Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Submitochondrial Particles”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,315 records · Page 73Linked to original sources
PubMed1991

Purification and characterisation of an archaebacterial succinate dehydrogenase complex from the plasma membrane of the thermoacidophile Sulfolobus acidocaldarius.

A succinate dehydrogenase complex was isolated in a three-step purification from plasma membranes of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. It consists of four subunits: a, 66 kDa; b, 31 kDa; c, 28 kDa and d, 12.8 kDa. In the 141-kDa native protein, the four subunits are present in an equimolar stoichiometry. The complex contains acid-non-extractable flavin, iron and acid-labile sulphide. Maximal succinate dehydrogenase activities were recorded at pH 6.5, which coincides with the internal pH of Sulfolobus cells. The temperature optimum of 81 degrees C defines the Sulfolobus succinate dehydrogenase as a thermophilic enzyme complex. The Km for succinate was found to be 1.42 mM (55 degrees C). Similar to the mitochondrial soluble succinate dehydrogenase, this enzyme is capable of transferring electrons to artificial electron acceptors, for instance phenazine methosulfate, N,N,N',N'-tetramethyl-p-phenylenediamine and ferricyanide. In contrast to the mitochondrial succinate dehydrogenase, the archaebacterial enzyme reduces 1,4-dichloroindophenol also in the absence of phenazine methosulfate. Caldariella quinone, the physiological electron mediator in the Sulfolobus respiratory chain, was only slowly reduced under adjusted conditions. The succinate--phenazine methosulfate-(1,4-dichloroindophenol) oxidoreductase of the isolated complex was strongly inhibited by tetrachlorobenzoquinone. In plasma membranes the complex reduces molecular oxygen in a cyanide-sensitive reaction. Polyclonal Sulfolobus anti-a antibodies crossreacted with 66-67-kDa polypeptides from membranes of Thermoplasma acidophilium, Sulfolobus solfataricus and beef heart submitochondrial particles.

Cell Membrane↗
PubMed1992

Presteady-state and steady-state kinetic properties of human cytochrome c oxidase. Identification of rate-limiting steps in mammalian cytochrome c oxidase.

Human cytochrome c oxidase was purified in a fully active form from heart and skeletal muscle. The enzyme was selectively solubilised with octylglucoside and KCl from submitochondrial particles followed by ammonium sulphate fractionation. The presteady-state and steady-state kinetic properties of the human cytochrome c oxidase preparations with either human cytochrome c or horse cytochrome c were studied spectrophotometrically and compared with those of bovine heart cytochrome c oxidase. The interaction between human cytochrome c and human cytochrome c oxidase proved to be highly specific. It is proposed that for efficient electron transfer to occur, a conformational change in the complex is required, thereby shifting the initially unfavourable redox equilibrium. The very slow presteady-state reaction between human cytochrome c oxidase and horse cytochrome c suggests that, in this case, the conformational change does not occur. The proposed model was also used to explain the steady-state kinetic parameters under various conditions. At high ionic strength (I = 200 mM, pH 7.4), the kcat was highly dependent on the type of oxidase and it is proposed that the internal electron transfer is the rate-limiting step. The kcat value of the 'high-affinity' phase, observed at low ionic strength (I = 18 mM, pH 7.4), was determined by the cytochrome c/cytochrome c oxidase combination applied, whereas the Km was highly dependent only on the type of cytochrome c used. Our results suggest that, depending on the cytochrome c/cytochrome c oxidase combination, either the dissociation of ferricytochrome c or the internal electron transfer is the rate-limiting step in the 'high-affinity' phase at low ionic strength. The 'low-affinity' kcat value was not only determined by the type of oxidase used, but also by the type of cytochrome c. It is proposed that the internal electron-transfer rate of the 'low-affinity' reaction is enhanced by the binding of a second molecule of cytochrome c.

Animals↗
PubMed1996

Involvement of cyclophilin D in the activation of a mitochondrial pore by Ca2+ and oxidant stress.

