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1.
The vacuolar pH and the trans-tonoplast ΔpH 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 31P NMR technique. The H +-ATPase induced an intravacuolar acidification as large as 0.8 pH unit, building a trans-tonoplast ΔpH 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 ΔpH 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 23Na 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 4Cl 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. 相似文献
2.
Microsomal vesicles from 24-hour-old radish ( Raphanus sativus L.) seedlings accumulate Ca 2+ upon addition of MgATP. MgATP-dependent Ca 2+ uptake co-migrates with the plasma membrane H +-ATPase on a sucrose gradient. Ca 2+ uptake is insensitive to oligomycin, inhibited by vanadate (IC 50 40 micromolar) and erythrosin B (IC 50 0.2 micromolar) and displays a pH optimum between pH 6.6 and 6.9. MgATP-dependent Ca 2+ uptake is insensitive to protonophores. These results indicate that Ca 2+ transport in these microsomal vesicles is catalyzed by a Mg 2+-dependent ATPase localized on the plasma membrane. Ca 2+ strongly reduces ΔpH generation by the plasma membrane H +-ATPase and increases MgATP-dependent membrane potential difference (Δψ) generation. These effects of Ca 2+ on ΔpH and Δψ generation are drastically reduced by micromolar erythrosin B, indicating that they are primarily a consequence of Ca 2+ uptake into plasma membrane vesicles. The Ca 2+-induced increase of Δψ is collapsed by permeant anions, which do not affect Ca 2+-induced decrease of ΔpH generation by the plasma membrane H +-ATPase. The rate of decay of MgATP-dependent ΔpH, upon inhibition of the plasma membrane H +-ATPase, is accelerated by MgATP-dependent Ca 2+ uptake, indicating that the decrease of ΔpH generation induced by Ca 2+ reflects the efflux of H + coupled to Ca 2+ uptake into plasma membrane vesicles. It is therefore proposed that Ca 2+ transport at the plasma membrane is mediated by a Mg 2+-dependent ATPase which catalyzes a nH +/Ca 2+ exchange. 相似文献
3.
The Ca 2+ transport system of corn ( Zea mays) leaf plasma membrane is composed of Ca 2+ pump and Ca 2+/H + antiporter driven by H + gradient imposed by a H + pump (M Kasai, S Muto [1990] J Membr Biol 114: 133-142). It is necessary for characterization of these Ca 2+ transporters to establish the procedure for their solubilization, isolation, and reconstitution into liposomes. We attempted to solubilize and reconstitute the Ca 2+ pump in the present study. A nonionic detergent octaethyleneglycol monododecyl ether (C 12E 8) was the most effective detergent for a series of extraction and functional reconstitution of the Ca 2+ pump among seven detergents examined. This was judged from activities of ATP-dependent 45Ca 2+ uptake into liposomes reconstituted with the respective detergent-extract of the plasma membrane by the detergent dilution method. C 12E 8-extract of the plasma membrane was subjected to high performance liquid chromatography using a DEAE anion exchange column. Ca 2+-ATPase was separated from VO 43−-sensitive Mg 2+-ATPase. These ATPases were separately reconstituted into liposomes, and their ATP-dependent Ca 2+ uptake was measured. The liposomes reconstituted with the Ca 2+-ATPase, but not with the VO 43−-sensitive Mg 2+-ATPase, showed ATP-dependent Ca 2+ uptake. Nigericin-induced pH gradient (acid inside) caused only a little Ca 2+ uptake into liposomes reconstituted with the Ca 2+-ATPase, suggesting that the Ca 2+/H + antiporter was not present in the preparation. These results indicate that the Ca 2+-ATPase actually functions as Ca 2+ pump in the corn leaf plasma membrane. 相似文献
4.
