Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.14 (ATP synthase)
7,042 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The rate of ATP hydrolysis catalyzed by the membrane-bound CF0F1 ATP synthase from chloroplasts served as a probe for the determination of the reduction grade of the enzyme treated with dithiothreitol (DTT) or thioredoxin. Rate constants for reduction were obtained. It turns out that reduction by thioredoxin is about a factor of 6,000 more effective than DTT reduction. The activation profiles with respect to delta pH were obtained for reduced and oxidized ATPases. The activation curve of reduced enzyme turns out to have its half-maximum degree of activation at delta pH = 1.65, which is considerably lower than reported hitherto. The corresponding value of the oxidized enzyme has been obtained from the rate of ATP hydrolysis in the case of incomplete reduced ATPases, taking into account the aforementioned rate constants, and comes to delta pH = 3.35.
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PMID:Kinetics and energetics of redox regulation of ATP synthase from chloroplasts. 849 39

The kinetics of thiol modulation of the chloroplast H+-ATPase (CF0CF1) in membrana were analyzed by employing thioredoxins that were kept reduced by 0.1 mM dithiothreitol. The kinetics of thiol modulation depend on the extent of the proton gradient. The process is an exponential function of the thioredoxin concentration and reaction time and can be described by an irreversible second order reaction. The results indicate that the formation of the complex between thioredoxin and CF0CF1 is slow compared with the subsequent reduction step. Furthermore we have compared the efficiencies of the Escherichia coli thioredoxin Trx and the two chloroplast thioredoxins Tr-m and Tr-f. The second order rate constants are 0.057 (Tr-f), 0.024 (Trx), and 0.010 s-1 microM-1 (Tr-m) suggesting that Tr-f rather than Tr-m is the physiological reductant for the chloroplast ATPase. The often employed artificial reductant dithiothreitol exhibits a second order rate constant in thiol modulation of 1.02.10(-6) s-1 microM-1.
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PMID:Kinetics and thioredoxin specificity of thiol modulation of the chloroplast H+-ATPase. 920 2

The mechanism of thiol modulation of the chloroplast ATP synthase by Escherichia coli thioredoxin was investigated in the isolated ATPase subcomplex and in the ATP synthase complex reconstituted in bacteriorhodopsin proteoliposomes. Thiol modulation was resolved kinetically by continuously monitoring ATP hydrolysis by the isolated subcomplex and ATP synthesis by proteoliposomes. The binding rate constant of reduced thioredoxin to the oxidized ATPase subcomplex devoid of its epsilon subunit could be determined. It did not depend on the catalytic turnover. Reciprocically, the catalytic turnover did not seem to depend on thioredoxin binding. Thiol modulation by Trx of the epsilon-bearing ATPase subcomplex was slow and favored the release of epsilon. The rate constant of thioredoxin binding to the membrane-bound ATP synthase increased with the protonmotive force. It was lower in the presence of ADP than in its absence, revealing a specific effect of the ATP synthase turnover on thioredoxin-gamma subunit interaction. These findings, and more especially the comparisons between the isolated ATPase subcomplex and the ATP synthase complex, can be interpreted in the frame of the rotational catalysis hypothesis. Finally, thiol modulation changed the catalytic properties of the ATP synthase, the kinetics of which became non-Michaelian. This questions the common view about the nature of changes induced by ATP synthase thiol modulation.
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PMID:Mechanism of activation of the chloroplast ATP synthase. A kinetic study of the thiol modulation of isolated ATPase and membrane-bound ATP synthase from spinach by Eschericia coli thioredoxin. 1078 30

Chloroplast ATP synthase is a thiol-modulated enzyme whose DeltamuH(+)-linked activation is strongly influenced by reduction and the formation of a disulphide bridge between Cys(199) and Cys(205) on the gamma subunit. In solubilized chloroplast coupling factor 1 (CF(1)), reduction of the disulphide bond elicits the latent ATP-hydrolysing activity. To assess the regulatory importance of the amino acid residues around these cysteine residues, we focused on the three negatively charged residues Glu(210)-Asp-Glu(212) close to the two cysteine residues and also on the following region from Leu(213) to Ile(230), and investigated the modulation of ATPase activity by chloroplast thioredoxins. The mutant gamma subunits were reconstituted with the alpha and beta subunits from F(1) of the thermophilic bacterium Bacillus PS3; the active ATPase complexes obtained were purified by gel-filtration chromatography. The complex formed with a mutant gamma subunit in which Glu(210) to Glu(212) had been deleted was inactivated rather than activated by reduction of the disulphide bridge by reduced thioredoxin, indicating inverse regulation. This complex was insensitive to the inhibitory CF(1)-epsilon subunit when the mutant gamma subunit was oxidized. In contrast, the deletion of Glu(212) to Ile(230) converted the complex from a modulated state into a highly active state.
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PMID:Inverse regulation of F1-ATPase activity by a mutation at the regulatory region on the gamma subunit of chloroplast ATP synthase. 1110 86

