The effect of glucagon on hepatic respiratory capacity

Data from numerous laboratories show that mitochondria isolated from livers treated acutely with glucagon have higher rates of state 3 respiration than control mitochondria. The purpose of the present study was to learn whether this phenomenon is an isolation artifact resulting from a stabilization of the mitochondrial membrane or whether it represents a real increase in the maximal respiratory capacity of liver cells due to glucagon treatment. Electron transport was measured through different spans of the electron transport chain by using ferricyanide as an alternate electron acceptor to O2. With isolated intact liver mitochondria, pretreatment with glucagon was found to cause an increase in electron flow, through both Complex I and Complex III, suggesting that the effect of glucagon was not specific for a single site in the electron transport chain. Using intact isolated hepatocytes, different results are obtained. Respiration was measured in isolated hepatocytes after quantitation of the hepatocyte mitochondrial content by assay of citrate synthase. Hepatocyte respiration could therefore be reported per mg of mitochondrial protein. By providing durohydroquinone to the cells, it was possible to measure electron flow from coenzyme Q to O2 in the absence of the physiological regulation of substrate supply. Likewise, the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone released the in situ mitochondria from control by the cytosolic ATP/ADP ratio and it was possible to measure maximal electron flow rates through Complex III. In the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, electron flow was higher in mitochondria in the cell than in isolated mitochondria. Glucagon caused no increase in mitochondrial respiration in situ either in the presence of the physiological substrates or in the presence of durohydroquinone. The data obtained do not support a role for the electron transport chain as a target of glucagon action in hepatocytes.     

Inhibition of electron transfer from ferrocytochrome b to ubiquinone, cytochrome c1 and duroquinone by antimycin.

The effect of antimycin on (i) the respiratory activity of the KCN-insensitive pathway of mitochondria of Neurospora grown on chloramphenicol (chloramphenicol-grown) with durohydroquinone and succinate or NADH as substrate, (ii) the electron transfer from the b-type cytochromes to ubiquinone with durohydroquinone as electron donor as well as (iii) the electron transfer from the b-type cytochromes to duroquinone with succinate as electron donor in chloramphenicol-grown Neurospora and beef heart submitochondrial particles was studied. All experiments were performed in the uncoupled state. 1. The respiratory chain of chloramphenicol-grown Neurospora mitochondria branches at ubiquinone into two pathways. Besides the cytochrome oxidase-dependent pathway, a KCN-insensitive branch equiped with a salicylhydroxamate-sensitive oxidase exists. Durohydroquinone, succinate or NADH are oxidized via both pathways. The durohydroquinone oxidation via the KCN-insensitive pathway is inhibited by antimycin, wheras the succinate or NADH oxidation is not. The titer for ful inhibition is one mol antimycin per mol cytochrome b-563 or cytochrome b-557. 2. The electron transfer from durohydroquinone to ubiquinone, which takes place in the KCN-inhibited state, does not occur in the antimycin-inhibited state. 3. The reduction of duroquinone by succinate in the presence of KCN is inhibited by antimycin. The titer for full inhibition is one mol antimycin per mol cytochrome b-566 or cytochrome b-562 for beef heart (or cytochrome b-563 or cytochrome b-557 for Neurospora). 4. When electron transfer from the b-type cytochromes to cytochrome C1, ubiquinone and duroquinone is inhibited by antimycin, the hemes of cytochrome b-566 and cytochrome b-562 (or cytochrome b-563 and cytochrome b-557) are in the reduced state. 5. The experimental results suggest that the two b-type cytochromes form a binary complex the electron transferring activity of which is inhibited by antimycin, the titer for full inhibition being one mol of antimycin per mol of complex. The electron transfer from the b-type cytochromes to ubiquinone is inhibited in a non-linear fashion.     

Mitochondrial respiration and triiodothyronine concentration in liver from postpubertal and adult rats.