We previously demonstrated that Saccharomyces cerevisiae vnx1Δ mutant strains displayed an almost total loss of Na + and K +/H + antiporter activity in a vacuole-enriched fraction. However, using different in vitro transport conditions, we were able to reveal additional K +/H + antiporter activity. By disrupting genes encoding transporters potentially involved in the vnx1 mutant strain, we determined that Vcx1p is responsible for this activity. This result was further confirmed by complementation of the vnx1Δ vcx1Δ nhx1Δ triple mutant with Vcx1p and its inactivated mutant Vcx1p-H303A. Like the Ca 2+/H + antiporter activity catalyzed by Vcx1p, the K +/H + antiporter activity was strongly inhibited by Cd 2+ and to a lesser extend by Zn 2+. Unlike as previously observed for NHX1 or VNX1, VCX1 overexpression only marginally improved the growth of yeast strain AXT3 in the presence of high concentrations of K + and had no effect on hygromycin sensitivity. Subcellular localization showed that Vcx1p and Vnx1p are targeted to the vacuolar membrane, whereas Nhx1p is targeted to prevacuoles. The relative importance of Nhx1p, Vnx1p, and Vcx1p in the vacuolar accumulation of monovalent cations will be discussed. 相似文献
5.
Artificial pH gradients across tonoplast vesicles isolated from storage tissue of red beet ( Beta vulgaris L.) were used to study the kinetics of a Ca 2+/H + antiport across this membrane. Ca 2+-dependent H + fluxes were measured by the pH-dependent fluorescence quenching of acridine orange. ΔpH-dependent Ca 2+ influx was measured radiometrically. Both H + efflux and Ca 2+ influx displayed saturation kinetics and an identical dependence on external calcium with apparent Km values of 43.9 and 41.7 micromolar, respectively. Calcium influx was unaffected by an excess of Mg 2+ but was inhibited by La 3+ > Mn 2+ > Cd 2+. The apparent Km for external calcium was greatly affected (5-fold) by internal pH in the range of 6.0 to 6.5 and a transmembrane effect of internal proton binding on the affinity for external calcium is suggested. 相似文献
6.
H +-pumping ATPases were detected in microsomal vesicles of oat ( Avena sativa L. var Lang) roots using [ 14C]methylamine distribution or quinacrine fluorescent quenching. Methylamine (MeA) accumulation into vesicles and quinacrine quench were specifically dependent on Mg,ATP. Both activities reflected formation of a proton gradient (ΔpH) (acid inside) as carbonyl cyanide m-chlorophenylhydrazone, nigericin (in the presence of K +), or gramicidin decreased MeA uptake or increased quinacrine fluorescence. The properties of H + pumping as measured by MeA uptake were characterized. The Kmapp for ATP was about 0.1 millimolar. Mg,GTP and Mg, pyrophosphate were 19% and 30% as effective as Mg,ATP. MeA uptake was inhibited by N,N′-dicyclohexylcarbodiimide and was mostly insensitive to oligomycin, vanadate, or copper. ATP-dependent MeA was stimulated by anions with decreasing order of potency of Cl − > Br − > NO 3− > SO 42−, iminodiacetate, benzene sulfonate. Anion stimulation of H + pumping was caused in part by the ability of permeant anions to dissipate the electrical potential and in part by a specific requirement of Cl − by a H + -pumping ATPase. A pH gradient, probably caused by a Donnan potential, could be dissipated by K + in the presence or absence of ATP. MeA uptake was enriched in vesicles of relatively low density and showed a parallel distribution with vanadate-insensitive ATPase activity on a continuous dextran gradient. ΔpH as measured by quinacrine quench was partially vanadate-sensitive. These results show that plant membranes have at least two types of H + -pumping ATPases. One is vanadate-sensitive and probably enriched in the plasma membrane. One is vanadate-resistant, anion-sensitive and has many properties characteristic of a vacuolar ATPase. These results are consistent with the presence of electrogenic H + pumps at the plasma membrane and tonoplast of higher plant cells. 相似文献
7.
Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca 2+ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment. 45Ca 2+ transport was assayed by membrane filtration technique. Results showed that Ca 2+ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca 2+ transport system had a high affinity for Ca 2+( K
m
(Ca 2+)=0.4 m) and ATP( K
m
(ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca 2+ uptake in the plasma membrane vesicles was stimulated in the presence of Cl – or NO
3
–
. Quenching of quinacrine fluorescence showed that these anions also induced H + transport into the vesicles. The Ca 2+ uptake stimulated by Cl – was dependent on the activity of H + transport into the vesicles. However, carbonylcyanide m-chlorophenylhydrazone (CCCP) and VO
4
3–
which is known to inhibit the H + pump associated with the plasma membrane, canceled almost all of the Cl –-stimulated Ca 2+ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca 2+ uptake into the vesicles. These results suggest that the Cl –-stimulated Ca 2+ uptake is caused by the efflux of H + from the vesicles by the operation of Ca 2+/H + antiport system in the plasma membrane. In Cl –-free medium, H + transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca 2+ uptake into the vesicles. These results suggest that two Ca 2+ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca 2+ transport system (Ca 2+ pump) and the other is a Ca 2+/H + antiport system. Little difference in characteristics of Ca 2+ transport was observed between the plasma membranes isolated from etiolated and green corn leaves. 相似文献
8.
Two types of ATP-dependent calcium (Ca 2+) transport systems were detected in sealed microsomal vesicles from oat roots. Approximately 80% of the total Ca 2+ uptake was associated with vesicles of 1.11 grams per cubic centimeter and was insensitive to vanadate or azide, but inhibited by NO 3−. The remaining 20% was vanadate-sensitive and mostly associated with the endoplasmic reticulum, as the transport activity comigrated with an endoplasmic reticulum marker (antimycin A-insensitive NADH cytochrome c reductase), which was shifted from 1.11 to 1.20 grams per cubic centimeter by Mg 2+. Like the tonoplast H+-ATPase activity, vanadate-insensitive Ca2+ accumulation was stimulated by 20 millimolar Cl− and inhibited by 10 micromolar 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid or 50 micromolar N,N′-dicyclohexylcarbodiimide. This Ca2+ transport system had an apparent Km for Mg-ATP of 0.24 millimolar similar to the tonoplast ATPase. The vanadate-insensitive Ca2+ transport was abolished by compounds that eliminated a pH gradient and Ca2+ dissipated a pH gradient (acid inside) generated by the tonoplast-type H+-ATPase. These results provide compelling evidence that a pH gradient generated by the H+-ATPase drives Ca2+ accumulation into right-side-out tonoplast vesicles via a Ca2+/H+ antiport. This transport system was saturable with respect to Ca2+ (Km apparent = 14 micromolar). The Ca2+/H+ antiport operated independently of the H+-ATPase since an artifically imposed pH gradient (acid inside) could also drive Ca2+ accumulation. Ca2+ transport by this system may be one major way in which vacuoles function in Ca2+ homeostasis in the cytoplasm of plant cells. 相似文献
9.
Mg:ATP-dependent H + pumping has been studied in microsomal vesicles from 24-hour-old radish ( Raphanus sativus L.) seedlings by monitoring both intravesicular acidification and the building up of an inside positive membrane potential difference (Δ ψ). ΔpH was measured as the decrease of absorbance of Acridine orange and Δ ψ as the shift of absorbance of bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent Δ pH and Δ ψ generation are completely inhibited by vanadate and insensitive to oligomycin; moreover, Δ pH generation is not inhibited by NO 3−. These findings indicate that this membrane preparation is virtually devoid of mitochondrial and tonoplast H +-ATPases. Both intravesicular acidification and Δ ψ generation are influenced by anions: Δ pH increases and Δ ψ decreases following the sequence SO 42−, Cl −, Br −, NO 3−. ATP-dependent H + pumping strictly requires Mg 2+. It is very specific for ATP (apparent Km 0.76 millimolar) compared to GTP, UTP, CTP, ITP. Δ pH generation is inhibited by CuSO 4 and diethylstilbestrol as well as vanadate. Δ pH generation is specificially stimulated by K + (+ 80%) and to a lesser extent by Na + and choline (+28% and +14%, respectively). The characteristics of H + pumping in these microsomal vesicles closely resemble those described for the plasma membrane ATPase partially purified from several plant materials. 相似文献
10.
