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PubMed1994

The influence of cryopreservation on parameters of energetic metabolism and motility of fowl spermatozoa.

The objective of this study was to estimate the effect of cryopreservation on the main pathways of energetic metabolism and motility of fowl spermatozoa. Sperm diluted 1:5 with the cryoprotective medium containing ethylene glycol (1.4 M final concentration) was frozen at the rate of 2-3 degrees C/min to -25 degrees C with a pause on the plateau of crystallization and then at an exponentially increasing rate to -196 degrees C. The frozen sperm was thawed in two successive water baths at 0 and at 41 degrees C. After cryopreservation, the rate of radioactive glucose oxidation to 14CO2 slightly decreased, the rate of labeled glutamate oxidation remained unchanged, and the rate of labeled succinate oxidation increased two-fold. After freeze-thawing, the rates of endogenous respiration with and without 2,4-dinitrophenol decreased; the oxidation rate of exogenous succinate in the presence of 2,4-dinitrophenol, rotenone, and digitonin slightly decreased; and the rate of respiration in the presence of ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine, antimycin A, 2,4-dinitrophenol, and digitonin did not differ from that seen in control. Sperm respiration was highly sensitive to rotenone; antimycin A and cyanide blocked oxygen consumption completely. Succinate, added after 2,4-dinitrophenol and rotenone, stimulated respiration of thawed spermatozoa, which indicated plasma membrane damage. The addition of exogenous malate in the presence of 2,4-dinitrophenol and digitonin restored the respiration rate of thawed spermatozoa to that of unfrozen cells. The rate of respiration of thawed spermatozoa with oligomycin was higher than that of control cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1975

Correlation of the effects of citric acid cycle metabolites on succinate oxidation by rat liver mitochondria and submitochondrial particles.

1. Succinate dehydrogenase is inhibited by citrate and beta-hydroxy-butyrate in a complex manner, both in mitochondria and submitochondrial particles. Kinetics of inhibition in the particles points to a competitive component in the mechanism involved. 2. Pyruvate, alpha-ketoglutarate, malate, and glutamate stimulate oxidation of succinate by mitochondria. 3. Stimulation by alpha-ketoglutarate and glutamate is not influenced by the presence of rotenone. 4. Stimulation by pyruvate is higher in the absence of rotenone and increases significantly in the presence of K+ and valinomycin. Pyruvate supplies in mitochondria reducing equivalents for malate dehydrogenase operating in the reverse direction-reduction of oxaloacetate to malate. 5. Stimulation by malate is higher in the presence of rotenone.

Animals↗
PubMed2005

A possible site of superoxide generation in the complex I segment of rat heart mitochondria.

We searched for possible sites of superoxide generation in the complex I segment of the respiratory chain by studying both forward and reverse electron transfer reactions in isolated rat heart mitochondria. Superoxide production was monitored by measuring the release of hydrogen peroxide from mitochondria with a fluorescence spectrophotometer using the Amplex red/horseradish peroxidase system. In the forward electron transfer, a slow superoxide production in the presence of glutamate and malate was enhanced by both rotenone and piericidin A (specific inhibitors at the end of the complex I respiratory chain). Both diphenileneiodonium and ethoxyformic anhydride (inhibitors for respiratory components located upstream of the respiratory chain) inhibited the enhancement by rotenone and piericidin A. In contrast, in reverse electron transfer driven by ATP, both diphenileneiodonium and ethoxyformic anhydride enhanced the superoxide production. Piericidin A also increased superoxide production. Rotenone increased it only in the presence of piericidin A. Our results suggest that the major site of superoxide generation is not flavin, but protein-associated ubisemiquinones which are spin-coupled with iron-sulfur cluster N2.

Animals↗
PubMed1991

Mercury-induced H2O2 production and lipid peroxidation in vitro in rat kidney mitochondria.

