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1.
Plantago species differ in their strategy towards salt stress, a major difference being the uptake and distribution of Na+ ions. A salt-sensitive ( Plantago media L.) and a salt-tolerant ( P. maritima L.) species were compared with respect to Na+/H+ antiport activities at the tonoplast. After exposure of the plants to 50 m M NaCl for 6 days isolated tonoplast vesicles of P. maritima showed Na+/H+ antiport activity with saturation kinetics and a Km of 2.4 m M Na+, NaCl-grown P. media and the control plants of both species showed no antiport activity. Selectivity of the antiport system for Na+ was high and was determined by adding different chloride salts after formation of a Δ pH in the vesicles. Specific tonoplast ATPase activities were similar in the two species and did not alter after exposure to NaCl stress.  相似文献   

2.
Abstract: Aspartate uptake by membrane vesicles derived from rat brain was investigated. The uptake is dependent on a Na+ gradient ([Na+] outside > [Na+] inside). Active transport of aspartate is strictly dependent upon the presence of sodium and maximal extent of transport is reached when both Na+ and Cl ions are present. The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. The uptake of aspartate is stimulated by a membrane potential (negative inside), as demonstrated by the effect of the ionophore carbonyl cyanide m -chlorophenylhydrazone and anions with different permeabilities. The presence of ouabain, an inhibitor of (Na++ K+)-ATPase, does not affect aspartate transport. The kinetic analysis shows that aspartate is accumulated by two systems with different affinities, showing K m and V max values of similar order to those found in slightly "cruder" preparations. Inhibition of the l -aspartate uptake by d -aspartate and d - and l -glutamate indicates that a common carrier is involved in the process, this being stereospecific for the d - and l -glutamate stereoisomers.  相似文献   

3.
In many cell types cytoplasmic alkalization is an early marker for cell activation. An amiloride-sensitive Na+/H+ exchanger is an important regulator of this process. However, in keratinocytes the existence of a Na+/H+ exchanger nor a proliferation-associated increase in intracellular pH (pHi) has been demonstrated.
The aim of this study was to investigate whether or not keratinocytes, derived from the BALB/MK cell line, contain a Na+/H+ exchanger and whether cytoplasmic alkalization is proliferation-associated in these cells. This mouse keratinocyte cell line can easily be switched between a proliferative and a quiescent state under defined culture conditions. The novel pH-sensitive dye seminaphthorhodafluor (SNARF)-calcein proved to be very suitable for flow cytometric pHi measurements in BALB/MK cells. Initial measurements of the pHi using a cocktail of the established fluorochromes 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and SNARF-1 failed because of the differential uptake and binding kinetics of these pH-sensitive dyes.
Using SNARF-calcein we were able to show proliferation to be associated with increased pHi. However, culture conditions were critical for these measurements. Our data indicate that the Na+/H+ exchanger is involved in this process, since acid load and pHi-recovery experiments showed the alkalization to be amiloride-sensitive.  相似文献   

4.
Abstract Washed invested vesicle preparations of Methanosarcina strain Gö1 catalyzed the formation of methyl-CoM from formaldehyde, H2 and CoM in the presence of tetrahydromethanopterin and 2-bromoethanesulfonate. The reaction was associated with the translocation of sodium ions into the lumen of the vesicles. This translocation was abolished by the Na+ ionophore ETH 157 but it was insensitive to the addition of the uncoupler SF6847 and the Na+/H+ antiport inhibitor amiloride and, therefore, is the result of a primary Na+ pump. Since the translocation of Na+ was also observed when formaldehyde + tetrahydromethanopterin was replaced by methyl-tetrahydromethanopterin, it follows that the methyl transfer from tetrahydromethanopterin to CoM is the sodium-motive reaction. Methyl-tetrahydromethanopterin could be replaced by methyl-tetrahydrofolate.  相似文献   

