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1.
In the thermohalophilic bacterium Rhodothermus marinus, the NADH:quinone oxidoreductase (complex I) is encoded by two single genes and two operons, one of which contains the genes for five complex I subunits, nqo10-nqo14, a pterin carbinolamine dehydratase, and a putative single subunit Na+/H+ antiporter. Here we report that the latter encodes indeed a functional Na+/H+ antiporter, which is able to confer resistance to Na+, but not to Li+ to an Escherichia coli strain defective in Na+/H+ antiporters. In addition, an extensive amino acid sequence comparison with several single subunit Na+/H+ antiporters from different groups, namely NhaA, NhaB, NhaC, and NhaD, suggests that this might be the first member of a new type of Na+/H+ antiporters, which we propose to call NhaE.  相似文献   

2.
Amiloride-sensitive Na+-H+ antiporter in Escherichia coli.   总被引:1,自引:0,他引:1       下载免费PDF全文
In everted vesicles of Escherichia coli, delta pH caused by H+ efflux through the Na+/H+ antiporter was measured by using a fluorescent dye. Amiloride inhibited the activity of the Na+/H+ antiporter. Kinetic studies showed that amiloride competed with Na+. The inhibition constant of 40 microM was obtained.  相似文献   

3.
A mutant of Escherichia coli with defective Na+/H+ antiporter was isolated. The rationale for its isolation was that cells possessing defective Na+/H+ antiporter, which is essential for establishment of a Na+ gradient, could not grow with a carbon source that was taken up with Na+. The mutant had no appreciable Na+/H+ antiporter activity, but its K+/H+ antiporter and Ca2+/H+ antiporter activities were normal. Judging from the reversion frequency, the defect seems to be due to a single mutation. The mutant could not grow at alkaline pH. Therefore, the Na+/H+ antiporter, but not the K+/H+ antiporter or the Ca2+/H+ antiporter, seems to be responsible for pH regulation in alkaline medium. This mutant will be useful for cloning the Na+/H+ antiporter gene and for detection of Na+-substrate cotransport systems.  相似文献   

4.
In our routine screening of chemicals that would inhibit cardiac sarcolemmal Na+/H+ antiporter, we discovered that some of the opioids produced inhibition of cardiac sarcolemmal Na+/H+ antiporter in micromolar concentrations. Using U-50,488H, a selective kappa-opioid agonist, we characterized the nature of interaction between opioids and the Na+/H+ antiporter. The inhibitory effect of U-50,488H on Na+/H+ antiporter was immediate and reversible, and was not mediated through the interaction with the opioid receptors but due to the direct interaction of U-50,488H with the Na+/H+ antiporter. The kinetic data show that in the presence of U-50,488H the Km for Na+ was increased from 2.5 +/- 0.2 to 5.0 +/- 0.3 mM, while the Vmax (52.0 +/- 5.0 nmol.mg-1.min-1) remained the same. These results suggest that U-50,488H and Na+ compete for the same site on the antiporter. When testing the effect of U-50,488H on other transport systems of cardiac sarcolemma, we found that U-50,488H also inhibited Na+/Ca2+ antiporter and Na+/K+ pump but at much higher concentrations suggesting that U-50,488H shows some degree of selectivity for cardiac sarcolemmal Na+/H+ antiporter. When we compared the inhibitory potency of U-50,488H with amiloride and its analog, namely 5-(N,N-hexamethylene)amiloride, we found that U-50,488H (IC50 = 100 +/- 15 microM) was threefold more potent than amiloride (IC50 = 300 +/- 20 microM) but it was three-fold less potent than the amiloride analog (IC50 = 30 +/- 10 microM) in inhibiting cardiac sarcolemmal Na+/H+ antiporter. These results show that although U-50,488H is more potent than amiloride, the inhibitory characteristics of U-50,488H on cardiac sarcolemmal Na+/H+ antiporter are similar to amiloride.  相似文献   

