Mitochondrial Efficiency-Dependent Viability of Saccharomyces cerevisiae Mutants Carrying Individual Electron Transport Chain Component Deletions

Mitochondria play a crucial role in eukaryotic cells; the mitochondrial electron transport chain (ETC) generates adenosine triphosphate (ATP), which serves as an energy source for numerous critical cellular activities. However, the ETC also generates deleterious reactive oxygen species (ROS) as a natural byproduct of oxidative phosphorylation. ROS are considered the major cause of aging because they damage proteins, lipids, and DNA by oxidation. We analyzed the chronological life span, growth phenotype, mitochondrial membrane potential (MMP), and intracellular ATP and mitochondrial superoxide levels of 33 single ETC component-deleted strains during the chronological aging process. Among the ETC mutant strains, 14 (sdh1Δ, sdh2Δ, sdh4Δ, cor1Δ, cyt1Δ, qcr7Δ, qcr8Δ, rip1Δ, cox6Δ, cox7Δ, cox9Δ, atp4Δ, atp7Δ, and atp17Δ) showed a significantly shorter life span. The deleted genes encode important elements of the ETC components succinate dehydrogenase (complex II) and cytochrome c oxidase (complex IV), and some of the deletions lead to structural instability of the membrane-F₁F₀-ATP synthase due to mutations in the stator stalk (complex V). These short-lived strains generated higher superoxide levels and produced lower ATP levels without alteration of MMP. In summary, ETC mutations decreased the life span of yeast due to impaired mitochondrial efficiency.     

Glibenclamide and HMR1098 normalize Cantú syndrome‐associated gain‐of‐function currents

Cantú syndrome (CS) is caused by dominant gain‐of‐function mutation in ATP‐dependent potassium channels. Cellular ATP concentrations regulate potassium current thereby coupling energy status with membrane excitability. No specific pharmacotherapeutic options are available to treat CS but I_KATP channels are pharmaceutical targets in type II diabetes or cardiac arrhythmia treatment. We have been suggested that I_KATP inhibitors, glibenclamide and HMR1098, normalize CS channels. I_KATP in response to Mg‐ATP, glibenclamide and HMR1098 were measured by inside‐out patch‐clamp electrophysiology. Results were interpreted in view of cryo‐EM I_KATP channel structures. Mg‐ATP IC₅₀ values of outward current were increased for D207E (0.71 ± 0.14 mmol/L), S1020P (1.83 ± 0.10), S1054Y (0.95 ± 0.06) and R1154Q (0.75 ± 0.13) channels compared to H60Y (0.14 ± 0.01) and wild‐type (0.15 ± 0.01). HMR1098 dose‐dependently inhibited S1020P and S1054Y channels in the presence of 0.15 mmol/L Mg‐ATP, reaching, at 30 μmol/L, current levels displayed by wild‐type and H60Y channels in the presence of 0.15 mmol/L Mg‐ATP. Glibenclamide (10 μmol/L) induced similar normalization. S1054Y sensitivity to glibenclamide increases strongly at 0.5 mmol/L Mg‐ATP compared to 0.15 mmol/L, in contrast to D207E and S1020P channels. Experimental findings agree with structural considerations. We conclude that CS channel activity can be normalized by existing drugs; however, complete normalization can be achieved at supraclinical concentrations only.     

Essential Role of TID1 in Maintaining Mitochondrial Membrane Potential Homogeneity and Mitochondrial DNA Integrity

The tumorous imaginal disc 1 (TID1) protein localizes mainly to the mitochondrial compartment, wherein its function remains largely unknown. Here we report that TID1 regulates the steady-state homogeneity of the mitochondrial membrane potential (Δψ) and maintains the integrity of mitochondrial DNA (mtDNA). Silencing of TID1 with RNA interference leads to changes in the distribution of Δψ along the mitochondrial network, characterized by an increase in Δψ in focal regions. This effect can be rescued by ectopic expression of a TID1 construct with an intact J domain. Chronic treatment with a low dose of oligomycin, an inhibitor of F₁F_o ATP synthase, decreases the cellular ATP content and phenocopies TID1 loss of function, indicating a connection between the disruption of mitochondrial bioenergetics and hyperpolarization. Prolonged silencing of TID1 or low-dose oligomycin treatment leads to the loss of mtDNA and the consequent inhibition of oxygen consumption. Biochemical and colocalization data indicate that complex I aggregation underlies the focal accumulation of Δψ in TID1-silenced cells. Given that TID1 is proposed to function as a cochaperone, these data show that TID1 prevents complex I aggregation and support the existence of a TID1-mediated stress response to ATP synthase inhibition.     