The mechanism of sucrose transport was investigated in plasma membrane (PM) vesicles isolated from spinach ( Spinacia oleracea L.) leaves. PM vesicles were isolated by aqueous two-phase partitioning and were equilibrated in pH 7.8 buffer containing K +. The vesicles rapidly accumulated sucrose in the presence of a transmembrane pH gradient (ΔpH) with external pH set at 5.8. The uptake rate was slow at pH 7.8. The K +-selective ionophore, valinomycin, stimulated uptake in the presence of a ΔpH, and the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), greatly inhibited ΔpH-dependent sucrose uptake. Addition of sucrose to the vesicles resulted in immediate alkalization of the medium. Alkalization was stimulated by valinomycin, was abolished by CCCP, and was sucrose-specific. These results demonstrate the presence of a tightly coupled H +/sucrose symporter in PM vesicles isolated from spinach leaves. 相似文献
11.
pH and Na + homeostasis in all cells requires Na +/H + antiporters. In most cases, their activity is tightly pH-regulated. NhaA, the main antiporter of Escherichia coli, has homologues in all biological kingdoms. The crystal structure of NhaA provided insights into the mechanism of action and pH regulation of an antiporter. However, the active site of NhaA remained elusive because neither Na + nor Li +, the NhaA ligands, were observed in the structure. Using isothermal titration calorimetry, we show that purified NhaA binds Li + in detergent micelles. This interaction is driven by an increase in enthalpy (Δ H of −8000 ± 300 cal/mol and Δ S of −15.2 cal/mol/degree at 283 K), involves a single binding site per NhaA molecule, and is highly specific and drastically dependent on pH; Li + binding was observed only at pH 8.5. Combining mutational analysis with the isothermal titration calorimetry measurements revealed that Asp-163, Asp-164, Thr-132, and Asp-133 form the Li + binding site, whereas Lys-300 plays an important role in pH regulation of the antiporter. 相似文献
12.
To determine whether the detergent-solubilized and purified vacuolar H +-ATPase from plants was active in H + transport, we reconstituted the purified vacuolar ATPase from oat roots ( Avena sativa var Lang). Triton-solubilized ATPase activity was purified by gel filtration and ion exchange chromatography. Incorporation of the vacuolar ATPase into liposomes formed from Escherichia coli phospholipids was accomplished by removing Triton X-100 with SM-2 Bio-beads. ATP hydrolysis activity of the reconstituted ATPase was stimulated twofold by gramicidin, suggesting that the enzyme was incorporated into sealed proteoliposomes. Acidification of K +-loaded proteoliposomes, monitored by the quenching of acridine orange fluorescence, was stimulated by valinomycin. Because the presence of K + and valinomycin dissipates a transmembrane electrical potential, the results indicate that ATP-dependent H + pumping was electrogenic. Both H + pumping and ATP hydrolysis activity of reconstituted preparations were completely inhibited by <50 nanomolar bafilomycin A 1, a specific vacuolar type ATPase inhibitor. The reconstituted H + pump was also inhibited by N,N′-dicyclohexylcarbodiimide or NO 3− but not by azide or vanadate. Chloride stimulated both ATP hydrolysis by the purified ATPase and H + pumping by the reconstituted ATPase in the presence of K + and valinomycin. Hence, our results support the idea that the vacuolar H +-pumping ATPase from oat, unlike some animal vacuolar ATPases, could be regulated directly by cytoplasmic Cl − concentration. The purified and reconstituted H +-ATPase was composed of 10 polypeptides of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. These results demonstrate conclusively that the purified vacuolar ATPase is a functional electrogenic H + pump and that a set of 10 polypeptides is sufficient for coupled ATP hydrolysis and H + translocation. 相似文献
13.
