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1.
Control of oxidation is the key mechanism in the regulation of energy metabolism. In glycolysis the oxidation of glyceraldehyde-3-phosphate is controlled by DPNH, which inhibits glyceraldehyde-3-phosphate dehydrogenase. In oxidative phosphorylation the inhibition of electron flow from DPNH to oxygen, called "respiratory control," is the subject of this paper. After a discussion of the physiological significance of the "tight coupling" between phosphorylation and oxidation, studies on "loosely coupled" submitochondrial particles are reported. These particles are capable of oxidative phosphorylation in the presence of a suitable phosphate acceptor system, but in contrast to controlled, intact mitochondria they oxidize DPNH in the absence of phosphate and ADP. The addition of o-phenanthroline to submitochondrial particles gives rise to an inhibition of respiration, which is partly reversed by phosphate and ADP or by dinitrophenol. The properties of this model system of respiratory control will be described.  相似文献   

2.
In order to measure the parameters of oxidative phosphorylation it is necessary to isolate physiologically intact mitochondria. The isolation of rat liver mitochondria by rate zonal centrifugation utilizing isoosmotic Ficoll gradients resulted in the uncoupling of oxidative phosphorylation in these organelles. Analysis of the Ficoll solutions used to construct the gradients indicated that the Ca2+ content (200–400 nmole Ca2+/mg protein) was sufficiently high to cause an uncoupling of oxidative phosphorylation. Treatment of the Ficoll solutions with Amberlite MB-3 resin reduced the Ca2+ content to levels below the limit of determination of the assay procedure. This resulted in the retention of respiratory control (1.42) in rate-zonally centrifuged mitochondria. The addition of bovine serum albumin (100 mg%) to the Ficoll gradients increased the respiratory control index to 2.10. The increase is due to an elevation in state 3 respiration rather than any change in state 4 respiration. The addition of 200 mg% bovine serum albumin to the Ficoll gradient did not further enhance the respiratory control index.Examination of subpopulations of rat liver mitochondria revealed that they are heterogeneous with regard to states 3 and 4 respiration, respiratory control indices, and ADP:O ratios. In mitochondrial subpopulations respiratory control indices ranged from 1.00 to 4.13 and ADP:O ratios ranged from 1.22 to 1.83. This investigation defined a procedure for the isolation of physiologically intact mitochondria from rat liver homogenates.  相似文献   

3.
A preparation that contained cytochrome c, mainly in the form of its ;dimer', was studied and compared with native cytochrome c with respect to its ability to support electron transfer and energy transformation in cytochrome c-depleted rat liver mitochondria. When the depleted mitochondria were titrated with either cytochrome c or the ;dimer', the extent of coupling between respiration and phosphorylation was enhanced, as manifested by an increase in the P/O ratio. The ;dimer' was relatively ineffective as an electron carrier in the respiratory system, but it was as effective as cytochrome c in reconstitution of oxidative phosphorylation in depleted mitochondria. Addition of ;dimer' to the depleted mitochondria, in the presence of a low, non-saturating concentration of cytochrome c, increased the P/O ratio without concomitant stimulation of respiration. Both cytochrome c and the ;dimer' stimulated spontaneous swelling and electron transport-driven proton translocation in depleted mitochondria. The pattern of action of cytochrome c and its ;dimer' is in accord with the assumption that they affect an early step in energy conservation.  相似文献   

4.
Energy coupling parameters of liver mitochondria from hibernating and arousing ground squirrels have been studied. In the oligomycin-treated mitochondria, carboxyatractylate, an inhibitor of the ATP/ADP-antiporter, is shown to decrease the respiration rate, to increase the membrane potential and to lower the rate of the membrane-potential discharge after the addition of cyanide to liver mitochondria from hibernating and arousing animals. BSA effectively substitutes for carboxyactactylate so that carboxyactactylate, added after BSA, has no effect. In mitochondria from hibernating animals, the maximal respiration rate in the presence of DNP and the rate of the membrane potential discharge in its absence are much lower than in those from arousing animals. It has been concluded that upon arousal of the animals from hibernation, the uncoupling of oxidative phosphorylation, mediated by free fatty acids and ATP/ADP-antiporter, parallels the respiratory chain activation.  相似文献   

