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.     

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 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.     

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.     

Oxidative Phosphorylation and the Response to an Osmotic Treatment of Liver Mitochondria of Rats Fed an Amino Acid Imbalanced Diet

Fatty livers were produced by feeding rats an amino acid imbalanced diet containing 8% of casein supplemented with 0.3% of DL-methionine, and the oxidative phosphorylation of liver mitochondria and homogenates was examined. In contrast to choline deficiency, the mitochondria and the homogenates of fatty liver resulting from such an amino acid imbalance showed almost normal P:O ratio. However, the response to an osmotic treatment of liver mitochondria of rats fed the amino acid imbalanced diet was different from that of normal rats.     

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.     

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.     

Circadian Rhythms of Oxidative Phosphorylation: Effects of Rotenone and Melatonin on Isolated Rat Brain Mitochondria

Mitochondrial experiments are of increasing interest in different fields of research. Inhibition of mitochondrian activities seems to play a role in Parkinson's disease and in this regard several animal models have used inhibitors of mitochondrial respiration such as rotenone or MPTP. Most of these experiments were done during the daytime. However, there is no reason for mitochondrial respiration to be constant during the 24h. This study investigated the circadian variation of oxidative phosphorylation in isolated rat brain mitochondria and the administration-time-dependent effect of rotenone and melatonin. The respiratory control ratio, state 3 and state 4, displayed a circadian fluctuation. The highest respiratory control ratio value (3.01) occurred at 04:00h, and the lowest value (2.63) at 08:00h. The highest value of state 3 and state 4 oxidative respiration occurred at 12:00h and the lowest one at 20:00h. The 24h mean decrease in the respiratory control ratio following incubation with melatonin and rotenone was 7 and 32%, respectively; however, the exact amount of the inhibition exerted by these agents varied according to the time of the mitochondria isolation. Our results show the time of mitochondrial isolation could lead to interindividual variability. When studies require mitochondrial isolation from several animals, the time between animal experiments has to be minimized. In oxidative phosphorylation studies, the time of mitochondria isolation must be taken into account, or at least specified in the methods section.     

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.     

大鼠肝线粒体中的脂肪酸分析

用双 2 乙基己基酚酞酸酯 (DEHP)诱导大鼠肝过氧化物酶体增殖 ,然后用蔗糖密度梯度离心法分离大鼠肝线粒体 ,用毛细管气相色谱法测定肝线粒体中的脂肪酸含量。测定结果 :所测 1 4种脂肪酸的总量 ,青年正常组大于青年诱导组 (P <0 .0 1 ) ,青年正常组大于老年正常组 (P <0 .0 5 )。不饱和脂肪酸与脂肪酸总量的比例 ,老年诱导组大于老年正常组 (P <0 .0 5 ) ,青年正常组大于老年正常组 (P <0 .0 5 )。长链脂肪酸与脂肪酸总量的比例 ,老年正常组小于老年诱导组 (P <0 .0 5 )。结果表明 ,用DEHP诱导大鼠肝过氧化物酶体增值 ,影响肝线粒体脂肪酸正常代谢 ,使线粒体膜结构发生变化 ,这种变化 ,青年鼠与老年鼠不同     

Feedback Regulation and Time Hierarchy of Oxidative Phosphorylation in Cardiac Mitochondria

To determine how oxidative ATP synthesis is regulated in the heart, the responses of cardiac mitochondria oxidizing pyruvate to alterations in [ATP], [ADP], and inorganic phosphate ([P_i]) were characterized over a range of steady-state levels of extramitochondrial [ATP], [ADP], and [P_i]. Evolution of the steady states of the measured variables with the flux of respiration shows that: (1) a higher phosphorylation potential is achieved by mitochondria at higher [P_i] for a given flux of respiration; (2) the time hierarchy of oxidative phosphorylation is given by phosphorylation subsystem, electron transport chain, and substrate dehydrogenation subsystems listed in increasing order of their response times; (3) the matrix ATP hydrolysis mass action ratio [ADP] × [P_i]/[ATP] provides feedback to the substrate dehydrogenation flux over the entire range of respiratory flux examined in this study; and finally, (4) contrary to previous models of regulation of oxidative phosphorylation, [P_i] does not modulate the activity of complex III.     

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.     

Study of DNA-Binding Proteins of Rat Liver Mitochondria

Acid-soluble proteins able to form DNA-protein complexes in the presence of physiological concentration of NaCl were isolated from rat liver mitochondria. Electrophoretic analysis of these proteins in 15% polyacrylamide gel showed that mitochondrial acid-soluble proteins include of approximately 20 polypeptides with molecular weight of 10–120 kDa. The fraction of acid-soluble proteins can be separated into basic and acidic proteins by chromatography on DEAE cellulose. Some of acidic proteins are tightly bound to the basic proteins and can be separated from them in the presence of 5 mM dithiothreitol. It is discovered that the fraction of acidic proteins contains proteases (including DNA-activated ones), which cleave different polypeptides of the basic proteins with different efficiency. Possibly, mitochondrial DNA-binding proteins and DNA-activated proteases are involved in the regulation of structural organization and functional activity of mitochondrial DNA.     

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.     