Regulation of Glycolytic Oscillations by Mitochondrial and Plasma Membrane H-ATPases

We investigated the coupling between glycolytic and mitochondrial membrane potential oscillations in Saccharomyces cerevisiae under semianaerobic conditions. Glycolysis was measured as NADH autofluorescence, and mitochondrial membrane potential was measured using the fluorescent dye 3,3′-diethyloxacarbocyanine iodide. The responses of glycolytic and membrane potential oscillations to a number of inhibitors of glycolysis, mitochondrial electron flow, and mitochondrial and plasma membrane H⁺-ATPase were investigated. Furthermore, the glycolytic flux was determined as the rate of production of ethanol in a number of different situations (changing pH or the presence and absence of inhibitors). Finally, the intracellular pH was determined and shown to oscillate. The results support earlier work suggesting that the coupling between glycolysis and mitochondrial membrane potential is mediated by the ADP/ATP antiporter and the mitochondrial F₀F₁-ATPase. The results further suggest that ATP hydrolysis, through the action of the mitochondrial F₀F₁-ATPase and plasma membrane H⁺-ATPase, are important in regulating these oscillations. We conclude that it is glycolysis that drives the oscillations in mitochondrial membrane potential.     

Aerobic Growth of Escherichia coli Is Reduced,and ATP Synthesis Is Selectively Inhibited when Five C-terminal Residues Are Deleted from the ? Subunit of ATP Synthase

F-type ATP synthases are rotary nanomotor enzymes involved in cellular energy metabolism in eukaryotes and eubacteria. The ATP synthase from Gram-positive and -negative model bacteria can be autoinhibited by the C-terminal domain of its ϵ subunit (ϵCTD), but the importance of ϵ inhibition in vivo is unclear. Functional rotation is thought to be blocked by insertion of the latter half of the ϵCTD into the central cavity of the catalytic complex (F₁). In the inhibited state of the Escherichia coli enzyme, the final segment of ϵCTD is deeply buried but has few specific interactions with other subunits. This region of the ϵCTD is variable or absent in other bacteria that exhibit strong ϵ-inhibition in vitro. Here, genetically deleting the last five residues of the ϵCTD (ϵΔ5) caused a greater defect in respiratory growth than did the complete absence of the ϵCTD. Isolated membranes with ϵΔ5 generated proton-motive force by respiration as effectively as with wild-type ϵ but showed a nearly 3-fold decrease in ATP synthesis rate. In contrast, the ϵΔ5 truncation did not change the intrinsic rate of ATP hydrolysis with membranes. Further, the ϵΔ5 subunit retained high affinity for isolated F₁ but reduced the maximal inhibition of F₁-ATPase by ϵ from >90% to ∼20%. The results suggest that the ϵCTD has distinct regulatory interactions with F₁ when rotary catalysis operates in opposite directions for the hydrolysis or synthesis of ATP.     

Late Sodium Current in Human Atrial Cardiomyocytes from Patients in Sinus Rhythm and Atrial Fibrillation

