Open-Loop Control of Oxidative Phosphorylation in Skeletal and Cardiac Muscle Mitochondria by Ca2+

In cardiac muscle, mitochondrial ATP synthesis is driven by demand for ATP through feedback from the products of ATP hydrolysis. However, in skeletal muscle at higher workloads there is an apparent contribution of open-loop stimulation of ATP synthesis. Open-loop control is defined as modulation of flux through a biochemical pathway by a moiety, which is not a reactant or a product of the biochemical reactions in the pathway. The role of calcium, which is known to stimulate the activity of mitochondrial dehydrogenases, as an open-loop controller, was investigated in isolated cardiac and skeletal muscle mitochondria. The kinetics of NADH synthesis and respiration, feedback from ATP hydrolysis products, and stimulation by calcium were characterized in isolated mitochondria to test the hypothesis that calcium has a stimulatory role in skeletal muscle mitochondria not apparent in cardiac mitochondria. A range of respiratory states were obtained in cardiac and skeletal muscle mitochondria utilizing physiologically relevant concentrations of pyruvate and malate, and flux of respiration, NAD(P)H fluorescence, and rhodamine 123 fluorescence were measured over a range of extra mitochondrial calcium concentrations. We found that under these conditions calcium stimulates NADH synthesis in skeletal muscle mitochondria but not in cardiac mitochondria.     

Electron Transport and Oxidative Phosphorylation in Arum Spadix Mitochondria

Arum spadix mitochondria exhibited a rapid cyanide-resistantoxygen uptake when oxidizing malate, NADH₂ or succinate, anda slower, cyanide-sensitive oxygen uptake when oxidizing ascorbate+tetramethylphenylenediamine(TMPD). Cytochrome oxidase does not therefore appear to functionas the terminal oxidase in the presence of cyanide, and therather low cytochrome c oxidase activity obtained using ascorbate+TMPDmay exclude it from possessing a major role even in the absenceof cyanide. ATP synthesis has been shown to accompany substrateoxidation. In the presence of antimycin A the P: O ratio accompanyingmalate oxidation was reduced by half, while phosphorylationaccompanying NADH₂ or succinate oxidation was almost completelyabolished. It is proposed that electrons from exogenous NADH₂enter the electron transport chain at a site after that whereendogenous NADH2 donates electrons and that electrons from exogenousNADH₂ are not coupled to ATP synthesis at site 1. The cyanide-resistant,non-phosphorylating electron-transport pathway may functionin the absence of cyanide and account for the low efficiencyof energy conservation observed in this tissue.     

Intermediate Reactions of Oxidative Phosphorylation in Mitochondria From Cabbage

Respiratory control ratios between 2.0 and 9.0 were obtained by comparison of the respiratory rates of cabbage mitochondria in the presence and in the absence of individual components of the system used to provide ADP and by comparing the rates before and after exhaustion of added ADP. These results indicate that respiration in cabbage mitochondria is controlled by the availability of ADP, which serves as the phosphate acceptor.Pentachlorophenol (PCP), 2,4-dinitrophenol (DNP), gramicidin and oleic acid inhibited phosphorylation to a greater extent than respiration in the cabbage mitochondria, but these reagents did not stimulate respiration in the absence of a phosphate acceptor. Respiration was stimulated by DNP only in the presence of added ATP.2,4-Dinitrophenol, pentachlorophenol, dicumarol and gramicidin did not stimulate ATPase activity either in the presence or absence of added Mg(2+). Oleic acid stimulated ATPase activity in the presence of added Mg(2+), but did not stimulate respiration even in the presence of added ATP.The ATP-(32)Pi exchange rate was increased many fold in the presence of added Mg(2+). Oleic acid and 2,4-dinitrophenol inhibited the exchange almost completely.     

