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Studies on energy-linked reactions. Energy-linked transhydrogenase reaction in Escherichia coli.

ABSTRACT: 1. Small particles from Escherichia coli catalyse an ATP-dependent reduction of NADP(+) by NADH. 2. The reaction was stimulated by Mg(2+) and only ATP and ITP could serve as energy donors. 3. Studies of the stoicheiometry of the reaction indicate that 1-2mol of ATP are utilized/mol of NADH produced. 4. The reaction is inhibited by uncoupling agents such as 2,4-dinitrophenol (200mum) and tetrachlorotrifluorobenzimidazole (2mum) but oligomycin does not inhibit. 5. The reaction is inhibited by relatively high concentrations (200mum) of piericidin A.

SUBMITTER: Sweetman AJ

PROVIDER: S-EPMC1176494 | biostudies-other | 1971 Jan

REPOSITORIES: biostudies-other

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The energy-linked transhydrogenase reaction in respiratory mutants of Escherichia coli K12.

Project description:1. Energy-linked and non-energy-linked transhydrogenase activities were assayed in membrane preparations from normal Escherichia coli K 12 and from various mutant strains. 2. The energy-linked transhydrogenase, which uses ATP as energy source, was dependent for activity on the presence of a functional Mg(2+)+Ca(2+)-stimulated adenosine triphosphatase. 3. Neither of the quinones formed by E. coli, namely ubiquinone-8 and menaquinone-8, was required for normal ATP-dependent energy-linked transhydrogenase activity. 4. The energy-linked transhydrogenase was inhibited by piericidin A at a site unrelated to the sites of inhibition of the electron-transport chain by piericidin A.

| S-EPMC1178083 | biostudies-other

Studies on energy-linked reactions. Energy-linked reduction of oxidized nicotinamide-adenine dinucleotide by succinate in Escherichia coli.

Project description:1. A method for the preparation of small particles from Escherichia coli is described. 2. These particles catalyse the ATP-driven reduction of NAD(+) by succinate. 3. ATP utilized/NADH produced ratios below 2.0 were measured and a stoicheiometry of 1 molecule of ATP utilized per molecule of NADH produced is proposed. 4. The reaction is not inhibited by 2,4-dinitrophenol or by oligomycin but is inhibited by other uncouplers such as pentabromophenol and dicoumarol. 5. The specificity of the energy source, the specificity of the electron acceptor, the effects of pH, Mg(2+), P(i) and temperature have been studied.

| S-EPMC1176493 | biostudies-other

Energy transfer between the nicotinamide nucleotide transhydrogenase and ATP synthase of Escherichia coli.

Project description:Membrane bound nicotinamide nucleotide transhydrogenase (TH) catalyses the hydride transfer from NADH to NADP+. Under physiological conditions, this reaction is endergonic and must be energized by the pmf, coupled to transmembrane proton transport. Recent structures of transhydrogenase holoenzymes suggest new mechanistic details, how the long-distance coupling between hydride transfer in the peripheral nucleotide binding sites and the membrane-localized proton transfer occurs that now must be tested experimentally. Here, we provide protocols for the efficient expression and purification of the Escherichia coli transhydrogenase and its reconstitution into liposomes, alone or together with the Escherichia coli F1F0 ATP synthase. We show that E. coli transhydrogenase is a reversible enzyme that can also work as a NADPH-driven proton pump. In liposomes containing both enzymes, NADPH driven H+-transport by TH is sufficient to instantly fuel ATP synthesis, which adds TH to the pool of pmf generating enzymes. If the same liposomes are energized with ATP, NADPH production by TH is stimulated > sixfold both by a pH gradient or a membrane potential. The presented protocols and results reinforce the tight coupling between hydride transfer in the peripheral nucleotide binding sites and transmembrane proton transport and provide powerful tools to investigate their coupling mechanism.

| S-EPMC8551311 | biostudies-literature

The udhA gene of Escherichia coli encodes a soluble pyridine nucleotide transhydrogenase.