5.
High concentrations of respiration inhibitors are known to sharply decrease the membrane potential in mitochondria. The effect of relatively low concentrations of oxidative phosphorylation inhibitors on the value of membrane potential of intact mitochondria and on the rate of respiration and phosphorylation as well was studied. It has been found that within a certain concentration range the inhibitors of oxidative phsophorylation--malonic acid, sodium cyanide m-chlorophenyl hydrozonecarbonylcyanide, sharply decrease the phosphorylation rate (by 70 divided by 90%) but do not practically a affect the membrane potential value of intact mitochondria in the state 3 according to Chance.  相似文献   

6.
The transmembrane potential (delta psi) of rabbit brain mitochondria was measured with the fluorescent dye dis--C2--5. During oxidative phosphorylation a fall in delta psi in the order of 20% was observed. In the presence of inhibitors of ATP synthesis, there was a good correlation between the fall in delta psi and the ADP-stimulated increase in respiration rate. The influence of endogenous calcium on the energetic metabolism of mitochondria was studied by measuring the changes of delta psi. An amount of 12 nmol Ca2+/mg protein cause half-inhibition of the ATP synthesis rate; 50 nmol/mg completely inhibits oxidative phosphorylation. The effect of the Ca2+ load on the ATPase activity of intact mitochondria was studied. It was found that endogenous calcium inhibits in a similar degree synthesis and hydrolysis of ATP. It was shown that both Ca ATP and Mg ATP can serve as a substrate for the mitochondrial ATPase.  相似文献   

7.
It has been found that mitochondria isolated from the flight muscle of the housefly, Musca domestica, are capable of effecting oxidative phosphorylation. A systematic investigation of the factors which regulate this coupling was undertaken. It was found: 1. The molarity of the isolation medium had considerable influence on the morphology of the mitochondria. These physical alterations were associated with changes in oxidation, phosphorylation, and ATPase activity. 2. In addition to an optimum isolation medium, the normal morphology of the mitochondria needed to be further stabilized by serum albumin. 3. A "latent" ATPase activity in insect mitochondria was demonstrated. An inverse relationship was found between oxidative phosphorylation and ATPase activity. 4. Oxygen consumption and the uptake of phosphate were linear with respect to time. 5. A respiratory substrate was necessary for phosphorylation and for maintenance of spatially organized mitochondria. 6. No differences in oxygen uptake were found in the presence or absence of inorganic phosphate. 7. Magnesium was required for optimal oxidative phosphorylation. Calcium and manganese inhibited both respiration and phosphorylation. 8. The addition of cytochrome c had no effect on either oxygen or phosphate uptake. 9. ATP, ADP, or AMP were capable of participating in oxidative phosphorylation, but the glucose-hexokinase trapping system was necessary. 10. Fluoride inhibited the phosphorylation of AMP, but increased P/O when ATP was used. This stimulation was not due to the inhibition of ATPase. 11. Neither arginine nor creatine was phosphorylated. 12. The addition of other isolated fractions of flight muscle to the mitochondrial system had no appreciable effect on respiration or phosphorylation.  相似文献   

8.
On the basis of polarographic data it is shown that protamine has a biphasic effect on the respiration of intact mitochondria. At lower protamine concentrations respiration is stimulated and this combined with a decrease of the respiratory control index; at higher ones respiration is inhibited and respiratory control is lost. In cytochrome c-depleted and restored mitochondria protamine effect on oxidative phosphorylation is only inhibitory. Increasing cytochrome c concentrations restore respiration in protamine-treated cytochrome c depleted mitochondria but not the respiratory control. Binding of cytochrome c to mitochondria is studied by determining from Scatchard plots the number of high affinity binding sites (n) and their stability constants (K). In absence of protamine in intact mitochondria n = 2.7 and K = 4.67-10(6) M-1; in cotochrome c depleted mitochondria n = 4.7 and K = 5.16-10(6) M-1. In both types of mitochondria protamine decreases significantly n as well as K. These data show that protamine may affect oxidative phosphorylation by causing desorption of cytochrome c from the inner mitochondrial membrane.  相似文献   