Mercuric ion (Hg(II)) causes oxidative tissue damage in kidney cortical cells. We studied the in vitro effects of Hg(II) on hydrogen peroxide (H2O2) production by rat kidney mitochondria, a principal intracellular target of Hg(II). In mitochondria supplemented with a respiratory chain substrate (succinate or malate/glutamate) and an electron transport inhibitor (antimycin A (AA) or rotenone), Hg(II) (30 nmol/mg protein) increased H2O2 formation approximately 4-fold at the ubiquinone-cytochrome b region (AA-inhibited) and 2-fold at the NADH dehydrogenase region (rotenone-inhibited). Concomitantly, Hg(II) increased iron-dependent lipid peroxidation 3.5-fold at the NADH dehydrogenase region, but only by 25% at the ubiquinone-cytochrome b region. The mitochondrial concentration of reduced glutathione (GSH) decreased both with incubation time and Hg(II) concentration. Hg(II), at a concentration of 12 nmol/mg protein, caused almost complete depletion of measurable GSH in substrate-supplemented mitochondria after a 30-min incubation. In electron transport-inhibited mitochondria, Hg(II) caused greater depletion of GSH in rotenone-inhibited than in AA-inhibited mitochondria, consistent with the effects of Hg(II) on lipid peroxidation. These results suggest that Hg(II) at low concentrations depletes mitochondrial GSH and enhances H2O2 formation in kidney mitochondria under conditions of impaired respiratory chain electron transport. The increased H2O2 formation by Hg(II) may lead to oxidative tissue damage, such as lipid peroxidation, observed in mercury-induced nephrotoxicity.

Animals↗
PubMed1996

Exposure to the parkinsonian neurotoxin 1-methyl-4-phenylpyridinium (MPP+) and nitric oxide simultaneously causes cyclosporin A-sensitive mitochondrial calcium efflux and depolarisation.

The effect of the parkinsonian neurotoxin, 1-methyl-4-phenylpyridinium (MPP+) together with nitric oxide donors on mitochondrial calcium homeostasis and membrane potential was investigated. Simultaneous exposure of calcium-loaded mitochondria to MPP+ and nitric oxide donors led to Cyclosporin A-sensitive mitochondrial calcium efflux and depolarisation. When MPP+ was replaced with the respiratory inhibitor rotenone, mitochondrial calcium efflux and depolarisation also occurred. As both MPP+ and rotenone induce mitochondrial superoxide formation, the possibility that calcium efflux and depolarisation were due to peroxynitrite formation from reaction of superoxide with nitric oxide was investigated. It was shown that simultaneous exposure of mitochondrial membranes to nitric oxide donors and rotenone led to peroxynitrite formation. The possible roles of nitric oxide, peroxynitrite, mitochondrial depolarisation, and calcium efflux in MPP+ toxicity are discussed.

1-Methyl-4-phenylpyridinium↗
PubMed1990

A pharmacological model for studying the role of Na+ gradients in the modulation of synaptosomal free [Ca2+]i levels and energy metabolism.

Lactate production (Jlac), oxygen consumption rate (QO2), plasma membrane potentials (Em) and cytosolic free calcium levels [Ca2+]i were studied on synaptosomes isolated from rat brains, incubated in presence of high doses of nicardipine (90 microM), diltiazem (0.5 mM) and verapamil (0.25 mM), and submitted to depolarizing stimulation or inhibition of mitochondrial respiration. Nicardipine was able to completely prevent the veratridine-induced stimulation of Jlac, QO2 and Em depolarization, whereas diltiazem and verapamil were less effective, although the concentrations used were 5 and 3 times higher, respectively, than nicardipine. Diltiazem, verapamil and nicardipine (9 microM) also prevented the veratridine-induced increase in [Ca2+]i, this effect being much less pronounced if the drugs were added after veratridine. Monensin (20 microM) was also able to increase [Ca2+]i but this effect was not affected by verapamil. Synaptosomes were also submitted to an inhibition of respiration of intrasynaptic mitochondria by incubation with rotenone (5 microM); in this condition of mimicked hypoxia Em was more positive of about 11 mV; none of the drugs utilized modified this situation. The rotenone-induced 3-fold increase in Jlac was barely modified by diltiazem and verapamil but it was completely abolished by nicardipine. The possible mechanism of the counteracting action of the drugs towards veratridine stimulation and rotenone inhibition and the involvement of Na+/Ca2+ exchanger in affecting [Ca2+]i are discussed.

