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5,5'-Diphenyl-2-thiohydantoin (DPTH) administered in vitro, inhibited state 3 oxidation, stimulated state 4 oxidation and decreased ADP:O ratio when 3-hydroxybutyrate and succinate were used as substrates. Considerably lower DPTH concentrations were required for the inhibition of 3-hydroxybutyrate oxidation (50% inhibition occurred at approximately 0.17 mumoles DPTH/mg protein) than were needed for inhibition of succinate oxidation (50% inhibition occurred at about 0.62 mumoles DPTH/mg protein). DPTH showed no inhibitory effects when ascorbate plus tetramethylphenylenediamine (TMPD) served as the substrate. The inhibition of state 3 respiration was not reversed by 2,4-dinitrophenol (DNP), although there was a slight increase in the DNP rate:state 3 rate suggesting the presence of a weak DPTH inhibotory site located within the Site I energy transport chain. Uncoupling, in the presence of DPTH, was observed with all substrates. In experiments utilizing sonicated mitochondria, DPTH inhibited NADH-linked oxidation, but did not inhibit succinate or ascorbate plus TMPD oxidation. The effects of DPTH were reversed by dilution and by addition of albumin. DPTH concentrations which produced inhibition of state 3 respiration in vitro were reached, in vivo, in the livers of rats receiving a single oral dose of 40 mg/kg of DPTH.  相似文献   

3.
In normal or thyroidectomized rat liver mitochondria, glucagon produced fast but transient stimulation of respiration rates in state 3 and state 4 whatever the substrates. Stimulation reached its maximum 20 to 30 minutes after glucagon injection. However, the effects of glucagon are less marked after removal of the thyroid gland, since the increases observed in the oxygen consumption and basal metabolic rates were only half those shown in normal rats. The activating effects of triiodothyronine and glucagon on the ADP phosphorylation rates were found to be additive. Pretreatment with cycloheximide blocked the activation induced by glucagon but not that induced by triiodothyronine. Both hormones therefore stimulate oxidative phosphorylation but by different mechanisms. Thyroidectomy did not alter the early rise in glycaemia observed in response to glucagon. It may therefore be assumed that the hypothyroid rat's sensitivity to glucagon is not directly connected with the change in cAMP metabolism.  相似文献   

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Effects of dietary copper deficiency in rats on respiratory enzymes of isolated rat liver mitochondria have been studied. After 2 weeks of Cu-depletion, cytochrome c oxidase (EC 1.9.3.1) activity had declined by 42% and between 4 and 8 weeks exhibited between 20 and 25% of the activity of control mitochondria. Activities of NADH cytochrome c reductase (EC 1.6.99.3) and succinate cytochrome c reductase (EC 1.3.99.1), were unaffected initially but declined by 32 and 46%, respectively, after 8 weeks of Cu-depletion. After 4 weeks there was a significant (34%) decline in succinate supported state 3 respiration with only a modest (18%) decline in state 4 respiration. The ADP:O ratio was unaffected by Cu-depletion after 6 and 8 weeks of dietary Cu-restriction. State 3 respiration was significantly reduced after 6 weeks when glutamate/malate or beta-hydroxybutyrate were used as substrates, whereas state 4 respiration and ADP:O ratios were unaffected. The fall in state 3 respiration was of sufficient magnitude at 8 weeks to cause a significant decline in the respiratory control ratio with all substrates. Comparisons between the relative activities of cytochrome c oxidase and reductase activities in Cu-deficient preparations, the relatively specific effect of the deficiency on state 3 respiration with all substrates tested and the ability to increase significantly oxygen consumption in excess of maximal state 3 respiration by the uncoupler 2,4-dinitrophenol suggest that the defect in Cu-deficient mitochondria cannot be attributed solely to the decreased activity of cytochrome c oxidase.  相似文献   

6.
Free fatty acids (FFA) are known to uncouple oxidative phosphorylation in mitochondria. However, their mechanism of action has not been elucidated as yet. In this study we have investigated in detail the patterns of uncoupling by the FFA oleate and palmitate in rat liver mitochondria and submitochondrial particles. The patterns of uncoupling by FFA were compared to uncoupling induced by the ionophores valinomycin (in the presence of K+) and gramicidin (in the presence of Na+) and the proton translocator carbonyl cyanide m-chlorophenylhydrazone (CCCP). The most striking difference in the pattern of uncoupling relates to the effect on the proton electrochemical potential gradient, delta mu H. Uncoupling by ionophores, particularly valinomycin, is associated with and most likely caused by a major reduction of delta mu H. In contrast, uncoupling by FFA is not associated with a significant reduction of delta mu H, indicating another mechanism of uncoupling. We suggest the use of the term decouplers for uncoupling agents such as FFA and general anesthetics that do not collapse the delta mu H [Rottenberg, H. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3313-3317]. The protonophore CCCP and to some extent the ionophore gramicidin indicate a mixed mode of uncoupling since their effect on delta mu H is moderate when compared to that of valinomycin. Another distinguishing feature of uncouplers that collapse the delta mu H is their ability to stimulate ADP-stimulated respiration (state 3) further. Decouplers such as FFA and general anesthetics do not stimulate state 3 respiration.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

