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Effect of Funiculosin and Antimycin A on the Redox‐Driven H+‐Pumps in Mitochondria: on the Nature of ‘Leaks’
Abstract
The effect of antimycin A and funiculosin, two inhibitors which block electron transfer in the b‐c 1 complex, on electron flow and electrochemical potential difference of H ⁺ ions in mitochondria at static head (state 4) is investigated. In addition, the respiratory control ratio is determined as the ratio between uncoupler stimulated and static‐head electron flow. Malonate, a competitive inhibitor of succinic dehydrogenase, is used for comparison.
All three inhibitors cause an extensive depression of static‐head electron flow but only a limited decrease in the electrochemical potential difference of H ⁺ ions. With the antimycin‐type of inhibitors, the respiratory control ratio slightly increases up to about 50% inhibition of electron flow and then steeply declines. With malonate, a strong decrease of the respiratory control ratio is observed in a concentration range where the electron flow is inhibited less than 10%.
It is shown that the data do not compiy with the generally accepted hypothesis of a leak conductance being regulated by the electrochemical potential difference of H ⁺ ions. They can be interpreted in terms of not tightly coupled redox‐driven H ⁺ ‐pumps. A non‐vanishing electron flow at static head then arises predominantly from molecular slipping in the pumps, and the (constant) leak conductance yields only a minor contribution.
... TH have a profound effect on the mitochondria, the organelles responsible for producing energy for the cell. Some studies have shown that an increased concentration of thyroid hormones can induce mitochondrial biogenesis, enhancing the ability of cells to generate the energy necessary for biological processes [3][4][5]. It is also known that there is a stimulation of the mitochondrial respiration rate of different organs [6][7][8] and a change in the activity of electron transport chain enzymes (ETC) [9]. ...
... The development of hyperthyroidism was confirmed by the determination of plasma T 3 and T 4 concentrations. There was about a 1.8-and 3.4-fold increase in T 3 and T 4 levels in HR compared with the values in the control group (Table 1). ...
... Despite the fact that the activity of most ETC complexes increased, there was no activation of respiration in the heart mitochondria of the HR (Tables 2 and 3). The deficiency of ATP synthesis could be a consequence of either the failure of the proton pumps when they transfer electrons with a reduced extrusion of protons out across the membrane or switching respiratory chain complexes to the idle mode [4,23]. Because the electron transport in the mitochondrial ETC is a chain reaction, the disorder/decrease in the electron flow at any step can become a rate-limiting stage. ...
In this work, the effect of thyroxine on energy and oxidative metabolism in the mitochondria of the rat heart was studied. Hyperthyroidism was observed in experimental animals after chronic administration of T4, which was accompanied by an increase in serum concentrations of free triiodothyronine (T3) and thyroxine (T4) by 1.8 and 3.4 times, respectively. The hyperthyroid rats (HR) had hypertrophy of the heart. In HR, there was a change in the oxygen consumption in the mitochondria of the heart, especially when using palmitoylcarnitine. The assay of respiratory chain enzymes revealed that the activities of complexes I, I + III, III, IV increased, whereas the activities of complexes II, II + III decreased in heart mitochondria of the experimental animals. It was shown that the level of respiratory complexes of the electron transport chain in hyperthyroid rats increased, except for complex V, the quantity of which was reduced. The development of oxidative stress in HR was observed: an increase in the hydrogen peroxide production rate, increase in lipid peroxidation and reduced glutathione. The activity of superoxide dismutase in the heart of HR was higher than in the control. At the same time, the activity of glutathione peroxidase decreased. The obtained data indicate that increased concentrations of thyroid hormones lead to changes in energy metabolism and the development of oxidative stress in the heart of rats, which in turn contributes to heart dysfunction.
... Originally described by Pietrobon et al. [44], alteration of the proton pumps expresses a change in the amount of protons stored in the intermembrane space (redox slipping) or the amount of protons returning to the matrix via ATP synthase (proton slipping), for the same amount of oxidized electrons. ...