Calcium transport into tomato ( Lycopersicon esculentum Mill, cv Castlemart) fruit tonoplast vesicles was studied. Calcium uptake was stimulated approximately 10-fold by MgATP. Two ATP-dependent Ca 2+ transport activities could be resolved on the basis of sensitivity to nitrate and affinity for Ca 2+. A low affinity Ca 2+ uptake system ( Km > 200 micromolar) was inhibited by nitrate and ionophores and is thought to represent a tonoplast localized H +/Ca 2+ antiport. A high affinity Ca 2+ uptake system ( Km = 6 micromolar) was not inhibited by nitrate, had reduced sensitivity to ionophores, and appeared to be associated with a population of low density endoplasmic reticulum vesicles that contaminated the tonoplast-enriched membrane fraction. Arrhenius plots of the temperature dependence of Ca 2+ transport in tomato membrane vesicles showed a sharp increase in activation energy at temperatures below 10 to 12°C that was not observed in red beet membrane vesicles. This low temperature effect on tonoplast Ca 2+/H + antiport activity could only by partially ascribed to an effect of low temperature on H +-ATPase activity, ATP-dependent H + transport, passive H + fluxes, or passive Ca 2+ fluxes. These results suggest that low temperature directly affects Ca 2+/H + exchange across the tomato fruit tonoplast, resulting in an apparent change in activation energy for the transport reaction. This could result from a direct effect of temperature on the Ca 2+/H + exchange protein or by an indirect effect of temperature on lipid interactions with the Ca 2+/H + exchange protein. 相似文献
14.
Summary In rabbit ileum, Ca 2+/calmodulin (CaM) appears to be involved in physiologically inhibiting the linked NaCl absorptive process, since inhibitors of Ca 2+/CaM stimulate linked Na + and Cl – absorption. The role of Ca 2+/CaM-dependent phosphorylation in regulation of the brush-border Na +/H + antiporter, which is believed to be part of the neutral linked NaCl absorptive process, was studied using purified brush-border membrane vesicles, which contain both the Na +/H + antiporter and Ca 2+/CaM-dependent protein kinase(s) and its phosphoprotein substrates. Rabbit ileal villus cell brush-border membrane vesicles were prepared by Mg precipitation and depleted of ATP. Using a freezethaw technique, the ATP-depleted vesicles were loaded with Ca 2+, CaM, ATP and an ATP-regenerating system consisting of creatine kinase and creatine phosphate. The combination of Ca 2+/CaM and ATP inhibited Na +/H + exchange by 45±13%. This effect was specific since Ca 2+/CaM and ATP did not alter diffusive Na + uptake, Na +-dependent glucose entry, or Na + or glucose equilibrium volumes. The inhibition of the Na +/H + exchanger by Ca 2+/CaM/ATP was due to an effect on the V
max and not on the K
m
for Na +. In the presence of CaM and ATP, Ca 2+ caused a concentration-dependent inhibition of Na + uptake, with an effect 50% of maximum occurring at 120 nm. This Ca 2+ concentration dependence was similar to the Ca 2+ concentration dependence of Ca 2+/CaM-dependent phosphorylation of specific proteins in the vesicles. The Ca 2+/CaM/ATP-inhibition of Na +/H + exchange was reversed by W 13, a Ca 2+/CaM antagonist, but not by a hydrophobic control, W 12, or by H-7, a protein kinase C antagonist. we conclude that Ca 2+, acting through CaM, regulates ileal brush-border Na +/H + exchange, and that this may be involved in the regulation of neutral linked NaCl absorption. 相似文献
15.