The purpose of this study was to investigate the decline in rat liver mitochondria respiration found in adult rats compared to younger ones, and to find a link between this respiratory impairment and a tissue hypothyroidism state. To this end, hepatic concentration and serum levels of triiodothyronine were measured in postpubertal rats (60 days old) and adult rats (180 days old). In addition, in these rats we measured oxidative phosphorylation in homogenate together with coupled and uncoupled respiration in isolated mitochondria using succinate or durohydroquinone as substrate. We found that mitochondria from adult rats consumed less oxygen compared to younger rats due to lower electron transport chain and phosphorylating system activity. In addition, we found that in state 4 condition, mitochondria from adult rats consumed less oxygen than mitochondria from young rats. Finally, we found a decrease in liver triiodothyronine concentration in adult rats. In conclusion, the results of this study show that hepatic mitochondria in adult rats have a decreased ATP synthesis capacity and proton permeability, both consistent with the tissue hypothyroidism found in the liver of adult rats.     

Elevated hepatic mitochondrial oxidative capacities in cold exposed rats

1. Hepatic mitochondrial oxidative capacities were studied in rats exposed to cold for periods ranging from 5 to 15 days. The mitochondria obtained in this study were well coupled as shown by RCR and ADP/O ratios. 2. The liver mitochondria of cold exposed rats showed significantly increased respiratory rates (ng atoms of oxygen consumed min-1 mg prot-1), starting from day 10 of cold exposure, using lipid and non-lipid substrates. 3. For non-lipid substrates, the elevated respiratory rates found in the mitochondria could indicate an increased capacity to oxidize these substrates. For the lipid substrate, on the other hand, an enhanced oxidation through Krebs-cycle of a part of acetyl-CoA otherwise utilized to form ketone bodies, could also occur. 4. Taken together the results suggest that, during cold exposure, liver mitochondria could participate in cold adaptation mechanisms, by improving ATP production.     

Shift in the localization of sites of hydrogen peroxide production in brain mitochondria by mitochondrial stress

We have determined the underlying sites of H(2)O(2) generation by isolated rat brain mitochondria and how these can shift depending on the presence of respiratory substrates, electron transport chain modulators and exposure to stressors. H(2)O(2) production was determined using the fluorogenic Amplex red and peroxidase system. H(2)O(2) production was higher when succinate was used as a respiratory substrate than with another FAD-dependent substrate, alpha-glycerophosphate, or with the NAD-dependent substrates, glutamate/malate. Depolarization by the uncoupler p-trifluoromethoxyphenylhydrazone decreased H(2)O(2) production stimulated by all respiratory substrates. H(2)O(2) production supported by succinate during reverse transfer of electrons was decreased by inhibitors of complex I (rotenone and diphenyleneiodonium) whereas in glutamate/malate-oxidizing mitochondria diphenyleneiodonium decreased while rotenone increased H(2)O(2) generation. The complex III inhibitors antimycin and myxothiazol decreased succinate-induced H(2)O(2) production but stimulated H(2)O(2) production in glutamate/malate-oxidizing mitochondria. Antimycin and myxothiazol also increased H(2)O(2) production in mitochondria using alpha-glycerophosphate as a respiratory substrate. In substrate/inhibitor experiments maximal stimulation of H(2)O(2) production by complex I was observed with the alpha-glycerophosphate/antimycin combination. In addition, three forms of in vitro mitochondrial stress were studied: Ca(2+) overload, cold storage for more than 24 h and cytochrome c depletion. In each case we observed (i) a decrease in succinate-supported H(2)O(2) production by complex I and an increase in succinate-supported H(2)O(2) production by complex III, (ii) increased glutamate/malate-induced H(2)O(2) generation by complex I and (iii) increased alpha-glycerophosphate-supported H(2)O(2) generation by complex III. Our results suggest that all three forms of mitochondrial stress resulted in similar shifts in the localization of sites of H(2)O(2) generation and that, in both normal and stressed states, the level and location of H(2)O(2) production depend on the predominant energetic substrate.     