... Arrow ( ) indicate increase, arrow ( ) indicate decrease, arrow ( ) indicate no changes.B. Changes in Proton Pumping StoichiometryOriginally described by Pietrobon et al.[44], alteration of the proton pumps expresses a change in the amount of protons stored in the intermembrane space (redox slipping) or the amount of protons returning to the matrix via ATP synthase (proton slipping), for the same amount of oxidized electrons. ...
The link between liver dysfunction and decreased mitochondrial oxidative phosphorylation in sepsis has been clearly established in experimental models. Energy transduction is plastic: the efficiency of mitochondrial coupling collapses in the early stage of sepsis but is expected to increase during the recovery phases of sepsis. Among the mechanisms regulating the coupling efficiency of hepatic mitochondria, the slipping reactions at the cytochrome oxidase and ATP synthase seem to be a determining element, whereas other regulatory mechanisms such as those involving proton leakage across the mitochondrial membrane have not yet been formally proven in the context of sepsis. If the dysfunction of hepatic mitochondria is related to impaired cytochrome c oxidase and ATP synthase functions, we need to consider therapeutic avenues to restore their activities for recovery from sepsis. In this review, we discussed previous findings regarding the regulatory mechanism involved in changes in the oxidative phosphorylation of liver mitochondria in sepsis, and propose therapeutic avenues to improve the functions of cytochrome c oxidase and ATP synthase in sepsis.
The effect of uridine (30 mg/kg for 7 days; intraperitoneally) on the functions of liver mi-tochondria in rats with experimentally induced hyperthyroidism (HT) (200 µg/100 g for 7 days, in-traperitoneally) is studied in this paper. An excess of thyroid hormones (THs) led to an intensifica-tion of energy metabolism, the development of oxidative stress, a significant increase in the biogen-esis, and changes in the content of proteins responsible for the fusion and fission of mitochondria. The injection of uridine did not change the concentration of THs in the blood of hyperthyroid rats (HRs) but normalized their body weight. The exposure to uridine improved the parameters of oxi-dative phosphorylation and corrected the activity of some complexes of the electron transport chain (ETC) in the liver mitochondria of HRs. The analysis of ETC complexes showed that the level of СI-CV did not change by the action of uridine in rats with the condition of HT. The application of uridine caused a significant increase in the activity of superoxide dismutase and lowered the rate of hydrogen peroxide production. It was found that uridine affected mitochondrial biogenesis by increasing the expression of the genes Ppargc1a and NRF1 and diminishing the expression of the Parkin gene responsible for mitophagy compared with the control animals. In addition, the mRNA level of the OPA1 gene was restored, which may indicate an improvement in the ETC activity and oxidative phosphorylation in the mitochondria of HR. As a whole, the results obtained demonstrate that uri-dine has a protective effect against HT-mediated functional disorders in the metabolism of rat liver mitochondria.
Mitochondria are double-membrane-enclosed organelles that ensure a number of central functions in the vast majority of eukaryotic cells. These include, among others, key steps in lipid and amino acid metabolism, biosynthesis of iron–sulfur clusters, heme, and further prosthetic groups, regulation of programmed cell death, and most prominently the final transformation of proteins, fats, and sugars into the cellular energy currency adenosine-5′-triphosphate (ATP) by oxidative phosphorylation (OXPHOS). Severe mitochondrial dysfunctions are associated with so-called mitochondrial diseases, and are largely associated with specific or broad OXPHOS defects. In addition, alterations of mitochondrial properties have been associated with other diseases associated with metabolic and/or physiologic alterations. This chapter aims to give a general introduction on mitochondrial properties and functions that will be further developed in subsequent chapters.