Heart and liver mitochondria contain a structure that is able to form a large non-selective pore in the inner membrane under conditions of high matrix Ca2+ and oxidant stress. The pore is blocked by cyclosporin A (CSA). In this study, rat liver mitochondria were covalently labelled with a photoactive CSA derivative in the presence and absence of the pore ligands Ca2+ and ADP. Photolabelling of a 21-kDa protein was selectively depressed by Ca2+ in a manner reversed by ADP. The protein exhibited peptidyl-prolyl cis-trans isomerase (PPIase) activity and was inhibited by CSA (Ki, 8 nM). The PPIase was associated with the outside of sonicated submitochondrial particles but dissociated in 0.5 M NaCl. When mitochondria were treated with increasing concentrations of digitonin, the 21-kDa PPIase fractionated with the matrix marker enzyme, malate dehydrogenase. A second PPIase of 18 kDa fractionated with the intermembrane-space marker, adenylate kinase. Photolabelling of the 18-kDa PPIase was unaffected by Ca2+ or ADP. The 21-kDa PPIase was digested with endoproteinase Asp-N and 11 of the peptides were N-terminally sequenced. The sequences were most similar to those of human cyclophilin-D, and it is concluded that this protein is probably the CSA receptor during pore blockade by CSA. The implications of these findings are discussed.

Adenosine Diphosphate↗
PubMed2005

Recognition memory impairment and brain oxidative stress induced by postnatal iron administration.

Iron accumulation in the brain has been implicated in the pathogenesis of neurodegenerative disorders. It is known that iron catalyses the formation of highly reactive hydroxyl radicals. Recent studies have implicated oxidative damage in memory deficits in rats and humans. The purpose of the present study was to investigate the long-term effects of iron treatment in four different phases of the neonatal period on recognition memory in rats. Additionally, parameters of oxidative stress in cerebral regions related to memory formation were evaluated. Male Wistar rats received vehicle or 10.0 mg/kg of Fe2+ orally at postnatal days 5-7, 12-14, 19-21 or 30-32. Animals given iron at any phase of the neonatal period showed impairments in long-term retention of object recognition memory, although only the group given iron from postnatal days 12-14 showed a complete memory blockade. Iron treatment induced oxidative damage in the brain as assessed by the thiobarbituric acid reactive species assay. Moreover, iron administration increased superoxide production in submitochondrial particles, suggesting impaired mitochondrial function; and there was an increase in superoxide dismutase activity in brain regions susceptible to iron administration. The results show that iron load in the early stages of life induces cognitive impairment possibly by inducing oxidative damage in the brain. These findings are consistent with the view that oxidative stress may be related to the cognitive decline observed in normal ageing.

Animals↗
PubMed1993

Reactivation of NADH dehydrogenase (complex I) inhibited by 1-methyl-4-(4'-alkylphenyl)pyridinium analogues: a clue to the nature of the inhibition site.

Expression of the neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, following oxidation to 1-methyl-4-phenylpyridinium ion (MPP+), is believed to involve inhibition of mitochondrial electron transport from NADH dehydrogenase (complex I) to ubiquinone. MPP+ and its analogues have been shown to block electron transport at or near the same site as two powerful inhibitors of mitochondrial respiration, rotenone and piericidin A. All three types of inhibitors combine at two sites on NADH dehydrogenase, a hydrophilic and hydrophobic one, and occupancy of both sites is required for complete inhibition. Tetraphenylboron anion (TPB-) in catalytic amounts is known to increase the effectiveness of positively charged MPP+ analogues in blocking mitochondrial respiration. A part of this effect involves facilitation of the entry of MPP+ congeners into the hydrophobic site by ion pairing, as has been demonstrated in studies with submitochondrial particles (electron transport particles). This communication documents the fact that TPB-, when present in molar excess over the MPP+ analogues, reverses the inhibition. This seems to involve again strong ion pairing, removal of the inhibitory analogue from one to the two binding sites, and concentration of the inhibitor in the membrane, so that only the hydrophobic binding site remains occupied, resulting in lowering of the inhibition to 30-40%.

1-Methyl-4-phenylpyridinium↗
PubMed2002

K(ATP) channel-independent targets of diazoxide and 5-hydroxydecanoate in the heart.