9.
The effect of heliomycin and known uncouplers of oxidative phosphorylation on respiration and oxidative phosphorylation was studied comparatively. Heliomycin, as well as 2,4-dinitrophenol, valinomycin and gramicidin S inhibited the mitochondrial synthesis of ATP. This process was inhibited completely by heliomycin at a concentration of 1.5 x 10(-5) M. The synthesis of inorganic pyrophosphate, the other macroergic compound, was also inhibited by heliomycin, ATPase and pyrophosphatase of uncoupled mitochondria being not inhibited by the antibiotic. Like 2,4-dinitrophenol, heliomycin stimulated the synthesis of ATPase and respiration in intact mitochondria. Probably, heliomycin inhibited the synthesis of ATP and pyrophosphate by uncoupling the processes of respiration and oxidative phosphorylation. It was shown earlier that heliomycin, a specific inhibitor of bacterial RNA synthesis, also affected energy metabolism of bacterial cells by inhibiting the synthesis of ATP and active transport.  相似文献   

10.
The bioenergetic interaction between glycolysis and oxidative phosphorylation in isolated nerve terminals (synaptosomes) from guinea-pig cerebral cortex is characterized. Essentially all synaptosomes contain functioning mitochondria. There is a tight coupling between glycolytic rate and respiration: uncoupler causes a tenfold increase in glycolysis and a sixfold increase in respiration. Synaptosomes contain little endogenous glycolytic substrate and glycolysis is dependent on external glucose. In glucose-free media, or following addition of iodoacetate, synaptosomes continue to respire and to maintain high ATP/ADP ratios. In contrast to glucose, the endogenous substrate can neither maintain high respiration in the presence of uncoupler nor generate ATP in the presence of cyanide. Pyruvate, but not succinate, is an excellent substrate for intact synaptosomes. The in-situ mitochondrial membrane potential (delta psi m) is highly dependent upon the availability of glycolytic or exogenous pyruvate; glucose deprivation causes a 20-mV depolarization, while added pyruvate causes a 6-mV hyperpolarization even in the presence of glucose. Inhibition of pyruvate dehydrogenase by arsenite or pyruvate transport by alpha-cyano-4-hydroxycinnamate has little effect on ATP/ADP ratios; however respiratory capacity is severely restricted. It is concluded that synaptosomes are valuable models for studying the control of mitochondrial substrate supply in situ.  相似文献   

11.
1. Uncoupled oxidative phosphorylation in isolated guinea pig brown-adipose-tissue mitochondria is reflected by a low phosphorylation state of adenosine phosphates in the mitochondrial matrix and in the extramitochondrial space during oxidation of succinate or glycerol 1-phosphate in the presence of serum albumin and 100 muM ADP. Recoupling of respiration and phosphorylation in the mitochondria is indicatdd by a dramatic increase in the phosphorylation state of adenine nucleotides in both compartments, when substrates inducing substrate level phosphorylation are respired. In this case ATP/ADP ratios in the extramitochondrial compartment are 10-15 times higher than in the mitochondrial matrix. 2. Recoupling mediated by substrate level phosphorylation depends on the presence of extramitochondrial adenosine phosphate and on intact adenine nucleotide translocation. In the presence of substrate level phosphorylation the amount of extramitochondrial ADP required to restore energy coupling can be extremely low (20 muM ADP or 10 nmol ADP/mg mitochondrial protein respectively). If substrate level phosphorylation is prevented by rotenone or in the presence of atractyloside, 20-50 times higher amounts of extramitochondrial adenine nucleotides are necessary to cause coupled oxidative phosphorylation. The recoupling effect of ATP is significantly stronger than that of ADP. 3. GDP (100 muM) causes a rapid increase of the ATP/ADP ratio in both compartments which is independent of substrate level phosphorylation as well as of the extramitochondrial adenosine phosphate concentration and the adenine nucleotide carrier. 4. The amount of extramitochondrial adenosine phosphate in guinea pig brown-adipose-tissue (18 nmol/mg mitochondrial protein or 2.5 mM respectively) would suffice for recoupling of oxidative phosphorylation mediated by substrate level phosphorylation under conditions in vitro; this suggests that substrate level phosphorylation is of essential importance in brown fat in vivo with respect to energy conditions in the tissue during different states of thermogenesis.  相似文献   

12.
The effect of 2,4-DNP and malonate on tissue-specific uncoupling of oxidative phosphorylation (OP) of rat liver and kidney mitochondria by homologous comutons has been studied. The addition of 2,4-DNP in the presence of comuton induced beta state of comuton regulation. Transfer of liver mitochondria from alpha to beta state also resulted from partial inhibition of succinate dehydrogenase activity of addition of 0.25-0.35 mM malonate. This suggests that the transfer to beta state may be caused by de-energization of mitochondria.  相似文献   