The protonmotive mitochondrial respiratory chain, comprising complexes I, III and IV, transduces free energy of the electron transfer reactions to an electrochemical proton gradient across the inner mitochondrial membrane. This gradient is used to drive synthesis of ATP and ion and metabolite transport. The efficiency of energy conversion is of interest from a physiological point of view, since the energy transduction mechanisms differ fundamentally between the three complexes. Here, we have chosen actively phosphorylating mitochondria as the focus of analysis. For all three complexes we find that the thermodynamic efficiency is about 80–90% and that the degree of coupling between the redox and proton translocation reactions is very high during active ATP synthesis. However, when net ATP synthesis stops at a high ATP/ADP.Pi ratio, and mitochondria reach “State 4” with an elevated proton gradient, the degree of coupling drops substantially. The mechanistic cause and the physiological implications of this effect are discussed. Wikström and Springett analyze the thermodynamic efficiency of redox reactions and proton translocation by the complexes of mitochondrial respiratory chain. They report that the thermodynamic efficiency is about 80–90% and that the degree of coupling between the redox and proton translocation reactions is very high during active ATP synthesis, but decreases when ATP synthesis stops.
The hypermetabolic effects of thyroid hormones (THs), the major endocrine regulators of metabolic rate, are widely recognized. Although, the cellular mechanisms underlying these effects have been extensively investigated, much has yet to be learned about how TH regulates diverse cellular functions. THs have a profound impact on mitochondria, the organelles responsible for the majority of cellular energy production, and several studies have been devoted to understand the respective importance of the nuclear and mitochondrial pathways for organelle activity. During the last decades, several new aspects of both THs (i.e., metabolism, transport, mechanisms of action, and the existence of metabolically active TH derivatives) and mitochondria (i.e., dynamics, respiratory chain organization in supercomplexes, and the discovery of uncoupling proteins other than uncoupling protein 1) have emerged, thus opening new perspectives to the investigation of the complex relationship between thyroid and the mitochondrial compartment. In this review, in the light of an historical background, we attempt to point out the present findings regarding thyroid physiology and the emerging recognition that mitochondrial dynamics as well as the arrangement of the electron transport chain in mitochondrial cristae contribute to the mitochondrial activity. We unravel the genomic and nongenomic mechanisms so far studied as well as the effects of THs on mitochondrial energetics and, principally, uncoupling of oxidative phosphorylation via various mechanisms involving uncoupling proteins. The emergence of new approaches to the question as to what extent and how the action of TH can affect mitochondria is highlighted. © 2016 American Physiological Society. Compr Physiol 6:1591-1607, 2016.
Using purified DNA gyrase to supercoil circular plasmid pBR322 DNA, we examined how the linking number attained at the steady
state (‘static head’) varies with the concentrations of ATP and ADP, both in the absence and presence of spermidine. In the
absence of spermidine at total adenine nucleotide concentrations between 0.35 and 1.4 mM, the static-head linking number was independent of the sum concentration of ATP and ADP, but depended strongly on the ratio
of their concentrations. We established that the same linking number was attained independent of the direction from which
the steady state was approached. The decrease in linking number at static head is more extensive when spermidine is present
in the incubation, but remains a function of the [ATP]-to-[ADP] ratio.
These results are discussed in terms of various kinetic schemes for DNA gyrase. We present one kinetic scheme that accounts
for the experimental observations. According to this scheme our experimental results imply that there is significant slip
in DNA gyrase when spermidine is absent. It is possible that spermidine acts through adjustment of the degree of coupling
of DNA gyrase.