Diazoxide and 5-hydroxydecanoate (5-HD; C10:0) are reputed to target specifically mitochondrial ATP-sensitive K(+) (K(ATP)) channels. Here we describe K(ATP) channel-independent targets of diazoxide and 5-HD in the heart. Using submitochondrial particles isolated from pig heart, we found that diazoxide (10-100 microM) dose-dependently decreased succinate oxidation without affecting NADH oxidation. Pinacidil, a non-selective K(ATP) channel opener, did not inhibit succinate oxidation. However, it selectively inhibited NADH oxidation. These direct inhibitory effects of diazoxide and pinacidil cannot be explained by activation of mitochondrial K(ATP) channels. Furthermore, application of either diazoxide (100 microM) or pinacidil (100 microM) did not decrease mitochondrial membrane potential, assessed using TMRE (tetramethylrhodamine ethyl ester), in isolated guinea-pig ventricular myocytes. We also tested whether 5-HD, a medium-chain fatty acid derivative which blocks diazoxide-induced cardioprotection, was 'activated' via acyl-CoA synthetase (EC 6.2.1.3), an enzyme present both on the outer mitochondrial membrane and in the matrix. Using analytical HPLC and electrospray ionisation mass spectrometry, we showed that 5-HD-CoA (5-hydroxydecanoyl-CoA) is indeed synthesized from 5-HD and CoA via acyl-CoA synthetase. Thus, 5-HD-CoA may be the active form of 5-HD, serving as substrate for (or inhibiting) acyl-CoA dehydrogenase (beta-oxidation) and/or exerting some other cellular action. In conclusion, we have identified K(ATP) channel-independent targets of 5-HD, diazoxide and pinacidil. Our findings question the assumption that sensitivity to diazoxide and 5-HD implies involvement of mitochondrial K(ATP) channels. We propose that pharmacological preconditioning may be related to partial inhibition of respiratory chain complexes.

Acyl Coenzyme A↗
PubMed1996

Use of transmitochondrial cybrids to assign a complex I defect to the mitochondrial DNA-encoded NADH dehydrogenase subunit 6 gene mutation at nucleotide pair 14459 that causes Leber hereditary optic neuropathy and dystonia.

A heteroplasmic G-to-A transition at nucleotide pair (np) 14459 within the mitochondrial DNA (mtDNA)-encoded NADH dehydrogenase subunit 6 (ND6) gene has been identified as the cause of Leber hereditary optic neuropathy (LHON) and/or pediatric-onset dystonia in three unrelated families. This ND6 np 14459 mutation changes a moderately conserved alanine to a valine at amino acid position 72 of the ND6 protein. Enzymologic analysis of mitochondrial NADH dehydrogenase (complex I) with submitochondrial particles isolated from Epstein-Barr virus-transformed lymphoblasts revealed a 60% reduction (P < 0.005) of complex I-specific activity in patient cell lines compared with controls, with no differences in enzymatic activity for complexes II plus III, III and IV. This biochemical defect was assigned to the ND6 np 14459 mutation by using transmitochondrial cybrids in which patient Epstein-Barr virus-transformed lymphoblast cell lines were enucleated and the cytoplasts were fused to a mtDNA-deficient (p 0) lymphoblastoid recipient cell line. Cybrids harboring the np 14459 mutation exhibited a 39% reduction (p < 0.02) in complex I-specific activity relative to wild-type cybrid lines but normal activity for the other complexes. Kinetic analysis of the np 14459 mutant complex I revealed that the Vmax of the enzyme was reduced while the Km remained the same as that of wild type. Furthermore, specific activity was inhibited by increasing concentrations of the reduced coenzyme Q analog decylubiquinol. These observations suggest that the np 14459 mutation may alter the coenzyme Q-binding site of complex I.

Cell Line, Transformed↗
PubMed1982

Sulphide as an inhibitor and electron donor for the cytochrome c oxidase system.