Animals↗
PubMed1995

Determination of structure-activity relationships of Annonaceous acetogenins by inhibition of oxygen uptake in rat liver mitochondria.

A new group of natural compounds, the Annonaceous acetogenins, have recently been determined to inhibit ATP production at a similar site of action and higher levels of potency as rotenone, i.e., at NADH-ubiquinone oxido-reductase, complex I of the mitochondrial electron-transport chain. The acetogenins had earlier been determined to be pesticidal, antimalarial, antimicrobial, anti-parasitic, cytotoxic, and in vivo active as potentially new antitumor agents. In order to determine structural activity relationships (SARs) among these compounds, at the subcellular level, several available acetogenins have been tested. Data obtained, from the inhibition of oxygen consumption by rat liver mitochondria, demonstrated that all of the twenty acetogenins tested are active with IC50 values in the range of 15-800 nM/mg protein. The IC50 value of rotenone was 17 nM/mg protein. The bis-adjacent THF ring acetogenins and the bis-nonadjacent THF ring compounds are about ten times more active than the mono-THF ring acetogenins. Overall, 30-OH and 31-OH-bullatacinone were the most active and were slightly more active than rotenone. The least active were the 4-deoxy bis-adjacent THF ring compounds followed by the mono-THF ring group. There was some variation between the groups, e.g., within the bis-adjacent and mono-THF ring groups, the alpha, beta-unsaturated-gamma-lactones were less active than the keto-lactones, but this observation was reversed for one of the pairs of bis-nonadjacent THF ring acetogenins. Additional hydroxylations, to a maximum of three, seemed to increase activity within all of the groups. Before final decisions on SARs can be made, additional comparisons of the results of this subcellular assay (as an in vitro assay) with the results of in vivo assays should be made. Also, future investigations into the exact site of action within complex I and other possible sites of action (such as the NADH oxidase of plasma membranes) need to be conducted for a more. complete understanding of the utility and potential of this new group of very potent compounds.

Acetylation↗
PubMed1982

Role of glucose metabolism in acid formation by isolated gastric glands.

The role placed by glucose in providing energy for acid formation was studied in isolated gastric glands from rabbit. The widely-used inhibitors of glycolysis, iodoacetic acid and iodoacetamide were found to inhibit glucose oxidation as well as the indicators of acid formation, respiration and accumulation of aminopyrine. However, the potent inhibitor of acid formation was found to involve a nonspecific mechanism other than the simple inhibition of glycolysis. An alternative approach involved use of the glucose transport inhibitor, phloretin. Phloretin blocked glucose oxidation and also inhibited functional responses. Acid formation was restored easily by the addition of pyruvate or various other oxidizable substrates. Measurement of lactate formation in the absence of exogenous glucose showed that the gastric glands contain very little glycogen. Addition of external glucose resulted in a 10-fold increase in lactate formation and this rate was stimulated further by histamine and rotenone. Rotenone also inhibited both respiration and aminopyrine accumulation; however, the inhibition was not complete. Phloretin treatment resulted in total inhibition of the residual aminopyrine accumulation after rotenone treatment. The results are interpreted to indicate that gastric glands are dependent almost totally on external substrate supply to support acid formation; and, that while anaerobic glucose metabolism can sustain a very low level of acid formation, the major role of glucose is to yield pyruvate equivalents for subsequent oxidation.

Animals↗
PubMed2003

Detection of mitochondrial electron chain carrier redox status by transhepatic light intensity during rat liver reperfusion.