Under non-phosphorylating conditions a high proton transmembrane gradient inhibits the rate of oxygen consumption mediated by the mitochondrial respiratory chain (state IV). Slow electron transit leads to production of reactive oxygen species (ROS) capable of participating in deleterious side reactions. In order to avoid overproducing ROS, mitochondria maintain a high rate of O(2) consumption by activating different exquisitely controlled uncoupling pathways. Different yeast species possess one or more uncoupling systems that work through one of two possible mechanisms: i) Proton sinks and ii) Non-pumping redox enzymes. Proton sinks are exemplified by mitochondrial unspecific channels (MUC) and by uncoupling proteins (UCP). Saccharomyces. cerevisiae and Debaryomyces hansenii express highly regulated MUCs. Also, a UCP was described in Yarrowia lipolytica which promotes uncoupled O(2) consumption. Non-pumping alternative oxido-reductases may substitute for a pump, as in S. cerevisiae or may coexist with a complete set of pumps as in the branched respiratory chains from Y. lipolytica or D. hansenii. In addition, pumps may suffer intrinsic uncoupling (slipping). Promising models for study are unicellular parasites which can turn off their aerobic metabolism completely. The variety of energy dissipating systems in eukaryote species is probably designed to control ROS production in the different environments where each species lives.
Mitochondrial bioenergetics is a broad field. In this chapter we will focus on the state of the art of oxidative phosphorylation regulation. The main theoretical basis of energy transfer processes was laid by Peter Mitchell in the 1960s. Since this pioneering work, great progress has been made in the description of the systems involved at the molecular level. Moreover, the three-dimensional structure of most of the proton pumps has now been ascertained. Our understanding of oxidative phosphorylation kinetic regulations has also greatly progressed. However, numerous experimental data cannot be explained in the framework of nonintegrative biology. We will summarize the structural and functional evidence showing a high degree of dynamic organization of the oxidative phosphorylation components in supramolecular states and discuss some of the functional consequences of such integrated systems.
In mitochondria isolated from the yeast Saccharomyces cerevisiae, under non-phosphorylating conditions, we have previously shown that there is a right of way for electrons coming from the external NADH dehydrogenase, Nde1p. In this work, we show that the electron competition process is identical under more physiological conditions i.e. oxidative phosphorylation. Such a competition generates a priority for cytosolic NADH reoxidation. Furthermore, this electron competition process is associated with an energy wastage (the "active leak") that allows an increase in redox equivalent oxidation when the redox pressure increases. When this redox pressure is decreased, i.e. under phosphorylating conditions, most of this energy wastage is alleviated. By studying mutant strains affected either in respiratory chain supramolecular organization or in electron competition activity, we show that the respiratory chain supramolecular organization is not responsible for the electron competition processes. Moreover, we show two distinct relationships between the respiratory rate and the quinone redox state that seem to indicate two quinone pools that are involved in the electron right of way. Indeed, the more reduced pool would be associated to the electron right of way for the external dehydrogenases whereas the less reduced pool would be associated to the electron right of way for the internal dehydrogenases.
The main function of mitochondria is energy transduction, from substrate oxidation to the free energy of ATP synthesis, through oxidative phosphorylation. For physiological reasons, the degree of coupling between these two processes must be modulated in order to adapt redox potential and ATP turnover to cellular needs. Such a modulation leads to energy wastage. To this day, two energy wastage mechanisms have been described: the membrane passive proton conductance (proton leak) and the decrease in the coupling efficiency between electrons transfer and proton extrusion at the proton pumps level (redox or proton slipping). In this paper, we describe a new energy wastage mechanism of interest. We show that in isolated yeast mitochondria, the membrane proton conductance is strictly dependent on the external dehydrogenases activity. An increase in their activity leads to an increase in the membrane proton conductance. This proton permeability is independent of the respiratory chain and ATP synthase proton pumps. We propose to name this new mechanism "active proton leak." Such a mechanism could allow a wide modulation of substrate oxidation in response to cellular redox constraints.
Mitochondrial mechanistic P/O ratios are still in question. The major studies since 1937 are summarized and various systematic errors are discussed. Values of about 2.5 with NADH-linked substrates and 1.5 with succinate are consistent with most reports after apparent contradictions are explained. Variability of coupling may occur under some conditions but is generally not significant. The fractional values result from the coupling ratios of proton transport. An additional revision of P/O ratios may be required because of a report of the structure of ATP synthase (D. Stock, A.G.W. Leslie, J.E. Walker, Science 286 (1999) 1700-1705) which suggests that the H+/ATP ratio is 10/3, rather than 3, consistent with P/O ratios of 2.3 with NADH and 1.4 with succinate, values that are also possible.