Slowly inactivating Na⁺ channels conducting “late” Na⁺ current (I_Na,late) contribute to ventricular arrhythmogenesis under pathological conditions. I_Na,late was also reported to play a role in chronic atrial fibrillation (AF). The objective of this study was to investigate I_Na,late in human right atrial cardiomyocytes as a putative drug target for treatment of AF. To activate Na⁺ channels, cardiomyocytes from transgenic mice which exhibit I_Na,late (ΔKPQ), and right atrial cardiomyocytes from patients in sinus rhythm (SR) and AF were voltage clamped at room temperature by 250-ms long test pulses to -30 mV from a holding potential of -80 mV with a 100-ms pre-pulse to -110 mV (protocol I). I_Na,late at -30 mV was not discernible as deviation from the extrapolated straight line IV-curve between -110 mV and -80 mV in human atrial cells. Therefore, tetrodotoxin (TTX, 10 μM) was used to define persistent inward current after 250 ms at -30 mV as I_Na,late. TTX-sensitive current was 0.27±0.06 pA/pF in ventricular cardiomyocytes from ΔKPQ mice, and amounted to 0.04±0.01 pA/pF and 0.09±0.02 pA/pF in SR and AF human atrial cardiomyocytes, respectively. With protocol II (holding potential -120 mV, pre-pulse to -80 mV) TTX-sensitive I_Na,late was always larger than with protocol I. Ranolazine (30 μM) reduced I_Na,late by 0.02±0.02 pA/pF in SR and 0.09±0.02 pA/pF in AF cells. At physiological temperature (37°C), however, I_Na,late became insignificant. Plateau phase and upstroke velocity of action potentials (APs) recorded with sharp microelectrodes in intact human trabeculae were more sensitive to ranolazine in AF than in SR preparations. Sodium channel subunits expression measured with qPCR was high for SCN5A with no difference between SR and AF. Expression of SCN8A and SCN10A was low in general, and lower in AF than in SR. In conclusion, We confirm for the first time a TTX-sensitive current (I_Na,late) in right atrial cardiomyocytes from SR and AF patients at room temperature, but not at physiological temperature. While our study provides evidence for the presence of INa,late in human atria, the potential of such current as a target for the treatment of AF remains to be demonstrated.     

ATPaseTb2, a Unique Membrane-bound FoF1-ATPase Component,Is Essential in Bloodstream and Dyskinetoplastic Trypanosomes

In the infectious stage of Trypanosoma brucei, an important parasite of humans and livestock, the mitochondrial (mt) membrane potential (Δψ_m) is uniquely maintained by the ATP hydrolytic activity and subsequent proton pumping of the essential F_oF₁-ATPase. Intriguingly, this multiprotein complex contains several trypanosome-specific subunits of unknown function. Here, we demonstrate that one of the largest novel subunits, ATPaseTb2, is membrane-bound and localizes with monomeric and multimeric assemblies of the FoF1-ATPase. Moreover, RNAi silencing of ATPaseTb2 quickly leads to a significant decrease of the Δψ_m that manifests as a decreased growth phenotype, indicating that the F_oF₁-ATPase is impaired. To further explore the function of this protein, we employed a trypanosoma strain that lacks mtDNA (dyskinetoplastic, Dk) and thus subunit a, an essential component of the proton pore in the membrane F_o-moiety. These Dk cells generate the Δψ_m by combining the hydrolytic activity of the matrix-facing F₁-ATPase and the electrogenic exchange of ATP⁴- for ADP³- by the ATP/ADP carrier (AAC). Surprisingly, in addition to the expected presence of F1-ATPase, the monomeric and multimeric F_oF₁-ATPase complexes were identified. In fact, the immunoprecipitation of a F₁-ATPase subunit demonstrated that ATPaseTb2 was a component of these complexes. Furthermore, RNAi studies established that the membrane-bound ATPaseTb2 subunit is essential for maintaining normal growth and the Δψ_m of Dk cells. Thus, even in the absence of subunit a, a portion of the F_oF₁-ATPase is assembled in Dk cells.     

Energetics of Sodium Transport in Frog Skin : II. The effects of electrical potential on oxygen consumption

Studies were made of the dependence of the rate of oxygen consumption, J_r, on the electrical potential difference, Δψ, across the frog skin. After the abolition of sodium transport by ouabain the basal oxygen consumption was independent of Δψ. In fresh skins J_r was a linear function of Δψ over a range of at least ±70 mv. Treatment with aldosterone stimulated the short-circuit current, I_o, and the associated rate of oxygen consumption, J_ro, and increased their stability; linearity was then demonstrable over a range of ±160 mv. Brief perturbations of Δψ (±30–200 mv) did not alter subsequent values of I_o. Perturbations for 10 min or more produced a "memory" effect both with and without aldosterone: accelerating sodium transport by negative clamping lowered the subsequent value of I_o; positive clamping induced the opposite effect. Changes in J_ro were more readily detectable in the presence of aldosterone; these were in the same direction as the changes in I_o. The linearity of J_r in Δψ indicates the validity of analysis in terms of linear nonequilibrium thermodynamics—brief perturbations of Δψ appear to produce no significant effect on either the phenomenological coefficients or the free energy of the metabolic driving reaction. Hence it is possible to evaluate this free energy.     