7.
Whole cells, homogenates and mitochondrial obtained from the livers of albino rats which were starved for 6 days or more showed a 50% decrease in oxidative activity. The decrease could be corrected by the addition of cytochrome c in vitro. The phosphorylative activity of mitochondria remained unaffected. The decrease in oxidative rate was not observed when starving animals were given the anti-hypercholesterolaemic drug clofibrate. The total cellular concentration of cytochrome c was not affected by starvation. However, the concentration of the pigment in hepatic mitochondria isolated from starving animals was less than half that in normal mitochondria. Clofibrate-treated animals did not show a decreased concentration of cytochrome c in hepatic mitochondria. Mitochondria isolated from starving animals, though deficient in cytochrome c, did not show any decrease in succinate dehydrogenase activity or in the rate of substrate-dependent reduction of potassium ferricyanide or attendant phosphorylation. In coupled mitochondria, ferricyanide may not accept electrons from the cytochrome c in the respiratory chain. Starvation decreases the concentration of high-affinity binding sites for cytochrome c on the mitochondrial membrane. The dissociation constant increases in magnitude.  相似文献   

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The reaction of fluorescamine with ammonia, benzylamine, o,p-dimethylbenzylamine, 2-phenylethylamine, p-aminobenzoic acid, and the mycosamine-containing macrolide antibiotic, amphotericin B, yield compounds which induce significant effects on mitochondrial activities. From their effects on energy-yielding processes which lead to transmembranous proton movements, the compounds may be divided into three classes. While all modifiers significantly inhibit proton movement induced by both ATP hydrolysis and electron transfer in mitochondria, their influence on the primary energy yielding steps are quite different. Class I modifiers, e.g., the compound made from amphotericin B, inhibit electron transfer but have no effect on the Pi release associated with ATP hydrolysis. Class II modifiers, e.g., the compound made from benzylamine, inhibit respiration but stimulate Pi release. Class III modifiers, e.g., the compound made from p-aminobenzoic acid, on the other hand, only slightly increase Pi release but have no effect on redox reactions. These and other effects of the modifiers are taken to mean that the proton movements and their associated energy-yielding processes are only linked indirectly. The effects of the modifiers on State 3 mitochondrial activities were also investigated. Although all the modifiers decrease the rates of both State 3 respiration and its coupled ATP synthesis, the efficiency of energy conversion measured by the P/O ratio remains unaltered.  相似文献   

10.
The dynamics of primary aliphatic amines (ethylamine, propylamine) effects on the processes of oxidative phosphorylation in rat liver mitochondria was estimated. The inhibiting action of ethylamine and propylamine on the oxidative phosphorylation processes in the rat liver mitochondria was revealed.  相似文献   

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Rats malnourished since birth and fed on a protein-free diet for 2 weeks showed a 23-27% decrease in the State-3 oxidation of glutamate, succinate and ascorbate + NNN' N'-tetramethyl-p-phenylenediamine by liver mitochondria compared with control fed animals. ATP synthesis and the respiratory control index were diminished at the three coupling sites, but significant alterations were not observed in ADP/O ratios. Vmax. for NADH oxidation in electron-transport particles was 40% lower. Mitochondrial cytochromes b and c1 remained unchanged, but cytochrome c was increased by 26%. Cytochromes a + a3 were diminished by 22%. Vmax. for mitochondrial ATPase was 23% lower. These results suggest that the lower content of cytochrome a + a3 at the rate-controlling step of oxidative phosphorylation in malnourished rats might be mainly responsible for the decrease in substrate oxidations as well as ATP synthesis at the three coupling sites. The decreased synthesis and hydrolysis of ATP suggests that other energy-dependent mitochondrial processes could be decreased during malnutrition.  相似文献   

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Isolated rat liver mitochondria have been treated with the general anaesthetic propofol (2,6-diisopropylphenol, 200 microM) and the physiological NO donor nitrosoglutathione (GSNO, 200 or 250 microM). The efficiency of the oxidative phosphorylation has been evaluated by measuring the respiration and ATP synthesis rates and the behavior of transmembrane electrical potential. In mitochondria energized by succinate, the simultaneous presence of both propofol and GSNO gives rise to a synergic action in affecting the resting and the ADP-stimulated respiration, the respiratory control ratio, the ATP synthesis, and the formation and utilization of the electrochemical transmembrane potential.  相似文献   

