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)

Active K+ secretion in isolated posterior midguts of Manduca sexta was studied by measuring the short-circuit current. One aim of this study was to verify the postulate from biochemical reports that the cooperative apical arrangement of a vacuolar-type H+-ATPase (V-ATPase) and a K+/H+ antiporter drive the short-circuit current. Hence, we tested several specific inhibitors of the V-ATPase on the in vitro midgut preparation. Nitrate, bafilomycin A1, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) and amiloride all reduced the short-circuit current. This suggests that the H+-ATPase is involved in transepithelial K+ secretion. However, even at relatively high doses of these inhibitors, the block of the short-circuit current was not complete. Two other agents, thallium ions (Tl+, at millimolar concentrations) and trimethyltin chloride (TMT, 50 µmol l-1), did abolish the short-circuit current. Apical, but not basal, use of the ionophore amphotericin B largely eliminated the short-circuit current. This supports the view that the current-generating source resides in the apical membranes. An apical (and probably intracellular) site of action for NO3-, Tl+ and TMT is suggested by the observation that basal amphotericin B is needed for blockage by NO3- but does not, however, influence the effect of Tl+ and TMT. Likely sites of action are the V-ATPase (for nitrate and TMT) and the K+/H+ antiporter (for Tl+).
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PMID:AN INVESTIGATION OF THE MIDGUT K+ PUMP OF THE TOBACCO HORNWORM (MANDUCA SEXTA) USING SPECIFIC INHIBITORS AND AMPHOTERICIN B 931 13

It was found that a facultatively anaerobic and halophilic alkaliphile, M-12 (Amphibacillus sp.), possesses a Na(+)-stimulated ATPase in the membrane. The ATPase activity was inhibited by NO3- and SCN- which are the inhibitors of V-type ATPase, but not by azide and vanadate, inhibitors of F-type ATPase and P-type ATPase, respectively. Upon the incubation of the membrane in buffer containing ATP and MgCl2, several polypeptides were released from the membrane. Among them, two major polypeptides with apparent molecular masses of 79 and 55 kDa crossreacted with an antiserum against the catalytic units (subunits A and B) of V-type ATPase from Enterococcus hirae. The N-terminal amino acid sequences of the 79 and 55 kDa polypeptides showed high similarity to those of subunits A and B of V-type ATPase from Enterococcus hirae, respectively. M-12 is likely to possess a V-type Na(+)-ATPase.
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PMID:Presence of Na(+)-stimulated V-type ATPase in the membrane of a facultatively anaerobic and halophilic alkaliphile. 978 52

In contrast to animal cells, plant cells contain approximately 5-50 mM nitrate in cytosol and vacuole. The lack of specific spectroscopic probes, or suitable isotopes, impedes in vitro studies of NO3- transport. Reconstitution of root cell plasma membrane (PM) proteins in mixed soybean lipid:egg phosphatidylcholine allowed for the generation of large K+-valinomycin diffusion potentials (Em), monitored with the oxonol VI dye. Nevertheless, Em was restricted to approximately 130 mV by capacitor properties of biological membranes. This caused an increasing discrepancy at higher K+-Nernst potentials used for calibration. Therefore, Em was determined directly from the fluorescence of the dye free in buffer, bound at zero Em, and bound upon Em generation. Then, an electrophysiological analysis of the NO3--dependent dissipation rate of Em gave the net passive flux (JN) and the permeability coefficient to NO3- (PN). The plant root cell PM exhibited a strikingly large PN (higher than 10(-9) m s-1) at high Em (90-100 mV) and pH 6.5. At low Em (50-60 mV) and pH 7.4, PN decreased by 70-fold and became similar to that of the lipid bilayer. This agreed with the previous observation that 15 mM NO3- short-circuits the plant root PM H+-ATPase at its optimal pH of 6.5.
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PMID:Electrophysiological study with oxonol VI of passive NO3- transport by isolated plant root plasma membrane. 987 48

Sucrose efflux from the vacuole of mobilizing red-beet (Beta vulgaris L.) hypocotyl cells was investigated using purified tonoplast vesicles. Tonoplast vesicle purity was assured by the immunoreactivity to antibodies raised against the vacuolar ATPase and by the strong inhibition exhibited by the H+-ATPase to bafilomycin-A and NO3-. Inhibition of the H+-ATPase by vanadate and azide was negligible. Sucrose was loaded into tonoplast vesicles by using the pH-jump method of energization. Addition of ATP to sucrose-loaded vesicles in the presence of bafilomycin-A resulted in efflux of a significant amount of sucrose. During ATP-induced sucrose efflux, bafilomycin-insensitive ATPase activity increased significantly with no increase in H+-translocating activity. The additional bafilomycin-A insensitive ATPase activity observed in sucrose-loaded vesicles was completely inhibited by vanadate as was the efflux of sucrose. Similar to vanadate, thapsigargin was also inhibitory to sucrose efflux and to the bafilomycin-A insensitive ATPase activity. The data indicate that vacuolar sucrose can be actively mobilized by a specific ATP-dependent efflux mechanism.
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PMID:ATP-induced sucrose efflux from red-beet tonoplast vesicles. 1092 6