Cold-Shock 310-kD Protein Uncouples Oxidative Phosphorylation in Plant Mitochondria

We studied the effects of cold-shock 310-kD protein (CSP310) isolated from winter rye seedlings on the energetic activity of plant mitochondria. CSP310 was shown to enhance nonphosphorylating respiration and uncoupled oxidative phosphorylation in isolated mitochondria. The uncoupling effect was enhanced with increasing protein concentration. An antibody against CSP310 interfered with the uncoupling effect of CSP310. Free fatty acids were not evidently involved in uncoupling. The physiological role of uncoupling between oxidation and phosphorylation during plant adaptation to low temperatures is discussed.     

Streptolydigin, an Inhibitor of Oxidative Phosphorylation in Rat Liver Mitochondria

Streptolydigin interferes with oxidative phosphorylation in rat liver mitochondria. The agent acts primarily as an uncoupler of respiration-associated phosphorylation but also impairs respiration to various degrees depending on the substrate. Streptolydigin partially inhibits electron flow at a point past the cytochrome b and prior to the cytochrome c reduction site. Streptolydigin also inhibits the function of the enzyme ribonucleic acid polymerase in whole bacterial cells and cell-free systems. The streptolydigin concentrations that cause effective inhibition of ribonucleic acid polymerase in cell-free systems are approximately 10 times less than those required to inhibit oxidative phosphorylation in mitochondria.     

Threshold Effects and Control of Oxidative Phosphorylation in Nonsynaptic Rat Brain Mitochondria

Abstract: The amount of control exerted by respiratory chain complexes in isolated nonsynaptic mitochondria prepared from rat brain on the rate of oxygen consumption was assessed using inhibitor titrations. Rotenone, myxothiazol, and KCN were used to titrate the activities of NADH:ubiquinone oxidoreductase (EC 1.6.5.3; complex I), ubiquinol:ferrocytochrome c oxidoreductase (EC 1.10.2.2; complex III), and cytochrome c oxidase (EC 1.9.3.1; complex IV), respectively. Complexes I, III, and IV shared some of the control of the rate of oxygen consumption in nonsynaptic mitochondria, having flux control coefficients of 0.14, 0.15, and 0.24, respectively. Threshold effects in the control of oxidative phosphorylation were demonstrated for complexes I, III, and IV. It was found that complex I activity could be decreased by ∼72% before major changes in mitochondrial respiration and ATP synthesis took place. Similarly, complex III and IV activities could be decreased by ∼70 and 60%, respectively, before major changes in mitochondrial respiration and ATP synthesis occurred. These results indicate that previously observed decreases in respiratory chain complex activities in some neurological disorders need to be reassessed as these decreases might not affect the overall capability of nonsynaptic mitochondria to maintain energy homeostasis unless a certain threshold of decreased complex activity has been reached. Possible implications for synaptic mitochondria and neurodegenerative disorders are also discussed.     

The Respiratory Chain of Plant Mitochondria. II. Oxidative Phosphorylation in Skunk Cabbage Mitochondria