Chloride or nitrate decreased a pH gradient (measured as [ 14C]methylamine accumulation) in tonoplast-enriched vesicles. The ΔpH decrease was dependent on the anion concentration. These effects are independent of the anion-sensitive H +-ATPase of the tonoplast, since the pH gradient (acid inside) was imposed artificially using a pH jump or a K + gradient and nigericin. 4,4′-Diisothiocyano-2,2′-stilbene disulfonic acid partially prevented the decrease in pH gradient induced by Cl −. Two possible models to account for this anion-dependent decrease of ΔpH are: (a) H + loss is accompanied by Cl − or NO 3− efflux from the vesicles via H +/anion symport systems on the tonoplast and (b) H + loss is accompanied by Cl − or NO 3− uptake into the vesicles via H +/anion antiport systems. Depending on the requirements and conditions of the cell, these two systems would serve to either mobilize Cl − and NO 3− stored in the vacuole for use in the cytoplasm or to drive anions into the vacuole. Chloride or nitrate also decreased a pH gradient in fractions containing plasma membrane and Golgi, implying that these membranes may have similar H +-coupled anion transport systems. 相似文献
16.
Using vesicles of symbiosome membrane (SM), it was shown that the Ca 2+-ATPase can function as an ATP-energized Ca 2+/H + antiporter. The initial rate of the acidic shift inside the vesicles, as well as the rate of the ITP-dependent alkalization of the medium inside them markedly increased in the presence of valinomycin. This process was rapidly stopped by eosin Y, a known inhibitor of the type IIB Ca 2+-ATPase. ITP-dependent uptake of Ca 2+ was blocked after the addition to the reaction mixture of nigericin in the presence of K +. Under these conditions, the alkaline shift of pH inside the vesicles occurred, leading to the inhibition of operation of the calcium pump in SM. Evaluation of the pH shifts inside the vesicles by using pH-indicator pyranine confirmed the ion-exchange mechanism of the Ca 2+-ATPase functioning in the SM. 相似文献
17.
We examined nitrate-dependent Fe 2+ oxidation mediated by anaerobic ammonium oxidation (anammox) bacteria. Enrichment cultures of “ Candidatus Brocadia sinica” anaerobically oxidized Fe 2+ and reduced NO 3− to nitrogen gas at rates of 3.7 ± 0.2 and 1.3 ± 0.1 (mean ± standard deviation [SD]) nmol mg protein −1 min −1, respectively (37°C and pH 7.3). This nitrate reduction rate is an order of magnitude lower than the anammox activity of “ Ca. Brocadia sinica” (10 to 75 nmol NH 4+ mg protein −1 min −1). A 15N tracer experiment demonstrated that coupling of nitrate-dependent Fe 2+ oxidation and the anammox reaction was responsible for producing nitrogen gas from NO 3− by “ Ca. Brocadia sinica.” The activities of nitrate-dependent Fe 2+ oxidation were dependent on temperature and pH, and the highest activities were seen at temperatures of 30 to 45°C and pHs ranging from 5.9 to 9.8. The mean half-saturation constant for NO 3− ± SD of “ Ca. Brocadia sinica” was determined to be 51 ± 21 μM. Nitrate-dependent Fe 2+ oxidation was further demonstrated by another anammox bacterium, “ Candidatus Scalindua sp.,” whose rates of Fe 2+ oxidation and NO 3− reduction were 4.7 ± 0.59 and 1.45 ± 0.05 nmol mg protein −1 min −1, respectively (20°C and pH 7.3). Co-occurrence of nitrate-dependent Fe 2+ oxidation and the anammox reaction decreased the molar ratios of consumed NO 2− to consumed NH 4+ (ΔNO 2−/ΔNH 4+) and produced NO 3− to consumed NH 4+ (ΔNO 3−/ΔNH 4+). These reactions are preferable to the application of anammox processes for wastewater treatment. 相似文献
18.