Anomalies both kinetic and equilibrium in nature are described for the inhibition of cytochrome c oxidase activity by sulphide in the isolated enzyme and in submitochondrial particles. These anomalies are related to the involvement of more than 1 mol of sulphide in the blockage of one cytochrome aa3 centre. Sulphide reduces resting cytochrome a3, a reaction that results in oxygen uptake and the loss of a sulphide molecule. Sulphide can also reduce cytochromes c and a; in the former case, a part of the one-equivalent oxidation product, presumed to be the SH radical, reacts with oxygen. Such oxygen uptake is also seen under aerobic conditions when ferricyanide reacts with sulphide. Three phases are identified in the inhibitory interaction of sulphide with the cytochrome c oxidase enzyme itself: an initial rapid reaction involving sulphide oxidation, oxygen uptake, and conversion of cytochrome aa3 into the low-spin "oxyferri" form; a subsequent step in which sulphide reduces cytochrome a; and the final inhibitory step in which a third molecule of sulphide binds the a3 iron centre in the cytochrome a2+ a3 3+ (oxy) species to give cytochrome a2+ a3 3+ H2S. the initial events parallel some of the events in the interaction of the cytochrome c-cytochrome aa3 system with monothiols; the final inhibitory event resembles that with cyanide.

Animals↗
PubMed1988

Lysosphingolipids and mitochondrial function. II. Deleterious effects of sphingosylphosphorylcholine.

Psychosine, sphingosylphosphorylcholine (52-104 microM), and other glycosphingolipids stimulate mitochondrial respiration (up to 500%) and inhibit oxidative phosphorylation to varying degrees. Above 104 microM these functions as well as uptake of Ca2+ are prevented. At 104 microM sphingosylphosphorylcholine inhibits the mitochondrial ATPase reaction in submitochondrial particles by 48%. Both sphingosylphosphorylcholine and psychosine enhance the active phosphate-dependent swelling of mitochondria. Passive swelling occurs in the presence of rotenone (when swelling does not normally occur) and under hypotonic conditions. A direct interaction of sphingosylphosphorylcholine with membranes is demonstrated by a discharge of the proton gradient across mitochondrial membranes, hemolysis of red blood cells, and binding to inner and outer mitochondrial membranes. Thus lysosphingolipids bind strongly to mitochondrial membranes and markedly alter mitochondrial function. This alteration would affect the ATP levels, thereby altering a wide range of ATP-dependent cellular functions. These results offer a partial explanation for the pathogenesis of representative lysosomal storage diseases.

Adenosine Triphosphatases↗
PubMed1990

Oxyradical production results from the Fe3(+)-doxorubicin complex undergoing self-reduction by its alpha-ketol group.

A variety of different measures have been used to compare the self-reduction of the Fe3+ complexes of doxorubicin and daunorubicin. The Fe3(+)-doxorubicin complex exhibited a much faster rate of (i) O2 consumption, (ii) self-reduction under Ar to the Fe2+ complex, (iii) aerobic reduction of ferricytochrome c, (iv) scavenging of Fe2+ by bipyridine, (v) hydroxyl radical production measured by electron paramagnetic resonance spin-trapping experiments, and (vi) inactivation of the cytochrome c oxidase activity of beef heart submitochondrial particles, than did the corresponding Fe3(+)-daunorubicin complex. In contrast to Fe3(+)-doxorubicin, the Fe3(+)-daunorubicin complex displayed only a fast phase of inhibition of the cytochrome c oxidase activity, indicating that the initial binding of these two Fe3(+)-drug complexes is very similar. All of these results indicate that Fe3(+)-doxorubicin undergoes a much faster self-reduction to the Fe2+ complex and hence a much greater rate of production of damaging oxyradicals when the Fe2+ is reoxidized by O2 or H2O2. The addition of the alpha-ketol acetol to Fe3(+)-daunorubicin resulted in greatly increased rates of (i) ferricytochrome c reduction, (ii) Fe2+ production, and (iii) hydroxyl radical production. These results support the hypothesis that the alpha-ketol functional group of doxorubicin (which is not present on daunorubicin and is the only structural difference between these two compounds) reduces the Fe3+ while undergoing oxidation itself.

Cytochrome c Group↗
PubMed1995

The Na+/H+ antiport in renal mitochondria.