13.
Preincubation of liver mitochondria (Mch) with Ca2+ ions at inorganic phosphate concentration less than I mM in the presence of liver cell soluble phase (CSP) induced rotenone-independent tissue-specific uncoupling of oxidative phosphorylation (beta state of comuton regulation) and rotenone-stimulated tissue-specific uncoupling (gamma state of comuton regulation). The reduction in K+ ion concentration in the incubation medium entirely inhibited the induction of beta state. Tissue-specific stimulation of the rat liver Mch respiration in substrate-containing medium was increased after rotenone addition. Ruthenium red was added to the medium before and after the end of Mch preincubation with Ca2+ in the presence of CSP. The results suggest that limited Ca2+ transport in Mch is necessary for the induction of beta and gamma states of comuton regulation. Ca2+ ejected from Mch also participates in the induction of beta state of comuton regulation. Comuton receptor on the mitochondrial membrane surface is devoid of glyco- and mucoprotein components bound by ruthenium red.  相似文献   

14.
We have examined the ultrastructure of mitochondria as it relates to energy metabolism in the intact cell. Oxidative phosphorylation was induced in ultrastructurally intact Ehrlich ascites tumor cells by rapidly generating intracellular adenosine diphosphate from endogenous adenosine triphosphate by the addition of 2-deoxyglucose. The occurrence of oxidative phosphorylation was ascertained indirectly by continuous and synchronous monitoring of respiratory rate, fluorescence of pyridine nucleotide, and 90° light-scattering. Oxidative phosphorylation was confirmed by direct enzymatic analysis of intracellular adenine nucleotides and by determination of intracellular inorganic orthophosphate. Microsamples of cells rapidly fixed for electron microscopy revealed that, in addition to oxidative phosphorylation, an orthodox → condensed ultrastructural transformation occurred in the mitochondria of all cells in less than 6 sec after the generation of adenosine diphosphate by 2-deoxyglucose. A 90° light-scattering increase, which also occurs at this time, showed a t ½ of only 25 sec which agreed temporally with a slower orthodox → maximally condensed mitochondrial transformation. Neither oxidative phosphorylation nor ultrastructural transformation could be initiated in mitochondria in intact cells by the intracellular generation of adenosine diphosphate in the presence of uncouplers of oxidative phosphorylation. Partial and complete inhibition of oxidative phosphorylation by oligomycin resulted in a positive relationship to partial and complete inhibition of 2-deoxyglucose-induced ultrastructural transformation in the mitochondria in these cells. The data presented reveal that an orthodox → condensed ultrastructural transformation is linked to induced oxidative phosphorylation in mitochondria in the intact ascites tumor cell.  相似文献   

15.
Effects of low and high 25 and 100 micrograms per 100 g of body weight doses of adrenaline on respiration and oxidative phosphorylation in rat liver mitochondria are compared. The high dose of adrenaline is shown to decrease activation of respiration and phosphorylation typical of the low doses. This decrease is caused by inhibition of succinate dehydrogenase and is accompanied by uncoupling of respiration and phosphorylation in mitochondria.  相似文献   

16.
In vivo thyroid hormones control the binding to mitochondria of low molecular weight water-soluble cytoplasmic mediators that are capable to induce oxidative phosphorylation uncoupling, by increasing the sensitivity of mitochondria to the effects of these mediators. In hyperthyroid rat liver mitochondria cytoplasmic mediators stimulate the phosphate-dependent transport of K+ and H+ in a greater degree than in liver mitochondria of control rats. The increase in the oxidative phosphorylation uncoupling by cytoplasmic mediators is one of mechanisms of thermogenesis stimulation by thyroid hormones.  相似文献   

17.
Nitrite incubation in mitochondria and nitrate intoxication of rats have been studied for their effect on aerobic energetic processes in the liver. Sodium nitrite in concentration of 2 mg/l causes an inhibition of ADP-stimulated respiration and provides uncoupling processes of oxidative phosphorylation and respiration in mitochondria, when adding succinate as a substrate. Low doses of nitrate in vivo promote oxygen economization in mitochondria. Intoxication of rats with nitrate in a dose of 50 mg/l for 30 days induces a decrease of the respiration rate after ADP-phosphorylation and an increase of the coefficient of oxidative phosphorylation efficiency (ADP/O). Intraperitoneal administration of adrenalin in a dose of 25 micrograms/100 g to rats after 30-day nitrate intoxication in a concentration of 10 mg/l induces no typical increase of ADP-stimulated respiration and rate of oxidative phosphorylation and succinate oxidation.  相似文献   