A treatment of leaves of Spinacia oleracea L. with light or with the thiol reagent dithiothreitol in the dark led to partly uncoupled thylakoids. After induction in intact leaves, the partial uncoupling was irreversible at the level of isolated thylakoids. We distinguish between uncoupling by proton slip, which means a decrease of the H+/e(-) -ratio due to less efficient proton pumping, and proton leak as defined by enhanced kinetics of proton efflux. Proton slip and proton leak made about equal contributions to the total uncoupling. The enhanced proton efflux kinetics corresponded to reduction of subunit CF1-gamma of the ATP synthase as shown by fluorescence labeling of thylakoid proteins with the sulfhydryl probe 5-iodoacetamido fluorescein. The maximum value of the fraction of reduced CF1-gamma was only 36%, which indicates that in vivo the reduction of CF1-gamma could be limited by fast reoxidation and/or restricted accessibility of CF1-gamma to thioredoxin. Measurements of the ratio ATP/2e indicated that only the uncoupling related to less efficient proton pumping led to a decrease in the ATP yield.
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PMID:Light-induced proton slip and proton leak at the thylakoid membrane. 1565 3

We have used the nervous system of the medicinal leech as a preparation to study the molecular basis of neural repair. The leech central nervous system, unlike mammalian CNS, can regenerate to restore function, and contains identified nerve cells of known function and connectivity. We have constructed subtractive cDNA probes from whole and regenerating ganglia of the ventral nerve cord and have used these to screen a serotonergic Retzius neuron library. This identifies genes that are regulated as a result of axotomy, and are expressed by the Retzius cell. This approach identifies many genes, both novel and known. Many of the known genes identified have homologues in vertebrates, including man. For example, genes encoding thioredoxin (TRX), Rough Endoplasmic Reticulum Protein 1 (RER-1) and ATP synthase are upregulated at 24 h postinjury in leech nerve cord. To investigate the functional role of regulated genes in neuron regrowth we are using microinjection of antisense oligonucleotides in combination with horseradish peroxidase to knock down expression of a chosen gene and to assess regeneration in single neurons in 3-D ganglion culture. As an example of this approach we describe experiments to microinject antisense oligonucleotide to a leech isoform of the structural protein, Protein 4.1. Our approach thus identifies genes regulated at different times after injury that may underpin the intrinsic ability of leech neurons to survive damage, to initiate regrowth programs and to remake functional connections. It enables us to determine the time course of gene expression in the regenerating nerve cord, and to study the effects of gene knockdown in identified neurons regenerating in defined conditions in culture.
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PMID:Hirudo medicinalis: a platform for investigating genes in neural repair. 1604 50

A single injection of monocrotaline produces a pulmonary insult in rats with a phenotype similar to human primary pulmonary hypertension. Although extensively used as a model, the mechanism(s) by which this chemical insult mimics a condition with genetic and environmental links remains an enigma, although formation of protein adducts has been implicated. Monocrotaline (MCT) is non-toxic and must undergo hepatic dehydrogenation to the soft electrophile monocrotaline pyrrole as prerequisite to damaging endothelial cells lining arterioles at remote pulmonary sites. In this report we extend our earlier investigation (J. Biol. Chem. 2000, 275, 29091-29099) by examining protein adducts to lower abundance adducts, a pI range not covered before, and subcellular localization of adduct-forming proteins associated with plasma membranes. Human pulmonary artery endothelial cells were exposed to [(14)C]MCT pyrrole (MCTP) and protein targets were identified using 2-DE with IPG 4-11. Adducted proteins were identified by pI, apparent molecular weight, and PMF using MALDI-TOF MS. Results of this study show that the majority of adducts form on proteins that contain reactive thiols in a CXXC motif, such as protein disulfide isomerase A(3) (ERp57), protein disulfide isomerase (PDI), and endothelial PDI. These same proteins were the major adduct-forming proteins associated with the plasma membrane. Other proteins found to be targets were thioredoxin, galectin-1, reticulocalbin 1 and 3, cytoskeletal tropomyosin, mitochondrial ATP synthase beta-chain, annexin A2 and cofilin-1. For the first time, MCTP adducts were observed on proteins not known to contain cysteine residues. However, known reactive proteins including nucleophosmin did not form detectable adducts, potentially indicating that MCTP did not reach the interior of nucleus to the same extent as other cellular sites. These findings suggest that molecular events underlying MCTP toxicity are initiated at the plasma membrane or readily accessible subcellular regions including the cytosol and membranes of the endoplasmic reticulum and mitochondria.
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PMID:Monocrotaline pyrrole targets proteins with and without cysteine residues in the cytosol and membranes of human pulmonary artery endothelial cells. 1622 22