Absence of Nitric-oxide Synthase in Sequentially Purified Rat Liver Mitochondria

Data, both for and against the presence of a mitochondrial nitric-oxide synthase (NOS) isoform, is in the refereed literature. However, irrefutable evidence has not been forthcoming. In light of this controversy, we designed studies to investigate the existence of the putative mitochondrial NOS. Using repeated differential centrifugation followed by Percoll gradient fractionation, ultrapure, never frozen rat liver mitochondria and submitochondrial particles were obtained. Following trypsin digestion and desalting, the mitochondrial samples were analyzed by nano-HPLC-coupled linear ion trap-mass spectrometry. Linear ion trap-mass spectrometry analyses of rat liver mitochondria as well as submitochondrial particles were negative for any peptide from any NOS isoform. However, recombinant neuronal NOS-derived peptides from spiked mitochondrial samples were easily detected, down to 50 fmol on column. The protein calmodulin (CaM), absolutely required for NOS activity, was absent, whereas peptides from CaM-spiked samples were detected. Also, l-[¹⁴C]arginine to l-[¹⁴C]citrulline conversion assays were negative for NOS activity. Finally, Western blot analyses of rat liver mitochondria, using NOS (neuronal or endothelial) and CaM antibodies, were negative for any NOS isoform or CaM. In conclusion, and in light of our present limits of detection, data from carefully conducted, properly controlled experiments for NOS detection, utilizing three independent yet complementary methodologies, independently as well as collectively, refute the claim that a NOS isoform exists within rat liver mitochondria.Nitric oxide (NO^·)² is a highly diffusible, hydrophobic, and gaseous free radical (1) that is responsible for autocrine and paracrine signaling activities (2). NO^· can readily partition into and through membranes (3–5) to influence biological functions such as blood pressure regulation, platelet aggregation and adhesion, neurotransmission, and cellular defense (4, 6–11). The mechanism by which NO^· influences biological functions is by binding to target proteins that contain heme and/or thiol(s). Alternatively, NO^· can combine with to produce the highly reactive species peroxynitrite.Mitochondria are highly compartmentalized, membranous organelles that contain abundant amounts of reactive hemoproteins and thiols (12, 13), to which NO^· may bind reversibly (14, 15) or irreversibly (16–18). Mitochondria also generate various amounts of during the process of cellular respiration (19, 20). Studies conducted during the past decade have suggested that NO^· can diffuse into mitochondria and cause mitochondrial dysfunction by reversibly inhibiting cytochrome c oxidase (14, 21, 22) and NADH dehydrogenase (23).In the mid-90s, a putative variant of NOS was proposed to reside within mitochondria. Initially, Kobzik et al. (24) and Hellsten and co-workers (25) observed an apparent endothelial NOS (eNOS) immunoreactivity in skeletal muscle mitochondria. Simultaneously, Bates et al. (26, 27) observed an apparent eNOS histochemical reactivity in inner mitochondrial membrane preparations, isolated from rat liver, brain, heart, skeletal muscle, and kidney. Tatoyan and Giulivi (28), acting on these initial observations, performed experiments in an attempt to confirm the identity of this putative mtNOS. Relying on immunochemical analysis, Tatoyan and Giulivi (28) claimed that inducible NOS (iNOS) was the NOS isoform present in rat liver mitochondria. This same group using mass spectrometry later presented data in support of the putative mtNOS being a variant of nNOS (29). Ghafourifar and Richter (30) had reported previously that the putative mtNOS was calcium-sensitive and constitutive in nature. Since these reports, different groups have reported the presence of each of the three main isoforms of NOS within mitochondria (29, 31, 32). Also, biochemical characterization of the putative mtNOS performed by Giulivi and co-workers (29) revealed certain post-translational modifications (myristoylation and phosphorylation of the protein) that are thought to be unique to eNOS. During the last decade, various reports have supported the presence of at least one of the three main isoforms of NOS residing in mitochondria. However, the more recent reports tend to question this claim (33–36). Because of the contradictory reports regarding the existence of a putative mtNOS, Brookes (33) compiled a critical and thorough review of the literature published up to 2003 dealing with the putative mtNOS. This review brought to light the diverse technical issues involved in the aforementioned studies. Major issues were the degree of purity of mitochondrial preparations (37, 38), shortcomings of measurement methodology (29, 39–41), use of inappropriate, or total lack of, experimental controls and confusing technical practices. Lacza et al. (42) has reviewed the more recent developments in the area of mitochondrial NO^· production and discussed some of the shortcomings of certain techniques still being used.In light of this ongoing controversy regarding the presence or absence of a mtNOS, we designed and carefully conducted properly controlled studies to either confirm or refute the existence of any NOS isoform within mitochondria. Ultrapure rat liver mitochondria were isolated using repeated differential centrifugation followed by Percoll gradient purification. Proteomic analyses were then performed using a nano-HPLC-coupled nanospray LTQ-MS. To avoid the interfering factors that are rampant in NO^· trapping assays (43), the NOS-catalyzed conversion of l-[¹⁴C]arginine to l-[¹⁴C]citrulline was used to probe for NOS activity in mitochondria. Appropriate controls were employed and, for inhibition studies, high concentrations of l-thiocitrulline (TC) (44) were used. Additionally, immunochemical analyses were performed with ultrapure mitochondria using nNOS, eNOS, and CaM antibodies. The problems faced with the commonly used techniques in mtNOS studies are discussed.     

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.     

nHAP对大鼠肝线粒体的生物活性作用

为了探讨羟基磷灰石纳米粒子(nHAP)对大鼠肝线粒体生物活性的影响,将nHAP直接作用于线粒体,在不同浓度和时间下测定线粒体标志酶琥珀酸脱氢酶(SDH)比活性,并与对照组进行比较。结果显示,当nHAP中水含量在10%以下时,线粒体生物活性未发现改变;当nHAP浓度递增时,在等时间段内,对线粒体SDH比活性呈逐步抑制作用;在不等时间段内,nHAP对线粒体SDH比活性的抑制作用与对照组相比较差异有显著性(p<0.05)。因此,nHAP对线粒体SDH比活性的抑制有浓度和时间的依赖性。