[Show abstract][Hide abstract] ABSTRACT: To characterize the role of supernumerary subunits of the mammalian FoF1 ATP synthase, crossreconstitution of mitochondrial and bacterial FoF1 complexes has been carried out. Escherichia coli F1 (EcF1) can be reconstituted with F1-stripped everted membranes of E. coli (UPEc) and of bovine heart mitochondria (USMP). Bovine heart mitochondrial F1 (BHF1) can also be reconstituted with both membranes. Both EcF1 and BHF1, when reconstituted with UPEc, exhibited oligomycin-insensitive ATP-hydrolase activity. Subunits of the mammalian Fo, in particular FoI-PVP protein, F6 and oligomycin-sensitivity-conferring protein (OSCP) conferred oligomycin sensitivity to the catalytic activity of EcF1 or BHF1 reconstituted with UPEc. Reaction of N, N′-dicyclohexylcarbodiimide and development of inhibition of passive H+ conduction was, in UPEc, considerably slower and exhibited a lower apparent affinity than in USMP. The ATP hydrolase activity of UPEc+EcF1 or UPEc+BHF1 was, also, less sensitive to inhibition by N, N′-dicyclohexylcarbodiimide than USMP+EcF1 or USMP+BHF1. Addition of mitochondrial FoI-PVP to UPEc enhanced the sensitivity of H+ conduction to oligomycin. FoI-PVP and OSCP added to UPEc, promoted inhibition by N, N′-dicyclohexylcarbodiimide of passive H+ conduction and increased its binding affinity to subunit c of E. coli Fo. The presence of FoI-PVP and OSCP also promoted inhibition by N, N′-dicyclohexylcarbodiimide of the ATP-hydrolase activity of EcF1 or BHF1 reconstituted with UPEc.
[Show abstract][Hide abstract] ABSTRACT: Using isolated polypeptides of the F0 sector of bovine heart mitochondrial H+-ATPase, antisera were developed detecting specifically two components of F0. These two components were identified as F0I and oligomycin-sensitivity-conferring protein (OSCP) respectively. Both F0I and OSCP were digested by mild trypsin treatment of submitochondrial particles depleted of the catalytic part of H+-ATPase (USMP). Proteolysis was largely prevented by binding of F1 to F0. Proteolysis of F0I resulted in the formation of three immunoreactive. membrane-bound fragments of apparently 26 kDa, 25.5 kDa and 18 kDa, respectively, indicating that F0I contains trypsin-accessible Arg or Lys residues located close to the end and the middle part of the protein, respectively, which are in intimate contact with F1. Digestion of USMP with trypsin resulted in depression of passive H+ conduction through F0 which could be ascribed to proteolysis of F0I.
[Show abstract][Hide abstract] ABSTRACT: We have investigated the effect of thyroid hormone on the mitochondrial membrane permeability properties in a hypothyroid rat model. The role played by calcium in affecting these properties has been also examined. Cyclosporin A-sensitive mitochondrial calcium efflux, swelling, and external release of matrix proteins are events that occur normally during the permeability transition process induced by calcium loading of mitochondria. We demonstrate that these events are impaired in mitochondria isolated from the liver of hypothyroid rats, even in the presence of high calcium content. However, after thyroid hormone administration to hypothyroid rats, the mitochondrial permeability transition process in response to calcium loading is restored. Consequently, mitochondrial calcium efflux, swelling, and release of matrix proteins, like glutamate dehydrogenase, malate dehydrogenase, and aspartate aminotransferase occur. These effects are abrogated by the concomitant administration of cyclosporin A. The results of the present study suggest that hypothyroidism may be a potential source of adverse effects in patients receiving cyclosporin A.