Nutritional effects on mitochondrial bioenergetics. Alterations in calcium uptake by rat liver mitochondria

The uptake of Ca2+ by energized liver mitochondria was compared in normal fed as well as in protein-energy malnourished rats. In the presence of phosphate, mitochondria obtained from both groups were able to accumulate Ca2+ from the suspending medium and eject H+ during oxidation of common substrates which activate different segments of the respiratory chain. The rate of Ca2+ uptake was significantly lower in mitochondria from protein-energy malnourished rats. The rates of oxygen consumption and H+ ejection were decreased by 20-30% during oxidation of substrates at the three coupling sites. Similarly, mitochondria from protein-energy malnourished rats exhibit a 34% decrease in the maximal rate of Ca2+ uptake and a 25% lower capacity for Ca2+ load. The stoichiometric relationship of Ca2+/2e- remained unaffected. In steady state, with succinate as a substrate in the presence of rotenone and N-ethylmaleimide, mitochondria from normal fed and protein-energy malnourished rats showed a similar rate of Ca2+ uptake. Furthermore in both groups the stoichiometry of the H+/O ratio was close to 8.0 (H+/site ratio close to 4.0), and of Ca2+/site was close to 2.0. The diminished rate of Ca2+ uptake observed in mitochondria from protein-energy malnourished rats could be explained on the basis of a depressed rate of electron transport in the respiratory chain rather than by an effect at the level of the Ca2+ or H+ transport mechanism per se.     

Mechanism of Cold Injury in Sweet Potatoes

The biochemical mechanism of cold injury occurring in sweet potatoes stored at 0°C was studied. Oxygen uptake and RC ratio of mitochondria from sweet potatoes kept at 0°C for about 15 days declined when succinate or malate was used as substrate. As sweet potatoes suffered slight cold injury, a decrease in the respiratory rate of state 3 of mitochondria was observed. This decrease could be restored approximately to the level of that of healthy sweet potato mitochondria by the addition of cytochrome c when succinate was used as substrate. When sweet potatoes suffered severe damage, only partial recovery was observed with cytochrome c. While it was found that the respiratory rate in state 3 of mitochondria from chilled sweet potatoes was less inhibited by cyanide than that of healthy sweet potato mitochondria, the inhibition could be restored to that of healthy sweet potato mitochondria by the addition of cytochrome c. When malate was used as substrate, no effect of cytochrome c and NADH₂ was observed. There was no difference between chilled and healthy sweet potato mitochondria in enzyme activities of the electron transport system except for malate dehydrogenase.     

Stimulation of the electron transport chain in mitochondria isolated from rats treated with mannoheptulose or glucagon

We investigated the kinetics of the mitochondrial respiratory chain, proton leak, and phosphorylating subsystems of liver mitochondria from mannoheptulose-treated and control rats. Mannoheptulose treatment raises glucagon and lowers insulin; it had no effect on the kinetics of the mitochondrial proton leak or phosphorylating subsystems, but the respiratory chain from succinate to oxygen was stimulated. Previous attempts to detect any stimulation of cytochrome c oxidase by glucagon are shown by flux control analysis to have used inappropriate assay conditions. To investigate the site of stimulation of the respiratory chain we measured the relationship between the thermodynamic driving force and respiration rate for the span succinate to coenzyme Q, the cytochrome bc1 complex and cytochrome c oxidase. Hormone treatment of rats altered the kinetics of electron transport from succinate to coenzyme Q in subsequently isolated mitochondria and activated succinate dehydrogenase. The kinetics of electron transport through the cytochrome bc1 complex were not affected. Effects on cytochrome c oxidase were small or nonexistent.     