Brief periods of myocardial ischemia prior to timely reperfusion result in prolonged, yet reversible, contractile dysfunction of the myocardium, or "myocardial stunning". It has been hypothesized that the delayed recovery of contractile function in stunned myocardium reflects damage to one or a few key sarcomeric proteins. However, damage to such proteins does not explain observed physiological alterations to myocardial oxygen consumption and ATP requirements observed following myocardial stunning, and therefore the impact of alterations to additional functional groups is unresolved. We utilized two-dimensional gel electrophoresis and mass spectrometry to identify changes to the protein profiles in whole cell, cytosolic- and myofilament-enriched subcellular fractions from isolated, perfused rabbit hearts following 15 min or 60 min low-flow (1 mL/min) ischemia. Comparative gel analysis revealed 53 protein spot differences (> 1.5-fold difference in visible abundance) in reperfused myocardium. The majority of changes were observed to proteins from four functional groups: (i) the sarcomere and cytoskeleton, notably myosin light chain-2 and troponin C; (ii) redox regulation, in particular several components of the NADH ubiquinone oxidoreductase complex; (iii) energy metabolism, encompassing creatine kinase; and (iv) the stress response. Protein differences appeared to be the result of isoelectric point shifts most probably resulting from chemical modifications, and molecular mass shifts resulting from proteolytic or physical fragmentation. This is consistent with our hypothesis that the time course for the onset of injury associated with myocardial stunning is too brief to be mediated by large changes to gene/protein expression, but rather that more subtle, rapid and potentially transient changes are occurring to the proteome. The physical manifestation of stunned myocardium is therefore the likely result of the summed functional impairment resulting from these multiple changes, rather than a result of damage to a single key protein.
The effects of ciprofloxacin on mitochondrial DNA (mtDNA) content, oxygen consumption, mitochondrial membrane potential, cellular
ATP formation, and capacitative Ca2+ entry into Jurkat cells were investigated. In cells incubated for several days with 25 μg/ml ciprofloxacin, a 60% reduction
of mtDNA content, inhibition of the respiratory chain, and a significant decrease in mitochondrial membrane potential were
observed. These changes led to a decrease in the calcium buffering capacity of mitochondria which, in turn, resulted in a
gradual inhibition of the capacitative Ca2+ entry. On days 4, 7, and 11 of incubation with ciprofloxacin, the initial rates of Ca2+ entry were reduced by 33%, 50%, and 50%, respectively. Ciprofloxacin caused a transient decrease in the cellular capability
for ATP formation. In cells incubated for 15 min with glucose, pyruvate, and glutamine as exogenous fuel, ciprofloxacin reduced
ATP content by 16% and 35% on days 4 and 7, respectively, of incubation with the drug. However, on day 11 of incubation with
ciprofloxacin, a recovery of cellular ATP formation was observed. In conclusion, long-term exposure of Jurkat cells to ciprofloxacin
at a concentration of 25 μg/ml seriously affects cellular energy metabolism and calcium homeostasis.