Mitochondria were prepared from the spadices of skunk cabbage (Symplocarpus foetidus) whose respiratory rate with succinate and malate showed 15% to 30% sensitivity to cyanide inhibition, and which showed respiratory control by added ADP. The observed respiratory control ratios ranged from 1.1 to 1.4. The change in pH of the mitochondrial suspension was recorded simultaneously with oxygen uptake: alkalinization of the medium, expected for phosphorylation of ADP, coincided with the period of acceleration in oxygen uptake caused by addition of an ADP aliquot. The ADP/O ratios obtained were 1.3 for succinate and 1.9 for malate. In the presence of 0.3 mm cyanide, the ADP/O ratio for succinate was zero, while that for malate was 0.7. These results are consistent with the existence of an alternate oxidase which interacts with the flavoprotein and pyridine nucleotide components of the respiratory chain and which, in the presence of cyanide, allows the first phosphorylation site to function with an efficiency of about 70%. In the absence of respiratory inhibitors, the efficiency of each phosphorylation site is also about 70%. This result implies that diversion of reducing equivalents through the alternate oxidase, thereby bypassing the 2 phosphorylation sites associated with the cytochrome components of these mitochondria, occurs to a negligible extent during the oxidative phosphorylation of ADP or State 3.Addition of ADP or uncoupler to skunk cabbage mitochondria respiring in the controlled state or State 4, results in reduction of cytochrome c and the oxidation of the cytochromes b, ubiquinone and pyridine nucleotide. A site of interaction of ADP with the respiratory chain between cytochromes b and cytochrome c is thereby identified by means of the crossover theorem. Flavoprotein measured by fluorescence is also oxidized upon addition of ADP or uncoupler, but flavoprotein measured by optical absorbance changes becomes more reduced under these conditions. Depletion of the mitochondria by pretreatment with ADP and uncoupler prevents reduction of most of the fluorescent flavoprotein by succinate. These results indicate that skunk cabbage mitochondria contain both high and low potential flavo-proteins characterized by different fluorescence/absorbance ratios similar to those demonstrated to be part of the respiratory chain in mitochondria from animal tissues.     

The Regulation of Pyruvate Dehydrogenase Activity in Pea Leaf Mitochondria (The Effect of Respiration and Oxidative Phosphorylation)

The regulation of the pea (Pisum sativum) leaf mitochondrial pyruvate dehydrogenase complex by respiratory rate and oxidative phosphorylation has been investigated by measuring the respiratory activity, the redox poise of the quinone pool (Q-pool), and mitochondrial pyruvate dehydrogenase (mtPDC) activity under various metabolic conditions. It was found that, under state 4 conditions, mtPDC activity was unaffected by either the addition of succinate, 2-oxoglutarate, or glycine or the overall respiratory rate and redox poise of the Q-pool but was partially inhibited by NADH due to product inhibition. In the presence of ADP significant inactivation of PDC, which was sensitive to oligomycin, was observed with all substrates, apart from pyruvate, suggesting that inactivation was due to ATP formation. Inactivation of PDC by ADP addition was observed even in the presence of carboxyatractyloside, an inhibitor of the ATP/ADP translocator, suggesting that other mechanisms to facilitate the entry of adenylates, in addition to the adenylate carrier, must exist in plant mitochondria.     

ADP Protects Cardiac Mitochondria under Severe Oxidative Stress

ADP is not only a key substrate for ATP generation, but also a potent inhibitor of mitochondrial permeability transition pore (mPTP). In this study, we assessed how oxidative stress affects the potency of ADP as an mPTP inhibitor and whether its reduction of reactive oxygen species (ROS) production might be involved. We determined quantitatively the effects of ADP on mitochondrial Ca²⁺ retention capacity (CRC) until the induction of mPTP in normal and stressed isolated cardiac mitochondria. We used two models of chronic oxidative stress (old and diabetic mice) and two models of acute oxidative stress (ischemia reperfusion (IR) and tert-butyl hydroperoxide (t-BH)). In control mitochondria, the CRC was 344 ± 32 nmol/mg protein. 500 μmol/L ADP increased CRC to 774 ± 65 nmol/mg protein. This effect of ADP seemed to relate to its concentration as 50 μmol/L had a significantly smaller effect. Also, oligomycin, which inhibits the conversion of ADP to ATP by F₀F₁ATPase, significantly increased the effect of 50 μmol/L ADP. Chronic oxidative stress did not affect CRC or the effect of 500 μmol/L ADP. After IR or t-BH exposure, CRC was drastically reduced to 1 ± 0.2 and 32 ± 4 nmol/mg protein, respectively. Surprisingly, ADP increased the CRC to 447 ± 105 and 514 ± 103 nmol/mg protein in IR and t-BH, respectively. Thus, it increased CRC by the same amount as in control. In control mitochondria, ADP decreased both substrate and Ca²⁺-induced increase of ROS. However, in t-BH mitochondria the effect of ADP on ROS was relatively small. We conclude that ADP potently restores CRC capacity in severely stressed mitochondria. This effect is most likely not related to a reduction in ROS production. As the effect of ADP relates to its concentration, increased ADP as occurs in the pathophysiological situation may protect mitochondrial integrity and function.     