Bioenergetic Mechanism for Nisin Resistance, Induced by the Acid Tolerance Response of Listeria monocytogenes

This study examined the bioenergetics of Listeria monocytogenes, induced to an acid tolerance response (ATR). Changes in bioenergetic parameters were consistent with the increased resistance of ATR-induced (ATR⁺) cells to the antimicrobial peptide nisin. These changes may also explain the increased resistance of L. monocytogenes to other lethal factors. ATR⁺ cells had lower transmembrane pH (ΔpH) and electric potential (Δψ) than the control (ATR⁻) cells. The decreased proton motive force (PMF) of ATR⁺ cells increased their resistance to nisin, the action of which is enhanced by energized membranes. Paradoxically, the intracellular ATP levels of the PMF-depleted ATR⁺ cells were ~7-fold higher than those in ATR⁻ cells. This suggested a role for the F_oF₁ ATPase enzyme complex, which converts the energy of ATP hydrolysis to PMF. Inhibition of the F_oF₁ ATPase enzyme complex by N′-N′-1,3-dicyclohexylcarbodiimide increased ATP levels in ATR⁻ but not in ATR⁺ cells, where ATPase activity was already low. Spectrometric analyses (surface-enhanced laser desorption ionization-time of flight mass spectrometry) suggested that in ATR⁺ listeriae, the downregulation of the proton-translocating c subunit of the F_oF₁ ATPase was responsible for the decreased ATPase activity, thereby sparing vital ATP. These data suggest that regulation of F_oF₁ ATPase plays an important role in the acid tolerance response of L. monocytogenes and in its induced resistance to nisin.     

THEORY AND MEASUREMENT OF VISUAL MECHANISMS : III. ΔI AS A FUNCTION OF AREA,INTENSITY, AND WAVE-LENGTH,FOR MONOCULAR AND BINOCULAR STIMULATION

Measurements of ΔI as a function of retinal area illuminated have been obtained at various levels of standard intensity I ₁, using "white" light and light of three modal wave-lengths (λ465, 525, 680), for monocular stimulation and for simultaneous excitation of the two eyes ("binocular"), using several methods of varying (rectangular) area and retinal location, with control of exposure time. For data homogeneous with respect to method of presentation, log ΔI_m = -Z log A + C, where ΔI = Ĩ ₂ – I ₁, A is area illuminated, and C is a terminal constant (= log ΔI_m for A = 1 unit) depending on the units in which ΔI and A are expressed, and upon I ₁. The equation is readily deduced on dimensional grounds, without reference to specific theories of the nature of ΔI or of retinal area in terms of its excitable units. Z is independent of the units of I and A. Experimentally it is found to be the same for monocular and binocular excitations, as is to be expected. Also as is expected it is not independent of λ, and it is markedly influenced by the scheme according to which A is varied; it depends directly upon the rate at which potentially excitable elements are added when A is made to increase. For simultaneous excitation of the two eyes (when of very nearly equivalent excitability), ΔĪ_B is less than for stimulation of either eye alone, at all levels of I ₁, A, λ. The mean ratio (ΔĪ_L + ΔĪ_R)/2 to ΔI^B was 1.38. For white light, doubling A on one retina reduces ΔI_m in the ratio 1.21, or a little less than for binocular presentation under the same conditions. These facts are consistent with the view that the properties of ΔI are quantitatively determined by events central to the retina. The measure σ_1ΔI of organic variation in discrimination of intensities and ΔI_m are found to be in simple proportion, independent of I ₁, A, λ (and exposure time). Variability (σ_1ΔI) is not a function of the mode of presentation, save that it may be slightly higher when both retinas are excited, and its magnitude (for a given level of ΔI_m) is independent of the law according to which the adjustable intensity I ₂ is instrumentally controlled.     