The aim of the study was to investigate mitochondrial electron transfer during rat liver reperfusion after cold storage and hypothermic machine perfusion. Livers from male Brown Norway rats were preserved (UW) for 10h either by cold storage (CS) or by hypothermic oxygenated perfusion extracorporal (HOPE). Transhepatic photometric analysis allowed determination of the redox status of mitochondrial cytochromes during preservation, rewarming and reperfusion. Mitochondrial electron chain carriers were inhibited at different sites with rotenone and cyanide in some experiments. reversed transcriptional polymerase chain reaction (RT-PCR) was performed after reperfusion concerning transcription of TNFalpha, caspase 9, and c-jun kinase (JNK). Increased superoxide anion formation as well as transcription of TNFalpha, caspase 9, and JNK during reperfusion after cold storage (CS) were related with completely reduced cytochromes before and during reperfusion. In contrast, hypothermic oxygenated livers (HOPE) showed oxygenated cytochromes as well as decreased superoxide anion formation and no detectable transcription of TNFalpha, caspase 9, and JNK. A similar low level of superoxide anion formation was found when electron chain transfer of cold stored livers was inhibited during reperfusion with rotenone but not with cyanide. After hypothermic oxygenation (HOPE) inhibition of mitochondrial electron chain with rotenone showed no change in formation of superoxide anion formation whereas inhibition with cyanide showed increased superoxide anion formation. Thus mitochondrial cytochrome redox status is suggested to be related: (i) with the release of reactive oxygen substances as well as (ii) with the expressions of TNFalpha, caspase 9, and JNK during reperfusion and may thus be usable as predictive marker of liver grafts.

Animals↗
PubMed2006

The effect of bovine serum albumin on the membrane potential and reactive oxygen species generation in succinate-supported isolated brain mitochondria.

Characteristics of the succinate-supported H(2)O(2) formation were compared in mitochondria prepared from guinea-pig brain either by Percoll gradient centrifugation or using digitonin. The high rate of H(2)O(2) generation measured in mitochondria prepared with digitonin (600.6+/-26.8pmol/min/mg protein) was inhibited by rotenone, consistently with a reverse flow of electrons via complex I. The rate of H(2)O(2) formation was significantly smaller in Percoll-purified mitochondria (252.6+/-17.3pmol/min/mg protein) and this was stimulated by rotenone. Since bovine serum albumin (BSA) is usually present in the isolation medium used in the digitonin method, systematic study was performed addressing the effect of BSA on H(2)O(2) formation. Mitochondria prepared by the digitonin method (BSA present in the isolation medium) were highly polarized (185+/-3.2mV) and addition of BSA (0.025%) to the assay medium increased H(2)O(2) generation by only 50%. In Percoll-purified mitochondria DeltaPsim was more depolarized (171+/-2mV) and BSA caused hyperpolarization by 10.7+/-1.9mV. H(2)O(2) formation, which was largely independent of DeltaPsim, was stimulated by 400%, became highly dependent on DeltaPsim and could be inhibited by rotenone in the presence of BSA. This shows that in Percoll-purified mitochondria ROS formation via reverse electron flow is preferred only when BSA is present in the assay medium. It is demonstrated that (i) the presence or absence of BSA could determine the mechanism by which ROS is generated in succinate-supported mitochondria and (ii) depolarization by about 10mV eliminates reverse electron flow and the remaining ROS formation, which is smaller but still significant, is no longer dependent on DeltaPsim.

Animals↗
PubMed1989

The pathway of electron transfer in NADH:Q oxidoreductase.