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial bioenergetic impairment has been found in the organelles isolated from rat liver during the prereplicative phase of liver regeneration. To gain insight into the mechanism underlying this impairment, we investigated mitochondrial ultrastructure and membrane permeability properties in the course of liver regeneration after partial hepatectomy, with special interest to the role played by Ca2+ in this process. The results show that during the first day after partial hepatectomy, significant changes in the ultrastructure of mitochondria in situ occur. Mitochondrial swelling and release from mitochondria of both glutamate dehydrogenase and aspartate aminotransferase isoenzymes with an increase in the mitochondrial Ca2+ content were also observed. Cyclosporin-A proved to be able to prevent the changes in mitochondrial membrane permeability properties. At 24 h after partial hepatectomy, despite alteration in mitochondrial membrane permeability properties, no release of cytochrome c was found. The ultrastructure of mitochondria, the membrane permeability properties and the Ca2+ content returned to normal values during the replicative phase of liver regeneration. These results suggest that, during the prereplicative phase of liver regeneration, the changes in mitochondrial ultrastructure observed in liver specimens were correlated with Ca2+-induced permeability transition in mitochondria.
European Journal of Biochemistry 08/2002; 269(13):3304-12. DOI:10.1046/j.1432-1033.2002.03010.x · 3.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hepatic steatosis is associated with mitochondrial oxidative alterations. This study aimed to characterize in a choline-deficient model of rat fatty liver whether this oxidative imbalance is related to an impairment of the capacity of ATP synthesis both under fed conditions and after starvation, which may sensitize mitochondria to oxidative injury. Mitochondria were isolated from normal and fatty livers of fed or 18-hour fasted rats. Oxidative injury was evaluated by measuring the mitochondrial content of thiobarbituric reactive substances, protein carbonyls, glutathione, and protein sulfhydryls. The mitochondrial F(0)F(1)-ATP synthase content, tissue ATP concentration, and liver histology were also determined. Compared with normal liver, under fed conditions, fatty livers showed a greater mitochondrial content of oxidized lipids and proteins together with a low concentration of sulfhydryls and glutathione. The mitochondrial catalytic beta-F(1) subunit of the F(0)F(1)-ATP synthase was about 35% lower in fatty livers. Hepatic ATP was also significantly reduced in fatty liver. Starvation exacerbated mitochondrial oxidative injury in both groups but to a greater extent in fatty livers. In the steatotic group, fasting induced a significant decrease of the ATP levels, which was accompanied by a 70% fall of the catalytic beta-F(1) subunit. These data indicate that the mitochondrial oxidative alterations in fatty livers are associated with an important reduction of the F(0)F(1)-ATP synthase. These changes, which are greatly exacerbated after starvation, may account for the reduced synthesis of the hepatic ATP observed in the presence of fatty infiltration.
[Show abstract][Hide abstract] ABSTRACT: Liver mitochondria were isolated from male rats exposed for 2 months to low doses of ethanol (3% v/v in drinking water), a condition not associated with tolerance or dependence. The results show no significant changes in the content of reduced or oxidized glutathione in the liver mitochondria of ethanol treated rats with respect to controls. However, a slight but significant increase in lipid peroxidation, accompanied by an increased content of oxidized proteins, was found in ethanol exposed animals. Mitochondrial content of cytochrome complexes was not significantly affected by ethanol intake. The specific enzymatic activity of cytochrome oxidase showed, however, a significant decrease in ethanol-treated rats. The slight mitochondrial alterations found in the liver of rats exposed chronically to low doses of ethanol might represent the beginning of a more extensive damage previously observed in rats exposed to high doses of this substance.
Life Sciences 02/2000; 66(6):477-84. DOI:10.1016/S0024-3205(99)00617-7 · 2.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In hypothyroid rats, partial hepatectomy does not induce liver regeneration until 120 h aftersurgical operation, when, instead, in normal rats a complete recovery of the liver mass, in thisinterval, is observed. In normal rats, a good efficiency of mitochondrial oxidative phosphorylationis needed as an energy source for liver regeneration (Guerrieri, F.et al., 1995); inhypothyroid rats the efficiency of mitochondrial oxidative phosphorylation is low in the 0–120h interval after partial hepatectomy. This low efficiency of oxidative phosphorylation appearsto be related to a low mitochondrial content of F0F1-ATP synthase, in liver of hypothyroidrats, which does not recover after partial hepatectomy. In the liver of hypothyroid rats, lowlevels of the nuclear-encoded mitochondrial catalytic F1 subunit and of its transcript areobserved and they do not increase, as occurs in normal rats, after partial hepatectomy.