Disturbance of Oxidative Phosphorylation in the Mitochondria of Late Post-Traumatic Epileptic Nidus

We measured the rate of oxygen consumption by the mitochondria from the brain tissues of rabbits within a remote period after light cranio-cerebral trauma. One and six months after traumatization, oxidative phosphorylation in rabbits of the experimental groups demonstrated no significant difference from that in the control group. Yet, after a 12-month-long interval, clear differences were observed within the cortical zone with post-traumatic epileptic nidus. The coefficient of energy production decreased, and the process of oxidative phosphorylation became uncoupled. When succinate was used as a substrate for oxidation, we observed significant decreases in the rate of oxygen consumption in ADP phosphorylation and in the coefficient of respiration control. A significant decrease in the rate of oxygen consumption in the resting state (V ₂), the absence of disturbances in the respiration control, and preservation of a sufficient reserve ATPase activity were characteristic features when glutamate was used as a substrate. It seems probable that such shifts in oxidative phosphorylation can result in creation of an excessive glutamate pool and provide excessive epileptogenic glutamatergic activation of the neurons.     

Effect of Chlorophenol Analogues on the Oxidative Phosphorylation in Rat Liver Mitochondria

The inhibition of chlorophenol analogues on oxidative phosphorylation in rat liver mitochondria was studied using polarographic technique and some new findings that not only pentachlorophenol (PCP) but also other analogues inhibited the oxidative phoshorylation in a similar manner were made. The inhibitory activity was found to be roughly correlated with its dissociation constant of the inhibitor, PCP being the strongest, varying with the number and position of chlorine atoms in the molecule. The mode of the inhibition was classified into three types and discussed in detail.     

Studies of Respiratory Components and Oxidative Phosphorylation in Mitochondria of mi-1 Neurospora crassa

Oxidative phosphorylation has been demonstrated with mitochondria of the mi-1 respiratory mutant of Neurospora crassa. The P/O ratios observed with these mitochondria were approximately 0.8 with citrate and 0.4 with either externally added reduced nicotinamide adenine dinucleotide (NADH), succinate, or ascorbate-tetramethyl-p-phenylenediamine (TPD). These P/O ratios suggest that there are only two sites of phosphorylation in mitochondria isolated from young (20 to 24 h) cultures of the mi-1 mutant. The energy-dependent reduction of NAD(+) with succinate and the phosphorylation associated with ascorbate-TPD oxidation indicate that the first and the third sites of energy coupling are present in this mutant. Difference spectra of mitochondria from young cultures of the mi-1 mutant revealed the presence of cytochrome c. Cytochromes b and a + a(3) were not detected. However, in the presence of antimycin A, a small peak in the Soret region at 430 nm was observed. A carbon monoxide difference spectrum revealed the presence of a component of the respiratory chain with a spectrum similar to that of cytochrome o. It is of interest that respiratory inhibitors such as antimycin A, 2-n-nonylhydroxyquinoline N-oxide, and cyanide abolished phosphorylation but only partially inhibited oxidation. It is postulated that the mi-1 respiratory system contains two pathways of electron transport-the first is associated with a phosphorylating pathway, whereas the second is a non-phosphorylating electron transport pathway.     

