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Results for “OXIDATIVE PHOSPHORYLATION”

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At least 1,009 records · Page 56Linked to original sources
PubMed2000

High level expression of full-length estrogen receptor in Escherichia coli is facilitated by the uncoupler of oxidative phosphorylation, CCCP.

The expression of high levels of full-length human estrogen receptor alpha (hERalpha) in Escherichia coli has proven difficult. We found that expression of the ER DNA binding domain is highly toxic to E. coli, resulting in rapid loss of the expression plasmid. Using a tightly regulated arabinose expression system and the antibiotic Timentin, we were able to overcome ER toxicity and express substantial levels of ER. The expressed ER exhibited protease cleavage at a single site near the N-terminus of the hinge region. Of the many measures we tested to eliminate ER cleavage, only addition of carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), an uncoupler of oxidative phosphorylation, completely blocked intracellular proteolysis of the ER. Using CCCP and our expression methods, full-length FLAG epitope-tagged hERalpha (fER) was expressed in E. coli at approximately 1 mg/l. The fER was purified to homogeneity in a single step by immunoaffinity chromatography with anti-FLAG monoclonal antibody. Purified full-length bacterial fER binds 17beta-estradiol with the same affinity as hER expressed in human cells (K(D) approximately 0.5 nM). At high concentrations of fER (20 nM), a bell-shaped estrogen binding curve with a Hill coefficient of 1.7 was seen. Bacterially-expressed fER exhibits a reduced affinity for the estrogen response element (ERE). Anti-FLAG antibody restores high affinity binding of the fER to the ERE, suggesting that impaired dimerization may be responsible for the reduced affinity of bacterially-expressed fER for the ERE. The use of Timentin and CCCP may provide a general method for high level bacterial expression of steroid/nuclear receptors and other proteins important in hormone action.

Base Sequence↗
PubMed2001

Induction of cyclosporin A-sensitive pore in mitochondria of intact neurons during uncoupling of oxidative phosphorylation.

Using primary cultures of cerebellar granule cells we showed that Ca(2+)transported into neurons under the effect of glutamate is accumulated and stored in mitochondria for a long time. Protonophore FCCP, an uncoupler of oxidative phosphorylation, stimulated the release of Ca(2+)from mitochondria in a calcium-free medium in 81% glutamate-treated cells. Cyclosporin A and ATP-synthase blocker oligomycin decreased the number of cells with FCCP-induced Ca(2+)release to 53 and 12%, respectively. Oligomycin partly prevented glutamate- and FCCP-induced decrease of intracellular ATP level.

Adenosine Triphosphate↗
PubMed1999

Further studies of diabetes-prone BHE/Cdb rats: increased sensitivity to calcium ion suppression of oxidative phosphorylation.

The responsiveness of hepatic mitochondria isolated from hyperthyroid and control Sprague-Dawley (SD) and diabetes-prone BHE/Cdb rats was studied. Hyperthyroidism was induced through the addition of thyroxine (T(4)) to the diet (2 mg/kg of diet). Oxidative phosphorylation (OXPHOS) with the addition of adenosine diphosphate (ADP) or an 8:1 mixture of adenosine monophosphate (AMP):ADP was studied. Dose response curves of state 3 and state 4 respiration, respiratory control (RC) ratio, and ADP:O ratio to calcium levels (0-7.5 microm) were generated. Mitochondria from BHE/Cdb rats were more sensitive to the addition of calcium than mitochondria from SD rats, as judged by losses in OXPHOS. T(4) treatment potentiated this strain difference and we conclude that the diabetes phenotype in the BHE/Cdb rat is probably related not only to the previously described mutation in the F(O)ATPase but also to a defect in the efflux of the calcium ion that, in turn, affected the regulation of OXPHOS.

Journal Article↗
PubMed2004

Effect of the oxidative phosphorylation uncoupler 2,4-dinitrophenol on hypoxia-inducible factor-regulated gene expression in bovine blastocysts.

In cattle embryos, development to the blastocyst stage is improved in the presence of 10 micro;m 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, coincident with an increase in glycolytic activity following embryonic genome activation. The present study examined redox-sensitive gene expression and embryo development in response to the addition of DNP post-compaction. 2,4-Dinitrophenol increased the expression of hypoxia-inducible factor 1alpha and 2alpha (HIF1alpha, HIF2alpha) mRNA. Although HIF1alpha protein remained undetectable in bovine blastocysts, HIF2alpha protein was localised within the nucleus of trophectoderm and inner cell mass (ICM) cells of blastocysts cultured in the presence or absence of DNP, with a slight increase in staining evident within the ICM in blastocysts cultured in the presence of DNP. However, the expression of GLUT1 and VEGF mRNA, genes known to be regulated by HIFs, was unaffected by the addition of DNP to the culture. Although the development of Grade 1 and 2 blastocysts was unaltered by the addition of DNP post compaction in the present study, a significant increase in the proportion of ICM cells was observed. Results indicate that 10 microm DNP improves the quality of bovine embryos, coincident with increased HIF2alpha protein localisation within ICM cells and increased HIFalpha mRNA levels. Therefore, the results demonstrate redox-regulated expression of HIF2.

2,4-Dinitrophenol↗
PubMed1987

[Mitochondrial oxidative phosphorylation in the rat kidney during the perinatal period].

