Adenine nucleotide status, phosphoglycerate reduction and photosynthetic phosphorylation in a reconstituted chloroplast system.
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ABSTRACT:
SUBMITTER: Carver KA
PROVIDER: S-EPMC1154216 | biostudies-other | 1983 Jan
REPOSITORIES: biostudies-other
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Project description:ADP was shown to inhibit phosphoglycerate-dependent O2 evolution in a simplified reconstituted chloroplast system containing 3-phosphoglycerate kinase and triose phosphate dehydrogenase. Rates of O2 evolution in the simplified system are comparable with those obtained by using stromal protein rather than purified enzymes. ADP does not inhibit O2 evolution with glycerate 1,3-biphosphate as substrate nor does it inhibit triose phosphate dehydrogenase. The inhibitory effect of ADP is attributed to an increase in the rate of conversion of glycerate biphosphate into phosphoglycerate. The results are discussed in terms of control by ADP of phosphoglycerate-dependent oxygen evolution.
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Project description:Phosphorylation of the light-harvesting chlorophyll protein (LHCP) by the thylakoid protein kinase has been examined in the reconstituted chloroplast system. The level of phosphorylation by [32P]Pi was maximal at high light intensity and in the absence of 3-phosphoglycerate; dephosphorylation resulted from a subsequent decrease in light intensity or from the addition of 3-phosphoglycerate. Addition of ribose 5-phosphate, which acts as an ATP 'sink', also caused dephosphorylation. It is concluded that the degree of phosphorylation is dependent on the redox state and energy state of the system, thereby providing a mechanism for adapting light harvesting to the demands of carbon assimilation.
| S-EPMC1154252 | biostudies-other
Project description:1. The ratio [ATP]/[ADP][P(i)], as measured by direct determination of the three components in rat liver, was found in various nutritional states to have approximately the same value as the ratio [ATP]/[ADP][P(i)] calculated from the concentrations of lactate, pyruvate, glyceraldehyde phosphate and 3-phosphoglycerate on the assumption that lactate dehydrogenase, glyceraldehyde phosphate dehydrogenase and 3-phosphoglycerate kinase are at near-equilibrium in the liver. This implies that the redox state of the NAD couple in the cytoplasm is linked to, and partially controlled by, the phosphorylation state of the adenine nucleotides. 2. The combined equilibrium constant of the glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate kinase reactions at 38 degrees C and I0.25, was found to be 5.9x10(-6). 3. The fall of the [NAD(+)]/[NADH] ratio in starvation and other situations is taken to be the consequence of a primary fall of the [ATP]/[ADP][HPO(4) (2-)] ratio.
| S-EPMC1178952 | biostudies-other
Project description:During photosynthesis, electrons travel from light-excited chlorophyll molecules along the electron transport chain to the final electron acceptor nicotinamide adenine dinucleotide phosphate (NADP) to form NADPH, which fuels the Calvin-Benson-Bassham cycle (CBBC). To allow photosynthetic reactions to occur flawlessly, a constant resupply of the acceptor NADP is mandatory. Several known stromal mechanisms aid in balancing the redox poise, but none of them utilizes the structurally highly similar coenzyme NAD(H). Using Arabidopsis (Arabidopsis thaliana) as a C3-model, we describe a pathway that employs the stromal enzyme PHOSPHOGLYCERATE DEHYDROGENASE 3 (PGDH3). We showed that PGDH3 exerts high NAD(H)-specificity and is active in photosynthesizing chloroplasts. PGDH3 withdrew its substrate 3-PGA directly from the CBBC. As a result, electrons become diverted from NADPH via the CBBC into the separate NADH redox pool. pgdh3 loss-of-function mutants revealed an overreduced NADP(H) redox pool but a more oxidized plastid NAD(H) pool compared to wild-type plants. As a result, photosystem I acceptor side limitation increased in pgdh3. Furthermore, pgdh3 plants displayed delayed CBBC activation, changes in nonphotochemical quenching, and altered proton motive force partitioning. Our fluctuating light-stress phenotyping data showed progressing photosystem II damage in pgdh3 mutants, emphasizing the significance of PGDH3 for plant performance under natural light environments. In summary, this study reveals an NAD(H)-specific mechanism in the stroma that aids in balancing the chloroplast redox poise. Consequently, the stromal NAD(H) pool may provide a promising target to manipulate plant photosynthesis.
