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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A H(+)-translocating inorganic pyrophosphatase (H(+)-PPase) was associated with low density membranes enriched in tonoplast vesicles of oat roots. The H(+)-PPase catalyzed the electrogenic transport of H(+) into the vesicles, generating a pH gradient, inside acid (quinacrine fluorescence quenching), and a membrane potential, inside positive (Oxonol V fluorescence quenching). Transport activity was dependent on cations with a selectivity sequence of Rb(+) = K(+) > Cs(+); but it was inhibited by Na(+) or Li(+). Maximum rates of transport required at least 20 millimolar K(+) and the K(m) for this ion was 4 millimolar. Fluoride inhibited both DeltapH formation and K(+)-dependent PPase activity with an I(50) of 1 to 2 millimolar. Inhibitors of the anion-sensitive, tonoplast-type H(+)-ATPase (e.g. a disulfonic stilbene or NO(3) (-)) had no effect on the PPase activity. Vanadate and azide were also ineffective. H(+)-pumping PPase was inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide, but its sensitivity to N,N'-dicyclohexylcarbodiimide was variable. The sensitivity to ions and inhibitors suggests that the tonoplast H(+)-PPase and the H(+)-ATPase are distinct activities and this was confirmed when they were physically separated after Triton X-100 solubilization and Sepharose CL-6B chromatography. H(+) pumping activity was strongly affected by Mg(2+) and pyrophosphate (PPi) concentrations. At 5 millimolar Mg(2+), H(+) pumping showed a K(m(aPP) ) for PPi of 15 micromolar. The rate of H(+) pumping at 60 micromolar PPi was often equivalent to that at 1.5 millimolar ATP. The results suggest PPi hydrolysis could provide another source of a proton motive force used for solute transport and other energy-requiring processes across the tonoplast and other membranes with H(+)-PPase.
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PMID:Electrogenic h-pumping pyrophosphatase in tonoplast vesicles of oat roots. 1666 45

Sealed tonoplast vesicles were isolated from single cells of Chara corallina with the aid of an intracellular perfusion technique in combination with a 3/10% Percoll two step gradient centrifugation. The isolated tonoplast fraction was free from plasmalemma and chloroplasts, and showed no activities of cytochrome c oxidase, and latent IDPase, but had about 10% of the NADH-cytochrome c reductase activity. The vesicles had both ATPase and PPase activities, which could be stimulated in the presence of 10 micromolar gramicidin by 170 and 130%, respectively, demonstrating the existence of sealed vesicles. Furthermore, ATP- and PPi-dependent H(+) pumping through the membrane into the vesicles was shown. Both ATPase and PPase had pH optima around pH 8.5. At the physiological pH, 7.3, they still had more than 80% of their maximal activities. Ammonium molybdate, azide, and vanadate had no or little effect on the activities of both enzymes or their associated H(+) pumping activities. N,N'-dicyclohexylcarbodiimide inhibited the ATPase strongly (I(50) = 20 micromolar) but the PPase only weakly. The ATPase was also more sensitive to N-ethylmaleimide than the PPase. 4,4'-Stilbenedisulfonic acid affected both enzyme activities and their associated H(+) pumping activities. This is in contrast to the H(+)-PPase of higher plants which is 4,4'-stilbenedisulfonic acid insensitive.
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PMID:Characterization of the H Translocating Adenosine Triphosphatase and Pyrophosphatase of Vacuolar Membranes Isolated by Means of a Perfusion Technique from Chara corallina. 1666 49