Oxidative metabolism in the developing rat kidney has been studied on isolated mitochondria. An increase of about 50% in state 3 respiration has been observed at birth, using succinate, glutamate, or palmitoyl-L-carnitine as a substrate. The rate of respiration in the presence of 2,4-dinitrophenol was found identical to state 3 respiration in all cases. Cytochrome oxidase activity did not change between the fetal and newborn stages. The increase of mitochondrial respiration revealed here, which is not linked to a modification of the respiratory chain, could be involved in the rise of kidney ATP level and energy charge observed at birth.

Aging↗
PubMed1977

Prevention of endotoxin-induced changes in oxidative phosphorylation in hepatic mitochondria.

E. coli endotoxemia affects hepatic energy linked function by uncoupling oxidation from phosphorylation. This study was done to determine whether a steroid, methylprednisolone sodium succinate (MPS), as well as excess substrate sodium succinate (SS), alters directly the effects of endotoxin on hepatic mitochondria. An assay system using alpha-ketoglutarate (alpha-Kg) was developed to test this hypothesis. Isolated rat hepatic mitochondria were first incubated in concentrations of MPS, ranging from 2.0 to 6.0 mg/ml. At these concentrations uncoupling identical to that occurring with addition of endotoxin resulted. However, a more dilute solution of MPS, 0.12 mg/ml, permitted normal mitochondrial function. Preincubation of MT in 0.12 mg/ml of MPS, as well as with sodium succinate, prevented endotoxin-induced uncoupling. Both endotoxin and steroid resulted in increased ATPase activity in the medium. While preincubation with MPS blocks the endotoxin effect, very high steroid concentrations alone are harmful. A direct action of steroids on mitochondria is evident, as well as a weaker protective effect due to excess substrate (alpha-Kg + SS). Since mitochondria are probably in direct communication with extracellular fluid, the assay system permits interaction of endotoxin, steroids, and substrates which mimic those which occur in vivo. The results of this study account for the previously reported variable effects obtained when steroids have been tested in vivo.

Adenosine Triphosphatases↗
PubMed1995

Modulation of 2-oxoglutarate dehydrogenase and oxidative phosphorylation by Ca2+ in pancreas and adrenal cortex mitochondria.

The effect of external free Ca2+ ([Ca2+]ex) on the 2-oxoglutarate dehydrogenase (2-OGDH) activity and the rate of state 3 respiration was studied in dog pancreas and adrenal cortex mitochondria. A method for the preparation of mitochondria from pancreas, free of zymogen granules, was developed. The mitochondria showed high respiratory control values and maintained adequate ADP/O ratios in the course of several hours. Half-maximal stimulation (K0.5) of 2-OGDH activity and state 3 respiration by Ca2+ was decreased twofold by spermine, in both pancreas and adrenal cortex mitochondria incubated with Mg2+; Na+ increased the K0.5 value for Ca2+ in both kinds of mitochondria. The matrix Ca2+ content was enhanced by spermine at different [Ca2+]ex. The Ca2+ gradient ([Ca2+]m/[Ca2+]ex) determined in Fluo 3-loaded pancreas mitochondria was found to be below 1, either in the presence of Na+ or in the absence of spermine. The protonmotive force was increased by 1 microM Ca2+, in state 3 conditions, by 6-8 mV; lower Ca2+ concentrations kept the H+ gradient rather constant. An increase in the spermine/Mg2+ ratio at constant external Ca2+ resulted in stimulation of the 2-OGDH activity and state 3 respiration. The data indicate that matrix Ca2+ may regulate the rate of ATP synthesis in pancreas and adrenal cortex mitochondria, through the modulation of 2-OGDH and the maintenance of a high H+ gradient. In addition to changes in external Ca2+, the variation in the spermine/Mg2+ ratio may also exert control of oxidative phosphorylation.

Adrenal Cortex↗
PubMed2001

Conjugative metabolism of 1,2-dibromoethane in mitochondria: disruption of oxidative phosphorylation and alkylation of mitochondrial DNA.

1,2-Dibromoethane (DBE) is an environmental contaminant that is metabolized by glutathione S-transferases to a haloethane-glutathione conjugate. Since haloethane-glutathione conjugates are known to alkylate nuclear DNA and cytoplasmic proteins, these effects were investigated in isolated rat liver mitochondria exposed to DBE by measuring guanine adducts and several aspects of oxidative phosphorylation including respiratory control ratios, respiratory enzyme activity, and ATP levels. Mitochondrial large-amplitude swelling and glutathione status were assessed to evaluate mitochondrial membrane integrity and function. When exposed to DBE, mitochondria became uncoupled rapidly, yet no large-amplitude swelling or extramitochondrial glutathione was observed. Mitochondrial GSH was depleted to 2-53% of controls after a 60-min exposure to micromolar quantities of DBE; however, no extramitochondrial GSH or GSSG was detected. The depletion of mitochondrial glutathione corresponded to an increase of an intramitochondrial GSH-conjugate which, based on HPLC elution profiles and retention times, appeared to be S,S'-(1,2-ethanediyl)bis(glutathione). Activities of the NADH oxidase and succinate oxidase respiratory enzyme systems were inhibited 10-74% at micromolar levels of DBE, with succinate oxidase inactivation occurring at lower doses. ATP concentrations in DBE-exposed mitochondria in the presence of succinate were 5-90% lower than in the controls. The DNA adduct S-[2-(N(7)-guanyl)ethyl]glutathione was detected by HPLC in mtDNA isolated from DBE-exposed mitochondria. The results suggest that respiratory enzyme inhibition, glutathione depletion, decreased ATP levels, and DNA alkylation in DBE-exposed mitochondria occur via the formation of an S-(2-bromoethyl)glutathione conjugate, the precursor of the episulfonium ion alkylating species of DBE.

Adenosine Triphosphate↗