The nature of the association between nitrate reductase (NR) and membranes was examined. Nitrate reductase activity (NRA) associated with the microsomal fraction of barley (Hordeum vulgare L.) roots amounted to 0.6 to 0.8% of soluble NRA following sonication in the presence of 250 mM KI and repeated osmotic shock. This treatment removed all contaminating soluble NRA from microsomes of uninduced barley roots that had been homogenized in a soluble extract from roots of NO3(-)-induced plants. On continuous sucrose gradients, NRA co-migrated specifically with VO4(-)-sensitive ATPase activity, a plasma membrane (PM) marker; activity of glucose-6-phosphate dehydrogenase, assayed as cytosolic marker, co-migrated with NRA. Microsomal NRA was absent in barley deficient in soluble NR. Perturbation and trypsinolysis experiments with PM vesicles isolated by aqueous two-phase partitioning indicated that NR is associated with the periphery of the cytoplasmic face of the bilayer. These results demonstrate that PM and soluble NRs are essentially the same protein but that the membrane-associated form is tightly bound. Although it is possible that PM-associated NR exists in vivo, unequivocal evidence for this has yet to be shown. However, PM NR is definitely present in vitro.
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PMID:Characterization of the association of nitrate reductase with barley (Hordeum vulgare L.) root membranes. 1153

Plasma membrane H+-ATPase was studied in maize (Zea mays L.) roots induced for NO3- uptake. Membrane vesicles were isolated by means of Suc density gradient from roots exposed for 24 h either to 1.5 mM NO3- or 1.5 mM SO4-. The two populations of vesicles had similar composition as shown by diagnostic inhibitors of membrane-associated ATPases. However, both ATP-dependent intravesicular H+ accumulation and ATP hydrolysis were considerably enhanced (60-100%) in vesicles isolated from NO3--induced roots. Km for Mg:ATP and pH dependency were not influenced by NO3- treatment of the roots. ATP hydrolysis in plasma membrane vesicles for both control and NO3--induced roots was not affected by 10 to 150 mM NO3- or Cl-. On the other hand, kinetics of NO3-- or Cl--stimulated ATP-dependent intravesicular H+ accumulation were modified in plasma membrane vesicles isolated from NO3-- induced roots. Immunoassays carried out with polyclonal antibodies against plasma membrane H+-ATPase revealed an increased steady-state level of the enzyme in plasma membrane vesicles isolated from NO3--induced roots. Results are consistent with the idea of an involvement of plasma membrane H+-ATPase in the overall response of roots to NO3-.
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PMID:Plasma Membrane H+-ATPase in Maize Roots Induced for NO3- Uptake. 1222 68

Various physiological and biochemical process like growth, NO3- -uptake, nitrate reductase, glutamine synthetase and ATPases (Mg2+ and Ca2+ dependent) in the cyanobacterium Anabaena 7120 were observed under iron stress. Growth was found to be maximum in 50 microM Fe3+ added cells however, 20 microM Fe3+ (the Fe3+ concentration generally used for routine culturing of cyanobacterial cell in Chu 10 medium) incubation resulted in lower growth. Fe3+ starvation on the other hand showed very poor growth up to 4th day but once the growth started it reached at significant level on 7th day. Higher Fe3+ concentration reflected reduced growth with lethality at 500 microM Fe3+. Chlorophyll a fluorescence under Fe3+ stress reflected almost the similar results as in case of growth. However, the pigment was found to be more sensitive as compared to protein under Fe3+ stress. Similar results have been observed in case of NO3-uptake with only 80% reduction in nutrient uptake in 500 microM Fe3+ incubated cells. Nitrate reductase activity was lower in Fe3+ starved cells as compared to significant enzyme activity in 20 and 50 microM Fe3+ incubated cells. Similar to nitrate reductase, glutamine synthetase also showed maximum level in 50 microM Fe3+ added cells, however, higher Fe3+ concentration (300-500 microM ) resulted in reduced enzymatic activity. Glutamine synthetase activity was less sensitivity as compared to nitrate reductase activity under Fe3+ stress. ATPase (Mg2+ and Ca2+ dependent) always showed higher level with increasing Fe3+ concentration.
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PMID:Physiological and biochemical alterations in Anabaena 7120 under iron stress. 1262 8

