Na+/H+ exchange activity in the alkaliphile halotolerant cyanobacterium Aphanothece halophytica (original) (raw)
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In Vivo pH Regulation by a Na+/H+ Antiporter in the Halotolerant Alga Dunaliella salina
PLANT PHYSIOLOGY, 1991
Na+/H+ exchange activity in whole cells of the halotolerant alga DunaIfeila saUna can be elicited by intracellular acidification due to addition of weak acids at appropriate external pH. The changes in both intracellular pH and Nat were followed. Following a mild intracellular acidification, intracellular Nat content increased dramatically and then decreased. We interpret the phase of Nat LiCI Treatment Cells were centrifuged, resuspended in growth medium in which 1 M LiCl replaced the NaCl, and incubated for 2 h in the light (21). They were then centrifuged, resuspended in the reaction medium, and treated with a weak acid as described above. The treatment with Li did not affect the ability of the cells to grow following resuspension in the regular growth medium. Vanadate Treatment Cells were treated with vanadate as described previously (17). The cells were transferred to phosphate-free growth medium and incubated under growth conditions for 24 h.
Intracellular PH Regulation by a Na+/H+ Exchanger in Cultured Bovine Trabecullar Cells
Acta Ophthalmologica, 1992
ACTA 0 P H T H A L M 0 LOG I CA 70 (1992) 772-779 lntracellular pH regulation by a Na+/H+ exchanger in cultured bovine trabecular cells Abstract. Intracellular pH (pH,) of cultured bovine trabecular cells was measured using video-imaging techniques with a pH-sensitive intracellular fluorescent dye, BCECF. In bicarbonate-rich Ringer at pH 7.4, pHi was 7.29 ?r. 0.03 (k SEM, n = 12 monolayers, 120 cells sampled). Exposure to 20 mM NH,Cl immediately alkalinized pHi: replacement with a Na+-rich solution acidxed pHi before recovery to resting levels. When NH,Cl was replaced by a low Na+ solution, acidification was sustained but pHi recovery occurred after Na+-rich solution. A pHi of 7.11 k 0.02 (n = 2 monolayers, 20 cells) occurred in pH 6.8 and pHi was 7.72 f0.03 (n = 2 monolayers, 20 cells) in pH 8.0. Amiloride (1 mM) acidified pHi but DIDS (1 mM) treatment, HCOS-free condition, 1 mM ouabain, 50 mM K+, and 2 mM BaCl, failed to change pHi. Hydrogen peroxide (1 mM) acidified pHi but no change occurred with 50 wM. Trabecular cells possess an Na+/H+ exchanger similar to that in other cell types.
Characterization of basolateral Na/H exchange (Na/H-1) in MDCK cells
Pfl�gers Archiv European Journal of Physiology, 1992
MDCK cells were grown to confluent monolayers on permeant filter supports; pH was analysed by using the pH-sensitive fluorescent probe 2'7'-biscarboxyethyl-5,6-carboxyfluorescein and a routine spectrofluorometer equipped with a perfusion cuvette [Krayer-Pawlowska et al. (1990) J Membr Biol 120:173-183]. Superfusion of the basolateral (but not apical) cell surface with Na+-containing solutions led to immediate recovery of pH i from an acid load (NH 4 prepulse). This pH i recovery was reversibly inhibited by ethylisopropylamiloride indicating Na/H exchange activity. Na/H exchange activity showed an apparent Km for Na + of about 25 mM Na + and an apparent Ki for inhibition by dimethylamiloride of around 0.2 gM; inhibition by dimethylamiloride was competitive with Na + interaction. Lowering pH i prior to analysis of Na/H exchange leads to sharp activation of Na/H exchange; the apparent Vmax for Na/H exchange is increased more than tenfold by lowering the pH i from 7.0 to 6.7 without an effect on apparent K m values for Na+interaction. It is concluded that MDCK cells (strain I) grown on a permeant support contain only basolateral Na/H exchange activity, most likely Na/H-I [for nomenclature see Kidney Int 40:$84-$89].