In isolated renal mitochondria, Na+ and Li+ stimulated H+ efflux from the mitochondrial matrix. In submitochondrial particles (SMP), Na+ flux was also coupled to H+ transport in the opposite direction. The overshoot of Na+ uptake in SMP with an outwardly directed H+ gradient indicated that downhill efflux of H+ through the mitochondrial membrane induced uphill transport of Na+. Similar to the Na+/H+ antiport in other types of mitochondria, the antiport in renal mitochondria was more sensitive to amiloride derivatives than to amiloride itself. Benzamil and ethylisopropylamiloride (EIPA), but not amiloride, inhibited the antiport, with 50% inhibition of 10(-4) M for both benzamil in mitochondria and EIPA in SMP. The Na+/H+ antiport in renal mitochondria had simple saturation kinetics for external Na+ [Michaelis-Menten constant (Km) = 3.27 +/- 0.63 mM; maximal velocity (Vmax) = 0.022 +/- 0.002 pH units/s] and Li+ (Km = 3.62 +/- 0.75 mM; Vmax = 0.022 +/- 0.002 pH units/s). NH4Cl and NH4 acetate stimulated Na+ efflux and inhibited Na+ uptake in SMP. Comparable results with NH4 acetate and chloride suggested that NH4+ modified Na+ transport through its direct interaction with the Na+/H+ antiport, rather than through the alkalinization of intra-SMP space from non-ionic diffusion of NH3. These results suggested that the Na+/H+ antiport may be a factor in the exit of NH4+ from renal mitochondria.

Amiloride↗
PubMed1998

Nitric oxide regulates oxygen uptake and hydrogen peroxide release by the isolated beating rat heart.

Isolated rat heart perfused with 1.5-7.5 microM NO solutions or bradykinin, which activates endothelial NO synthase, showed a dose-dependent decrease in myocardial O2 uptake from 3.2 +/- 0.3 to 1.6 +/- 0.1 (7.5 microM NO, n = 18, P < 0.05) and to 1.2 +/- 0.1 microM O2.min-1.g tissue-1 (10 microM bradykinin, n = 10, P < 0.05). Perfused NO concentrations correlated with an induced release of hydrogen peroxide (H2O2) in the effluent (r = 0.99, P < 0.01). NO markedly decreased the O2 uptake of isolated rat heart mitochondria (50% inhibition at 0.4 microM NO, r = 0.99, P < 0.001). Cytochrome spectra in NO-treated submitochondrial particles showed a double inhibition of electron transfer at cytochrome oxidase and between cytochrome b and cytochrome c, which accounts for the effects in O2 uptake and H2O2 release. Most NO was bound to myoglobin; this fact is consistent with NO steady-state concentrations of 0.1-0.3 microM, which affect mitochondria. In the intact heart, finely adjusted NO concentrations regulate mitochondrial O2 uptake and superoxide anion production (reflected by H2O2), which in turn contributes to the physiological clearance of NO through peroxynitrite formation.

Animals↗
PubMed2004

Excessive ATP hydrolysis in ischemic myocardium by mitochondrial F1F0-ATPase: effect of selective pharmacological inhibition of mitochondrial ATPase hydrolase activity.

Mitochondrial F(1)F(0)-ATPase normally synthesizes ATP in the heart, but under ischemic conditions this enzyme paradoxically causes ATP hydrolysis. Nonselective inhibitors of this enzyme (aurovertin, oligomycin) inhibit ATP synthesis in normal tissue but also inhibit ATP hydrolysis in ischemic myocardium. We characterized the profile of aurovertin and oligomycin in ischemic and nonischemic rat myocardium and compared this with the profile of BMS-199264, which only inhibits F(1)F(0)-ATP hydrolase activity. In isolated rat hearts, aurovertin (1-10 microM) and oligomycin (10 microM), at concentrations inhibiting ATPase activity, reduced ATP concentration and contractile function in the nonischemic heart but significantly reduced the rate of ATP depletion during ischemia. They also inhibited recovery of reperfusion ATP and contractile function, consistent with nonselective F(1)F(0)-ATPase inhibitory activity, which suggests that upon reperfusion, the hydrolase activity switches back to ATP synthesis. BMS-199264 inhibits F(1)F(0) hydrolase activity in submitochondrial particles with no effect on ATP synthase activity. BMS-199264 (1-10 microM) conserved ATP in rat hearts during ischemia while having no effect on preischemic contractile function or ATP concentration. Reperfusion ATP levels were replenished faster and necrosis was reduced by BMS-199264. ATP hydrolase activity ex vivo was selectively inhibited by BMS-199264. Therefore, excessive ATP hydrolysis by F(1)F(0)-ATPase contributes to the decline in cardiac energy reserve during ischemia and selective inhibition of ATP hydrolase activity can protect ischemic myocardium.