The distal-convoluted tubule (DCT) of the kidney absorbs NaCl mainly via an Na+-Cl- cotransporter located at the apical membrane, and Na+, K+ ATPase at the basolateral side. Cl- transport across the basolateral membrane is thought to be conductive, but the corresponding channels have not yet been characterized. In the present study, we investigated Cl- channels on microdissected mouse DCTs using the patch-clamp technique. A channel of approximately 9 pS was found in 50% of cell-attached patches showing anionic selectivity. The NPo in cell-attached patches was not modified when tubules were preincubated in the presence of 10-5 M forskolin, but the channel was inhibited by phorbol ester (10-6 M). In addition, NPo was significantly elevated when the calcium in the pipette was increased from 0 to 5 mM (NPo increased threefold), or pH increased from 6.4 to 8.0 (NPo increased 15-fold). Selectivity experiments conducted on inside-out patches showed that the Na+ to Cl- relative permeability was 0.09, and the anion selectivity sequence Cl(-)--I(-) > Br(-)--NO3(-) > F(-). Intracellular NPPB (10-4 M) and DPC (10-3 M) blocked the channel by 65% and 80%, respectively. The channel was inhibited at acid intracellular pH, but intracellular ATP and PKA had no effect. ClC-K Cl- channels are characterized by their sensitivity to the external calcium and to pH. Since immunohistochemical data indicates that ClC-K2, and perhaps ClC-K1, are present on the DCT basolateral membrane, we suggest that the channel detected in this study may belong to this subfamily of the ClC channel family.
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PMID:A chloride channel at the basolateral membrane of the distal-convoluted tubule: a candidate ClC-K channel. 1266 33

Our previous investigations have established that Na+ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na+-pump, Na+-ATPase, is accompanied by H+ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402-406]. The hypothesis that the Na+-ATPase of T. viridis operates as an Na+/H+ exchanger is tested in the present work. The study of Na+ and H+ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO3- caused (i) an increase in ATP-driven Na+ uptake by the vesicles, (ii) an increase in (Na(+)+ATP)-dependent vesicle lumen alkalization resulting from H+ efflux out of the vesicles and (iii) dissipation of electrical potential, deltapsi, generated across the vesicle membrane by the Na+-ATPase. The (Na(+)+ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K+ abolished deltapsi at the vesicle membrane. The fact that the Na+-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane deltapsi is consistent with a primary role of the Na+-ATPase in driving H+ outside the vesicles. The findings allowed us to conclude that the Na+-ATPase of T. viridis directly performs an exchange of Na+ for H+. Since the Na+-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa+/nH+, where m> n.
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PMID:The Na+-translocating ATPase in the plasma membrane of the marine microalga Tetraselmis viridis catalyzes Na+/H+ exchange. 1499 93

The effect of NO3- uptake on cellular pH was studied in maize roots by an in vivo 31P-NMR technique. In order to separate the effects on cytoplasmic pH due to NO3- uptake from those due to NO3- reduction, tungstate was used to inhibit nitrate reductase (NR). The results confirm that in maize roots tungstate inhibited NR activity. 15N-NMR in vivo experiments demonstrated the cessation of nitrogen flux from nitrate to organic compounds. Tungstate affected neither NO3- uptake nor the levels of the main phosphorylated compounds. Slight changes in cytoplasmic pH were observed during NO3- uptake and reduction (i.e. control). By contrast, in the presence of tungstate, a consistent decrease in cytoplasmic pH occurred. The vacuolar pH did not change in any of the conditions tested. These data show that NO3- uptake is an acidifying process and suggest a possible involvement of NO3- reduction in pH homeostasis. In the presence of NO3-, a transient depolarization of transmembrane electric potential difference (Em) was observed in all the conditions analysed. However, in tungstate-treated roots, a lesser depolarization accompanied by a greater ability to recover Em was found. This was related to a higher activity of the plasma membrane (PM) H+-ATPase. When NO3- was administered as potassium salt, its uptake increased and a greater depolarization of Em took place, whilst the changes in cytoplasmic pH were remarkably reduced, according to the central role played by K+ in the control of plasma membrane activities and cell pH homeostasis. A possible involvement of cytoplasmic pH in the control of PM H+-ATPase expression during nitrate exposure is suggested.
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PMID:Effect of NO3- transport and reduction on intracellular pH: an in vivo NMR study in maize roots. 1531 Aug 18


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