Journal of Bioenergetics 01/2000; 32(2):183-191. DOI:10.1023/A:1005564031119 · 3.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mitochondria, isolated from rat livers during the early phase of liver regeneration (7-24 h after partial hepatectomy), show: (i) decrease in the rate of ATP synthesis; (ii) increase of malondialdehyde and of oxidized protein production; (iii) decrease of the content of intramitochondrial glutathione and of protein thiols on mitochondrial proteins; (iv) increase of the glutathione bound to mitochondrial proteins by disulfide bonds. These observations suggest an increase of production of oxygen radicals in liver mitochondria, following partial hepatectomy, which can alter the function of the enzymes involved in the oxidative phosphorylation. Blue-native gel electrophoresis of rat liver mitochondria, isolated after partial hepatectomy, shows, during the early phase of liver regeneration (0-24 h after partial hepatectomy), a progressive decrease of the content of F0F1-ATP synthase complex. The amount of glutathione bound to the F0F1-ATP synthase, electroeluted from the blue-native gels, progressively increased during the early phase of liver regeneration. It is concluded that partial hepatectomy causes mitochondrial oxidative stress that, in turn, modifies proteins (such as F0F1-ATP synthase) involved in the mitochondrial oxidative phosphorylation.
Free Radical Biology and Medicine 02/1999; 26(1-2):34-41. DOI:10.1016/S0891-5849(98)00145-2 · 5.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In liver mitochondria isolated from hypothyroid rats, the rate of ATP synthesis is lower than in mitochondria from normal rats. Oligomycin-sensitive ATP hydrolase activity and passive proton permeability were significantly lower in submitochondrial particles from hypothyroid rats compared to those isolated from normal rats. In mitochondria from hypothyroid rats, the changes in catalytic activities of F0F1-ATP synthase are accompanied by a decrease in the amount of immunodetected beta-F1, F0 1-PVP, and OSCP subunits of the complex. Northern blot hybridization shows a decrease in the relative cytosolic content of mRNA for beta-F1 subunit in liver of hypothyroid rats. Administration of 3,5,3'-triodo-L-thyronine to the hypothyroid rats tends to remedy the functional and structural defects of F0F1-ATP synthase observed in the hypothyroid rats. The results obtained indicate that hypothyroidism leads to a decreased expression of F0F1-ATP synthase complex in liver mitochondria and this contributes to the decrease of the efficiency of oxidative phosphorylation.
Journal of Bioenergetics 07/1998; 30(3):269-76. DOI:10.1023/A:1020548904384 · 3.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 70% partial hepatectomy (PH) in the rat causes a release, into the cytosolic fraction, of mitochondrial matrix proteins, namely the mitochondrial isoform of aspartate aminotransferase (mAAT) and malate dehydrogenase (MDH), during the first 24 h after PH, when no growth of the residual liver is observed. After this time interval, the weight of the liver starts to increase and the normal weight is reached at 96 h after PH. This proliferative phase is characterized by a progressive recovery of the normal levels of intramitochondrial activities of mAAT and MDH. Mitochondria isolated at 24 h after PH show a membrane permeabilization to sucrose accompanied by a release of matrix enzymes; both are blocked by cyclosporin A. These results suggest an alteration of mitochondrial membrane integrity, during the prereplicative phase of liver regeneration, with the occurrence of an increased permeability that allows the passage into the cytosol of matrix enzymes.