During daily torpor in the dwarf Siberian hamster, Phodopus sungorus, metabolic rate is reduced by 65% compared with the basal rate, but the mechanisms involved are contentious. We examined liver mitochondrial respiration to determine the possible role of active regulated changes and passive thermal effects in the reduction of metabolic rate. When assayed at 37 degrees C, state 3 (phosphorylating) respiration, but not state 4 (nonphosphorylating) respiration, was significantly lower during torpor compared with normothermia, suggesting that active regulated changes occur during daily torpor. Using top-down elasticity analysis, we determined that these active changes in torpor included a reduced substrate oxidation capacity and an increased proton conductance of the inner mitochondrial membrane. At 15 degrees C, mitochondrial respiration was at least 75% lower than at 37 degrees C, but there was no difference between normothermia and torpor. This implies that the active regulated changes are likely more important for reducing respiration at high temperatures (i.e., during entrance) and/or have effects other than reducing respiration at low temperatures. The decrease in respiration from 37 degrees C to 15 degrees C resulted predominantly from a considerable reduction of substrate oxidation capacity in both torpid and normothermic animals. Temperature-dependent changes in proton leak and phosphorylation kinetics depended on metabolic state; proton leakiness increased in torpid animals but decreased in normothermic animals, whereas phosphorylation activity decreased in torpid animals but increased in normothermic animals. Overall, we have shown that both active and passive changes to oxidative phosphorylation occur during daily torpor in this species, contributing to reduced metabolic rate.
Nonalcoholic fatty liver disease (NAFLD) has become common liver disease in Western countries. There is accumulating evidence that mitochondria play a key role in NAFLD. Nevertheless, the mitochondrial consequences of steatohepatitis are still unknown. The bioenergetic changes induced in a methionine- and choline-deficient diet (MCDD) model of steatohepatitis were studied in rats. Liver mitochondria from MCDD rats exhibited a higher rate of oxidative phosphorylation with various substrates, a rise in cytochrome oxidase (COX) activity, and an increased content in cytochrome aa3. This higher oxidative activity was associated with a low efficiency of the oxidative phosphorylation (ATP/O, i.e., number of ATP synthesized/natom O consumed). Addition of a low concentration of cyanide, a specific COX inhibitor, restored the efficiency of mitochondria from MCDD rats back to the control level. Furthermore, the relation between respiratory rate and protonmotive force (in the nonphosphorylating state) was shifted to the left in mitochondria from MCDD rats, with or without cyanide. These results indicated that, in MCDD rats, mitochondrial ATP synthesis efficiency was decreased in relation to both proton pump slipping at the COX level and increased proton leak although the relative contribution of each phenomenon could not be discriminated. MCDD mitochondria also showed a low reactive oxygen species production and a high lipid oxidation potential. We conclude that, in MCDD-fed rats, liver mitochondria exhibit an energy wastage that may contribute to limit steatosis and oxidative stress in this model of steatohepatitis.
A technique is described, based on the distribution of rubidium, acetate and methylammonium ions, for the simultaneous estimation of membrane potential and pH gradient across the inner membrane of mitochondria. The technique requires less than 0.5 mg mitochondrial protein and is independent of many factors which interfere with electrode determinations of protonmotive force (Δ p ).
With a limiting matrix volume of 0.4 μl/mg mitochondrial protein, the indicated value of Δ p for rat liver mitochondria is 228 mV in state 4, 170 mV in state 3, and –0.6 mV in the presence of rotenone and uncoupler. The relative contributions of the pH gradient and membrane potential are dependent on the availability of electrophoretically and electroneutrally translocatable species in the incubation medium.
In a sucrose‐based medium containing 0.5 mM KCl, rotenone and uncoupler, the technique indicated a membrane potential of + 85 mV and a pH gradient of + 1.46 (acidic in the matrix compartment). In state 4, under no conditions examined did the pH gradient contribute more than 50% of the total protonmotive force.
The hydrolysis of ATP generates an optimal Δ p of 220 mV.
The proton conductance of the inner membrane is potential dependent, increasing when Δ p is greater than 200 mV.
The extra‐mitochondrial phosphate potential sustainable by respiration was found to change in parallel to Δ p , but to exceed the latter parameter when based upon a stoichiometry of two protons translocated per ATP synthesised.