The pre-steady-state reduction by NADPH of NADH:Q oxidoreductase, as present in submitochondrial particles, has been further investigated with the rapid-mixing, rapid-freezing technique. It was found that trypsin treatment, that had previously been used to inactivate the transhydrogenase activity (Bakker, P.T.A. and Albracht, S.P.J. (1986) Biochim. Biophys. Acta 850, 413-422), considerably affected the stability at pH 6.2 of the NAD(P)H oxidation activity of submitochondrial particles. Use of the inhibitor butadione circumvented this problem, thus allowing a more careful investigation of the kinetics at pH 6.2. In the presence of the inhibitor rotenone it was found that 50% of the Fe-S clusters 3 and all of the Fe-S clusters 2 and 4 could be reduced by NADPH within 30 ms at pH 6.2. The remainder of the Fe-S clusters 3 and all of the Fe-S clusters 1 were reduced slowly (complete reduction only after more than 60 s). It was concluded that these latter Fe-S clusters play no role in the NADPH oxidation activity. In the absence of rotenone at pH 6.2 only 50% of the Fe-S clusters 2-4 could be reduced within 30 ms, while Fe-S cluster 1 was again not reduced. This difference was attributed to the fast reoxidation of part of the Fe-S clusters 2 and 4 by ubiquinone. At pH 8.0, where the NADPH oxidation activity is almost zero, 50% of the Fe-S clusters 2-4 could still be reduced by NADPH within 30 ms, while Fe-S cluster 1 was not reduced. The presence of rotenone had no effect on this reduction. From these observations it is concluded that the Fe-S clusters 2 and 4, which were rapidly reduced by NADPH and reoxidised by ubiquinone at pH 6.2, could not be reduced by NADPH at 8.0. This provides an explanation why NADH:Q oxidoreductase was not able to oxidise NADPH at pH 8.0, while part of the Fe-S clusters were still rapidly reduced. As a working hypothesis a dimeric structure for NADH:Q oxidoreductase is proposed. One protomer (B) contains FMN and Fe-S clusters 1-4 in equal amounts; the other protomer (A) is identical except for the absence of Fe-S cluster 1. NADH is able to react with both protomers, while NADPH only reacts with protomer A. A pH-dependent electron transfer from protomer A to protomer B is proposed, which would allow the reduction of Fe-S clusters 2 and 4 of protomer B by NADPH at pH 6.2, which is required for NADPH:Q oxidoreductase activity.

Animals↗
PubMed1998

Review article number 135 biosynthesis in the rotenoid group of natural products: applications of isotope methodology.

Using the natural rotenoids rotenone, amorphigenin, deguelin, rotenonic acid, dalpanol and munduserone as examples, their phytochemical biosynthesis has been examined in Derris elliptica, Amorpha fruticosa and Tephrosia vogelii. The rotenoids are advanced isoflavonoids, and construction of their angular A&z.drule;B&z.drule;C&z.drule;D-ring systems has been studied experimentally starting out from simple primary metabolites and passing through a series of mainly oxidative phases. The oxidative reactions of rot-2'-enonic acid which biosynthetically form the E-rings of rotenone, amorphigenin, dalpanol and deguelin have been studied in both chemical and stereochemical detail using seedling preparations and the enzyme deguelin cyclase. This investigation has extensively employed substrates isotopically labelled with (14)C, (13)C, (2)H and (3)H and, particularly in connection with E-ring biosynthesis, required new approaches to the demanding chemical and stereochemical requirements of the experimentation.

Journal Article↗
PubMed2002

Acute mitochondrial and chronic toxicological effects of 1-methyl-4-phenylpyridinium in human neuroblastoma cells.

At low micromolar concentrations, 1-methyl-4-phenylpyridinium (MPP+), the toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) selectively kills nigrostriatal dopaminergic neurons by mechanisms believed to involve impairment of mitochondrial complex I. A human neuroblastoma cell line expressing the dopamine transporter (DAT) was utilized to examine the effects of MPP+ on acute physiologic responses and subsequent cell death. Acute responses were measured by microphysiometry and by monitoring mitochondrial membrane potential with [3H]tetraphenylphosphonium (TPP+) uptake. MPP+ (10 microM) increased extracellular proton excretion in DAT-expressing cells within 2-3 min, but had no effect in untransfected cells. The lipophilic complex I inhibitor, rotenone, increased proton excretion in both cell lines. In DAT-expressing cells, mitochondrial membrane potential was reduced within I h of 10 microM MPP+ exposure. Rotenone reduced mitochondrial membrane potential in both cell lines. MPP+ caused apoptotic death of DAT-transfected cells 2-3 days after drug application, but did not kill untransfected cells. Thus, MPP+ produces immediate mitochondrial impairment only in cells that express DAT, and these changes occur days before overt cellular toxicity. The magnitude, time course and nature of these changes were similar to those produced by rotenone, confirming the site of action of MPP+ as mitochondrial complex I. These immediate mitochondrial effects appear to be an accurate predictor of subsequent cell death.