[Show abstract][Hide abstract] ABSTRACT: Cancer cells, despite growing aerobically, have the propension to utilize the glycolytic pathway as energy source. This biochemical phenotype is accompanied by a decreased content of mitochondria and, paradoxically, by enhanced transcription of nuclear and mitochondrial-encoded genes for the enzymes of oxidative phosphorylation (OXPHOS). The role of OXPHOS enzymes in normal and neoplastic cell growth has been studied in liver regeneration and human hepatocellular carcinoma. In early liver regeneration characterized by active mtDNA replication, a decrease in the content and activity of ATP synthase occurs while transcription of the ATPsyn beta nuclear gene is activated. Translation of ATP synthase subunits seems, on the contrary, to be less effective in this phase. In the second replicative phase of liver regeneration, the repression of ATPsyn beta translation is relieved and normal cell growth starts. In this replicative phase the recovery of the liver mass appears to be directly related to the recovery of the OXPHOS capacity. Mitochondria isolated from biopsies of human hepatocellular carcinoma exhibit a decreased rate of respiratory ATP synthesis (OXPHOS) and a decreased ATPase activity. The decline in the activity of the ATP synthase is found to be associated with a decreased content of the ATPsyn beta in the inner mitochondrial membrane. In neoplastic tissue the ATPase inhibitor protein (IF1) is overexpressed. This could contribute to prevent hydrolysis of glycolytic ATP in cancer cells. A peptide segment of IF1 (IF1-(42-58)-peptide), constructed by chemical synthesis, proved to be equally effective as IF1 in inhibiting the ATPase activity of the ATP synthase complex in the mitochondrial membrane deprived of IF1. The synthetic peptide might turn out to be a useful tool to develop immunological approaches for the control of neoplastic growth.
Journal of Bioenergetics 09/1997; 29(4):379-84. DOI:10.1023/A:1022402915431 · 3.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Evidence is available showing that the coupling efficiency of the proton pump in cytochrome c oxidase of mitochondria can under certain conditions decrease significantly below the maximum attainable value. The view is developed that slips in the proton pump of cytochrome c oxidase represent an intrinsic switch mechanism which regulates the relative contribution of energy transfer and respiratory protection against oxygen toxicity by the oxidase.
[Show abstract][Hide abstract] ABSTRACT: Changes in cell energy metabolism and mitochondrial dysfunction have been observed after acetaminophen administration. Because consumption of hepatic glutathione is closely related to acetaminophen toxicity, we investigated the kinetics of: 1. glutathione depletion in liver mitochondria and cytosol; 2. State 3 and 4 respiratory rates of succinate-supplemented mitochondria; 3. rate of ATP synthesis; 4. oligomycin-sensitive ATP hydrolase activity and passive proton conductivity of inside-out vesicles of the inner mitochondrial membrane; and 5. changes in hepatic and mitochondrial malondialdehyde in the rat after in vivo acetaminophen administration. Two hours after acetaminophen injection, hepatic glutathione decreased and malondialdehyde increased. In the same interval, an increase in both State 3 and 4 respiratory rates of succinate-supplemented mitochondria was observed. This was accompanied by a decrease in the rate of ATP synthesis and the P/O ratio and by an increase in the passive proton permeability of the inner mitochondrial membrane, which was insensitive to oligomycin. No significant change in oligomycin-sensitive ATP hydrolase activity was observed. Four hours after APAP injection, the respiratory rates, as well as the proton conductivity, decreased, the rate of ATP synthesis was restored, and the mitochondrial glutathione started to increase; the cytosolic levels of glutathione were still low and the cytosolic and mitochondrial levels of malondialdehyde remained high for 2 more hr. The concentrations of these indices were completely restored 24 hr postdosing. Our findings suggest that acetaminophen administration selectively depletes (within 2 hr) mitochondrial glutathione, and produces local toxicity by altering membrane permeability and decreasing the efficiency of oxidative phosphorylation. This renders mitochondria more susceptible to oxidative damage, especially during increased free radical production, as in the case of enhanced mitochondrial respiration in State 4. The concomitant restoration of mitochondrial respiration, oxidative phosphorylation, membrane permeability, and glutathione levels is consistent with the importance of the mitochondrial glutathione pool for the protection of the mitochondrial membrane against oxidative damage.
