addition do not have any effect, ii) the light-reconstituted system containing ferredoxin, ferredoxin-thioredoxin reductase, thioredoxins and thylakoids is not effective either. However, light-dark experiments indicate that phosphophycolate phosphatase can be subjected to a fine tuning of its activity.All these data suggest that light cannot induce a modification of the protein but could exert a tight control of its activity by the intermediate of Mg²⁺ and substrate concentrations and the levels of metabolites such as NADP(H), ATP, ADP, AMP. So, the regulation of the activity shown, in vitro, by energy charge and NADP(H) might be of physiological significance.Abbreviations AEC adenylate energy charge - DTT dithiothreitol - FBPase fructose 1,6-bisphosphatase - Fd ferredoxin - FTR ferredoxin-thioredoxin reductase - NADP-MDH NADP-malate dehydrogenase - P glycolate-phosphoglycolate - P glycolate phosphatase-phosphoglycolate phosphatase - PSII photosystem II - PPDK pyruvate, Pi dikinase - Rubisco Ribulose 1,5-bisphosphate carboxylase/oxygenase     

The physiological state of the phytoplankton community of three types of lakes as estimated by its adenylate energy charge

Variations in physiological state and biomass of the phytoplankton community were examined in three different types of lakes, namely Lake Barato, Lake Akan, and Lake Shikaribetu. When the physiological state of the phytoplankton community was estimated by its adenylate energy charge (AEC), low biomass and low physiological state co-appeared gradually in the metalimnion and hypolimnion during stratification. The physiological state of the phytoplankton as estimated by its AEC value did not always correspond to its biomass, estimated by chlorophyll-a and ATP in these three lakes. A high physiological state of the community was usually observed in the euphotic zone, but the low AEC value observed in the euphotic zone of Lake Barato was not identified in the euphotic zones of the other lakes. Thus, the relationship between the value of AEC, and biomass of phytoplankton is a complex variable, which is further discussed in this paper.     

Changes in the adenylate energy charge and the induction of sporulation in Saccharomyces diastaticus

The induction of sporulation in yeast is generally accompanied by a sharp increase in energy metabolism which is evidenced by a rise of the adenylate energy charge by that time. The energy charge can be held at a low level by limitation of the phosphate supply in the growth medium. Ascus formation remains unaffected by this treatment. This suggests that the rise in ATP production normally encountered during early sporulation is not essential for the initiation of sporulation.     

A comparison between aerobic growth of Bacillus licheniformis in continuous culture and partial-recycling fermentor,with contributions to the discussion on maintenance energy demand

Energy costs of biomass synthesis are relatively higher at low than at high specific growth rates () because of an increased protein content of the cell and increased costs of protein synthesis as such at low values. A comparison of aerobic, glucose limited cultures of Bacillus licheniformis in a chemostat and in a partial-recycling fermentor indicated that pulse-wise nutrient addition increased the maintenance energy demand (m). In the chemostat experiments, we also found a striking deviation from linearity between substrate consumption and , with large implications for the maintenance coefficient. The deviation is mainly due to a large shift in metabolic carbon flows at specific growth rates between 50 and 100% of _max. At those growth rates, uncoupled growth occurs, presumably as a necessary condition for faster growth, since uncoupling results in a faster energysupply for biosynthetic purposes.The maintenance coefficient as determined by chemostat studies should be regarded as a compounds parameter, constituted of maintenance energy demands like ppGpp accumulation, variable costs of mRNA and protein accumulation, kinetic proofreading etc. and influenced by fermentor operation parameters like the substrate addition rate; moreover, both constancy of m and a linear relation between m and appear quite unlikely.     