Mitochondrial respiration at low levels of oxygen and cytochrome c

In the intracellular microenvironment of active muscle tissue, high rates of respiration are maintained at near-limiting oxygen concentrations. The respiration of isolated heart mitochondria is a hyperbolic function of oxygen concentration and half-maximal rates were obtained at 0.4 and 0.7 microM O(2) with substrates for the respiratory chain (succinate) and cytochrome c oxidase [N,N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)+ascorbate] respectively at 30 degrees C and with maximum ADP stimulation (State 3). The respiratory response of cytochrome c-depleted mitoplasts to external cytochrome c was biphasic with TMPD, but showed a monophasic hyperbolic function with succinate. Half-maximal stimulation of respiration was obtained at 0.4 microM cytochrome c, which was nearly identical to the high-affinity K(')(m) for cytochrome c of cytochrome c oxidase supplied with TMPD. The capacity of cytochrome c oxidase in the presence of TMPD was 2-fold higher than the capacity of the respiratory chain with succinate, measured at environmental normoxic levels. This apparent excess capacity, however, is significantly decreased under physiological intracellular oxygen conditions and declines steeply under hypoxic conditions. Similarly, the excess capacity of cytochrome c oxidase declines with progressive cytochrome c depletion. The flux control coefficient of cytochrome c oxidase, therefore, increases as a function of substrate limitation of oxygen and cytochrome c, which suggests a direct functional role for the apparent excess capacity of cytochrome c oxidase in hypoxia and under conditions of intracellular accumulation of cytochrome c after its release from mitochondria.     

Evaluation of Fe(III) reduction by mitochondria induced with a respiratory substrate NADH or succinate,using a Fe(II)-specific chelator bathophenanthroline disulfonate in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Mitochondria are the centers of the cellular iron metabolism. Iron utilization by mitochondria is deeply related to their respiratory chain activity. We isolated mitochondria from Saccharomyces cerevisiae and examined Fe(III) reduction induced by a respiratory substrate (NADH or succinate), using a Fe(II)-specific chelator (bathophenanthroline disulfonate). In the presence of either 50 μM NADH or 5 mM succinate, the amount of reduced Fe(III) was linearly correlated with the amount of mitochondria. As the concentration of the substrate increased, the rate of the mitochondrial Fe(III) reduction reached a plateau. In the presence of 1 mM ADP or 1 mM ATP, the extramitochondrial Fe(III) reduction was repressed when succinate was used as the substrate, but not when NADH was used. ADP had an inhibitory effect even under low concentration of succinate, suggesting that ADP and ATP acted in a manner of both competitive and uncompetitive inhibition.     

The role of succinate in the respiratory chain of Trypanosoma brucei procyclic trypomastigotes.

Trypanosoma brucei procyclic trypomastigotes were made permeable by using digitonin (0-70 micrograms/mg of protein). This procedure allowed exposure of coupled mitochondria to different substrates. Only succinate and glycerol phosphate (but not NADH-dependent substrates) were capable of stimulating oxygen consumption. Fluorescence studies on intact cells indicated that addition of succinate stimulates NAD(P)H oxidation, contrary to what happens in mammalian mitochondria. Addition of malonate, an inhibitor of succinate dehydrogenase, stimulated NAD(P)H reduction. Malonate also inhibited intact-cell respiration and motility, both of which were restored by further addition of succinate. Experiments carried out with isolated mitochondrial membranes showed that, although the electron transfer from succinate to cytochrome c was inhibitable by antimycin, NADH-cytochrome c reductase was antimycin-insensitive. We postulate that the NADH-ubiquinone segment of the respiratory chain is replaced by NADH-fumarate reductase, which reoxidizes the mitochondrial NADH and in turn generates succinate for the respiratory chain. This hypothesis is further supported by the inhibitory effect on cell growth and respiration of 3-methoxyphenylacetic acid, an inhibitor of the NADH-fumarate reductase of T. brucei.     

