Homeostasis of the protonmotive force in phosphorylating mitochondria

The relationship between the respiration rate and the magnitude of the electrochemical proton potential (Δμ_H⁺) in rat liver mitochondria was investigated. (1) Under the active-state conditions, the action of inhibitors of either phosphorylation (oligomycin) or respiration (rotenone, malonate) on the respiration and Δμ_H⁺ was measured. Both inhibitors diminished the respiration, whereas rotenone resulted in a decrease of Δμ_H⁺, and oligomycin produced an increase of this potential. The effect of the inhibitors was much more pronounced on the respiration rate than on Δμ_H⁺; for example, the excess of oligomycin produced a 90% inhibition of the respiration while Δμ_H⁺ was changed only by 9%. (2) Under the resting-state conditions, small concentrations of the uncoupler stimulated the respiration while changing Δμ_H⁺ to a relatively small extent. The uncoupler concentrations which doubled and tripled the respiration rate produced only 5 and 9% decrease of Δμ_H⁺, respectively. (3) The present results enabled us to propose a model describing the interrelationship between respiration and Δμ_H⁺.     

Flow-force relationships during energy transfer between mitochondrial proton pumps.

The effect of inhibitors of proton pumps, of uncouplers and of permeant ions on the relationship between input force, delta mu H+, and output flows of the ATPase, redox and transhydrogenase H(+)-pumps in submitochondrial particles was investigated. It is concluded that: (1) The decrease of output flow of the transhydrogenase proton pump, defined as the rate of reduction of NADP+ by NADH, is linearily correlated with the decrease of input force, delta mu H+, in an extended range of delta mu H+, independently of whether the H(+)-generating pump is the ATPase or a redox pump, or whether delta mu H+ is depressed by inhibitors of the H(+)-generating pump such as oligomycin or malonate, or by uncouplers. (2) The output flows of the ATPase and of the site I redox H(+)-pumps exhibit a steep dependence on delta mu H+. The flow-force relationships differ depending on whether the depression of delta mu H+ is induced by inhibitors of the H(+)-generating pump, by uncouplers or by lipophilic anions. (3) With the ATPase as H(+)-consuming pump, at equivalent delta mu H+ values, the output flow is more markedly inhibited by malonate than by uncouplers; the latter, however, are more inhibitory than lipophilic anions such as ClO4-. With redox site I as proton-consuming pump, at equivalent delta mu H+ values, the output flow is more markedly inhibited by oligomycin than by uncouplers; again, uncouplers are more inhibitory than ClO4-. (4) The results provide further support for a delocalized interaction of transhydrogenase with other H(+)-pumps.     

Therapeutic doses of menadione reduce the rotenone-induced inhibition of respiration and membrane potential generation in mitochondria

Menadione restores the rotenone-inhibited respiration of diaphragm muscle pieces in approximately the same degree as the respiration of heart mitochondria, i.e., to 30-40%. The respiration of heart mitochondria induced by 2-5 microM menadione (after its inhibition by rotenone) is partly coupled with ATP synthesis whose rate is much lower than that of oxidation of NAD-dependent substrates. The effects of menadione and mitochondrial energetics inhibitors on lymphocyte respiration and rhodamine 123 fluorescence in individual lymphocytes and their suspensions were compared. Menadione (2--5 microM) increased the rotenone + oligomycin suppressed delta psi m in lymphocytes. At 5-40 microM menadione did not act as an uncoupler and had little effect on the uncoupled lymphocyte respiration. All these effects were observed at menadione concentrations close to therapeutic ones. Vicasol, a water-soluble analog of menadione, exerted a similar effect.     

The effect of delta mu H+ on the interaction of rotenone with complex I of submitochondrial particles.