Influence of NO3 - and NH4 + nutrition on fermentation,nitrate reductase activity and adenylate energy charge of roots of Carex pseudocyperus L. and Carex sylvatica Huds. exposed to anaerobic nutrient solutions

The adenylate energy charge, production of ethanol and lactate, and nitrate reductase activity were determined in order to study the influence of different nitrogen sources on the metabolic responses of roots of Carex pseudocyperus L. and Carex sylvatica HUDS. exposed to anaerobic nutrient solutions. Determination of adenylates was carried out by means of a modified HPLC technique. Total quantity of adenylates was higher in Carex pseudocyperus than in Carex sylvatica under all conditions. In contrast, the adenylate energy charge was only slightly different between the species and decreased more or less in relation to the applied nitrogen source under oxygen deficiency. The adenylate energy charge in roots of plants under nitrate nutrition showed a smaller decrease under anaerobic environmental conditions than plants grown with ammonium or nitrate/ammonium. Roots of nitrate-fed plants showed a lower ethanol and lactate production than ammonium/nitrate- and ammonium-fed plants. Ethanol production was higher in C. pseudocyperus, formation of lactate was lower compared to that in Carex sylvatica. The activity of enzymes involved in fermentation processes (ADH, LDH and PDC) was enhanced significantly after 24 hours of exposure to anaerobic nutrient solutions in roots of both species. The induction of these enzymes was only slightly influenced by different nitrogen supply. In vivo nitrate reductase activity increased almost 3-fold compared to the aerobic treatment in both species and overcompensated loss of NADH reoxidation capacity caused by decrease of ethanol and lactate development. Induction of in vitro nitrate reductase activity was enhanced 313% in C. pseudocyperus and 349% in C. sylvatica under anaerobic environmental conditions and nitrate supply. These results indicate that nitrate may serve as an alternative electron acceptor in anaerobic plant root metabolism and that the nitrate-supported energy charge may be due to an accelerated glycolytic flux resulting from a more effective NADH reoxidation capacity by nitrate reduction plus fermentation than by fermentation alone.Abbreviations ADH alcohol dehydrogenase - AEC adenylate energy charge - DMSO dimethyl sulfoxide - EDTA ethylen diamine tetraacetic acid - HPLC high performance liquid chromatography - LDH lactate dehydrogenase - NRA nitrate reductase activity - PCA perchloric acid - PDC pyruvate decarboxylase - PVP polyvinylpyrrolidone - PVPP polyvinylpolypyrrolidone - TCA trichloroacetic acid, Tris-tris(hydroxymethyl)aminomethane     

Influence of specific growth limitation and dilution rate on the phosphorylation efficiency and cytochrome content of mitochondria of Candida utilis NCYC 321

With Candida utilis cells that had been removed directly from a 61 chemostat culture, in steady state, well-coupled mitochondria generally could be isolated. This required a modified snail-gut enzyme procedure that allowed the total processing time to be decreased to 3 h, or less. Examination of these mitochondria in an oxygraph showed the presence of 3 sites of energy conservation when the cells were grown at various dilution rates between 0.1 and 0.45 h^-1 in environments that were, successively, glucose-, ammonia-, magnesium-, phosphate- and sulphate-limited. Potassium-limited cells also apparently possessed 3 sites of oxidative phosphorylation when growing at dilution rates greater than 0.2 h^-1, but only 2 sites when growing at lower dilution rates. Analysis of cytochrome spectra obtained with these intact mitochondria revealed large quantitative (but not qualitative) differences, depending on the environmental conditions under which the yeast had been cultured. In particular, comparison of the ratio of cytochrome b to cytochrome a showed a pattern of change with dilution rate in mitochondria from potassium-limited cells that was distinctly different from those evident in mitochondria from cells that had been limited in their growth by the availability of other nutrients.     