5.
6.
Using in vivo assays, we show that the Na+/H+ antiporter activity of the Escherichia coli mutant HIT-1 is reduced dramatically compared with activity in wild-type cells. An isogenic nhaA (formerly antA) deletion strain, however, is not significantly different from wild type in this respect. We call the locus affecting Na+/H+ antiporter activity of the HIT-1 mutant nhaB. The nhaB activity exhibits no pH dependence in the range between 7.0 and 8.5, whereas that of the nhaA gene increases considerably at pH levels above 8.0. Mutants with defects in nhaB grow normally on agar media containing 0.5 M NaCl, but nhaA mutants are sensitive to 0.5 M NaCl. We have mapped the nhaB mutation of HIT-1 to 25.6 min on the E. coli map. It is unlinked to the nhaA region, which is located at about 0.5 min. Since a cell with a mutation in nhaB alone is essentially Na+/H+ antiporter negative up to pH 8.0, we conclude that nhaB is required for the major Na+/H+ antiporter activity in the usual physiological pH range.  相似文献   

7.
Sha (also known as Mrp/Mnh/Pha) is a Na+/H+ antiporter encoded by a cluster of six or seven genes that probably form a multisubunit transport complex. The Sha system is important for the homeostasis of H+, Na+, and other monovalent cations and plays a critical role in various functions, including alkaliphily, sporulation, and symbiosis. Here, we characterized the sha homologue genes from the opportunistic pathogen Pseudomonas aeruginosa, which exist as a cluster of six genes (PA1054 to PA1059). The gene cluster PA1054 to PA1059, but not the cluster with a deletion of PA1054, complemented a growth defect in the presence of 0.2 M NaCl and a defect in Na+/H+ antiport activity of the Escherichia coli TO114 mutant lacking the three major Na+/H+ antiporters, indicating that genes PA1054 to PA1059 are responsible for Na+/H+ antiport activity. We disrupted PA1054 (a shaA homologue gene) and determined its effect on Na+ tolerance during growth, Na+ efflux, and pathogenicity in mice. Disruption of PA1054 resulted in severe Na+ sensitivity during growth and decreased Na+ efflux activity. In mice, the deletion mutant of PA1054 also exhibited an attenuated virulence in systemic, pulmonary, and urinary tract infections and also a decrease in colonization of the infected organs. From these results, we conclude that the genes PA1054 to PA1059 encode a Na+/H+ antiporter that is largely responsible for Na+ extrusion in P. aeruginosa and has a role in the infection of the pathogen. We propose to designate PA1054 to PA1059 as the sha (sodium hydrogen antiporter) genes, shaABCDEFG.  相似文献   

8.
A Na+/H+ antiporter coded by the nhaA (ant) gene of Escherichia coli has been overproduced and purified. The amino-terminal sequence of the protein has been determined and shown to correlate with initiation at a GUG codon, 75 bases upstream from the previously suggested AUG initiation codon. The purified protein, when reconstituted into proteoliposomes, has Na+/H+ antiport activity. It can mediate sodium uptake when a transmembrane pH gradient is applied. Downhill sodium efflux is shown to be highly dependent on pH and is accelerated by a transmembrane pH gradient. An imposed membrane potential negative inside accelerates Na+ efflux at all pH values tested. These findings suggest that the antiporter is electrogenic both at acid and alkaline pH. The activation at alkaline pH values (2000-fold increase) is consistent with the proposed role of the antiporter in regulation of internal pH at the alkaline pH range.  相似文献   

9.
The full-length cDNA (3612 bp) of OsNHA1 was cloned by RT-PCR approach from rice (Oryza sativa L.), which encodes a putative plasma membrane Na+/H+ antiporter. Its deduced protein, OsNHA1, has 11 transmembrane domains and a significant similarity to a plasma membrane Na+/H+ antiporter AtNHA1 from Arabidopsis thaliana. Phylogenetic analysis showed that the OsNHA1 clusters with the plasma membrane Na+/H+ antiporters from various organisms. The semi-quantitative RT-PCR assay revealed that the expression of OsNHA1 was up-regulated in both shoots and roots of rice seedlings under salt stress, whereas it was not induced in the rice seedlings treated by drought stress.  相似文献   