18.
The regulation of oxidative phosphorylation was studied with digitonin-treated epididymal bull spermatozoa in which mitochondria are directly accessible to low molecular compounds in the extracellular medium. Due to the high extramitochondrial ATPase activity in this cell preparation, it was possible to stimulate respiration to a small extent only by added hexokinase in the presence of glucose and adenine nucleotides. Added pyruvate kinase plus phosphoenol pyruvate, however, strongly suppressed the respiration. Under these conditions, the respiration was found to depend on the extramitochondrial [ATP]/[ADP] ratio in the range of 1-100. The contribution of the adenine nucleotide translocator to this dependence was determined by titration with the irreversible inhibitor carboxyatractyloside in the presence of ADP. Using lactate plus malate as substrate, the active state respiration was controlled to about 30% by the translocator, whereas 12 and 4% were determined in the presence of L-glycerol-3-phosphate and malate alone, respectively. In order to compare the results with those for intact cells, the adenine nucleotide patterns were determined in intact and digitonin-treated spermatozoa under conditions of controlled respiration in the presence of vanadate and carboxyatractyloside, respectively. About 21% of total cellular adenine nucleotides were found in digitonin-treated cells representing the mitochondrial compartment. While allowing for the intramitochondrial amount of adenine nucleotides, the cytosolic [ATP]/[ADP] ratio was estimated to be 6-times higher than the mitochondrial ratio in intact cells. It is concluded from the data presented that the principal mechanism by which oxidative phosphorylation in sperm mitochondria is regulated via the extramitochondrial [ATP]/[ADP] ratio is the same as that demonstrated for other isolated mitochondria.  相似文献   

19.
Cellular energy metabolism is fundamental for all biological functions. Cellular proliferation requires extensive metabolic reprogramming and has a high energy demand. The Kv1.3 voltage-gated potassium channel drives cellular proliferation. Kv1.3 channels localise to mitochondria. Using high-resolution respirometry, we show Kv1.3 channels increase oxidative phosphorylation, independently of redox balance, mitochondrial membrane potential or calcium signalling. Kv1.3-induced respiration increased reactive oxygen species production. Reducing reactive oxygen concentrations inhibited Kv1.3-induced proliferation. Selective Kv1.3 mutation identified that channel-induced respiration required an intact voltage sensor and C-terminal ERK1/2 phosphorylation site, but is channel pore independent. We show Kv1.3 channels regulate respiration through a non-conducting mechanism to generate reactive oxygen species which drive proliferation. This study identifies a Kv1.3-mediated mechanism underlying the metabolic regulation of proliferation, which may provide a therapeutic target for diseases characterised by dysfunctional proliferation and cell growth.Subject terms: Cell growth, Energy metabolism  相似文献   

20.
The potential importance of lipids and ketone bodies as fuels in the African lungfish, Protopterus dolloi, and the role of oxidative metabolism, were examined under control, fasted and aestivated conditions. In aestivating but not fasting lungfish, the activities of citrate synthase (CS) and cytochrome c oxidase (CCO) (enzymes of oxidative metabolism) showed tissue-specific changes. Significant reductions in CS activity occurred in the kidney, heart, gill and muscle, and in CCO in the liver and kidney tissues. Aestivation, but not fasting, also had a tissue-specific effect on mitochondrial state 3 respiration rates (using succinate as a substrate), with a >50% reduction in the liver, yet no change within muscle mitochondria. There is no indication that enzymes involved in lipid catabolism are up-regulated during periods of fasting or aestivation; however, both 3-hydroxyacyl CoA dehydrogenase (HOAD) and carnitine palmitoyl CoA transferase (CPT) activities were sustained in the liver despite the approximately 42% reduction in CCO activity, potentially indicating lipid metabolism is of importance during aestivation. Lungfish are able to utilize both the d- and l-stereoisomers of the ketone body beta-hydroxybutyrate (beta-HB); however, beta-HB does not appear to be an important fuel source during aestivation or fasting as no changes were observed in beta-HB tissue levels. This study demonstrates that an important aspect of metabolic depression during aestivation in lungfish is the tissue-specific down regulation of enzymes of aerobic metabolism while maintaining the activities of enzymes in pathways that supply substrates for aerobic metabolism.  相似文献   

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