Respiration of Mitochondria Isolated from Leaves and Protoplasts of Avena sativa

Mitochondria isolated from mesophyll protoplasts differed from mitochondria isolated directly from leaves of Avena sativa in that protoplast mitochondria (a) had a lower overall respiratory capacity, (b) were less able to use low concentrations of exogenous NADH, (c) did not respond rapidly or strongly to added NAD, (d) appeared to accumulate more oxaloacetate, and (e) oxidized both succinate and tetramethyl-p-phenylene-diamine (an electron donor for cytochrome oxidase) more slowly than did leaf mitochondria. It is concluded that cytochrome oxidase activity was inhibited, the external NADH dehydrogenase had a reduced affinity for NADH, succinate oxidation was inhibited, NAD and oxaloacetate porters were probably inhibited, and accessibility to respiratory paths may have been reduced in protoplast mitochondria. The results also suggest that there was a reduced affinity of a succinate porter for this substrate in oat mitochondria. In addition, all oat mitochondria required salicylhydroxamic acid (SHAM) as well as cyanide to block malate and succinate oxidation. Malate oxidation that did not appear to saturate the cytochrome pathway was sensitive to SHAM in the absence of cyanide, suggesting that the oat mitochondria studied had concomitant alternative and subsaturating cytochrome oxidase pathway activity.     

The stimulation of hepatic oxidative phosphorylation following dexamethasone treatment of rats

The effect of short-term treatment of rats with the synthetic glucocorticoid, dexamethasone, on mitochondrial oxidative phosphorylation has been examined. Treatment of rats for 3 h increased the oxidative capacity of the subsequently isolated mitochondria such that they displayed increased uncoupled and State 3 rates of respiration with NAD-linked substrates, succinate or durohydroquinone. The oxidation of ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine was unaffected. No change was apparent in the activity of a variety of dehydrogenase enzymes nor was there any increase in the mitochondrial content of cytochromes a, b, c1 or c. The uncoupler-dependent ATPase activity of the mitochondria was slightly enhanced following hormone treatment, but not the basal or the total ATPase activity measured in the presence of Triton X-100 plus Mg2+. The mitochondria prepared from dexamethasone-treated rats also displayed increased intramitochondrial concentrations of Mg2+, K+ and exchangeable adenine nucleotides but not Ca2+. It is suggested that the effect of glucocorticoids on mitochondrial respiration may be both the result of a direct activation of the respiratory chain within Complex III and an elevated intramitochondrial adenine nucleotide concentration. The evidence for the de novo synthesis of mitochondrial proteins which mediate the response remains inconclusive.     

Factors limiting respiration by isolated cauliflower mitochondria

Oxygen consumption by isolated cauliflower mitochondria oxidising malate, succinate or NADH was less than when a combination of any two substrates was supplied. The rates obtained with any two were less than the aggregate of the individual rates. Only with the combination of malate plus succinate did the presence of the NADH result in faster rates of oxygen uptake. Conversely, malate and succinate separately, or in combination, inhibited the oxidation of erogenous NADH. The rate-limiting step for the oxidation of these substrates lies within the respiratory chain but on that part used exclusively by each substrate. The addition of NADH with either malate or succinate, or both, saturates the chain and makes the rate limiting step a component common to all pathways. Malate and isocitrate oxidation were considerably greater in disrupted, than in intact mitochondria, eliminating substrate dehydrogenases as rate-limiting factors. Substrate entry was also eliminated in the case of malate oxidation. It is suggested that the tricarboxylate transporter may restrict the rate of isocitrate oxidation.     

Extramitochondrial release of hydrogen peroxide from insect and mouse liver mitochondria using the respiratory inhibitors phosphine, myxothiazol, and antimycin and spectral analysis of inhibited cytochromes