The inhibition by rotenone of the forward (NADH-oxidase) and reverse (delta mu H(+)-dependent succinate-NAD+ reductase activities of submitochondrial vesicles was measured. The inhibition of NADH-oxidase, measured in the presence of uncoupler, followed a monophasic inhibition curve with Ki < or = 2 nM. The reverse electron flow was only partially (40%) inhibited at these rotenone concentrations. The rest of the activity was less sensitive to the inhibitor (Ki approximately 30 nM). The lower affinity for the inhibitor of the reverse electron flow is a consequence of enhanced rate of rotenone dissociation caused by the high delta mu H+ value required for this reaction. The analysis of the results indicates that the AS-SMP preparation consists of two subpopulations: one with a relatively low degree of coupling, which exhibits high sensitivity to rotenone and the other which is highly coupled with lower affinity to the inhibitor.     

Uncoupler-inhibitor titrations of ATP-driven reverse electron transfer in bovine submitochondrial particles provide evidence for direct interaction between ATPase and NADH:Q oxidoreductase

From the chemiosmotic hypothesis it follows that no change is expected in potency of an uncoupler to inhibit an energy-driven reaction in an energy-transducing membrane if the energy-requiring part of the reaction, the so-called secondary proton pump, is partially inhibited by a specific, tightly bound inhibitor. An increase in potency upon inhibition of the primary pump may be expected, due to a lower rate of the total proton flow that can be used by the secondary pump and dissipated by the uncoupler. Contrary to this prediction several uncouplers (S13, SF6847, 2,4-dinitrophenol, valinomycin + nigericin) show an increase in uncoupling efficiency in ATP-driven reverse electron transfer (reversal) upon inhibition of the secondary pump in this reaction, the NADH:Q oxidoreductase, by rotenone. The increase in uncoupling efficiency is proportional to the decrease in the rate of reversal, that is to the decrease in concentration of active secondary pump. Similarly, upon inhibition of the primary pump, the ATPase, with oligomycin, an increase in uncoupling efficiency was found, also proportional to the decrease in the rate of reversal. When the pore-forming uncoupler gramicidin was used, no change in uncoupling potency was found upon inhibition of NADH:Q oxidoreductase. Inhibition of the ATPase, however, resulted in a proportionally lower uncoupling titre for gramicidin, just as was found for S13 in the presence of oligomycin. A difference was also found in the relative concentrations of S13 and gramicidin required to stimulate ATP hydrolysis or to inhibit reversal. The amount of S13 needed to stimulate ATP hydrolysis was clearly higher than the amount needed to inhibit reversal. On the contrary, the titre of gramicidin for both actions was about the same. To explain these results we propose that gramicidin uncouples via dissipation of the bulk delta mu H+, whereas the carrier-type uncouplers preferentially interfere with the direct energy transduction between the ATPase and redox enzymes. This is in accordance with the recently developed collision hypothesis.     

Free energy coupling between H+-generating and H+-consuming pumps. Ratio between output and input forces

The delta Gp/delta mu H ratio has been measured in mitochondria close to state 4 in the presence of various uncoupler or K+/valinomycin concentrations in media containing either 1 mM or 50 mM Pi. Care has been taken to control the factors affecting delta Gp and delta mu H which could lead to an artefactual increase of the delta Gp/delta mu H ratio above the highest accepted value for the H+/ATP stoichiometry (n = 4, synthesis + transport). In particular, to avoid overestimation of delta Gp due to inactivation of the ATPases at low delta mu H or to the presence of adenylate kinase, the static head state was approached from the side of net ATP synthesis and delta Gp was measured in a state close to static head but still maintaining a residual rate of aerobic phosphorylation. For each concentration of uncoupler or K+, the Pi concentration and/or the adenylate energy charge (EC) as a function of time have been measured as indicators of net ATP synthesis. Only the values of delta Gp measured during a decrease in Pi concentration and/or an increase in EC have been considered to be meaningful for calculations of delta Gp/delta mu H ratios. Both uncouplers and K+ transport cause a marked depression of delta mu H and a parallel depression of the rate of ATP synthesis. However the low rate of ATP synthesis taking place under conditions of low delta mu H eventually results, especially at high Pi concentrations, in a relatively large delta Gp. The delta Gp/delta mu H ratios obtained at the lower delta mu H values exceed 4 and approach 6. Although slightly higher delta Gp/delta mu H ratios are obtained with valinomycin-treated than with uncoupler-treated mitochondria, the pattern of the rise of the force ratio as delta mu H decreases is similar in both cases. An increase of the delta Gp/delta mu H ratio above 4, the maximal accepted H+/ATP stoichiometry is thermodynamically incompatible with the delocalized protonic coupling model.     