The Inhibitory Effect of 3-Nitropropionate on the Oxidative Phosphorylation in Rat Mitochondria

The effect of 3-nitropropionate (3-NPA)on oxidative phosphorylation by using mitochondria prepared from both rat liver and brain were investigated in connection with the toxicity of this material. It was found that 3-NPA inhibited oxidative phosphorylation. In this inhibition, the uptake of inorganic phosphate was blocked but the oxygen uptake was not influenced at all. Furthermore, increase in ATPase activity of intact mitochondria was shown by the addition of 3-NPA. Results showed that 3-NPA disturbed oxidative phosphorylation as an uncoupler. However, the degree of inhibition by 3-NPA was not so high in comparison with other well-known uncouplers.

Thus the toxicity of 3-NPA is not due to the inhibition of oxidative phosphorylation. 3-NPA also does not affect on cytochrome oxidase activity.     

The Coupling Factor of Oxidative Phosphorylation from the Mitochondria of Mung Bean

The coupling factor F1 from the mitochondria of the mung bean etiolated seedlings had been isolated and preliminarily purified. The results showed that the coupling factor F1 we obtained had ATPase activity. The activity in the preliminary purifying preparation was about 54 times as high as that of ATPase activity of the original mitochondria, and the activity to hydrolyze ATP had reached 2.14 μmole/min/mg protein. The optimum pH of the coupling factor F1 from mung bean seedlings was about 8.5, and the optimum temperature was 45 ℃. The coupling factor F1 from mung bean mitochondria was cold labile. When the F1 was resolved from the mitoehondria inner-membrane and was in soluble form, it lost the sensitivety to DCCD. The coupling factor F1 of mung bean mitoehondria was Mg++-dependent, and it was activated by DNP, but the activation by Ca++, NaCl and KC1 were not observed. The molecular weight of the coupling factor F1 was about 380,000 as shown by gel electrophorsis.     

The Effect of Temperature on Oxidative Phosphorylation with Insect Flight Muscle Mitochondria

The effect of different temperatures on the biochemical activity and morphology of insect flight muscle mitochondria was examined. It was found that respiration and phosphorylation have the same thermal response at temperatures of 25°C. and below. The energy of activation for both systems is approximately 12,300 calories. Oxidation and phosphorylation can be uncoupled effectively by temperature, for at temperatures above 25°C. there is more rapid heat inactivation of phosphorylation. This is evident from reduced P/O values as well as from morphological deterioration in the mitochondrial population. The thermal response of both this sarcosomal enzyme system and the respiration in the living fly are quantitatively similar.     

Response of Isolated Rat Liver Mitochondria to Variation of External Osmolarity in KCl Medium: Regulation of Matrix Volume and Oxidative Phosphorylation

When isolated rat liver mitochondria are incubated in KCl medium, matrix volume, flux, and forces in both hypo- and hyperosmolarity are time-dependent. In hypoosmotic KCl medium, matrix volume is regulated via the K⁺/H⁺ exchanger. In hyperosmotic medium, the volume is regulated in such a manner that at steady state, which is reached within 4 min, it is maintained whatever the hyperosmolarity. This regulation is Pi- and -dependent, indicating Pi-K salt entry into the matrix. Under steady state, hyperosmolarity has no effect on isolated rat liver mitochondria energetic parameters such as respiratory rate, proton electrochemical potential difference, and oxidative phosphorylation yield. Hypoosmolarity decreases the NADH/NAD⁺ ratio, state 3 respiratory rate, and , while oxidative phosphorylation yield is not significantly modified. This indicates kinetic control upstream the respiratory chain. This study points out the key role of potassium on the regulation of matrix volume, flux, and forces. Indeed, while matrix volume is regulated in NaCl hyperosmotic medium, flux and force restoration in hyperosmotic medium occurs only in the presence of external potassium.     

Effect of Pyrrolnitrin on Electron Transport and Oxidative Phosphorylation in Mitochondria Isolated from Neurospora crassa

Pyrrolnitrin, at low concentrations, uncouples oxidative phosphorylation in Neurospora mitochondria. At higher concentrations, pyrrolnitrin inhibits electron transport both in the flavine region and through cytochrome oxidase.