[Show abstract][Hide abstract] ABSTRACT: The DCCD-sensitive proton permeability of chromatophores, from a green strain of Rhodobacter Capsulatus is potentiometrically detected following the proton release induced by a transmembrane diffusion potential imposed by a valinomycin-mediated potassium influx with a procedure already used for bovine heart submitochondrial particles (ESMP) and vesicles from Escherichia coli (Zanotti et al. (1994) Eur. J. Biochem. 222, 733-741). In the photosynthetic system, addition of increasing amounts of DCCD inhibits, with a similar titre, both proton permeability and MgATP-dependent ATPase activity as detected in the dark. The titre for 50% inhibition coincides with that obtained measuring proton permeability and ATP hydrolysis in ESMP. Upon removal of F1, the passive proton permeability is much less sensitive to DCCD in chromatophores than in USMP, suggesting that in chromatophores the F1-Fo interaction shapes the DCCD-sensitive proton conducting pathway. Addition of the purified mitochondrial FoI-PVP and oligomycin sensitivity-conferring (OSCP) proteins to the F1 stripped chromatophores restored the sensitivity of proton permeability to DCCD detected in untreated chromatophores. Analysis of the binding of 14C[DCCD] on F1 stripped chromatophores shows that the increase of DCCD sensitivity of proton permeability, caused by addition of mitochondrial Fo proteins, is related to an increase of the binding of the inhibitor to subunit c of Fo sector of ATP synthase complex.
[Show abstract][Hide abstract] ABSTRACT: The time course of changes in mitochondrial energy metabolism during liver regeneration, following partial hepatectomy, is analyzed. For 24 h after surgical operation, a lag phase in the time course of the growth of liver is observed. In this period mitochondria showed a decrease of: (1) the respiratory control index; (2) the rate of oxidative phosphorylation; (3) the amount of immunodetected beta-F1 and F01-PVP subunits of F0F1-ATP synthase. No decrease, but instead a small increase in the content of mRNA for beta-F1 was observed in this phase. After this lag phase the growth of liver started, the content of mRNA for beta F1, as well as the level of immunodetected mitochondrial beta-F1 and F01-PVP subunits, increased and oxidative phosphorylation recovered. Analysis of the relative beta F1 protein/mRNA ratio indicates a decrease of beta F1 translational efficiency which remained low up to 72 h after partial hepatectomy and reached the same ratio of control at 96 h. It is concluded that the regenerating capability of rat liver is correlated with the efficiency of oxidative phosphorylation.
[Show abstract][Hide abstract] ABSTRACT: Earlier studies by Rouslin and coworkers showed that, during myocardial ischemia in slow heart-rate species which include rabbits and all larger mammals examined including humans, there is an IF1-mediated inhibition of the mitochondrial ATPase due to an increase in the amount of IF1 bound to the ATPase (Rouslin, W., and Pullman, M.E., J. Mol. Cell. Cardiol. 19,661-668, 1987). Earlier work by Guerrieri and colleagues demonstrated that IF1 binding to bovine heart ESMP was accompanied by parallel decreases in ATPase activity and in passive proton conduction (Guerrieri, F., et al., FEBS Lett. 213, 67-72, 1987). In the present study rabbit was used as the slow heart-rate species and rat as the fast heart-rate species. Rat is a fast heart-rate species that contains too little IF1 to down regulate the ATPase activity present. Mitochondria were prepared from control and ischemic hearts and ESMP were made from aliquots by sonication at pH 8.0 with 2 mM EDTA. Oligomycin-sensitive ATPase activity and IF1 content were measured in SMP prepared from the control and ischemic mitochondrial samples. After identical incubation procedures, oligomycin-sensitive ATPase activity, oligomycin-sensitive proton conductivity, and IF1 content were also measured in ESMP samples. The study was undertaken to corroborate further what appear to be fundamental differences in ATPase regulation between slow and fast heart-rate mammalian hearts evident during total myocardial ischemia. Thus, passive proton conductivity was used as an independent measure of these regulatory differences. The results show that, consistent with the low IF1 content of rat heart cardiac muscle mitochondria, control rat heart ESMP exhibit approximately twice as much passive proton conductivity as control rabbit heart ESMP regardless of the pH of the incubation and assay. Moreover, while total ischemia caused an increase in IF1 binding and a commensurate decrease in passive proton conductivity in rabbit heart ESMP regardless of pH, neither IF1 content nor proton conductivity changed significantly in rat heart ESMP as a result of ischemia.