1-Methyl-4-phenylpyridinium↗
PubMed1998

Development of infrared imaging to measure thermogenesis in cell culture: thermogenic effects of uncoupling protein-2, troglitazone, and beta-adrenoceptor agonists.

PURPOSE: Although the effects of thermogenic agents in cell culture can be measured by direct microcalorimetry, only a few samples can be analyzed over several hours. In this report, we describe a robust non-invasive technique to measure real-time thermogenesis of cells cultured in microtiter plates using infrared thermography. METHODS: Yeast were transformed with uncoupling protein-2 (UCP2) or exposed to carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) or rotenone. Adipocytes were exposed to rotenone, FCCP, cycloheximide. troglitazone, or CL316243. Thermogenesis was measured using infrared thermography. RESULTS: Thermogenesis increased after exposing yeast to the mitochondrial uncoupler, FCCP, or transforming the cells with UCP2. Further, thermogenesis in adipocytes was stimulated by CL316243, a beta3-adrenoceptor agonist being developed to treat obesity. The protein synthesis inhibitor, cycloheximide, did not inhibit CL316243-mediated thermogenesis. In contrast, the mitochondrial proton transport inhibitor, rotenone, inhibited thermogenesis in yeast and adipocytes. Similarly, the antidiabetic agent, troglitazone, suppressed thermogenesis in adipocytes. Although increased UCP synthesis resulted in increased thermogenesis in yeast, UCP expression did not correlate with thermogenesis in adipocytes. CONCLUSIONS: The results, taken together with the high resolution (0.002 degrees C) and robustness (384-well format) of the approach, indicate infrared-imaging is a rapid and effective method for measuring thermogenesis in vitro.

Adipocytes↗
PubMed2002

Isolation and characterization of tightly coupled mitochondria from wild type and nap mutant Neurospora crassa.

A fast and reproducible procedure was elaborated for isolation of tightly coupled mitochondria from wild type and nap-mutant Neurospora crassa cells harvested at different growth stages. The isolated mitochondrial preparations had controlled metabolic states and were tightly coupled, i.e., displayed good respiratory control and had close to the theoretically expected maximal ADP/O ratios upon oxidation of Krebs cycle intermediates and exogenous NADH. They contained the fully competent respiratory chain with all three points of energy conservation. Oxidation of all examined substrates by mitochondria from both wild type and mutant cells was mediated by two alternative terminal oxidative systems, albeit to varying extent, with the more pronounced engagement of the alternative oxidase in the stationary growth phase and with a minor contribution of this non-phosphorylating pathway in the substrate oxidation by mutant mitochondria. Oxidation of NAD-dependent substrates by mitochondria from the two cell types was accommodated via both rotenone-sensitive and rotenone-insensitive pathways, while the level of rotenone-insensitive pathway in mutant cells was lower than in wild type cells. It is suggested that a more limited contribution of alternative non-phosphorylating oxidative pathways to the total respiration in mutant cells, as compared with wild type cells, could, at least partially, explain an elevated ATP level in these cells. However, the absence of principal differences in the arrangement of the respiratory chain in mitochondria of wild type and mutant cells implies that the elevated ATP level in the nap mutant is largely related to reduced ATP expenses for transport processes in these cells.

Adenosine Diphosphate↗
PubMed2001

Involvement of matrix NADP turnover in the oxidation of NAD-linked substrates by pea leaf mitochondria.

The involvement of the internal rotenone-insensitive NADPH dehydrogenase on the inner surface of the inner mitochondrial membrane [NDin(NADPH)] in the oxidation of strictly NAD+-linked substrates by pea (Pisum sativum L.) leaf mitochondria was measured. As estimated by the inhibition caused by 5 µM diphenyleneiodonium (DPI) in the presence of rotenone to inhibit complex I, the activity of NDin(NADPH) during glycine oxidation (measured both as O2 uptake and as CO2 release) was 40-50 nmol mg-1 protein min-1. No significant activity of NDin(NADPH) could be detected during the oxidation of 2-oxoglutarate, another strictly NAD+-linked substrate; this was possibly due to its relatively low oxidation rate. Control experiments showed that, even at 125 µM, DPI had no effect on the activity of glycine decarboxylase complex (GDC) and lipoamide dehydrogenase. The relative activity of complex I, NDin(NADPH), and NDin(NADH) during glycine oxidation, estimated using rotenone and DPI, differed depending on the pyridine nucleotide supply in the mitochondrial matrix. This was shown by loading the mitochondria with NAD+ and NADP+, both of which were taken up by the organelle. We conclude that the involvement of NADP turnover during glycine oxidation is not due to the direct production of NADPH by GDC but is an indirect result of this process. It probably occurs via the interconversion of NADH to NADPH by the two non-energy-linked transhydrogenase activities recently identified in plant mitochondria.