The vacuolar pH and the trans-tonoplast DeltapH modifications induced by the activity of the two proton pumps H(+)-ATPase and H(+)-PPase and by the proton exchanges catalyzed by the Na(+)/H(+) and Ca(2+)/H(+) antiports at the tonoplast of isolated intact vacuoles prepared from Catharanthus roseus cells enriched in inorganic phosphate (Y Mathieu et al 1988 Plant Physiol [in press]) were measured using the (31)P NMR technique. The H(+)-ATPase induced an intravacuolar acidification as large as 0.8 pH unit, building a trans-tonoplast DeltapH up to 2.2 pH units. The hydrolysis of the phosphorylated substrate and the vacuolar acidification were monitored simultaneously to estimate kinetically the apparent stoichiometry between the vectorial proton pumping and the hydrolytic activity of the H(+)-ATPase. A ratio of H(+) translocated/ATP hydrolyzed of 1.97 +/- 0.06 (mean +/- standard error) was calculated. Pyrophosphate-treated vacuoles were also acidified to a significant extent. The H(+)-PPase at 2 millimolar PPi displayed hydrolytic and vectorial activities comparable to those of the H(+)-ATPase, building a steady state DeltapH of 2.1 pH units. Vacuoles incubated in the presence of 10 millimolar Na(+) were alkalinized by 0.4 to 0.8 pH unit. It has been shown by using (23)Na NMR that sodium uptake was coupled to the H(+) efflux and occurred against rather large concentration gradients. For the first time, the activity of the Ca(2+)/H(+) antiport has been measured on isolated intact vacuoles. Ca(2+) uptake was strongly inhibited by NH(4)Cl or gramicidin. Vacuoles incubated with 1 millimolar Ca(2+) were alkalinized by about 0.6 pH unit and this H(+) efflux was associated to a Ca(2+) uptake as demonstrated by measuring the external Ca(2+) concentration with a calcium specific electrode. Steady state accumulation ratios of Ca(2+) as high as 100 were reached for steady state external concentrations about 200 micromolar. The rate of Ca(2+) uptake appeared markedly amplified in intact vacuoles when compared to tonoplast vesicles but the antiport displayed a much lower affinity for calcium. The different behavior of intact vacuoles compared to vesicles appears mainly to be due to differences in the surface to volume ratio and in the rates of dissipation of the pH gradient. Despite its low affinity, the Ca(2+)/H(+) antiport has a high potential capacity to regulate cytoplasmic concentration of calcium.
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PMID:Regulation of Vacuolar pH of Plant Cells: II. A P NMR Study of the Modifications of Vacuolar pH in Isolated Vacuoles Induced by Proton Pumping and Cation/H Exchanges. 1666 25

Tonoplast-enriched membranes were prepared from maize (Zea mays L. cv LG 11) primary roots, using sucrose nonlinear gradients. The functional molecular size of the tonoplast ATP-and PPi-dependent proton pumps were analyzed by radiation inactivation. Glucose-6-phosphate dehydrogenase (G6PDH) was added as an internal standard. Frozen samples (-196 degrees C) of the membranes were irradiated with (60)Co for different periods of time. After thawing the samples, the activities of G6PDH, ATPase, and PPase were tested. By applying target theory, the functional sizes of the ATPase and PPase in situ were found to be around 540 and 160 kilodaltons, respectively. The two activities were solubilized and separated by gel filtration chromatography. The different polypeptides copurifying with the two pumps were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two bands (around 59 and 65 kilodaltons) were associated with the ATPase activity, whereas a double band (around 40 kilodaltons) was recovered with the PPase activity.
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PMID:Target Molecular Size and Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Analysis of the ATP-and Pyrophosphate-Dependent Proton Pumps from Maize Root Tonoplast. 1666

Using pH-sensitive microelectrodes (in vitro) and acridine orange photometry (in vivo), the actions of the two tonoplast phosphatases, the tp-ATPase and the tp-PPase, were investigated with respect to how effectively they could generate a transtonoplast pH-gradient. Under standard conditions the vacuoles of the aquatic liverwort Riccia fluitans have an in vivo pH of 4.7 to 5.0. In isolated vacuoles a maximal vacuolar pH (pH(v)) of 4.74 +/- 0.1 is generated in the presence of 0.1 millimolar PP(i), but only 4.93 +/- 0.13 in the presence of 2.5 millimolar ATP. Both substrates added together approximate the value for PP(i). Cl(-)-stimulates the H(+)-transport driven by the tp-ATPase, but has no effect on the tp-PPase. The transport activity of the tp-ATPase approximates saturation kinetics (K((1/2)) approximately 0.5 millimolar), whereas transport by the tp-PPase yields an optimum around 0.1 millimolar PP(i). The transtonoplast pH-gradient is dissipated slowly by weak bases, from which a vacuolar buffer capacity of roughly 300 to 400 millimolar/pH(v) unit has been estimated. From the free energy (-11.42 kilojoules per mole) for the hydrolysis of PP(i) under the given experimental conditions, we conclude that the PPase-stoichiometry (transported H(+) per hydrolyzed substrate molecule) must be 1, and that in vivo this enzyme works as a H(+)-pump rather than as a pyrophosphate synthetase.
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PMID:Proton Gradient Across the Tonoplast of Riccia fluitans as a Result of the Joint Action of Two Electroenzymes. 1666 81