31P nuclear magnetic resonance study on changes in phosphocreatine and the intracellular pH in rat skeletal muscle during exercise at various inspired oxygen contents

We measured ATP, phosphocreatine (PCr), inorganic phosphate (P_i), and the intracellular pH in rat hindlimb muscles during submaximal isometric exercise with various O₂ deliveries using³¹P nuclear magnetic resonance spectroscopy (³¹P NMR) to evaluate changes in energy metabolism in relation to O₂ availability. Delivery of O₂ to muscles was altered by controlling the fractional concentration of inspired oxygen (F _IO₂) at 0.50, 0.28, 0.21, 0.11 and 0.08 with monitoring partial pressure of oxygen and carbon dioxide, and bicarbonate at the femoral artery. The steady-state ratio of PCr : (PCr + P_i) during exercise decreased as a function ofF _IO₂ even at 0.21. Significant acidification of the intracellular pH during exercise occurred at 0.08F _IO₂. Change in the PCr : (PCr + P_i) ratio demonstrated that the oxidative capacity, i.e. the maximal rate of the oxidative phosphorylation reaction, in muscle was not limited by O₂ delivery at 0.50F _IO₂, but was significantly limited at 0.21F _IO₂ or below. Change in the intracellular pH at 0.08F _IO₂ could be interpreted as an increase in lactate, suggesting activation of glycolysis. Correlation between the PCr : (PCr + P_i) ratio and the intracellular pH revealed the existence of a critical PCr : (PCr + P_i) ratio and pH for glycolysis activation at around 0.4 and 6.7, respectively.     

Modeling of oxygen transport and cellular energetics explains observations on in vivo cardiac energy metabolism

Observations on the relationship between cardiac work rate and the levels of energy metabolites adenosine triphosphate (ATP), adenosine diphosphate (ADP), and phosphocreatine (CrP) have not been satisfactorily explained by theoretical models of cardiac energy metabolism. Specifically, the in vivo stability of ATP, ADP, and CrP levels in response to changes in work and respiratory rate has eluded explanation. Here a previously developed model of mitochondrial oxidative phosphorylation, which was developed based on data obtained from isolated cardiac mitochondria, is integrated with a spatially distributed model of oxygen transport in the myocardium to analyze data obtained from several laboratories over the past two decades. The model includes the components of the respiratory chain, the F₀F₁-ATPase, adenine nucleotide translocase, and the mitochondrial phosphate transporter at the mitochondrial level; adenylate kinase, creatine kinase, and ATP consumption in the cytoplasm; and oxygen transport between capillaries, interstitial fluid, and cardiomyocytes. The integrated model is able to reproduce experimental observations on ATP, ADP, CrP, and inorganic phosphate levels in canine hearts over a range of workload and during coronary hypoperfusion and predicts that cytoplasmic inorganic phosphate level is a key regulator of the rate of mitochondrial respiration at workloads for which the rate of cardiac oxygen consumption is less than or equal to approximately 12 μmol per minute per gram of tissue. At work rates corresponding to oxygen consumption higher than 12 μmol min⁻¹ g⁻¹, model predictions deviate from the experimental data, indicating that at high work rates, additional regulatory mechanisms that are not currently incorporated into the model may be important. Nevertheless, the integrated model explains metabolite levels observed at low to moderate workloads and the changes in metabolite levels and tissue oxygenation observed during graded hypoperfusion. These findings suggest that the observed stability of energy metabolites emerges as a property of a properly constructed model of cardiac substrate transport and mitochondrial metabolism. In addition, the validated model provides quantitative predictions of changes in phosphate metabolites during cardiac ischemia.     