The fumigant insecticide phosphine (PH3) is known to inhibit cytochrome c oxidase in vitro. Inhibition of the respiratory chain at this site has been shown to stimulate the generation of superoxide radicals (O2-), which dismutate to form hydrogen peroxide (H2O2). This study was performed in order to investigate the production of H2O2 by mitochondria isolated from granary weevil (Sitophilus granarius) and mouse liver on exposure to PH3. Other respiratory inhibitors, antimycin, myxothiazol, and rotenone were used with insect mitochondria. Hydrogen peroxide was measured spectrophotometrically using yeast cytochrome c peroxidase as an indicator. Insect and mouse liver mitochondria, utilizing endogenous substrate, both produced H2O2 after inhibition by PH3. Insect organelles released threefold more H2O2 than did mouse organelles, when exposed to PH3. Production of H2O2 by PH3-treated insect mitochondria was increased significantly on addition of the substrate alpha-glycerophosphate. Succinate did not enhance H2O2 production, however, indicating that the H2O2 did not result from the autoxidation of ubiquinone. NAD(+)-linked substrates, malate and pyruvate also had no effect on H2O2 production, suggesting that NADH-dehydrogenase was not the source of H2O2. Data obtained using antimycin and myxothiazol, both of which stimulated the release of H2O2 from insect mitochondria, lead to the conclusion that glycerophosphate dehydrogenase is a source of H2O2. The effect of combining PH3, antimycin, and myxothiazol on cytochrome spectra in insect mitochondria was also recorded. It was observed that PH3 reduces cytochrome c oxidase but none of the other cytochromes in the electron transport chain. There was no movement of electrons to cytochrome b when insect mitochondria are inhibited with PH3. The spectral data show that the inhibitors interact with the respiratory chain in a way that would allow the production of H2O2 from the sites proposed previously.     

Rotenone-like action of the branched-chain phytanic acid induces oxidative stress in mitochondria

Phytanic acid (Phyt) increase is associated with the hereditary neurodegenerative Refsum disease. To elucidate the still unclear toxicity of Phyt, mitochondria from brain and heart of adult rats were exposed to free Phyt. Phyt at low micromolar concentrations (maximally: 100 nmol/mg of protein) enhances superoxide (O(2)(.))(2) generation. Phyt induces O(2)(.) in state 3 (phosphorylating), as well as in state 4 (resting). Phyt stimulates O(2)(.) generation when the respiratory chain is fed with electrons derived from oxidation of glutamate/malate, pyruvate/malate, or succinate in the presence of rotenone. With succinate alone, Phyt suppresses O(2)(.) generation caused by reverse electron transport from succinate to complex I. The enhanced O(2)(.) generation by Phyt in state 4 is in contrast to the mild uncoupling concept. In this concept uncoupling by nonesterified fatty acids should abolish O(2)(.) generation. Stimulation of O(2)(.) generation by Phyt is paralleled by inhibition of the electron transport within the respiratory chain or electron leakage from the respiratory chain. The interference of Phyt with the electron transport was demonstrated by inhibition of state 3- and p-trifluoromethoxyphenylhydrazone (FCCP)-dependent respiration, inactivation of the NADH-ubiquinone oxidoreductase complex in permeabilized mitochondria, decrease in reduction of the synthetic electron acceptor 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in state 4, and increase of the mitochondrial NAD(P)H level in FCCP-uncoupled mitochondria. Thus, we suggest that complex I is the main site of Phyt-stimulated O(2)(.) generation. Furthermore, inactivation of aconitase and oxidation of the mitochondrial glutathione pool show that enhanced O(2)(.) generation with chronic exposure to Phyt causes oxidative damage.     

Deficiency of the complex I of the mitochondrial respiratory chain but improved adenylate control over succinate-dependent respiration are human gastric cancer-specific phenomena

The purpose of study was to comparatively characterize the oxidative phosphorylation (OXPHOS) and function of respiratory chain in mitochondria in human gastric corpus mucosa undergoing transition from normal to cancer states and in human gastric cancer cell lines, MKN28 and MKN45. The tissue samples taken by endobiopsy and the cells were permeabilized by saponin treatment to assess mitochondrial function in situ by high-resolution oxygraphy. Compared to the control group of endobiopsy samples, the maximal capacity of OXPHOS in the cancer group was almost twice lower. The respiratory chain complex I-dependent respiration, normalized to complex II-dependent respiration, was reduced that suggests deficiency of complex I, but the respiratory control by ADP in the presence of succinate was increased. Similar changes were observed also in mucosa adjacent to cancer tissue. The respiratory capacity of MKN45 cells was higher than that of MKN28 cells, but both types of cells exhibited a deficiency of complex I of the respiratory chain which appears to be an intrinsic property of the cancer cells. In conclusion, human gastric cancer is associated with decreased respiratory capacity, deficiency of the respiratory complex I of mitochondria, and improved coupling of succinate oxidation to phosphorylation in tumor tissue and adjacent atrophic mucosa. Detection of these changes in endobiopsy samples may be of diagnostic value.     