Project description:The udhA gene of Escherichia coli was cloned and expressed in E. coli and found to encode an enzyme with soluble pyridine nucleotide transhydrogenase activity. The N-terminal end of the enzyme contains the fingerprint motif of a dinucleotide binding domain, not present in published E. coli genome sequences due to a sequencing error. E. coli is hereby the first organism reported to possess both a soluble and a membrane-bound pyridine nucleotide transhydrogenase.

| S-EPMC93474 | biostudies-literature

Reconstitution of the energy-linked transhydrogenase activity in membranes from a mutant strain of Escherichia coli K12 lacking magnesium ion- or calcium ion-stimulated adenosine triphosphatase.

Project description:1. We have isolated a mutant of Escherichia coli K12 (strain AN295) that forms de-repressed amounts of Mg(2+),Ca(2+)-stimulated adenosine triphosphatase. 2. The Mg(2+),Ca(2+)-stimulated triphosphatase activity was separated from membrane preparations from strain AN295 by extraction with 5mm-Tris-HCl buffer containing EDTA and dithiothreitol, resulting in a loss of the ATP-dependent transhydrogenase activity. The non-energy-linked transhydrogenase activity remained in the membrane residue. 3. The solubilized Mg(2+),Ca(2+)-stimulated adenosine triphosphatase activity from strain AN295 was partially purified by repeated gel filtration. The addition of the purified Mg(2+),Ca(2+)-stimulated adenosine triphosphatase to the membrane residue from strain AN295 reactivated the ATP-dependent transhydrogenase activity. 4. Strain AN296, lacking Mg(2+),Ca(2+)-stimulated adenosine triphosphatase activity, was derived by transducing the mutant allele, uncA401, into strain AN295. The ATP-dependent transhydrogenase activity was lost but the non-energy linked transhydrogenase was retained. 5. The ATP-dependent transhydrogenase activity in membrane preparations from strain AN296 (uncA(-)) could not be re-activated by the purified Mg(2+),Ca(2+)-stimulated adenosine triphosphatase from strain AN295. However, after extraction by 5mm-Tris-HCl buffer containing EDTA and dithiothreitol, the ATP-dependent transhydrogenase activity could be re-activated by the addition of the purified Mg(2+),Ca(2+)-stimulated adenosine triphosphatase from strain AN295 to the membrane residue from strain AN296 (uncA(-)).

| S-EPMC1177644 | biostudies-other

Studies of energy-linked reactions. Inhibition of oxidative phosphorylation by DL-8-methyldihydrolipoate.

Project description:1. DL-8-Methyldihydrolipoate was shown to be a potent inhibitor of mitochondrial oxidative phosphorylation and ATP-driven energy-linked reactions. 2. ADP-stimulated respiration utilizing pyruvate + malate and succinate in both ox heart and rat liver mitochondria is inhibited; oxidative phosphorylation using pyruvate + malate, succinate and ascorbate + NNN'N'-tetramethyl-p-phenylenediamine as substrates is also inhibited; uncoupler-stimulated respiration is unaffected regardless of the substrate used. 3. Mitochondrial oligomycin-sensitive adenosine triphosphatase is inhibited in both the membrane-bound form and the purified detergent-dispersed preparation. 4. ATP-driven transhydrogenase and the ATP-driven energy-linked reduction of NAD+ by succinate in ox heart submitochondrial particles are inhibited, whereas the respiratory-chain-driven transhydrogenase is unaffected. 5. DL-8-Methyl-lipoate has no immediate effect on the above reactions, demonstrating the requirement for the reduced form for inhibition. 6. The inhibitory properties of DL-8-methyldihydrolipoate are analogous to those of oligomycin and provide further evidence of a role for lipoic acid in oxidative phosphorylation.

| S-EPMC1164849 | biostudies-other

The soluble transhydrogenase UdhA affecting the glutamate-dependent acid resistance system of Escherichia coli under acetate stress.