ON THE VARIABILITY OF CRITICAL ILLUMINATION FOR FLICKER FUSION AND INTENSITY DISCRIMINATION

From the data of experiments with bees in which threshold response is employed as a means of recognizing visual discrimination between stripes of equal width alternately illuminated by intensities I ₁ and I ₂, it is shown that the detectable increment of intensity ΔI, where ΔI = I ₂ - I ₁, is directly proportional to σ_I2 (I ₁ being fixed). From tests of visual acuity, where I ₁ = 0 and the width of the stripes is varied, σ_I2 = kI ₂ + const.; here I ₂ = ΔI, and ΔI/I ₂ = 1. When the visual excitability of the bee is changed by dark adaptation, λI ≡ kΔI (= k'' σ_ΔI) = k'''' I + const. For the measurements of critical illumination at threshold response to flicker, σ_I2 (= σ_ΔI) = k I ₂ = k'' ΔI + const. The data for critical illumination producing threshold response to flicker in the sun-fish Lepomis show for the rods σ_I2 = K I ₂ for the cones σ_I2 = K''(I ₂ + const.). The data thus indicate that in all these experiments essentially the same visual function is being examined, and that the recognition of the production of a difference in effect by alternately illuminated stripes takes place in such a way that d (ΔI)/d (σ_I2) = const., and that ΔI is directly proportional to I (or "I ₂," depending on the nature of the experiment). It is pointed out that the curve for each of the cases considered can be gotten equally well if mean I or σ_I is plotted as a function of the independent variable involved in the experiment. Certain consequences of these and related facts are important for the treatment of the general problem of intensity discrimination.     

Regulation of the mitochondrial ATPasein situ in cardiac muscle: Role of the inhibitor subunit

The mitochondrial F₁-ATPase inhibitor protein, IF₁, binds to subunits of the F₁-ATPase bothin vitro andin situ under nonenergizing conditions, i.e., under conditions that allow a net hydrolysis of ATP by the mitochondrial ATPase to take place. This reversible IF₁ binding occurs in a wide variety of cell types including (anaerobic) baker's yeast cells and (ischemic) mammalian cardiomyocytes under conditions that limit oxidative phosphorylation. The binding of inhibitor results in a marked slowing of ATP hydrolysis by the undriven mitochondrial ATP synthase. An apparent main function of this reversible IF₁ binding, at least in cells that undergo aerobic-anaerobic switching, is the mitigation of a wasteful hydrolysis of ATP produced by glycolysis during anoxic or ischemic intervals, by the mitochondrial ATPase. While this apparent main function is probably of considerable importance in cells that normally either can or must undergo aerobic-anaerobic switching such as baker's yeast cells and skeletal myocytes, one wonders why a full complement of IF₁ has been retained in certain cells that normally do not undergo such aerobic-anaerobic switching, cells such as adult mammalian cardiomyocytes of many species. While some mammalian species have, indeed, not retained a functional complement of IF₁ in their cardiomyocytes, those that have can benefit significantly from its presence during intervals of myocardial ischemia.This mini-review is dedicated to the memory of Professor Efraim Racker.     

The uniqueness of the plant mitochondrial potassium channel

The ATP-inhibited Plant Mitochondrial K⁺ Channel (PmitoK_ATP) was discovered about fifteen years ago in Durum Wheat Mitochondria (DWM). PmitoKATP catalyses the electrophoretic K⁺ uniport through the inner mitochondrial membrane; moreover, the co-operation between PmitoK_ATP and ⁺/H⁺ antiporter allows such a great operation of a K⁺ cycle to collapse mitochondrial membrane potential (ΔΨ) and ΔpH, thus impairing protonmotive force (Δp). A possible physiological role of such ΔΨ control is the restriction of harmful reactive oxygen species (ROS) production under environmental/oxidative stress conditions. Interestingly, DWM lacking Δp were found to be nevertheless fully coupled and able to regularly accomplish ATP synthesis; this unexpected behaviour makes necessary to recast in some way the classical chemiosmotic model. In the whole, PmitoK_ATP may oppose to large scale ROS production by lowering ΔΨ under environmental/oxidative stress, but, when stress is moderate, this occurs without impairing ATP synthesis in a crucial moment for cell and mitochondrial bioenergetics. [BMB Reports 2013; 46(8): 391-397]     

Kinetics of Oxygen Consumption after a Flash of Light in the Lateral Ocellus of the Barnacle