5.
F1F0 ATP synthases use the electrochemical potential of H+ or Na+ across biological membranes to synthesize ATP by a rotary mechanism. In bacteria, the enzymes can act in reverse as ATP-driven ion pumps creating the indispensable membrane potential. Here, we demonstrate that the F0 parts of a Na+- and H+-dependent enzyme display major asymmetries with respect to their mode of operation, reflected by the requirement of ∼100 times higher Na+ or H+ concentrations for the synthesis compared with the hydrolysis of ATP. A similar asymmetry is observed during ion transport through isolated F0 parts, indicating different affinities for the binding sites in the a/c interface. Together with further data, we propose a model that provides a rationale for a differential usage of membrane potential and ion gradient during ATP synthesis as observed experimentally. The functional asymmetry might also reflect an important property of the ATP synthesis mechanism in vivo . In Escherichia coli , we observed respiratory chain-driven ATP production at pH 7–8, while P -site pH values < 6.5 were required for ATP synthesis in vitro . This discrepancy is discussed with respect to the hypothesis that during respiration lateral proton diffusion could lead to significant acidification at the membrane surface.  相似文献   

6.
Abstract: The mechanism of unidirectional transport of sodium from blood to brain in pentobarbital-anesthetized rats was examined using in situ perfusion. Sodium transport followed Michaelis-Menten saturation kinetics with a V max of 50.1 nmol/g/min and a K m of 17.7 m M in the left frontal cortex. The kinetic analysis indicated that, at a physiologic sodium concentration, ∼26% of sodium transport at the blood-brain barrier (BBB) was carrier mediated. Dimethylamiloride (25 µ M ), an inhibitor of Na+/H+ exchange, reduced sodium transport by 28%, whereas phenamil (25 µ M ), a sodium channel inhibitor, reduced the transfer constant for sodium by 22%. Bumetanide (250 µ M ) and hydrochlorothiazide (1.5 m M ), inhibitors of Na+-K+-2Cl/NaCl symport, were ineffective in reducing blood to brain sodium transport. Acetazolamide (0.25 m M ), an inhibitor of carbonic anhydrase, did not change sodium transport at the BBB. Finally, a perfusate pH of 7.0 or 7.8 or a perfusate P co 2 of 86 mm Hg failed to change sodium transport. These results indicate that 50% of transcellular transport of sodium from blood to brain occurs through Na+/H+ exchange and a sodium channel in the luminal membrane of the BBB. We propose that the sodium transport systems at the luminal membrane of the BBB, in conjunction with Cl/HCO3 exchange, lead to net NaCl secretion and obligate water transport into the brain.  相似文献   

7.
The genes encoding the Na+/H+/L-glutamate symport proteins of the thermophilic organisms Bacillus stearothermophilus (gltTBs) and Bacillus caldotenax (gltTBc) were cloned by complementation of Escherichia coli JC5412 for growth on glutamate as sole source of carbon, energy and nitrogen. The nucleotide sequences of the gltTBs and gltTBc genes were determined. In both cases the translated sequences corresponded with proteins of 421 amino acid residues (96.7% amino acid identity between GltTBs and GltTBc). Putative promoter, terminator and ribosome-binding-site sequences were found in the flanking regions. These expression signals were functional in E. coli. The hydropathy profiles indicate that the proteins are hydrophobic and could form 12 membrane-spanning regions. The Na+/H+ coupled L-glutamate symport proteins GltTBs and GltTBc are homologous to the strictly H+ coupled L-glutamate transport protein of E. coli K-12 (overall 57.2% identity). Functional expression of glutamate transport activity was demonstrated by uptake of glutamate in whole cells and membrane vesicles. In accordance with previous observations (de Vrij et al., 1989; Heyne et al., 1991), glutamate uptake was driven by the electrochemical gradients of sodium ions and protons.  相似文献   

8.
A vacuolar Na^ /H^ antiporter cDNA gene was successfully isolated fromHordeum brevisubulatum (Trin.) Link using the rapid amplification ofcDNA ends (RACE) method. The gene was named HbNHXI and was found to consist of 1 916 bp encoding a predicted polypeptide of 540 amino acids with a conserved amiloride-binding domain. Phylogenetic tree analysis of the Na^ /H^ antiporters showed that the HbNHXI gene shares 55.3%-74.8% similarity with the vacuolar-type Na^ /H^ antiporters. Transgenic tobaccos that contain the HbNHXI gene, integrated by forward insertion into the tobacco genome, were obtained via Agrobacterium tumerfaciens and characterized for the determination of the concentration of Na^ and K^ ions, as well as proline, in the presence of 300 mmol/L NaCl. The T1 transgenic plants showed more tolerance to salt and drought than did wild-type plants. Our data suggest that overexpression of the HbNHXI gene could improve the tolerance of transgenic tobaccos to salt and drought through the function of the vacuolar Na^ /H^ antiporter.  相似文献   