The development of the photosynthetic apparatus and energy transduction in malate-limited phototrophic cultures of Rhodobacter capsulatus

The question was studied whether limited availability of the carbon source controls the development of the photosynthetic apparatus in Rhodobacter capsulatus. The organisms were grown phototrophically in a chemostat limited by malate as the sole source of reducing equivalents and carbon. The incident light-energy flux, representing the only energy source, was kept constant. Steady state levels of protein and dry weight of cells as well as molar growth yield coefficients (Y) decreased with increasing dilution rate (D, representing the growth rate, ) up to about D=0.14 h^-1. At higher D-values biomass levels as well as Y stayed largely constant. The specific rate of malate consumption leading to biomass production increased linearly while the rate representative of processes other than conversion of carbon into biomass increased almost exponentially with . Specific bacteriochlorophyll (Bchl) contents of cells as well as the specific rate of Bchl synthesis were rather low at low D-values. They increased as D was increased. Light energy fluxes required to half-maximally saturate proton extrusion by whole cells decreased when D was increased up to 0.1 h^-1; at higher D-values, however, they reached constancy. Maximal rates of proton extrusion as well as of photophosphorylation calculated on a Bchl basis decreased when D was increased up to 0.14 h^-1 and reached constancy at higher D-values. The results suggest that the availability of the growth limiting substrate controls the formation of the photosynthetic apparatus and, consequently, its functional properties including the efficiency of light-energy transduction. A relationship is assumed between malate conversion into biomass, i.e. Y-values, and the efficiency of light-energy transduction.Abbreviations ALA 5-aminoleyulinic acid - Bchl bacteriochlorophyll - D dilution rate [h^-1] - R Rhodobacter - Y molar growth yield coefficient - growth rate [h^-1]     

ATP and NADPH as the driving force of carbon reduction in leaves in relation to thylakoid energization by light

Carbon assimilation of spinach (Spinacia oleracea L.) leaves was measured in the presence of 2000l· l^–1CO₂ and 2% O₂ in the gas phase to suppress photorespiratory reactions and to reduce stomatal diffusion resistance. Simultaneously, membrane parameters such as modulated chlorophyll fluorescence, oxidation of P₇₀₀ in the reaction centre of photosystem I, and apparent changes in absorbance at 535 nm were recorded. After light-regulated enzymes were activated at a high irradiance, illumination was changed. About 3 min later (to maintain the previous activation state of enzymes), leaves were shock-frozen and freeze-dried. Chloroplasts were isolated nonaqueously and analysed for ATP, ADP, inorganic phosphate, NADPH and NADP. Observations made under the chosen conditions differed in some important aspects from those commonly observed when leaves are illuminated in air. (i) Not only assimilation, but also the phosphorylation potential [ATP]/([ADP]·[Pi]) increased hyperbolically with irradiance towards saturation. In contrast, the ratio of NADPH to NADP did not change much as irradiances increased from low to high photon flux densities. When ATP, the phosphorylation potential and the assimilatory force, F_A (the product of phosphorylation potential and NADPH/NADP ratio), were plotted against assimilation, ATP increased relatively less than assimilation, whereas the phosphorylation potential increased somewhat more steeply than assimilation did. A linear relationship existed between assimilation and F_A at lower irradiances. The assimilatory force F_A increased more than assimilation did when irradiances were very high. Differences from previous observations, where F_A was under some conditions higher at low than at high rates of carbon assimilation, are explained by differences in flux resistances caused not only by stomatal diffusion resistance but also by differences in the activity of light-regulated enzymes, (ii) The relationship between P₇₀₀ oxidation and a fast absorption change with a maximum close to 520 nm on one hand and carbon assimilation on the other hand was largely linear under the specific conditions of the experiments. A similar linear relationship existed also between the quantum efficiency of electron flow through photosystem II and the quantum efficiency of photosystem I electron transport. (iii) Whereas the increase in non-photochemical fluorescence quenching, q_N, was similar to the increase in assimilation, the relationship between light scattering and assimilation was distinctly sigmoidal. Light scattering appeared to be a better indicator of control of photosystem II activity under excessive irradiation than q_N. (iv) The results are discussed in relation to the relative significance of chloroplast levels of ATP and NADPH and of the assimilatory force F_A in driving carbon assimilation. From the observations, the proton/electron (H⁺/e^–) ratio of linear electron transport is suggested to be 3 and the H⁺/ATP ratio to be 4 in leaves. An H⁺/e^– ratio of 3 implies the existence of an obligatory Q-cycle in leaves.Abbreviations F_A assimilatory force - F_o fluorescence after long dark adaptation - F_m maximum fluorescence level - F_s steady-state fluorescence - PGA 3-phosphoglycerate - PFD photon flux density - P₇₀₀ (P₇₀₀+) electron-donor pigment in the reaction center of PSI (its oxidized form) - Q_A primary quinone acceptor of PSII - q_P photochemical quenching - q_N non-photochemical quenching - _PSII relative quantum efficiency of energy conversation at the level of photosystem II - _PSI relative quantum efficiency of photosystem II This research was supported by the Sonderforschungsbereich 251 of the University of Würzburg and the Stiftung Volkswagenwerk. U.G. is a member of the Graduate College of the Julius-von-Sachs Institut für Biowissenschaften, University of Würzburg, being on leave from Tartu University, Tartu, Estonia. The authors are grateful to Prof. A. Laisk, Chair of Plant Physiology, Tartu University, for stimulating discussions.     