10.
We have deleted the chromosomal ant gene from Escherichia coli by substitution with the kan gene, which encodes kanamycin resistance. The delta ant strains obtained cannot adapt to high sodium concentrations (700 mM, pH 6.8), which do not affect the wild type. The Na+ sensitivity of delta ant is pH dependent, increasing at alkaline pH. Thus at pH 8.5, 100 mM NaCl retard growth of delta ant with no effect on the wild type. The delta ant strains also cannot challenge the toxic effects of Li+ ions, a substrate of the Na+/H+ antiporter system. However, growth of these strains is normal on carbon sources which require Na+ ions for transport and growth. Moreover, antiporter activity, as measured in everted membrane vesicles, is not significantly impaired. A detailed analysis of the remaining antiporter activity in a delta ant strain reveals kinetic properties which differ from those displayed by the ant protein: (a) Km for transport of Li+ ions is about 15 times higher and (b) the activity is practically independent of intracellular pH. Our results demonstrate the presence of an alternative Na+/H+ antiporter(s) in E. coli, additional to ant system.  相似文献   

11.
Abstract An alkaliphilic cyanobacterium characterized as a Synechocystis species was purified from a soil sample taken from a village in Java, Indonesia, by its preferential growth at elevated pH; it grew optimally at pH 9.5. Phosphorus nuclear magnetic resonance studies showed that the organism can maintain a ΔpH of over 2 pH units at an external pH of 10. It was observed that the viability of the organism in the dark was dependent on sodium ions. Evidence from experiments in which the extrusion of Na+ was measured from cells subjected to an alkali shock suggests that the organism possesses a Na+ / H+ electrogenic antiporter which is used for the maintenance of pH homeostasis.  相似文献   

12.
The Na+/H+ antiporter is a ubiquitous membrane-bound enzyme involved in pH regulation of vertebrate cells. We cloned the human gene capable of complementing antiporter-deficient mouse fibroblasts and isolated an exon-containing genomic DNA fragment. Using this genomic probe, we mapped the putative structural gene of the amiloride-sensitive Na+/H+ antiporter to the human chromosome region 1p35----p36.1 by in situ hybridization.  相似文献   

13.
This study characterized the activation of the regulatory activity of the Na+/H+ antiporter during fertilization of hamster embryos. Hamster oocytes appeared to lack any mechanism for the regulation of intracellular pH in the acid range. Similarly, no Na+/H+ antiporter activity could be detected in embryos that were collected from the reproductive tract between 1 and 5 h post-egg activation (PEA). Activity of the Na+/H+ antiporter was first detected in embryos collected at 5.5 h PEA and gradually increased to reach maximal activity in embryos collected at 7 h PEA. Parthenogenetically activated one-cell and two-cell embryos demonstrate Na+/H+ antiporter activity, indicating that antiporter activity is maternally derived and initiated by activation of the egg. The inability of cycloheximide, colchicine, or cytochalasin D to affect initiation of antiporter activity indicates that antiporter appearance is not dependent on the synthesis of new protein or recruitment of existing protein to the cell membrane. In contrast, incubation of one-cell embryos with sphingosine did inhibit the appearance of Na+/H+ antiporter activity, showing that inhibition of normal protein kinase C activity is detrimental to antiporter function. Furthermore, incubation of oocytes with a phorbol ester which stimulates protein kinase C activity induced Na+/H+ antiporter activity in oocytes in which the activity was previously absent. Incubation with an intracellular calcium chelator also reduced the appearance of antiporter activity. Taken together, these data indicate that the appearance of Na+/H+ antiporter activity following egg activation may be due, at least in part, to regulation by protein kinase C and intracellular calcium levels.  相似文献   