Project description:The soluble transhydrogenase (UdhA) is one of two transhydrogenases that play a role in maintaining the balance between NAD(H) pools and NADP(H) pools in Escherichia coli Although UdhA has been extensively used in metabolic engineering and biocatalysis for cofactor regeneration, its role in acid resistance has not been reported. Here we used DNA microarray to explore the impact of UdhA on transcript levels. We demonstrated that during growth on acetate, the expression of genes involved in the respiratory chain and Gad acid resistance system was inhibited in the udhA-knockout strain. The deletion of udhA significantly repressed the expression of six genes (gadA, gadB, gadC, gadE, hdeA and hdeB) which are involved in Gad acid resistance and resulted in low survival of the bacterium at a low pH of 4.9. Moreover, UdhA was essential for NADH production which is important for the adaptive growth of E. coli on acetate, while NADH concentration in the udhA-knockout strain was quite low and supplemental NADH significantly increased the expression of acid resistance genes and survival of the udhA-knockout strain. These results demonstrated that UdhA is an important source of NADH of E. coli growth on acetate and affects Gad acid resistance system under acetate stress.

| S-EPMC6176936 | biostudies-literature

Studies of energy-linked reactions. Localization of the site of action of trialkyltin in yeast mitochondria.

Project description:Ligand-binding studies with labelled triethyltin on yeast mitochondrial membranes showed the presence of high-affinity sites (KD = 0.6 micronM; 1.2 +/- 0.3 nmol/mg of protein) and low-affinity sites (KD less than 45 micronM; 70 +/- 20 nmol/mg of protein). The dissociation constant of the high-affinity site is in good agreement with the concentration of triethyltin required for inhibition of mitochondrial ATPase (adenosine triphosphatase) and oxidative phosphorylation. The high-affinity site is not competed for by oligomycin or venturicidin, indicating that triethyltin reacts at a different site from these inhibitors of oxidative phosphorylation. Fractionation of the mitochondrial membrane shows a specific association of the high-affinity sites with the ATP synthase complex. During purification of ATP synthase (oligomycin-sensitive ATPase) there is a 5-6-fold purification of oligomycin- and triethyltin-sensitive ATPase activity concomitant with a 7-9-fold increase in high-affinity triethyltin-binding sites. The purified yeast oligomycin-sensitive ATPase complex contains approximately six binding sites for triethyltin/mol of enzyme complex. It is concluded that specific triethyltin-binding sites are components of the ATP synthase complex, which accounts for the specific inhibition of ATPase and oxidative phosphorylation by triethyltin.

| S-EPMC1164640 | biostudies-other

IGERS: inferring Gibbs energy changes of biochemical reactions from reaction similarities.

Project description:Mathematical analysis and modeling of biochemical reaction networks requires knowledge of the permitted directionality of reactions and membrane transport processes. This information can be gathered from the standard Gibbs energy changes (DeltaG(0)) of reactions and the concentration ranges of their reactants. Currently, experimental DeltaG(0) values are not available for the vast majority of cellular biochemical processes. We propose what we believe to be a novel computational method to infer the unknown DeltaG(0) value of a reaction from the known DeltaG(0) value of the chemically most similar reaction. The chemical similarity of two arbitrary reactions is measured by the relative number (T) of co-occurring changes in the chemical attributes of their reactants. Testing our method across a validated reference set of 173 biochemical reactions with experimentally determined DeltaG(0) values, we found that a minimum reaction similarity of T = 0.6 is required to infer DeltaG(0) values with an error of <10 kJ/mol. Applying this criterion, our method allows us to assign DeltaG(0) values to 458 additional reactions of the BioPath database. We believe our approach permits us to minimize the number of DeltaG(0) measurements required for a full coverage of a given reaction network with reliable DeltaG(0) values.

| S-EPMC2877342 | biostudies-literature

The Escherichia coli transcriptome linked to growth fitness.

Project description:A series of Escherichia coli strains with varied genomic sequences were subjected to high-density microarray analyses to elucidate the fitness-correlated transcriptomes. Fitness, which is commonly evaluated by the growth rate during the exponential phase, is not only determined by the genome but is also linked to growth conditions, e.g., temperature. We previously reported genetic and environmental contributions to E. coli transcriptomes and evolutionary transcriptome changes in thermal adaptation. Here, we describe experimental details on how to prepare microarray samples that truly represent the growth fitness of the E. coli cells. A step-by-step record of sample preparation procedures that correspond to growing cells and transcriptome data sets that are deposited at the GEO database (GSE33212, GSE52770, GSE61739) are also provided for reference.

| S-EPMC4778597 | biostudies-literature

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