Until recently, polarographic methods for measuring the time course of transient changes in the rate of oxygen consumption (ΔQO₂) have been applied only to tissue preparations containing thousands of cells. Here, we describe ΔQO₂ measurements on the lateral ocellus of the barnacle (Balanus eburneus) which contains only three photoreceptor cells. The decrement of partial pressure of oxygen (ΔPO₂) elicited by an 80 ms flash of light was measured near the cells with a microelectrode and the ΔQO₂ was calculated from the ΔPO₂ using a model of diffusion with spherical symmetry. As shown by mathematical simulation, the exact shape of the preparation is not crucial for our measurements of the time course of the ΔQO₂. For a given ΔQO₂, the model describes correctly the attenuation of the ΔPO₂ measured at increased distances from the preparation. To know more about the mechanisms controlling the ΔQO₂, we compared it with the electrical response of the photoreceptor cells: both responses have a similar spectral dependence, but only the ΔQO₂ was abolished by a 10-min exposure to 50 μM dinitrophenol or to 3 mM amytal. We conclude that the ΔQO₂ reflects an increase in mitochondrial respiration and that it is initiated by the phototransformation of rhodopsin, as was already found in the honeybee drone retina (Dimitracos and Tsacopoulos, 1985; Jones and Tsacopoulos, 1987).     

Purification and Characterization of the Soluble F(1)-ATPase of Oat Root Mitochondria

The properties of the soluble moiety (F₁) of the mitochondrial H⁺-ATPase from oat roots were examined and compared to those of the native mitochondrial membrane-bound enzyme. The chloroform soluble preparation was purified by Sephadex G-200 and DEAE-cellulose chromatography. The purified F₁ preparation contained major polypeptides corresponding to α, β, γ, δ, and ε of apparent molecular mass 58, 55, 35, 22, and 14 kilodaltons, respectively. The purified F₁-ATPase, like the native enzyme, was inhibited by azide (I₅₀ = 10 micromolar), nitrate (I₅₀ = 7-10 millimolar), 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (I₅₀ = 1-3 micromolar), and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (I₅₀ = 3 micromolar). F₁-ATPase activity was stimulated by bicarbonate but not by chloride. In both the native and the F₁-form of the ATPase, ATP was hydrolyzed in preference to GTP. The results indicate that these properties of the native membrane-bound mitochondrial ATPase have been conserved in the purified F₁. In contrast to the membrane-bound enzyme, the F₁-ATPase was not inhibited by oligomycin or by N,N′-dicyclohexylcarbodiimide. The mitochondrial F₁-ATPase from oat roots is analogous to other known F₁F₀-ATPases.     

Mitochondrial F1FO ATP synthase determines the local proton motive force at cristae rims

The classical view of oxidative phosphorylation is that a proton motive force (PMF) generated by the respiratory chain complexes fuels ATP synthesis via ATP synthase. Yet, under glycolytic conditions, ATP synthase in its reverse mode also can contribute to the PMF. Here, we dissected these two functions of ATP synthase and the role of its inhibitory factor 1 (IF1) under different metabolic conditions. pH profiles of mitochondrial sub‐compartments were recorded with high spatial resolution in live mammalian cells by positioning a pH sensor directly at ATP synthase’s F₁ and F_O subunits, complex IV and in the matrix. Our results clearly show that ATP synthase activity substantially controls the PMF and that IF1 is essential under OXPHOS conditions to prevent reverse ATP synthase activity due to an almost negligible ΔpH. In addition, we show how this changes lateral, transmembrane, and radial pH gradients in glycolytic and respiratory cells.     

Time requirement for hypoxic acclimation to anoxia tolerance in rice coleoptiles

Rice (Oriza sativa L.) seedlings were subjected tohypoxic pretreatment (H-PT; incubated in 5% O₂ atmosphere) forvarious lengths of time followed by a 24-h anoxic stress. Anoxiatolerance of rice coleoptiles was improved with increasing duration of H-PT, butH-PT longer than 6 h gave no additional improvement. ATP andethanol concentrations in the coleoptiles were increased by H-PT, and the timeand pattern of increase in ATP level and ethanol production rate were similar tothose of increase in the anoxia tolerance. These results suggest that the H-PTmay increase anoxia tolerance due to maintenance of anaerobic glycolysis withinduction of ethanolic fermentation to generate ATP, and hypoxic acclimation toanoxic stress in rice coleoptiles may occur within 6 h.