Polarographic study of cell respiration in a tissue culture

A simple procedure for measuring the cells respiration without their transformation into the suspension, is suggested: seven glass slides with the cell culture of PKEV (pork, kidney, embryo, versen) are placed into the polarographic cell. Under these conditions the oxygen electrode easily records the fall of oxygen tension in the polarographic cell due to the respiration of 10(5) cells. Oxygen consumption is linear in time at the O2 concentration within the range from 480 to 200 nanoatoms per 1 ml of medium. The respiration rate is found to be enhanced by uncoupler and suppressed by the inhibitors of the electron flow chain (NaCN, antimycin A, amytal, rotenone) and mitochondrial ATPase (oligomycin and diciclohexylcarbodiimide). The above approach enables to evaluate the mitochondrial respiration of any cell monolayer keeping the cells intact.     

Control processes in oxidative phosphorylation: kinetic constraints and stoichiometry

Control processes in oxidative phosphorylation have been studied in three experimental models. (1) In isolated yeast mitochondria, external ATP is a regulatory effector of cytochrome-c oxidase activity. In phosphorylating or uncoupling states, the relationships between respiratory rate and delta mu H+, and the respiratory rate and cytochrome-c oxidase reduction level are dependent on this kinetic regulation. (2) In rat liver mitochondria, the response of the respiratory rate to uncoupler addition is age-dependent: liver mitochondria isolated from young rats maintain a greater delta mu H+ than liver mitochondria isolated from adults, with the same respiratory rate obtained with the same concentration of uncoupler. This behaviour is linked to redox proton pump properties, i.e., to the degree of intrinsic uncoupling induced by uncoupler addition. (3) The effect of almitrine, a new kind of ATPase/ATPsynthase inhibitor, was studied in mammalian mitochondria. (i) Almitrine inhibits oligomycin-sensitive ATPase - it decreases the ATPase/O value without any change in delta mu H+; (ii) almitrine increased the mechanistic H+/ATP stoichiometry of ATPase/ATPsynthase; (iii) almitrine-induced changes in H+/ATPase stoichiometry depend on the flux magnitude through ATPase. These results are discussed in terms of the following interdependent parameters; flux value, force, pump efficiency and control coefficient.     

Unique relationships between the rates of oxidation and phosphorylation and the protonmotive force in rat-liver mitochondria

The rate of ATP synthesis (JP) in isolated rat-liver mitochondria was strongly dependent on the magnitude of the protonmotive force (delta mu H+) across the mitochondrial inner membrane. Addition of different concentrations of various uncouplers or malonate to mitochondrial incubations in State 3 led to a depression of delta mu H+ and a concomitant decrease in JP. A unique relationship between JP and delta mu H+ was obtained, which was independent of the way in which delta mu H+ was varied. This unique relationship was observed when K+ (in the presence of valinomycin) was used as a probe for delta psi. Different relationships between JP and delta mu H+ were observed when K+ was used as a probe for delta psi and when K+ was measured after separation of the mitochondria by centrifugation without silicone oil. This led to a serious underestimation of delta psi, specifically when uncouplers were present, and non-unique flow-force relationships were thus obtained. Anomalous relationships between JP and delta mu H+ were also found when TPMP+ was used as a probe for delta psi. However, in uncoupler incubations the presence of TBP- strongly affected the TPMP+ accumulation ratio without any effect on the K+ accumulation or on JP and in the presence of TBP- unique relationships between JP and delta mu H+ were again obtained. This indicates that the accumulation of TPMP+ inside the mitochondria is not a straightforward function of delta psi but also depends on conditions like the presence of TBP- or uncouplers. We conclude that there is a unique relationship between the rate of phosphorylation and the protonmotive force in mitochondria and that under some conditions the behaviour of TPMP+ is anomalous.     