Adenosine Triphosphate↗
PubMed1994

Mitochondrial F1-ATPase activity of canine myocardium: effects of hypoxia and stimulation.

Recent studies have suggested that modifications in mitochondrial F1-adenosinetriphosphatase (ATPase) activity may play an important role in the regulation of myocardial oxidative phosphorylation. The goal of the present study was to develop and characterize an assay of F1-ATPase activity that could be performed repeatedly on an intact heart under various physiological states. With the use of submitochondrial particles prepared from biopsy samples of canine myocardium, we found reproducible F1-ATPase activity when normalized to the activity of the intramitochondrial enzyme citrate synthase. The oligomycin-sensitive component of the ATPase activity was found to be mainly F1-ATPase. F1-ATPase activity of normal myocardium increased by incubation in high salt-pH buffer, suggesting baseline inhibition. Five minutes after global ischemia, F1-ATPase activity decreased to 60% of baseline. Hypoxia for 10 min resulted in no significant change in F1-ATPase activity. With phenylephrine infusion, myocardial oxygen consumption more than doubled, whereas F1-ATPase activity increased by approximately 30%. Both returned to baseline levels after discontinuation of the drug. With the use of an assay developed to measure F1-ATPase activity of intact myocardium, changes of the enzyme activity were found during both ischemia and at increased work loads. These data suggest that alterations of F1-ATPase activity may contribute to the regulation of myocardial oxidative phosphorylation.

Adenosine Triphosphatases↗
PubMed1998

Preconditioning in rat hearts is independent of mitochondrial F1F0 ATPase inhibition.

Mitochondrial F1F0 adenosinetriphosphatase (ATPase) is responsible for the majority of ATP synthesis during normoxic conditions, but under ischemic conditions it accounts for significant ATP hydrolysis. A previous study showed that preconditioning in isolated rat hearts is mediated by inhibition of this ATPase during ischemia. We tested this hypothesis in our isolated rat heart model of preconditioning. Preconditioning was accomplished by three 5-min periods of global ischemia separated by 5 min of reperfusion. This was followed by 20 min of global ischemia and 30 min of reperfusion. Preconditioning significantly enhanced reperfusion contractile function and reduced lactate dehydrogenase release but paradoxically reduced the time to onset of contracture during global ischemia. Myocardial ATP was depleted at a faster rate during the prolonged ischemia in preconditioned than in sham-treated hearts, which is consistent with the reduced time to contracture. ATP during reperfusion was repleted more rapidly in preconditioned hearts, which is consistent with their enhanced contractile function. Preconditioning significantly reduced lactate accumulation during the prolonged ischemia. We were not able to demonstrate that mitochondrial F1F0 ATPase (measured in submitochondrial particles) was inhibited by preconditioning before or during the prolonged ischemia. The mitochondrial ATPase inhibitor oligomycin significantly conserved ATP during ischemia and increased the time to the onset of contracture, which is consistent with inhibition of the mitochondrial ATPase. Our results show that preconditioning in rat hearts can be independent of mitochondrial ATPase inhibition as well as ATP conservation.

Adenosine Triphosphate↗
PubMed1975

ATPase activity of kidney mitochondria stimulated by sodium.