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Project description:1. The effects of atractyloside and carboxyatractyloside (between 5 and 40mum) on O(2) uptake, glucose synthesis, urea synthesis, the adenine nucleotide content and the intracellular K(+) concentration were measured in isolated hepatocytes. 2. Urea synthesis was much less inhibited than glucose synthesis by both atractylosides. Measurements of intermediary metabolites of carbohydrate metabolism in freeze-clamped liver after injection of atractyloside into rats indicate that inhibition of gluconeogenesis is due to interference at the cytosolic reactions requiring ATP (phosphoenolpyruvate carboxykinase and 3-phosphoglycerate kinase). 3. The decrease in [ATP]/[ADP]x[P(i)] after addition of atractyloside or carboxyatractyloside was restricted to the cytosol. 4. Dihydroxyacetone can be converted either into glucose with the consumption of 2mol of ATP (per mol of glucose) or into lactate with the production of 2mol of ATP. In the presence of high concentrations of atractyloside and carboxyatractyloside more ATP was produced than was used for the synthesis of glucose from dihydroxyacetone, probably for the maintenance of intracellular [K(+)]. 5. When the rates of respiration were altered by changing substrates, the degrees of inhibition of respiration and translocation by a given concentration of the atractylosides were the same, whereas at a given concentration of HCN the degree of inhibition was high at higher initial rates, and low at lower initial rates. 6. Inhibition of a complex series of reactions by atractyloside does not necessarily indicate that the translocator is a rate-limiting step in that sequence as Th. P. M. Akerboom, H. Bookelman & J. M. Tager [(1977) FEBS. Lett.74, 50-54] assume. This point is discussed.
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Project description:To analyze the impact of photosynthetic redox signals, light sources with spectral qualities that preferentially excite either Photosystem I (PSI light) or Photosystem II (PSII light) were used. The light sources have been described in (Wagner et al, Planta 2008). Strong reduction signals were induced by light shifts from PSI to PSII light (PSI-II). In order to find primary regulated genes the acclimation responses were monitored at 30 min and 60 min after a light shift. The control was continuous Psi light at the same time. We used stn7 (a thylakoid redox regulated kinase) to specifically block transduction of photosynthetic redox signal in order to compare “real” redox regulated with that of other light acclimation pathways. Keywords: photosynthesis, redox regulation, light acclimation, retrograde signalling, long term response
2015-03-23 | GSE42710 | GEO
Project description:In pathological conditions, F(0)F(1)-ATPase hydrolyzes ATP in an attempt to maintain mitochondrial membrane potential. Using thermodynamic assumptions and computer modeling, we established that mitochondrial membrane potential can be more negative than the reversal potential of the adenine nucleotide translocase (ANT) but more positive than that of the F(0)F(1)-ATPase. Experiments on isolated mitochondria demonstrated that, when the electron transport chain is compromised, the F(0)F(1)-ATPase reverses, and the membrane potential is maintained as long as matrix substrate-level phosphorylation is functional, without a concomitant reversal of the ANT. Consistently, no cytosolic ATP consumption was observed using plasmalemmal K(ATP) channels as cytosolic ATP biosensors in cultured neurons, in which their in situ mitochondria were compromised by respiratory chain inhibitors. This finding was further corroborated by quantitative measurements of mitochondrial membrane potential, oxygen consumption, and extracellular acidification rates, indicating nonreversal of ANT of compromised in situ neuronal and astrocytic mitochondria; and by bioluminescence ATP measurements in COS-7 cells transfected with cytosolic- or nuclear-targeted luciferases and treated with mitochondrial respiratory chain inhibitors in the presence of glycolytic plus mitochondrial vs. only mitochondrial substrates. Our findings imply the possibility of a rescue mechanism that is protecting against cytosolic/nuclear ATP depletion under pathological conditions involving impaired respiration. This mechanism comes into play when mitochondria respire on substrates that support matrix substrate-level phosphorylation.