14.
Vibrio cholerae is the infectious agent of the deadly diarrheal disease, cholera. Na+ ion homeostasis is believed to play a key role in both physiology and pathogenicity of this bacterium. However, molecular mechanisms of sodium exchange in V. cholerae are still poorly understood. In the present work a gene encoding an unusual Na+/H+ antiporter, nhaD, was identified in the V. cholerae genome. nhaD was cloned from Vibrio cholerae and expressed in Escherichia coli. The antiporter functioned in an E. coli nhaAnhaB mutant strain to confer resistance to LiCl and NaCl. When assayed in inside-out subbacterial vesicles, V. cholerae NhaD demonstrated high affinity for Na+ ions (1.1 mM Na+ was required for the half-maximal response at the pH-optimum). The most striking feature of Vc-NhaD is a unique pH-profile of its activity with a sharp maximum at pH 8.0, different from that of any bacterial sodium-proton antiporter described so far. The difference is rationalized as being the result of a His to Arg substitution in a putative pH sensing residue.  相似文献   

15.
The conditions for optimal solubilization and reconstitution of bovine brain synaptic plasma membrane Na+/Ca2+ exchange activity were examined and a series of chromatographic procedures were used for the isolation of a protein involved in this transport activity. The zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in the presence of 20% (vol/vol) glycerol led to optimal solubilization, and soybean phospholipids in low-pH medium were found to produce optimal reconstitution of activity after dialysis to remove the detergent. Sequential chromatography steps involving the use of gel filtration on Sephacryl S-400 HR, ion exchange on diethylaminoethyl-Sephacel, and metal chelate chromatography on tris-(carboxymethyl)ethylenediamine loaded with LaCl3 led to the isolation of a fraction highly enriched in both Na+/Ca2+ exchange activity and two protein bands identified by denaturing electrophoresis. The estimated molecular masses of the two proteins were 50 and 36 kDa. Development of polyclonal antibodies to the 36-kDa protein permitted immunoextraction of greater than 95% of the antiporter activity from solubilized synaptic plasma membranes. These antibodies cross-reacted with the electroeluted 50-kDa protein on enzyme-linked immunosorbent assays, suggesting a close relationship between the two proteins. These results indicate that the 36-kDa protein is at least a component of the brain membrane Na+/Ca2+ antiporter.  相似文献   

16.
17.
18.
An alkali-sensitive mutant, 38154, of the alkalophilic Bacillus sp. strain C-125 could not grow at an alkaline pH. The nucleotide sequence of a 3.7 kb parental DNA fragment that recovers the growth of 38154 at alkaline pH has four open reading frames (ORF1–4). By sub-cloning the fragment, we demonstrated that a 0.25 kb DNA region is responsible for the recovery. Direct sequencing of the mutant's corresponding region revealed a G to A substitution. The mutation resulted in an amino acid substitution from Gly-393 to Arg of the putative 0RF1 product, which was deduced to be an 804-amino-acid polypeptide with a molecular weight of 89 070. The N-terminal part of the putative ORF1 product showed amino acid similarity to those of the chain-5 products of eukaryotic NADH quinine oxidoreductases. Membrane vesicles prepared from 38154 did not show membrane potential (δψ)-driven Na+/H+ antiporter activity. Antiporter activity was resumed by introducing a parental DNA fragment which recovered the mutant's alkalophily. These results indicate that the mutation in 38154 affects, either directly or indirectly, the electrogenic Na+/H+ antiporter activity. This is the first report which shows that a gene responsible for the Na+/H+ anti-porter system is important in the alkalophily of alkalo-philic microorganisms.  相似文献   