9.
N-System Amino Acid Transport at the Blood-CSF Barrier   总被引:1,自引:1,他引:0  
Abstract: Despite l -glutamine being the most abundant amino acid in CSF, the mechanisms of its transport at the choroid plexus have not been fully elucidated. This study examines the role of L-, A-, ASC-, and N-system amino acid transporters in l -[14C]glutamine uptake into isolated rat choroid plexus. In the absence of competing amino acids, approximately half the glutamine uptake was via a Na+-dependent mechanism. The Na+-independent uptake was inhibited by 2-amino-2-norbornane carboxylic acid, indicating that it is probably via an L-system transporter. Na+-dependent uptake was inhibited neither by the A-system substrate α-(methylamino)isobutyric acid nor by the ASC-system substrate cysteine. It was inhibited by histidine, asparagine, and l -glutamate γ-hydroxamate, three N-system substrates. Replacement of Na+ with Li+ had little effect on uptake, another feature of N-system amino acid transport. These data therefore indicate that N-system amino acid transport is present at the choroid plexus. The V max and K max for glutamine transport by this system were 8.1 ± 0.3 nmol/mg/min and 3.3 ± 0.4 m M , respectively. This system may play an important role in the control of CSF glutamine, particularly when the CSF glutamine level is elevated as in hepatic encephalopathy.  相似文献   

10.
Abstract: The excitatory amino acid glutamate was previously shown to stimulate aerobic glycolysis in astrocytes by a mechanism involving its uptake through an Na+-dependent transporter. Evidence had been provided that Na+,K+-ATPase might be involved in this process. We have now measured the activity of Na+,K+-ATPase in cultured astrocytes, using ouabain-sensitive 86Rb uptake as an index. l -Glutamate increases glial Na+,K+-ATPase activity in a concentration-dependent manner with an EC50 = 67 µ M . Both l - and d -aspartate, but not d -glutamate, produce a similar response, an observation that is consistent with an uptake-related effect rather than a receptor-mediated one. Under basal conditions, concentration-dependent inhibition of Na+,K+-ATPase activity in astrocytes by ouabain indicates the presence of a single catalytic site with a low affinity for ouabain ( K 0.5 = 113 µ M ), compatible with the presence of an α1 isozyme. On stimulation with glutamate, however, most of the increased activity is inhibited by low concentrations of ouabain ( K 0.5 = 20 n M ), thus revealing a high-affinity site akin to the α2 isozyme. These results suggest that astrocytes possess a glutamate-sensitive isoform of Na+,K+-ATPase that can be mobilized in response to increased neuronal activity.  相似文献   

11.
Barnea caridida oocytes release acid (1.35 pmole H+/oocyte) upon fertilization. After artificial activation by an excess of KCl, germinal vesicle breakdown (GVBD) occurs normally and a quite similar, but not identical, acid release is recorded (1.10 pmole H+/oocyte). KCl activation of Barnea oocytes is completely inhibited in 100 mM sodium-acetate sea water at pH 6.5 and fertilization does not result in activation when the oocytes are transferred after one minute into 100 mM sodium-acetate sea water at pH 6.3. When D–600, a calcium transmembrane fluxes inhibitor, is added 20 seconds after fertilization, GVBD is inhibited but a normal acid release is recorded. The presence of at least 10 mM sodium ions in the external medium is required for 100% activation of these oocytes by an excess of KCl. These results suggest that while an intracellular pH increase may be a requisite for GVBD, this can not be a sufficient condition to trigger it unless a calcium influx is allowed to occur. Moreover, the acid release does not result from a Ca++-H+ exchange transport but appears more likely to be due to a Na+-H* exchange as it has been demontrated in sea urchin eggs.  相似文献   

12.
Abstract Many species of bacteria isolated from saline environments require Na+ specifically for membrane transport. Transport occurs by a Na+ symport process energized by an electrochemical gradient of Na+ ions. The gradient at neutral pH appears to be produced by a primary electrogenic extrusion of protons coupled to a secondary, outwardly directed Na+ pump, a Na+/proton antiporter. At alkaline pH Vibrio alginolyticus may also produce the gradient by an energy-dependent primary extrusion of Na+ ions. Alteromonas haloplanktis and Vibrio costicola require salts in the medium to retain intracellular solutes. For A. haloplanktis the effects of the salts are primarily osmotic. For V. costicola , only NaCl is effective in retaining solutes and Na+ is required by this organism to maintain the membrane potential. In Escherichia coli a single substitution in the nucleotide sequence of the gene coding for the melibiose transport protein changed the cation specificity of the transport system. The possible ecological significance of this finding has been considered.  相似文献   