On the relationship between rate of ATP synthesis and H+ electrochemical gradient in rat-liver mitochondria

The relationship between rate of ATP synthesis, JATP, and value of the proton electrochemical gradient, delta mu H, has been analyzed in intact mitochondria. Onset of phosphorylation causes a depression of delta mu H of 1.5 kJ/mol. There is a close parallelism between inhibition of JATP and restoration of delta mu H to its state-4 value during titrations with oligomycin or atractyloside. Titrations with ionophores display the following features: (a) delta mu H can be depressed by 3-4 kJ/mol by valinomycin + K+ without affecting the rate of ATP synthesis; (b) uncouplers abolish JATP completely while depressing delta mu H by 3 kJ/mol; (c) complete abolition of ATP synthesis by inhibitors of electron transport is accompanied by a depression of delta mu H of only 1 kJ/mol. The results indicate that: (a) there is a close functional relationship between redox and ATPase H+ pumps, whereby inhibition of electron transfer is accompanied by simultaneous inhibition of the ATPase H+ pumps; and (b) uncoupling of oxidative phosphorylation is not due to depression of delta mu H per se. The consistence of the present data with either a chemiosmotic model where delta mu H is the sole and obligatory intermediate for energy coupling, or models where there is a direct transfer of energy between the two pumps is discussed.     

Characterization of a H+-ATPase in rat brain synaptic vesicles. Coupling to L-glutamate transport

Synaptic vesicles contain a H+-ATPase that generates a proton electrochemical gradient (delta mu H+) required for the uptake of neurotransmitters into the organelles. In this study, the synaptic vesicle H+-ATPase was examined for structural and functional similarities with other identified ATPases that generate a delta mu H+ across membranes. The synaptic vesicle H+-ATPase displayed immunological similarity with the 115-, 72-, and 39-kDa subunits of a vacuolar-type H+-ATPase purified from chromaffin granules. Functionally, the ATP-dependent H+ pumping across synaptic vesicles and ATP hydrolysis were sensitive to the sulfhydryl-modifying reagents, N-ethylmaleimide and 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, at concentrations known to affect vacuolar-type H+-ATPases. In addition, as with vacuolar-type H+-ATPases, the presence of NO3-, SO4(2-), or F- inhibited the generation of a delta mu H+, but addition of vanadate or oligomycin had no effect. The delta mu H+ is a function of the pH gradient (delta pH) and membrane potential (delta psi sv) across the synaptic vesicle. Acidification (delta pH) of the synaptic vesicle interior was enhanced in the presence of permeant anions, such as Cl-, or the K+ ionophore, valinomycin. In the absence of permeant anions, the H+-ATPase generated a delta psi sv that effected the transport of L-glutamate into the synaptic vesicles. Dissipation of delta psi sv by incubation with increased external Cl- or nigericin resulted in the abolition of glutamate uptake, despite the continued maintenance of a delta mu H+ across the synaptic vesicle as a substantial delta pH. The results suggest that the synaptic vesicle H+-ATPase is of a vacuolar type and energizes the uptake of anionic glutamate by virtue of the delta psi sv component of the delta mu H+ it generates.     

Methanogenesis and ATP synthesis in methanogenic bacteria at low electrochemical proton potentials. An explanation for the apparent uncoupler insensitivity of ATP synthesis