19.
The binding of [3H]nimodipine to purified synaptic plasma membranes (SPM) isolated from sheep brain cortex was characterized, and the effects of nimodipine, nifedipine, and (+)-verapamil on the [3H]nimodipine binding were compared to the effects on 45Ca2+ translocation under conditions that separate 45Ca2+ fluxes through Ca2+ channels from 45Ca2+ uptake via Na+/Ca2+ exchange. [3H]Nimodipine labels a single class of sites in SPM, with a KD of 0.64 +/- 0.1 nM, a Bmax of 161 +/- 27 fmol X mg-1 protein, and a Hill slope of 1.07, at 25 degrees C. Competition of [3H]nimodipine binding to purified SPM with unlabelled Ca2+ channel blockers shows that: nifedipine and nimodipine are potent competitors, with IC50 values of 4.7 nM and 5.9 nM, respectively; verapamil and (-)-D 600 are partial competitors, with biphasic competition behavior. Thus, (+)-verapamil shows an IC50 of 708 nM for the higher affinity component and the maximal inhibition is 50% of the specific binding, whereas for (-)-verapamil the IC50 is 120 nM, and the maximal inhibition is 30%; (-)-D 600 is even less potent than verapamil in inhibiting [3H]nimodipine binding (IC50 = 430 nM). However, (+)-verapamil, nifedipine, and nimodipine are less potent in inhibiting depolarization-induced 45Ca2+ influx into synaptosomes in the absence of Na+/Ca2+ exchange than in competing for [3H]nimodipine binding. Thus, (+)-verapamil inhibits Ca2+ influx by 50% at about 500 microM, whereas it inhibits 50% of the binding at concentrations 200-fold lower, and the discrepancy is even larger for the dihydropyridines. The Na+/Ca2+ exchange and the ATP-dependent Ca2+ uptake by SPM vesicles are also inhibited by the Ca2+ channel blockers verapamil, nifedipine, and d-cis-diltiazem, with similar IC50 values and in the same concentration range (10(-5)-10(-3) M) at which they inhibit Ca2+ influx through Ca2+ channels. We conclude that high-affinity binding of the Ca2+ blockers by SPM is not correlated with inhibition of the Ca2+ fluxes through channels in synaptosomes under conditions of minimal Na+/Ca2+ exchange. Furthermore, the relatively high concentrations of blockers required to block the channels also inhibit Ca2+ translocation through the Ca2+-ATPase and the Na+/Ca2+ exchanger. In this study, clear differentiation is made of the effects of the Ca2+ channel blockers on these three mechanisms of moving Ca2+ across the synaptosomal membrane, and particular care is taken to separate the contribution of the Na+/Ca2+ exchange from that of the Ca2+ channels under conditions of K+ depolarization.  相似文献   

20.
The effects of short- and long-chain fatty acids on the cerebromicrovascular (Na+ + K+)-ATPase were investigated using specific [3H]ouabain binding to the enzyme. Specific binding increased linearly with total microvessel protein (37-110 micrograms) and was time-dependent with maximum binding obtained by 10 min. Arachidonic acid, but not palmitic acid, stimulated [3H]ouabain binding in a dose-dependent manner, with a 105% increase over basal levels at 100 microM arachidonic acid. Preincubation of the microvessels with arachidonic acid did not alter the stimulation observed. 4-Pentenoic acid stimulated [3H]ouabain binding only at high concentrations (10 mM). Scatchard analysis of [3H]ouabain binding to untreated microvessels yielded a single class of "high-affinity" binding sites with an apparent binding affinity (KD) of 64.7 +/- 2.0 nM and a binding capacity (Bmax) of 10.1 +/- 1.5 pmol/mg protein. In the presence of 100 microM arachidonic acid, a monophasic Scatchard plot also was obtained, but the KD significantly decreased to 51.9 +/- 2.7 nM (p less than 0.01), whereas the Bmax remained virtually unchanged (12.5 +/- 1.2 pmol/mg protein). The stimulation of [3H]ouabain binding in the presence of arachidonic acid was potentiated by 4-pentenoic acid, but not by indomethacin or eicosatetraynoic acid. These data suggest that long-chain polyunsaturated fatty acids may be involved in the regulation of blood-brain barrier (Na+ + K+)-ATPase and may play a role in the cerebral dysfunction associated with diseases in which plasma levels of nonesterified fatty acids are elevated.  相似文献   

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