13.
Abstract A Na+/H+ antiporter catalyses coupled Na+ extrusion and H+ uptake across the membranes of extremely alkalophilic bacilli. This exchange is electrogenic, with H+ translocated inward > Na+ extruded. It is energized by the Δψ 2 component of the ΔμH+ that is established during primary proton pumping by the alkalophile respiratory chain complexes. These complexes abound in the membranes of extreme alkalophiles. Combined activity of the respiratory chain, the antiporter, and solute transport systems that are coupled to Na+ re-entry, allow the alkalophiles to maintain a cytoplasmic pH that is several pH units more acidic than optimal external pH values for growth. There is no compelling evidence for a specific and necessary role for any ion other than sodium in pH homeostasis, and although there is very high cytoplasmic buffering capacity in the alkaline range, active mechanisms for pH homeostasis are crucial. Energization of the antiporter as well as the proton translocating F 1 F 0-ATPase that catalyses ATP synthesis in the extreme alkalophiles must accommodate the problem of the low net ΔμH+ and the very low concentrations of protons, per se, in the external medium. This problem is by-passed by other bioenergetic work functions, such as solute uptake or motility, that utilize sodium ions for energy-coupling in the place of protons.  相似文献   

14.
As water and nutrient uptake should be related in the response of plants to salinity, the aim of this paper is to establish whether or not aquaporin functionality is related to H+-ATPase activity in root cells of pepper ( Capsicum annuum L.) plants. Thus, H+-ATPase activity was measured in plasma membrane vesicles isolated from roots and aquaporin functionality was measured using a cell pressure probe in intact roots. Salinity was applied as 60 m M NaCl or 60 m M KCl, to determine which ion (Na+, K+ or Cl) is producing the effects. We also investigated whether the effects of both salts were ameliorated by Ca2+. Similar results were obtained for cell hydraulic conductivity, Lpc, and H+-ATPase activity, large reductions in the presence at NaCl or KCl and an ameliorative effect of Ca2+. However, fusicoccin (an activator of H+-ATPase) did not alter osmotic water permeability of protoplasts isolated from roots. Addition of Hg2+ inhibited both ATPase and aquaporins, but ATPase also contains Hg-binding sites. Therefore, the results indicate that H+-ATPase and aquaporin activities may not be related in pepper plants.  相似文献   

15.
16.
Spore germination of Bacillus stearothermophilus was progressively inhibited as the concentrations of sodium bicarbonate (NaHCO3) in the germination media increased from 0% to 1·0% (w/v). The inhibitory effect of NaHCO3 was attributed to the release of HCO3 and its alkaline properties, each of which played a different role. At low concentrations (< 0·3%), the inhibitory effect of NaHCO3 was mainly due to bicarbonate. As NaHCO3 increased from 0·3% to higher concentrations, the effect of HCO3 reached a plateau while the alkalinating effect became the more dominant inhibitory factor. Fourier transform infrared (FTIR) analysis reveals that sodium bicarbonate reacted with the carboxyl group (1570 cm−1) of some acidic amino-acid residues of protein in the spore, leading to a less orientated structure. A shift of two units towards the longer frequency for carboxyl groups indicates that a stronger interaction was formed between the carboxyl group and the Na+ ion. The largest ratio of peak height between the absorbance of carboxylate (1570 cm−1) and of amide II (1546 cm−1) of spores after pretreatment with 0·3% sodium bicarbonate reflects the biggest structural alterations of keratin-like proteins in the spore. The role of NaHCO3 in enhancing the sporicidal effect of glutaraldehyde is discussed.  相似文献   