Journal of Bioenergetics 09/1995; 27(4):459-66. DOI:10.1007/BF02110008 · 3.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The rate of mitochondrial oxidative phosphorylation and the cytosolic and mitochondrial total and oxidized glutathione concentrations were studied in regenerating rat livers after partial (70%) hepatectomy. The rate of mitochondrial oxidative phosphorylation progressively decreased during the early prereplicative phase of liver regeneration. This was accompanied by a progressive decrease in mitochondrial, but not cytosolic, glutathione concentration. Twenty-four hours after partial hepatectomy, both the rate of adenosine triphosphate (ATP) synthesis and the amount of mitochondrial glutathione were depressed by 50% with respect to controls (sham-operated animals). During the second replicative phase, both the oxidative phosphorylation rate and mitochondrial glutathione concentration were recovered; however, the kinetics of the recovery were different, being the total amount of mitochondrial glutathione completely restored 48 hours after partial hepatectomy, whereas 72 hours were needed for the recovery of oxidative phosphorylation. The decrease in the rate of oxidative phosphorylation, during the early phase of liver regeneration, appeared to be secondary to the decreased content of the catalytic subunit beta-F1 of the ATP synthase complex, which in turn was shown to be linearly related to the decrease of intramitochondrial glutathione. These observations suggest that the two phenomena may be due to the previously reported increased free radical production during the early phase of liver regeneration. The depression of mitochondrial glutathione after partial hepatectomy may play a contributory role in structural and functional alterations of mitochondria occurring in the first retrodifferential phase of liver regeneration.
[Show abstract][Hide abstract] ABSTRACT: To characterize the role of supernumerary subunits of the mammalian F0F1 ATP synthase, cross-reconstitution of mitochondrial and bacterial F0F1 complexes has been carried out. Escherichia coli F1 (EcF1) can be reconstituted with F1-stripped everted membranes of E. coli (UPEc) and of bovine heart mitochondria (USMP). Bovine heart mitochondrial F1 (BHF1) can also be reconstituted with both membranes. Both EcF1 and BHF1, when reconstituted with UPEc, exhibited oligomycin-insensitive ATP-hydrolase activity. Subunits of the mammalian F0, in particular F0I-PVP protein, F6 and oligomycin-sensitivity-conferring protein (OSCP) conferred oligomycin sensitivity to the catalytic activity of EcF1 or BHF1 reconstituted with UPEc. Reaction of N,N'-dicyclohexylcarbodiimide and development of inhibition of passive H+ conduction was, in UPEc, considerably slower and exhibited a lower apparent affinity than in USMP. The ATP hydrolase activity of UPEc+EcF1 or UPEc+BHF1 was, also, less sensitive to inhibition by N,N'-dicyclohexylcarbodiimide than USMP+EcF1 or USMP+BHF1. Addition of mitochondrial F0I-PVP to UPEc enhanced the sensitivity of H+ conduction to oligomycin. F0I-PVP and OSCP added to UPEc, promoted inhibition by N,N'-dicyclohexylcarbodiimide of passive H+ conduction and increased its binding affinity to subunit c of E. coli F0. The presence of F0I-PVP and OSCP also promoted inhibition by N,N'-dicyclohexylcarbodiimide of the ATP-hydrolase activity of EcF1 or BHF1 reconstituted with UPEc.
European Journal of Biochemistry 07/1994; 222(3):733-41. · 3.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effect of aging on rat liver regeneration and on the FoF1-ATP synthase complex of isolated liver mitochondria was followed after partial (70%) hepatectomy. ATP hydrolase activity in submitochondrial particles prepared from regenerating liver was first depressed; the time needed to reach the lowest activity was age dependent. This decrease was accompanied by parallel decrease of i) the respiratory rate of succinate supplemented mitochondria in state III; ii) the respiratory control index; iii) the rate of synthesis of ATP in succinate supplemented submitochondrial particles. This first phase of liver regeneration, characterized at all ages by a lag phase in the growth, was followed by a second phase in which the tissue mass was restored and the enzyme activities normalized. Immunoblot analysis showed that the changes in the catalytic activities of the FoF1-ATP synthase observed during liver regeneration were accompanied by parallel changes in the amount of subunits of both the catalytic (F1) and the membrane (Fo) sector of the complex.
Biochemistry and molecular biology international 06/1994; 33(1):117-29.