The biologically important processes of respiratory-chain phosphorylation and of photophosphorylation involve the transduction of redox or light energy, respectively, for the synthesis of ATP from ADP and Pi against an energy gradient. In chemical or physical processes, a continuous entropy production occurs. It is this entropy change that may be considered as the irreversible component of such processes. Mitochondria are the subcellular organelles that catalyze the synthesis of ATP from ADP and phosphate, coupled to the oxidation of substrates by molecular oxygen. They consist of an outer membrane that allows free permeation of all low-molecular weight solutes, an extensively folded inner membrane that functions as the energy transducer, and an inner space. Energy transduction from light to ATP in chloroplasts is catalyzed by the membranes of the grana stacks or thylakoids. These thylakoid membranes contain light-harvesting pigments, reaction centers, carotenoids, redox components, and an ATPase complex comparable to that of mitochondria
The stimulation of respiration by uncoupling agents, called respiratory control by analogy with the stimulation of respiration by ADP plus phosphate, has been very useful in studies of the mechanism of energy coupling in oxidative phosphorylation. LOOMIS and LIPMANN (1948) originally reported that the rate of respiration of isolated mitochondria is increased by addition of 2,4-dinitrophenol. This led to the concept that such “uncoupling” agents break down a high-energy intermediate and thus relieve the back pressure of the coupling reactions on the oxidation reactions of the respiratory chain (SLATER, 1953). LEE and ERNSTER (1966) extended these observations by demonstrating that sonically fragmented vesicles of mitochondria, submitochondrial particles, also showed a stimulation of respiration by uncoupling agents, and that the controlled respiration of such preparation corresponded to the ability to couple respiration to various energy-linked functions.
The relationship between translocation of K ⁺ and respiration has been studied under conditions where large variations of the transmembrane K ⁺ concentration gradient were obtained. It is found that the energy expenditure is a function of the number of ions translocated and not of the transmembrane concentration gradient. The K ⁺ /∼ ratio is slightly below 3 when measured on the total oxygen uptake and between 3 and 4 when measured on the Δ oxygen uptake.
The maximal [K ⁺ ] i /[K ⁺ ] o ratio maintained by respiring liver mitochondria is about 1,600 corresponding to an equilibrium potential of 192 mV. When the [K ⁺ ] i /[K ⁺ ] o ratio exceeds 2,000 times addition of valinomycin under aerobic conditions causes an efflux instead of an uptake of K ⁺ .
Mitochondria behave as perfect osmometers in a wide range of osmolarities. It can be calculated that the concentration of osmotically active internal K ⁺ corresponds to 45% of the total osmolarity of the medium.
The relationship between oxidative phosphorylation and the energy-linked nicotinamide-adenine dinucleotide transhydrogenase reaction catalyzed by phosphorylating submitochondrial particles from beef heart has been investigated. The efficiency of the particles in exhibiting oxidative phosphorylation (P/O ratio) and NADH-linked NADP+ reduction (NADP+/O ratio) was measured, using succinate as oxidizable substrate and rotenone as inhibitor of NADH oxidase, under conditions where the phosphorylating and transhydrogenating systems were operating one by one or in parallel. When succinate oxidation was limited by increasing concentrations of malonate, the P/O ratio remained unchanged in the absence of an active phosphorylating system, and decreased in its presence. The NADP+/O ratio increased with increasing concentrations of malonate, both in the absence and presence of an active phosphorylating system. The (P + NADP+)/O ratio in the combined system approached 2, i.e., the generally accepted maximal ∼/O ratio for succinate oxidase. Active transhydrogenation caused an increase in the apparent Km for Pi in the phosphorylating system from 1.7 to 4.5 mM, and active phosphorylation caused an increase in the apparent Km for NADH in the transhydrogenating system from 21 to 40 μM. It is concluded that oxidative phosphorylation and the energy-linked transhydrogenase reaction are competing for a common nonphosphorylated high-energy intermediate generated by the respiratory chain. The kinetic evidence presented further suggests that the two systems interact with two different moieties of this high-energy intermediate.