Journal Article↗
PubMed1980

Respiration-dependent calcium ion uptake by two preparations of cardiac mitochondria. Effects of palmitoyl-coenzyme A and palmitoylcarnitine on calcium ion cycling and nicotinamide nucleotide reduction state.

Ca(2+) uptake and the effect of the uptake inhibitors palmitoyl-CoA and palmitoylcarnitine were examined in two preparations of dog cardiac mitochondria. Mitochondria prepared by using the Nagarse technique was 2.5-fold more active in respiration-dependent Ca(2+) uptake than were mitochondria isolated by using the Polytron procedure. Palmitoyl-CoA and palmitoylcarnitine inhibited Ca(2+) uptake in both preparations uncompetitively, with K(i,app) 0.4 and 20mum. Ca(2+)-uptake rates were related to, or influenced by, the concentration of mitochondrial reduced nicotinamide nucleotides, with uptake slowing as this concentration decreased. When most of the nicotinamide nucleotides was oxidized, Ca(2+) release and respiratory stimulation were observed. In the presence of Ruthenium Red and palmitoyl-CoA, oxidation of nicotinamide nucleotides was abolished and the time to Ca(2+) release was shortened corresponding to the time of onset of nicotinamide nucleotide oxidation in the absence of Ruthenium Red. The results suggest that NAD(P)H oxidation in the presence of rotenone was a consequence of Ca(2+) re-uptake and that net Ca(2+) release could be observed as reduced nicotinamide nucleotide concentrations declined. Although nicotinamide nucleotide oxidation occurred in the presence of rotenone, it was not linked in an apparent manner to acyl-group metabolism (palmitoylcarnitine was less effective than palmitoyl-CoA). Therefore either a by-pass of the rotenone block or a direct interaction of NAD(P)H with the Ca(2+)-uptake process was possible. Loss of NADH occurred before respiratory stimulation, and this loss may relate to decreased coupling efficiency at sites 2 and 3 of the respiratory chain, as suggested by others [Bhuvaneswaran & Wadkins (1978) Biochem. Biophys. Res. Commun.82, 648-654].

Acyl Coenzyme A↗
PubMed1980

Lipid peroxidation and the reduction of ADP-Fe3+ chelate by NADH-ubiquinone reductase preparation from bovine heart mitochondria.

The NADH-ubiquinone reductase preparation (Complex I) of bovine hart mitochondria catalysed in the presence of reduced coenzymes and ADP-Fe3+ the lipid peroxidation of liposomes prepared from mitochondrial lipids. The apparent Km values for the coenzymes and the optimal pH of the reactions agreed well with those of the lipid peroxidation of the submitochondrial particles treated with rotenone. On assay of the reduction of ADP-Fe3+ chelate by the reduction of cytochrome c in the presence of superoxide dismutase and antimycin A or by the oxidation of reduced coenzymes, the reactions were not affected by rotenone but were inhibited by thiol-group inhibitors. The properties of the ADP-Fe3+ reductase activity were highly consistent with those of the lipid-peroxidation reaction. These observations suggest that electrons from reduced coenzymes are transferred to ADP-Fe3+ chelate from a component between a mercurial-sensitive site and the rotenone-sensitive one of the NADH dehydrogenase and that the reduction of ADP-Fe3+ chelate by the NADH dehydrogenase is an essential step in the lipid peroxidation.

Adenosine Diphosphate↗