The rate of methane formation from H2 and CO2, the intracellular ATP content and the electrochemical proton potential (delta mu H+) were determined in cell suspensions of Methanobacterium thermoautotrophicum, which were permeabilized for K+ with valinomycin (1.2 mumol/mg protein). In the absence of extracellular K+ the cells formed methane at a rate of 4 mumol min-1 (mg protein)-1, the intracellular ATP content was 20 nmol/mg protein and the delta mu H+ was 200 mV (inside negative). When K+ was added to the suspensions the measured delta mu H+ decreased to the value calculated from the [K+]in/[K+]out ratio. Using this method of delta mu H+ adjustment, it was found that lowering delta mu H+ from 200 mV ([K+]in/[K+]out = 1000) to 100 mV ([K+]in/[K+]out = 40) had no effect on the rate of methane formation and on the intracellular ATP content. At delta mu H+ values below 100 mV ([K+]in/[K+]out less than 40) both the rate of methanogenesis and the ATP content decreased. Methanogenesis completely ceased and the ATP content was 2 nmol/mg when delta mu H+ was adjusted to values lower 50 mV ([K+]in/[K+]out less than 7). The data show that methanogenesis from H2 and CO2 and ATP synthesis in M. thermoautotrophicum are possible at relatively low electrochemical proton potentials. Similar results were obtained with Methanosarcina barkeri. Protonophoric uncouplers like 3,5,3',4'-tetrachlorosalicylanilide (TCS) or 3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile (SF 6847) were found not to dissipate delta mu H+ below 100 mV in M. thermoautotrophicum even when used at high concentrations (400 nmol/mg protein). This finding explains the observed uncoupler insensitivity of methanogenesis and ATP synthesis in this organism.     

Endogenous energy supply to the plasma membrane of dark aerobic cyanobacterium Anacystis nidulans: ATPase-independent efflux of H+ and Na+ from respiring cells

The ejection of protons from oxygen-pulsed cells and the gradients of Na+ concentration (Na+o/Na+i at 150 mM external NaCl) and proton electrochemical potential (delta mu H+) across the plasma membrane of Anacystis nidulans were studied in response to dark endogenous energy supply. Saturating concentrations of the F0F1-ATPase inhibitors dicyclohexylcarbodiimide (F0) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (F1) eliminated oxidative phosphorylation and lowered the ATP level from 2.6 +/- 0.15 to 0.7 +/- 0.1 nmol/mg dry wt while overall O2 uptake and delta mu H+ were much less affected. H+ efflux was inhibited only 60 to 75%. Aerobic Na+o/Na+i ratios (5.9 +/- 0.6) under these conditions remained 50% above the anaerobic level (2.1 +/- 0.2). Increasing concentrations of the electron transport inhibitors CO and KCN depressed H+ efflux and O2 uptake in parallel, with a pronounced discontinuity of the former at inhibitor concentrations, which reduced ATP levels from 2.6 to 0.8 nmol/mg dry wt, resulting in an abrupt shift of the apparent H+/O ratios from 4.0 +/- 0.3 to 1.9 +/- 0.2. Similarly, with KCN and CO the Na+o/Na+i ratios paralleled decreasing respiration rates more closely than decreasing ATP pool sizes. Ejection of protons also was observed when intact spheroplasts were pulsed with horse heart ferrocytochrome c or ferricyanide; the former reaction was inhibited, the latter was increased, by 1 mM KCN. Measurements of the proton motive force (delta mu H+) across the plasma membrane showed a strong correlation with respiration rates rather than ATP levels. It is concluded that the plasma membrane of intact A. nidulans can be directly energized by proton-translocating respiratory electron transport in the membrane and that part of this energy may be used by a Na+/H+ antiporter for the active exclusion of Na+ from the cell interior.     

Control of oxidative phosphorylation in rat heart mitochondria. The role of the adenine nucleotide carrier

Inhibitor titration experiments carried out with carboxyatractyloside, oligomycin and rotenone show that in the case of heart mitochondria the membrane-bound ATPase and the respiratory chain are the major factors controlling the rate of oxidative phosphorylation whereas the adenine nucleotide carrier exhibits no control strength. As shown by carboxyatractyloside titration curves under different conditions, the relative importance of the adenine nucleotide carrier depends on the mode of regeneration (F1-ATPase or glucose plus hexokinase) of ADP from ATP exported outside mitochondria, on the total concentration of adenine nucleotides present in the medium and on the mode of limitation of the rate of respiration (cyanide, rotenone, oligomycin or mersalyl). Concomitantly with the inhibition of O2 consumption, carboxyatractyloside brings about a rise in membrane potential. The inverse relationship between the two processes is observed for carboxyatractyloside concentrations ranging between 0.7 and 1.5 nmol per mg protein. Carboxyatractyloside concentrations below and above this range increase the membrane potential without affecting significantly the rate of respiration. Titration experiments aimed at comparing the effects of ADP, carboxyatractyloside and the uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, corroborate the conclusion that in heart mitochondria a major limiting factor in oxidative phosphorylation is the capacity of the respiratory chain.     