The relationship between the rate of substrate oxidation and the protonmotive force (electrochemical proton gradient) generated by bovine heart submitochondrial particles has been examined. Unexpectedly, oxidation of succinate generated a higher protonmotive force than the oxidation of NADH, although the rate of proton translocation across the membrane was inferred to be considerably lower with succinate as substrate. The data suggest that the flow of electrons through site 1 of the respiratory chain may increase the conductance of the mitochondrial membrane for protons. Upon reduction of the rate of succinate oxidation by titration with malonate, the protonmotive force remained essentially constant until the extent of inhibition was greater than 75%. The general conclusion from this work is that a constant passive membrane conductance for protons cannot be assumed.
The antibiotic funiculosin mimics the action of antimycin in several ways. It inhibits the oxidation of NADH and succinate, but not TMPD+ascorbate. The titer for maximal inhibition in Mg2+-ATP particles (0.4-0.6 nmol/mg protein) is close to the concentrations of cytochromes b and cc1. Funiculosin also induces the oxidation of cytochromes cc1 and an extra reduction of cytochrome b in the aerobic steady state, and it inhibits duroquinol-cytochrome c reductase activity in isolated Complex III. The location of the funiculosin binding site is clearly similar to that of antimycin. In addition, funiculosin, like antimycin, prevents electron transport from duroquinol to cytochrome b in isolated Complex III if the complex is pre-reduced with ascorbate. Funiculosin and antimycin differ, however, in the manner in which they modulate the reduction of cytochrome b by ascorbate+TMPD.
The correlation between deltamuH, the proton electrochemical potential difference, and the rate of controlled respiration is analyzed. deltamuH (the proton concentration gradient) is measured on the distribution of [3H]acetate, and deltapsi (the membrane potential) on the distribution of 86Rb+, 45Ca2+ and [3H]triphenylmethylphosphonium used either alone or simultaneously. The effects of the addition of ADP + hexokinase (state-3 ADP) and of carbonylcyanide trifluoromethoxyphenylhydrazone (state-3 uncoupler) on respiration and deltamuH are not equivalent: the uncoupler depresses deltamuH more than ADP at equivalent respiratory rates. The effects of the additions of nigericin-valinomycin and of ionophore A23187 (state-3 cation transport) and of carbonylcyanide trifluoromethoxy-phenylhydrazone (state 3-uncoupler) on respiration and deltamuH are also not equivalent: the uncoupler depresses deltamuH more than A23187 and nigericin + valinomycin at equivalent respiratory rate. A23187 is very efficient in stimulating respiration with negligible deltamuH changes.
The extent of K+ uptake in aerobic mitochondria is dependent on the valinomycin concentration. Also the extent of uptake of organic cations, such as tetrapropylammonium and tetraethylammonium, is dependent on the tetraphenylboron concentration. The results do not support the hypothesis that permeant cations are distributed in mitochondria in the steady state at electrochemical equilibrium and are in accord with a pump and leak mechanism of ion uptake.
The H+/Ca++ ratio during the aerobic uptake of Ca++ by rat liver mitochondria, oxidizing endogenous substrates or succinate, increased with the increase of the Ca++/protein ratio. At high protein concentrations an apparent “reuptake” of the H+ ejected was observed. Mitochondrial aging also resulted in a decrease of the H+/Ca++ ratio.
Succinate caused a decrease of the H+ ejection. The effect of succinate was dependent on the pH and on the concentration of Ca++. A binding of succinate to the mitochondria was observed parallel to the decrease of the H+/Ca++ ratio from 2 to 1.
Addition of Ca++ caused a release of K+ which was slow in fresh mitochondria and rapid in valinomycin treated mitochondria. The K+/Ca++ ratio was 2 in both cases.
A H+/Ca++ ratio of about 2 was usually observed when the aerobic uptake of Ca++ was started either by the addition of mitochondria to a Ca++ containing medium or by the addition of succinate to Ca++-and rotenone-treated mitochondria.
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