ATPase activity of intact mitochondria may be induced by the presence of sodium. Mitochondria obtained from kidney cortex or medulla demonstrate a higher sodium-stimulated ATPase than mitochondria prepared from liver. This difference is not due to a greater capacity of kidney mitochondria for ATP hydrolysis. Sodium-stimulated ATPase activity of mitochondria is not further enhanced by magnesium or potassium, nor is it inhibited by ouabain. Furthermore, the ATPase of submitochondrial particles is not enhanced by the presence of sodium. These results indicate that the ATPase activity of mitochondria which is stimulated by sodium is unrelated to the microsomal Na-K-ATPase. mitochondrial sodium-stimulated ATPase is affected by pH, anionic composition, furosemide, and ethacrynic acid. The demonstration that mitochondrial ATP hydrolysis can be activated by sodium suggests that intracellular sodium may directly influence intracellular energy metabolism.

Adenosine Triphosphatases↗
PubMed2003

Mitochondrial complex I, aconitase, and succinate dehydrogenase during hypoxia-reoxygenation: modulation of enzyme activities by MnSOD.

Both NADH dehydrogenase (complex I) and aconitase are inactivated partially in vitro by superoxide (O2-.) and other oxidants that cause loss of iron from enzyme cubane (4Fe-4S) centers. We tested whether hypoxia-reoxygenation (H-R) by itself would decrease lung epithelial cell NADH dehydrogenase, aconitase, and succinate dehydrogenase (SDH) activities and whether transfection with adenoviral vectors expressing MnSOD (Ad.MnSOD) would inhibit oxidative enzyme inactivation and thus confirm a mechanism involving O2-. Human lung carcinoma cells with alveolar epithelial cell characteristics (A549 cells) were exposed to <1% O2-5% CO2 (hypoxia) for 24 h followed by air-5% CO2 for 24 h (reoxygenation). NADH dehydrogenase activity was assayed in submitochondrial particles; aconitase and SDH activities were measured in cell lysates. H-R significantly decreased NADH dehydrogenase, aconitase, and SDH activities. Ad.MnSOD increased mitochondrial MnSOD substantially and prevented the inhibitory effects of H-R on enzyme activities. Addition of alpha-ketoglutarate plus aspartate, but not succinate, to medium prevented cytotoxicity due to 2,3-dimethoxy-1,4-naphthoquinone. After hypoxia, cells displayed significantly increased dihydrorhodamine fluorescence, indicating increased mitochondrial oxidant production. Inhibition of NADH dehydrogenase, aconitase, and SDH activities during reoxygenation are due to excess O2-. produced in mitochondria, because enzyme inactivation can be prevented by overexpression of MnSOD.

Aconitate Hydratase↗
PubMed1990

Effect of acute pH change on mitochondrial glutamine transport.

The uptake of [3H]glutamine in submitochondrial particles (SMP) was measured at varying medium pH. Glutamine transport, but not glutamine binding, was inversely related to medium pH (range 6.5-8.5). Glutamine uptake was highest at medium pH of 6.5 (4.59 +/- 0.4 pmol.mg-1.30 s-1) and lowest at medium pH of 8.0 (1.99 +/- 0.3 pmol.mg-1.30 s-1). The effect of medium pH on glutamine transport was rapidly reversible. Changes in pH gradient (delta pH) had no influence on the rate of glutamine transport. Kinetics of mitochondrial glutamine transport was studied at pH 6.5 and 8.5 to further elucidate the mechanism by which pH alters glutamine transport. Glutamine concentration at half-maximal velocity ([S]0.5) was 9.07 and 13.32 mM at pH 6.5 and 8.5, respectively (P less than 0.02). The maximal velocity (Vmax) was 1,417.72 +/- 185.69 pmol.mg-1.15 s-1 at pH 6.5 and 910.95 +/- 192.85 pmol.mg-1.15 s-1 at pH 8.5 (P less than 0.01). Thus both the affinity and the Vmax of the transport system were enhanced in acidic pH. These data imply that intracellular pH has influence over glutamine transport into the mitochondrial matrix and, consequently, renal NH3 production. Glutamine delivery to the matrix via changes in cytosolic pH may be one of the regulators for ammoniagenesis during acute acid-base disorders.

Acid-Base Imbalance↗