We analyzed the effects of ABA, auxin, and gibberellin on the expression of two genes (HVP1 and HVP10) for vacuolar H(+)-inorganic pyrophosphatase (EC 3.6.1.1) and one (HvVHA-A) for the catalytic subunit (subunit A) of vacuolar H(+)-ATPase (EC 3.6.1.3) by quantification of the transcript levels, to identify the hormones responsible for regulating the expression of these genes in barley (Hordeum vulgare L.) in response to environmental changes such as salt stress. ABA markedly induced the expression of HVP1 and slightly increased the expression of HVP10 and HvVHA-A. In contrast, 2,4-D only increased the expression of HVP1, and GA(3) had no significant effects on any gene. The maximum level of HVP1 transcripts in response to these hormones was also much higher than the levels of HVP10 and HvVHA-A transcripts. In addition, we also analyzed the expression of one gene (HvNHX1) for vacuolar Na(+)/H(+) antiporter, and HvNHX1 expression changed in a pattern similar to that of the HVP1 expression. Furthermore, treatment with ABA and 2,4-D increased Na(+)/H(+) antiport activity and proton-translocating activities by H(+)-PPase and H(+)-ATPase in tonoplast vesicles, and treatment with ABA also increased the amount of V-PPase protein of tonoplast vesicles. These results suggest that the hormones ABA and 2,4-D regulate the expression of the H(+)-pump and Na(+)/H(+) antiporter genes and are thus important effectors that regulate the expression of HVP1and HvNHX1.
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PMID:Effects of ABA, auxin, and gibberellin on the expression of genes for vacuolar H+ -inorganic pyrophosphatase, H+ -ATPase subunit A, and Na+/H+ antiporter in barley. 1680 58

Inorganic phosphate (Pi) uptake across the vacuolar membrane of intact vacuoles isolated from Catharanthus roseus suspension-cultured cells was measured. Under low Pi status, Pi uptake into the vacuole was strongly activated compared to high Pi status. Since Pi uptake across the vacuolar membrane is correlated with H+ pumping, we examined the dependency of H+ pumping on plant Pi status. Both H+ pumping and the activities of the vacuolar H+-pumps, the V-type H+-ATPase and the H+-PPase were enhanced under low Pi status. Despite this increase in H+ pumping, Western blot analysis showed no distinct increase in the amount of proton pump proteins. Possible mechanisms for the activation of Pi uptake into the vacuole under low Pi status are discussed.
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PMID:Inorganic phosphate uptake in intact vacuoles isolated from suspension-cultured cells of Catharanthus roseus (L.) G. Don under varying Pi status. 1695 72

pH-homeostasis in the endomembrane system requires the activity of proton-pumps. In animals, the progressive acidification of compartments along the endocytic and secretory pathways is critical for protein sorting and vesicle trafficking, and is achieved by the activity of the vacuolar H(+)-ATPase (V-ATPase). Plants have an additional endomembrane pump, the vacuolar H(+)-pyrophosphatase (V-PPase), and previous research was largely focused on the respective functions of the two pumps in secondary active transport across the tonoplast. Recent approaches, including reverse genetics, have not only provided evidence that both enzymes play unique and essential roles but have also highlighted the important functions of the two proton pumps in endocytic and secretory trafficking.
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PMID:Endomembrane proton pumps: connecting membrane and vesicle transport. 1700 21

Chemiosmotic circuits of plant cells are driven by proton (H(+)) gradients that mediate secondary active transport of compounds across plasma and endosomal membranes. Furthermore, regulation of endosomal acidification is critical for endocytic and secretory pathways. For plants to react to their constantly changing environments and at the same time maintain optimal metabolic conditions, the expression, activity and interplay of the pumps generating these H(+) gradients have to be tightly regulated. In this review, we will highlight results on the regulation, localization and physiological roles of these H(+)- pumps, namely the plasma membrane H(+)-ATPase, the vacuolar H(+)-ATPase and the vacuolar H(+)-PPase.
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PMID:Plant proton pumps. 1741 24

Mitochondria are subcellular organelles with an essentially oxidative type of metabolism. The production of reactive oxygen species (ROS) in it increases under stress conditions and causes oxidative damage. In the present study, effects of exogenous sodium nitroprusside (SNP), a nitric oxide (NO) donor, on both the ROS metabolism in mitochondria and functions of plasma membrane (PM) and tonoplast were studied in cucumber seedlings treated with 100mM NaCl. NaCl treatment induced significant accumulation of H(2)O(2) and led to serious lipid peroxidation in cucumber mitochondria, and the application of 50muM SNP stimulated ROS-scavenging enzymes and reduced accumulation of H(2)O(2) in mitochondria of cucumber roots induced by NaCl. As a result, lipid peroxidation of mitochondria decreased. Further investigation showed that application of SNP alleviated the inhibition of H(+)-ATPase and H(+)-PPase in PM and/or tonoplast by NaCl. While application of sodium ferrocyanide (an analog of SNP that does not release NO) did not show the effect of SNP, furthermore, the effects of SNP were reverted by addition of hemoglobin (a NO scavenger).
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PMID:Exogenous nitric oxide protect cucumber roots against oxidative stress induced by salt stress. 1760 79


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