15.
Linoleate hydropepoxide, purified by silica gel chromatography and at concentrations 70-100 nmol/mg mitochondrial protein, activated state 4 respiration and Mg-ATPase activity of mitochondria to levels of 80% and 25%, respectively, of those induced by 300 microM DNP, and completely inhibited oxidative phosphorylation. These effects are the same as those caused by linoleate, but the hydroperoxide caused more rapid degeneration of the activated respiration of mitochondria than linoleate. Further addition of the hydroperoxide induced oligomycin-insensitive Mg-ATPase to a level 3 times that obtained with DNP, accompanied by clearing of the mitochondrial suspension and release of malate dehydrogenase from the matrix. The extent of the effects caused by the methyl ester of linoleate hydroperoxide was much less than by the free acid.  相似文献   

16.
While moderate caloric restriction has beneficial effects on animal health state, fasting may be harmful. The present investigation was designed to test how fasting affects oxidative stress, and to find out whether the effects are opposite to those previously found in caloric restriction studies. We have focused on one of the main determinants of aging rate: the rate of mitochondrial free radical generation. Different parameters related to lipid and protein oxidative damage were also analyzed. Liver mitochondria from rats subjected to 72 h of fasting leaked more electrons per unit of O2 consumed at complex III, than mitochondria from ad libitum fed rats. This increased leak led to a higher free radical generation under state 3 respiration using succinate as substrate. Regarding lipids, fasting altered fatty acid composition of hepatic membranes, increasing the double bond and the peroxidizability indexes. In accordance with this, we observed that hepatic membranes from the fasted animals were more sensitive to lipid peroxidation. Hepatic protein oxidative damage was also increased in fasted rats. Thus, the levels of oxidative modifications, produced either indirectly by reactive carbonyl compounds (Nepsilon- malondialdehyde-lysine), or directly through amino acid oxidation (glutamic and aminoadipic semialdehydes) were elevated due to the fasting treatment in both liver tissue and liver mitochondria. The current study shows that severe food deprivation increases oxidative stress in rat liver, at least in part, by increasing mitochondrial free radical generation during state 3 respiration and by increasing the sensitivity of hepatic membranes to oxidative damage, suggesting that fasting and caloric restriction have different effects on liver mitochondrial oxidative stress.  相似文献   

17.
While moderate caloric restriction has beneficial effects on animal health state, fasting may be harmful. The present investigation was designed to test how fasting affects oxidative stress, and to find out whether the effects are opposite to those previously found in caloric restriction studies. We have focused on one of the main determinants of aging rate: the rate of mitochondrial free radical generation. Different parameters related to lipid and protein oxidative damage were also analyzed. Liver mitochondria from rats subjected to 72 h of fasting leaked more electrons per unit of O2 consumed at complex III, than mitochondria from ad libitum fed rats. This increased leak led to a higher free radical generation under state 3 respiration using succinate as substrate. Regarding lipids, fasting altered fatty acid composition of hepatic membranes, increasing the double bond and the peroxidizability indexes. In accordance with this, we observed that hepatic membranes from the fasted animals were more sensitive to lipid peroxidation. Hepatic protein oxidative damage was also increased in fasted rats. Thus, the levels of oxidative modifications, produced either indirectly by reactive carbonyl compounds (Nepsilon- malondialdehyde-lysine), or directly through amino acid oxidation (glutamic and aminoadipic semialdehydes) were elevated due to the fasting treatment in both liver tissue and liver mitochondria. The current study shows that severe food deprivation increases oxidative stress in rat liver, at least in part, by increasing mitochondrial free radical generation during state 3 respiration and by increasing the sensitivity of hepatic membranes to oxidative damage, suggesting that fasting and caloric restriction have different effects on liver mitochondrial oxidative stress.  相似文献   

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Preincubation of newborn rat liver mitochondria with ATP increases their state 3 respiration rate [J. K. Pollak (1975) Biochem. J. 150, 477-488; J. R. Aprille, and G. K. Asimakis (1980) Arch. Biochem. Biophys. 201, 564-575]. To determine which reactions contribute to control the rate of succinate oxidation with and without prior exposure to ATP, the effects of inhibitors specific for various reactions were studied. The adenine nucleotide translocator does not control the respiration in newborn more than in the adult mitochondria. The supply of reducing equivalents to the respiratory chain is an important step controlling the rate of oxidative phosphorylation by mitochondria from newborn rat liver, especially after preincubation with ATP. On the contrary, titrations with oligomycin show that the preincubation with ATP markedly decreases the control exerted by the ATPase-ATP synthase complex. That the rate of ATP synthesis is one of the steps controlling the rate of oxidative phosphorylation in newborn rat liver mitochondria is in striking contrast to the behavior of adult rat liver mitochondria. Other differences include a greater permeability to protons and a marked increase in sensitivity to mersalyl, indicating an easier accessibility of the proteins involved in oxidative phosphorylation to the thiol reagent.  相似文献   

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