A reaction cycle for cytochrome c oxidase as an electron-transport-driven proton pump: The effect of electrochemical potential and slips

A detailed reaction cycle for cytochrome oxidase, an electron-transport-driven proton pump, has been presented earlier by our research group. The essential feature of the model is that both cytochrome a and Cu_A must be reduced in order to allow the transition from the electron and proton input state to the output state. The model is thus based on an indirect coupling between electron transfer and proton translocation.In this study, the same model is examined with respect to (1) intrinsic electron and proton leaks and (2) the effect of applying an electrochemical potential gradient on the pump incorporated in a membrane, both with respect to the electrical and chemical components.The model is successfully used to simulate various experimental results. Comparisons of experimental results with simulations based on the model support the existence of electron and proton leaks. The analysis of electron leaks suggests that electron gating is best achieved by varying the reorganization energy rather than by varying the reduction potentials.It is also suggested that both the electrical and chemical components of the electrochemical potential gradient are responsible for the regulation of the enzyme activity. Furthermore, an attempt is made to interpret the seemingly contradictory results obtained when measuring the pH dependence of the reduction potential of cytochrome a. In addition, the simulations support the assumption that protons are pumped by a mechanism that combines a membrane Bohr effect with the transition-state mechanism.Abbreviations R molar gas constant - k _B Boltzmann contant - F Faraday constant - e elementary charge - T absolute temperature - transmembrane electrochemical potential gradient - pH transmembrane pH difference - pH₁ and pH₂ inside (matrix) and outside (cytosol) pH, respectively - transmembrane electrical potential - E _m midpoint potential     

Studies on 9-amino-6-chloro-2-methoxy acridine fluorescence quenching and enhancement in relation to energy transduction in spheroplasts and intact cells of the cyanobacterium Plectonema boryanum

The fluorescent probe 9-amino-6-chloro-2-methoxy acridine was used to study the energy transduction in the thylakoid and cell membranes of the cyanobacterium Plectonema boryanum. Apart from light-driven electron transfer, the dark endogenous respiration also leads to energization resulting in an ACMA fluorescence response, that is sensitive to the electron flow inhibitor 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, to the energy transfer inhibitors dicyclohexylcarbodiimide and venturicidine and to the uncoupler 5-chloro-3-t-butyl-2-chloro-4-nitrosalicylanilide.In spheroplasts, in which the cell membranes have lost their capacity to maintain a proton gradient, the respiration-and light-induced ACMA fluorescence changes (quenching) are similar to those in chloroplasts. In intact cells a combination of reversible quenching and enhancement of ACMA fluorescence was found. This dualistic behaviour is supposedly caused by an opposite orientation of the thylakoid and cell membranes. ACMA quenching at the level of the thylakoids was obtained either by respiratory or photosynthetic electron transfer and gave similar responses to those obtained in the spheroplasts. The slower ACMA fluorescence enhancement, only observed in cells with intact cell membranes, also evoked by both respiration and light-induced energization is sensitive to the compounds mentioned above and in addition to KCN.Our results support the view [8] that dark oxidation of substrates by O₂ proceeds via the thylakoid membrane and terminates at a CN^- sensitive oxidase located in the cell membrane which requires the involvement of a mobile cytoplasmic redox mediator.Abbreviations ACMA 9-amino-6-chloro-2-methoxy acridine - chl a chlorophyll a - DBMIB 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCCD dicyclohexylcarbodiimide - DNP dinitrophenol - DNP-INT dinitrophenyl ether of 2-iodo-4-nitrothymol - FCCP carbonylcyanide-p-trifluoro-methoxy phenylhydrazone - S-13 5-chloro-3-t-butyl-2-chloro-4-nitrosalicylanilide - tricine N-2 (2-Hydroxy-1, 1-bis (hydroxymethyl) ethyl)-glycine - Tris Tris (hydroxymethyl) amino methane     