The light-dependent regulation of chloroplast ATP synthase activity depends on an intricate but ill defined interplay between the proton electrochemical potential across the thylakoid membrane and thioredoxin-mediated redox modulation of a cysteine bridge located on the ATP synthase gamma-subunit. The abnormal light-dependent regulation of the chloroplast ATP synthase in the Arabidopsis thaliana cfq (coupling factor quick recovery) mutant was caused by a point mutation (G to A) in the atpC1 gene, which caused an amino acid substitution (E244K) in the vicinity of the redox modulation domain in the gamma-subunit of ATP synthase. Equilibrium redox titration revealed that this mutation made the regulatory sulfhydryl group energetically much more difficult to reduce relative to the wild type (i.e. raised the Em,7.9 by 39 mV). Enzymatic studies using isolated chloroplasts showed significantly lower light-induced ATPase and ATP synthase activity in the mutant compared with the wild type. The lower ATP synthesis capacity in turn restricted overall rates of leaf photosynthesis in the cfq mutant under low light. This work provides in situ validation of the concept that thioredoxin-dependent reduction of the gamma-subunit regulatory disulfide modulates the proton electrochemical potential energy requirement for activation of the chloroplast ATP synthase and that the activation state of the ATP synthase can limit leaf level photosynthesis.
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PMID:A point mutation in atpC1 raises the redox potential of the Arabidopsis chloroplast ATP synthase gamma-subunit regulatory disulfide above the range of thioredoxin modulation. 1795 6

Redox active proteins in plant mitochondria were examined using 2-D oxidant/reductant diagonal-SDS-PAGE to separate and identify proteins with intermolecular or intramolecular disulphide bonds using diamide in the first dimension and DTT in the second dimension. Eighteen proteins spots were resolved either above or below the diagonal and these were in-gel digested and identified by MS/MS. This analysis revealed intermolecular disulphide bonds in alternative oxidase, O-acetylserine (thiol) lyase, citrate synthase and between subunits of the ATP synthase. Intramolecular disulphide bonds were observed in a range of mitochondrial dehydrogenases, elongation factor Tu, adenylate kinase and the phosphate translocator. Many of the soluble proteins found were known glutaredoxin/thioredoxin targets in other plants, but the membrane proteins were not found by these methods nor were the nature of the disulphides able to be investigated. The accessibility of thiols involved in disulphide bonds to modification by a lipid derived aldehyde gave an insight into the potential impact of Cys modification on redox-functions in mitochondria during lipid peroxidation. Comparison of the protein sequences of the identified proteins with homologs from other species has identified specific Cys residues that may be responsible for plant-specific redox modulations of mitochondrial proteins.
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PMID:Identification of intra- and intermolecular disulphide bonding in the plant mitochondrial proteome by diagonal gel electrophoresis. 1799 21

Francisella tularensis is a gram-negative, highly infectious, aerosolizable facultative intracellular pathogen that causes the potentially life-threatening disease tularemia. To date there is no approved vaccine available, and little is known about the molecular mechanisms important for infection, survival, and dissemination at different times of infection. We report the first whole-genome screen using an inhalation mouse model to monitor infection in the lung and dissemination to the liver and spleen. We queried a comprehensive library of 2,998 sequence-defined transposon insertion mutants in Francisella novicida strain U112 using a microarray-based negative-selection screen. We were able to track the behavior of 1,029 annotated genes, equivalent to a detection rate of 75% and corresponding to approximately 57% of the entire F. novicida genome. As expected, most transposon mutants retained the ability to colonize, but 125 candidate virulence genes (12%) could not be detected in at least one of the three organs. They fell into a variety of functional categories, with one-third having no annotated function and a statistically significant enrichment of genes involved in transcription. Based on the observation that behavior during complex pool infections correlated with the degree of attenuation during single-strain infection we identified nine genes expected to strongly contribute to infection. These included two genes, those for ATP synthase C (FTN_1645) and thioredoxin (FTN_1415), that when mutated allowed increased host survival and conferred protection in vaccination experiments.
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PMID:Genome-wide screen in Francisella novicida for genes required for pulmonary and systemic infection in mice. 1895 78


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