The effects of calmodulin (CaM) on ATPase activity and ATP-dependent formation of a proton gradient (ΔpH) were studied in tonoplast membrane vesicles from corn ( Zea mays L.) roots. At 0.6 micromolar, CaM stimulated ATPase activity by about 20% in the absence of an uncoupler, but by only 4% in its presence. Thus, the uncoupler-dependent increment of activity was decreased 30 to 45% by CaM. The formation of a proton gradient across the membrane vesicle, measured by quinacrine fluorescence quench, was inhibited about 20% by CaM. Its effect was additive to the effect of Ca 2+ and was completely abolished by EGTA. These effects of CaM could be due to stimulation of H + efflux or due to inhibition of the H +-ATPase. To distinguish between these possibilities, we examined the effect of CaM on dissipation of preformed ΔpH after the ATPase was inhibited. CaM stimulated the dissipation of a preformed ΔpH by 40% after the H +-ATPase was inhibited with NO 3−. This indicates that CaM facilitates the recycling of protons across the tonoplast membranes and does not regulate the H +-ATPase by direct inhibition. 相似文献
19.
Summary We have examined the effect of second messengers on ATP-driven H + transport in an H + ATPase-bearing endosomal fraction isolated from rabbit renal cortex. cAMP (0.1 mm) had no effect on H + transport. Acridine orange fluorescence in the presence of 0.5 mm Ca 2+ (+1 mm EGTA) was 19±6% of control. Inhibition of ATP-driven H + transport by Ca 2+ was concentration dependent; 0.25 and 0.5 mm Ca 2+ (+1 mm EGTA) inhibited acridine orange fluorescence by 50 and 80%, respectively. Ca 2+ also produced a concentration-dependent increase in the rate of pH-gradient dissipation. Ca 2+ did not affect ATP hydrolysis. ATP-dependent Br – uptake was virtually unchanged in the presence of 0.5 mm Ca 2+ (+1 mm EGTA). These vesicles were also shown to transport Ca 2+ in an ATP-dependent mode. Inositol 1, 4, 5-trisphosphate had no effect on ATP-dependent Ca 2+ uptake. These results are consistent with the co-existence of an H + ATPase and an H +/Ca 2+ exchanger on these endosomes, the latter transport system using the H + gradient to energize Ca 2+ uptake. Attempts to demonstrate an H +/Ca 2+ antiporter in the absence of ATP have been unsuccessful. Yet, when a pH gradient was established by preincubation with ATP and residual ATP was subsequently removed by hexokinase + glucose, stimulation of Ca 2+ uptake could be demonstrated. A Ca 2+-dependent increase in H + permeability and an ATP-dependent Ca 2+ uptake might have important implications for the regulation of vacuolar H + ATPase activity as well as the homeostasis of cytosolic Ca 2+ concentration. 相似文献
20.
Using roots from Arabidopsis thaliana expressing the recombinant calcium indicator aequorin, we show that NH 3 uptake and alkalisation of plant cells act as a stimulus which induces transient elevations of the cytoplasmic free calcium concentration ([Ca 2+] c). The magnitudes of these [Ca 2+] c elevations are dependent on the concentration of the membrane permeable form, NH 3, and hence, particularly dependent on the pH in the external medium. EGTA and La 3+ are able to significantly suppress the [Ca 2+] c transients showing that Ca 2+ influx through the plasma membrane is likely to be involved. Verapamil and nifedipine had no inhibitory effects, which suggests that Ca 2+ release from internal stores might not contribute significantly to the NH 3‐triggered [Ca 2+] c response. Pre‐incubation in l ‐methionine‐dl ‐sulphoximine – an inhibitor of the glutamine synthetase – did not alter the NH 3‐induced [Ca 2+] c responses at all. These results are consistent with previous studies where NH 3‐induced changes of cytoplasmic and vacuolar pH were investigated in maize roots. Furthermore, the similarity between the kinetics of NH 3‐driven cellular pH changes demonstrated in previous studies and the [Ca 2+] c transients shown here suggests a direct relationship between [Ca 2+] c and cellular alkalisation (cytoplasmic pH and/or vacuolar pH). However, the mechanism behind this possible causal relation remains to be elucidated. 相似文献
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