Depolarization of In Situ Mitochondria Due to Hydrogen Peroxide-Induced Oxidative Stress in Nerve Terminals

Mitochondrial membrane potential (delta psi(m)) was determined in intact isolated nerve terminals using the membrane potential-sensitive probe JC-1. Oxidative stress induced by H2O2 (0.1-1 mM) caused only a minor decrease in delta psi(m). When complex I of the respiratory chain was inhibited by rotenone (2 microM), delta psi(m) was unaltered, but on subsequent addition of H2O2, delta psi(m) started to decrease and collapsed during incubation with 0.5 mM H2O2 for 12 min. The ATP level and [ATP]/[ADP] ratio were greatly reduced in the simultaneous presence of rotenone and H2O2. H2O2 also induced a marked reduction in delta psi(m) when added after oligomycin (10 microM), an inhibitor of F0F1-ATPase. H2O2 (0.1 or 0.5 mM) inhibited alpha-ketoglutarate dehydrogenase and decreased the steady-state NAD(P)H level in nerve terminals. It is concluded that there are at least two factors that determine delta psi(m) in the presence of H2O2: (a) The NADH level reduced owing to inhibition of alpha-ketoglutarate dehydrogenase is insufficient to ensure an optimal rate of respiration, which is reflected in a fall of delta psi(m) when the F0F1-ATPase is not functional. (b) The greatly reduced ATP level in the presence of rotenone and H2O2 prevents maintenance of delta psi(m) by F0F1-ATPase. The results indicate that to maintain delta psi(m) in the nerve terminal during H2O2-induced oxidative stress, both complex I and F0F1-ATPase must be functional. Collapse of delta psi(m) could be a critical event in neuronal injury in ischemia or Parkinson's disease when H2O2 is generated in excess and complex I of the respiratory chain is simultaneously impaired.     

Further comments on the logic of the application of uncoupler- inhibitor titrations for the elucidation of the mechanisms of energy coupling

Following the appearance of two papers in this journal commenting on the logic of the application of uncoupler-inhibitor titrations as a means of discriminating between 'delocalized' and 'localized' chemiosmotic mechanisms [(1984) FEBS Lett. 176,79-82; (1985) FEBS Lett. 186, 8-10], and in contrast with the arguments presented there and elsewhere, we show that in a linear model the increase in delta mu H which accompanies partial inhibition of the ATPases always leads to a relatively higher decrease of the rate of ATP synthesis by a given concentration of uncoupler in the presence of an ATPase inhibitor than in its absence. This is due to the fact that the same titre of uncoupler leads to a higher dissipative H+ flow in the presence of inhibitor, since the driving force delta mu H is higher.     

Regulation of the heart mitochondrial respiration rate. Comparison of oxidation of succinate and NAD-dependent substrates]