Phosphate is required to maintain the outer membrane integrity and membrane potential of respiring yeast mitochondria.

The buffer requirements to maintain mitochondrial intactness and membrane potential in in vitro studies were investigated, using gradient purified yeast mitochondria. It was found that the presence of phosphate is crucial for generation of a stable membrane potential and for preserving the intactness of the outer membrane, as assessed by probing the accessibility of Tom40p to trypsin and the leakage of cytochrome b2 from the intermembrane space. Upon addition of respiratory substrate in the absence of phosphate, mitochondria generate a membrane potential that collapses within 1 min. Under the same conditions, the mitochondrial outer membrane is disrupted. The presence of phosphate prevents both phenomena. The DeltapH component of the proton motive force appears to be responsible for the compromised outer membrane integrity. The collapse of the membrane potential is reversible to a limited extent. Only when phosphate is added soon enough after the addition of exogenous respiratory substrate can a stable membrane potential be obtained again. Within a few minutes, this capacity is lost. The presence of Mg(2+) prevents rupture of the outer membrane, but does not prevent rapid dissipation of the membrane potential. Similar results were obtained for mitochondria isolated and stored in the presence of dextran or bovine serum albumin.     

Regulation of oxidative phosphorylation,the mitochondrial membrane potential,and their role in human disease

Thirty years after Peter Mitchell was awarded the Nobel Prize for the chemiosmotic hypothesis, which links the mitochondrial membrane potential generated by the proton pumps of the electron transport chain to ATP production by ATP synthase, the molecular players involved once again attract attention. This is so because medical research increasingly recognizes mitochondrial dysfunction as a major factor in the pathology of numerous human diseases, including diabetes, cancer, neurodegenerative diseases, and ischemia reperfusion injury. We propose a model linking mitochondrial oxidative phosphorylation (OxPhos) to human disease, through a lack of energy, excessive free radical production, or a combination of both. We discuss the regulation of OxPhos by cell signaling pathways as a main regulatory mechanism in higher organisms, which in turn determines the magnitude of the mitochondrial membrane potential: if too low, ATP production cannot meet demand, and if too high, free radicals are produced. This model is presented in light of the recently emerging understanding of mechanisms that regulate mammalian cytochrome c oxidase and its substrate cytochrome c as representative enzymes for the entire OxPhos system.     

The effect of venturicidin on light and oxygen-dependent electron transport,proton translocation,membrane potential development and ATP synthesis in intact cells of Rhodopseudomonas capsulata

Venturicidin behaves as an orthodox energy transfer inhibitor in intact cells of Rhodopseudomonas capsulata as judged by the following criteria. 1. It led to inhibition of respiration. Inhibition was relieved by low concentrations of uncoupling agent. 2. It enhanced light-induced and oxygen dependent H⁺ efflux. 3. It stimulated light-induced and oxygen dependent carotenoid band shifts. The rate of decay of the band shifts after short flash excitation was decreased in the presence of venturicidin. 4. It stimulated light-induced and oxygen dependent butyltriphenylphosphonium uptake. 5. It inhibited the rise in cellular ATP concentration accompanying either photosynthesis or respiration.