Characterisation of the control of respiration in potato tuber mitochondria using the top-down approach of metabolic control analysis.

Control over oxidative phosphorylation by purified potato mitochondria was determined using the top-down approach of metabolic control analysis. The control over the respiration rate, phosphorylation rate, proton-leak rate and proton motive force exerted by the respiratory chain, phosphorylation reactions and the proton leak were measured over a range of phosphorylation rates from resting (state 4) to maximal (state 3). These rates were obtained by adding different amounts of hexokinase in the presence of glucose, or different amounts of oligomycin in the presence of ADP. The respiratory substrate was NADH or succinate, both of which feed electrons directly to ubiquinone. The rate of oxygen consumption by the alternative oxidase pathway was negligible with NADH as substrate but was measurable with succinate and was subtracted. Control over the respiration rate in potato mitochondria was predominantly exerted by the respiratory chain at all rates except close to state 4, where control by the proton leak was equally or more important. For oxidation of NADH, the flux control coefficient over the respiration rate exerted by the respiratory chain in state 3 was between 0.8 and 1.0, while in state 4, control over the respiration rate was shared about equally between the chain and the proton leak. The control over the phosphorylation rate was predominantly exerted by the respiratory chain, although at low rates control by the phosphorylation system was also important. For oxidation of NADH, the flux control coefficient over the phosphorylation rate exerted by the respiratory chain in state 3 was 0.8-1.0, while near state 4 the flux control coefficients over the phosphorylation rate were about 0.8 for the phosphorylation system and 0.25 for the chain. Control over the proton leak rate was shared between the respiratory chain and the proton leak; the phosphorylation system had negative control. For oxidation of NADH, the flux control coefficients over the leak rate in state 3 were 1.0 for the leak, 0.4 for the chain and -0.4 for the phosphorylation system, while in state 4 the flux control coefficients over leak rate were about 0.5 for the leak and 0.5 for the chain. Control over the magnitude of the protonmotive force was small, between -0.2 and +0.2, reflecting the way the system operates to keep the protonmotive force fairly constant; the respiratory chain and the phosphorylation system had equal and opposite control and there was very little control by the proton leak except near state 4.     

On the mechanism of action of polyether XXVIII at site I of the electron-transfer chain in rat liver mitochondria

In rat liver mitochondria, the macrocyclic polyether, dibenzo-18-crown-6 (polyether XXVIII) inhibits the oxidation of NAD-dependent substrates, as stimulated by ADP, uncouplers and valinomycin plus K⁺. It does not inhibit the oxidation of succinate. It is concluded that polyether XXVIII inhibits electron transfer in the NADH-CoQ span of the respiratory chain. This is a process that is reversed by menadione. Inhibition of oxidation of NAD-dependent substrates in K⁺-depleted mitochondria induced by the polyether is reversed by concentrations of K⁺ higher than 60 mM, and also by Li⁺, a cation that does not complex with polyether XXVIII. As assayed by swelling mitochondria, reversal of the inhibition of electron transfer is accompanied by influx of monovalent cations. Polyether XXVIII also inhibits in submitochondrial particles the aerobic oxidation of NADH, but not that of succinate; this inhibition is also reversed by K⁺ at high concentrations, and Li⁺. The data are consistent with the hypothesis that a monovalent cation is required for maximal rates of electron transport in the NADH-CoQ span of the respiratory chain.