17.
In embryos of the sea urchin, Hemicentrotus pulcherrimus , as well as in cultured cells derived from isolated micromeres, spicule formation was inhibited by allylisothiocyanate, an inhibitor of H+, K+-ATPase, at above 0.5 μM and was almost completely blocked at above 10 μM. Amiloride, an inhibitor of Na+, H+ antiporter, at above 100 μM exerted only slight inhibitory effect, if any, on spicule formation. Intravesicular acidification, determined using [ dimethylamine -14C]-aminopyrine as a pH probe, was observed in the presence of ATP and 200 mM KCl in microsome fraction obtained from embryos at the post gastrula stage, at which embryos underwent spicule calcification. Intravesicular acidification and K+-dependent ATPase activity were almost completely inhibited by allylisothiocyanate at 10 μM. Allylisothiocyanate-sensitive ATPase activity was found mainly in the mesenchyme cells with spicules isolated from prisms. H+, K+-ATPase, an H+ pump, probably mediates H+ release to accelerate CaCO3 deposition from Ca2+, CO2 and H2O in the primary mesenchyme cells. Intravesicular acidification was stimulated by valinomycin at the late gastrula and the prism stages but not at the pluteus stage. K+ permeability probably increases after the prism stage to activate H+ release.  相似文献   

18.
Abstract This review deals with work on some aspects of halotolerance carried out in our laboratory. The organism used throughout these investigations is a halotolerant, obligate aerobic rod, designated as Ba1. Attention is focused on the effect of osmotic stress on respiratory rate, since under the conditions tested the latter is rate-limiting for growth. Under hyperosmotic conditions there is a steep drop in the rate of oxygen uptake, but the inhibition can be relieved by betaine. Two major factors seem to be involved in this effect of betaine: (a) Iso-osmotic adaptation due to its accumulation in the cytosol (deplasmolysis); (b) facilitation of the penetration of Na+ into plasmolysed cells. Na+ is required for the proper functioning of the respiratory chain. Na+ probably enters the cells in symport with betaine, whereas an excess of this cytotoxic cation is extruded by a powerful primary Na+ pump which is found to operate in Ba1. Such a pump has also been identified in another halotolerant organism, Vibrio alginolyticus . In both micro-organisms the site of Na+-stimulation corresponds to the site of Na+ translocation.  相似文献   

19.
In cultured cells derived from micromeres isolated at the 16-cell stage of sea urchin embryos, the activity of H+, K+-ATPase became detectable after 15 hr of culture, when the cells started to form spicules, and then increased reaching a plateau from 25 hr of culture. The Na+, K+-ATPase activity of isolated micromeres increased to a maximum at 20 hr of culture and thereafter decreased gradually. Allylisothiocyanate, an inhibitor of H+, K+-ATPase, caused a decrease in intracellular pH (pHi) accompanied by blockage of 45Ca deposition in spicule rods in spicule-forming cells at 30 hr of culture. Ouabain and amiloride had scarcely any effect on the pHi or 45, deposition. In cultured cells exposed to nifedipine, which blocked 45Ca deposition in spicule rods, allylisothiocyanate did not cause any decrease in pHi. These results show that H+, which is generated in the overall reaction to produce CaCO3 from Ca2+ and HCO3, is probably released from the cells mainly in the reaction catalyzed by H+, K+-ATPase to maintain successive production of CaCO3.  相似文献   

20.
A tomato ( Lycopersicon esculentum Mill. cv. Pera) callus culture tolerant to NaCl was obtained by successive subcultures of NaCl-sensitive calli in medium supplemented with 50 m M NaCl. NaCl-tolerant calli grew better than NaCl-sensitive calli in media supplemented with 50 and 100 m M NaCl. Analysis of callus ion content showed a strong increase in Na+ and Cl both in NaCl-tolerant and -sensitive calli grown in media containing NaCl for one subculture. Cells from NaCl-tolerant calli showed a higher H+ extrusion activity than those from NaCl-sensitive calli grown for one subculture in the presence of NaCl. The inhibition of H+ extrusion by NaCl-sensitive cells was correlated with an inhibition of microsomal vanadate-sensitive H+-ATPase (EC 3.6.1.35) and ATP-dependent H+ transport, while the stimulation of H+ extrusion by cells tolerant to 50 m M NaCl was correlated with an increase in plasma membrane ATP-dependent H+ transport. The increase of ATP-dependent H+ extrusion in plasma membranes isolated from 50 m M NaCl-tolerant calli was not a result of stimulation of a vanadate-sensitive ATP hydrolytic activity or an increase in passive permeability to H+. Relative to NaCl-sensitive calli, plasma membrane H+-ATPase from calli tolerant to 50 m M NaCl showed a lower Km for Mg2+-ATP. Our results indicate that tolerance of tomato calli to 50 m M NaCl increases the affinity of plasma membrane H+-ATPase for the substrate ATP and stimulates the H+-pumping activity of this enzyme without modifying its phosphohydrolytic activity.  相似文献   

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