| S-EPMC2887268 | biostudies-literature
Project description:Adenine nucleotide translocases (ANT in mammals, AAC in yeast) mediate the 1:1 electrogenic exchange of ADP into and ATP out of the mitochondrial matrix and is thus a required component of the oxidative phosphorylation machinery. Originally modeled to function in isolation, the major isoform in Saccharomyces cerevisiae, AAC2, was recently demonstrated to associate with a number of protein complexes, including other mitochondrial solute carriers, higher order respiratory complexes (termed supercomplexes), and itself. As very little is known about the mammalian ANT interactome, we sought to define it using a proteomics-based approach. Here, we show that human ANT1 and ANT2, like yeast AAC2, do not function in isolation but have numerous binding partners including the respiratory supercomplex, Despite the differences in the organization of respiratory supercomplexes in yeast and mammals, wherein the latter additionally involves Complex I, the capacity of both AACs and ANTs to associate with it suggests an evolutionarily conserved interaction that has a functional importance.
2022-03-01 | PXD004356 | Pride
Project description:Phosphorylation of adenine nucleotide translocator 1 (ANT1) at residue Y194, which is part of the aromatic ladder located within the lumen of the carrier, critically regulates mitochondrial metabolism. Recent data support the concept that members of the Src family of nonreceptor tyrosine kinases are constitutively present in mitochondria and key to regulation of mitochondrial function. Herein, we demonstrate that site mutations of ANT1 (Y190-->F190, Y194-->F194) mimicking dephosphorylation of the aromatic ladder resulted in loss of oxidative growth and ADP/ATP exchange activity in respiration-incompetent yeast expressing mutant chimeric yN-hANT1. ANT1 is phosphorylated at Y194 by the Src family kinase members Src and Lck, and increased phosphorylation is tightly linked to reduced cell injury in preconditioned protected vs. unprotected cardiac mitochondria. Molecular dynamics simulations find the overall structure of the phosphorylated ANT1 stable, but with an increased steric flexibility in the region of the aromatic ladder, matrix loop m2, and four helix-linking regions. Combined with an analysis of the putative cytosolic salt bridge network, we reason that the effect of phosphorylation on transport is likely due to an accelerated transition between the main two conformational states (c<-->m) of the carrier during the transport cycle. Since "aromatic signatures" are typical for other mitochondrial carrier proteins with important biological functions, our results may be more general and applicable to these carriers.
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Project description:Several ATP-depending reactions take place in the endoplasmic reticulum (ER). Although in Saccharomyces cerevisiae ER the existence of a Sac1p-dependent ATP transport system was already known, its direct involvement in ATP transport was excluded. Here we report an extensive biochemical characterization of a partially purified adenine nucleotide transport system (ANTS) not dependent on Sac1p. Highly purified ER membranes from the wild-type and Δsac1 yeast strains reconstituted into liposomes transported ATP with the same efficiency. A chromatography on hydroxyapatite was used to partially purify ANTS from Δsac1 ER extract. The two ANTS-enriched transport activity eluted fractions showed essentially the presence of four bands, one having an apparent MW of 56 kDa, similar to that observed for ANTS identified in rat liver ER. The two fractions reconstituted into liposomes efficiently transported, by a strict counter-exchange mechanism, ATP and ADP. ATP transport was saturable with a Km of 0.28 mM. The ATP/ADP exchange mechanism and the kinetic constants suggest that the main physiological role of ANTS is to catalyse the transport of ATP into ER, where it is used in several energy-requiring reactions and to export back to the cytosol the ADP produced.
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