Regulation of respiration at all rates between State 4 and State 3 was studied in heart mitochondria oxidizing FAD- and NAD-dependent substrates (succinate, pyruvate + + malate and palmitoylcarnitine). The creatine phosphokinase ADP-regenerating system was used which allows to fix the concentrations of extramitochondrial adenine nucleotides in such a way that the rate of respiration is controlled by mitochondrial processes alone. It was shown that respiration is controlled by delta mu(H+)-utilizing system within the respiration rate interval from State 4 till 70-80% of the maximal rate in State 3 (corresponding to physiological rates) both for NAD- and FAD-dependent substrates. The main step in the control of respiration near State 4 is proton leakage through the inner mitochondrial membrane, whereas in all the other parts of the mentioned interval this role is assigned to the adenine nucleotide translocator (ANT). The control coefficient for ANT is higher, while that of proton leakage is lower at the same relative rates of respiration with NAD-dependent substrates compared with succinate. These differences were found to be related to much higher values of the membrane potential generated at the same relative rates of succinate oxidation in comparison with the case with pyruvate + + malate. The contribution of delta mu(H+)-utilizing system to respiration control sharply decreases, whereas that of the delta mu(H+)-generating system increases at maximal rates of respiration near State 3. This phenomenon in more characteristic of succinate. In this case the control coefficient of ANT drops to zero, while that of succinate dehydrogenase rises to 0.7.     

氧化磷酸化抑制剂对光滑球拟酵母糖酵解速度的影响

研究了不同浓度电子传递链抑制剂 ( 鱼藤酮和抗霉素 A) 和 F_OF₁-ATPase 抑制剂 ( 寡霉素 ) 对光滑球拟酵母胞内 ATP 水平、葡萄糖消耗速度、糖酵解途径关键酶的影响 . 在培养液中添加 10 mg/L 鱼藤酮和抗霉素 A ,相对于对照组,胞内 ATP 分别下降了 43% 和 27.7% ,使糖酵解关键酶磷酸果糖激酶 (PFK) 的活性分别提高 340% 和 230% ,从而导致葡萄糖消耗速度增加 360% 和 240% ,丙酮酸生成速度提高了 17% 和 8.5%. 改变胞内 ATP 水平并不影响糖酵解途径其他关键酶 HK 、 PK 活性 . 微量的寡霉素 (0.05 mg/L) 可使胞内 ATP 含量下降 64.3% ,当培养液中寡霉素浓度达到 0.4 mg/L 时,细胞不能继续生长,葡萄糖消耗速度和丙酮酸的生成速度却随着寡霉素浓度 ( 小于 0.6 mg/L) 的增加而增加 . 表明氧化磷酸化途径中, ATPase 决定着 ATP 的生成 . 降低胞内 ATP 含量能显著提高 PFK 活性 (r²=0.9971) ,葡萄糖消耗速度 (r²= 0.9967) 以及丙酮酸生产速度 (r²= 0.965) ,葡萄糖消耗速度的增加是糖酵解途径中关键酶 PFK 活性 (r² = 0.9958) 和 PK 活性 (r²= 0.8706) 增加所导致的 . 这一结果有利于揭示真核微生物细胞中氧化磷酸化与中心代谢途径 ( 酵解 ) 的关系 .     

Control of oxidative phosphorylation in rat heart mitochondria: The role of the adenine nucleotide carrier

1. Inhibitor titration experiments carried out with carboxyatractyloside, oligomycin and rotenone show that in the case of heart mitochondria the membrane-bound ATPase and the respiratory chain are the major factors controlling the rate of oxidative phosphorylation whereas the adenine nucleotide carrier exhibits no control strength. 2. As shown by carboxyatractyloside titration curves under different conditions, the relative importance of the adenine nucleotide carrier depends on the mode of regeneration (F₁-ATPase or glucose plus hexokinase) of ADP from ATP exported outside mitochondria, on the total concentration of adenine nucleotides present in the medium and on the mode of limitation of the rate of respiration (cyanide, rotenone, oligomycin or mersalyl). 3. Concomitantly with the inhibition of O₂ consumption, carboxyatractyloside brings about a rise in membrane potential. The inverse relationship between the two processes is observed for carboxyatractyloside concentrations ranging between 0.7 and 1.5 nmol per mg protein. Carboxyatractyloside concentrations below and above this range increase the membrane potential without affecting significantly the rate of respiration. 4. Titration experiments aimed at comparing the effects of ADP, carboxyatractyloside and the uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, corroborate the conclusion that in heart mitochondria a major limiting factor in oxidative phosphorylation is the capacity of the respiratory chain.     

Proton electrochemical gradient and rate of controlled respiration in mitochondria

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.