FEMS Microbiology Letters, 2007
Aphanothece cells could take up Na 1 and this uptake was strongly inhibited by the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells preloaded with Na 1 exhibited Na 1 extrusion ability upon energizing with glucose. Na 1 was also taken up by the plasma membranes supplied with ATP and the uptake was abolished by gramicidin D, monensin or Na 1 -ionophore. Orthovanadate and CCCP strongly inhibited Na 1 uptake, whereas N, N 0 -dicyclohexylcarbodiimide (DCCD) slightly inhibited the uptake. Plasma membranes could hydrolyse ATP in the presence of Na 1 but not with K 1 , Ca 21 and Li 1 . The K m values for ATP and Na 1 were 1.66 AE 0.12 and 25.0 AE 1.8 mM, respectively, whereas the V max value was 0.66 AE 0.05 mmol min À1 mg À1 . Mg 21 was required for ATPase activity whose optimal pH was 7.5. The ATPase was insensitive to N-ethylmaleimide, nitrate, thiocyanate, azide and ouabain, but was substantially inhibited by orthovanadate and DCCD. Amiloride, a Na 1 /H 1 antiporter inhibitor, and CCCP showed little or no effect. Gramicidin D and monensin stimulated ATPase activity. All these results suggest the existence of a P-type Na 1 -stimulated ATPase in Aphanothece halophytica. Plasma membranes from cells grown under salt stress condition showed higher ATPase activity than those from cells grown under nonstress condition.
Applied and Environmental Microbiology, 2005
Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium which can grow at NaCl concentrations up to 3.0 M and at pH values up to 11. The genome sequence revealed that the cyanobacterium Synechocystis sp. strain PCC 6803 contains five putative Na ؉ /H ؉ antiporters, two of which are homologous to NhaP of Pseudomonas aeruginosa and three of which are homologous to NapA of Enterococcus hirae. The physiological and functional properties of NapA-type antiporters are largely unknown. One of NapA-type antiporters in Synechocystis sp. strain PCC 6803 has been proposed to be essential for the survival of this organism. In this study, we examined the isolation and characterization of the homologous gene in Aphanothece halophytica. Two genes encoding polypeptides of the same size, designated Ap-napA1-1 and Ap-napA1-2, were isolated. Ap-NapA1-1 exhibited a higher level of homology to the Synechocystis ortholog (Syn-NapA1) than Ap-NapA1-2 exhibited. Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 complemented the salt-sensitive phenotypes of an Escherichia coli mutant and exhibited strongly pH-dependent Na ؉ /H ؉ and Li ؉ /H ؉ exchange activities (the highest activities were at alkaline pH), although the activities of Ap-NapA1-2 were significantly lower than the activities of the other polypeptides. Only one these polypeptides, Ap-NapA1-2, complemented a K ؉ uptake-deficient E. coli mutant and exhibited K ؉ uptake activity. Mutagenesis experiments suggested the importance of Glu129, Asp225, and Asp226 in the putative transmembrane segment and Glu142 in the loop region for the activity. Overexpression of Ap-NapA1-1 in the freshwater cyanobacterium Synechococcus sp. strain PCC 7942 enhanced the salt tolerance of cells, especially at alkaline pH. These findings indicate that A. halophytica has two NapA1-type antiporters which exhibit different ion specificities and play an important role in salt tolerance at alkaline pH.
Intracellular pH regulation by a Na+/H+ exchanger in cultured bovine trabecular cells
Acta Ophthalmologica, 2009
ACTA 0 P H T H A L M 0 LOG I CA 70 (1992) 772-779 lntracellular pH regulation by a Na+/H+ exchanger in cultured bovine trabecular cells Abstract. Intracellular pH (pH,) of cultured bovine trabecular cells was measured using video-imaging techniques with a pH-sensitive intracellular fluorescent dye, BCECF. In bicarbonate-rich Ringer at pH 7.4, pHi was 7.29 ?r. 0.03 (k SEM, n = 12 monolayers, 120 cells sampled). Exposure to 20 mM NH,Cl immediately alkalinized pHi: replacement with a Na+-rich solution acidxed pHi before recovery to resting levels. When NH,Cl was replaced by a low Na+ solution, acidification was sustained but pHi recovery occurred after Na+-rich solution. A pHi of 7.11 k 0.02 (n = 2 monolayers, 20 cells) occurred in pH 6.8 and pHi was 7.72 f0.03 (n = 2 monolayers, 20 cells) in pH 8.0. Amiloride (1 mM) acidified pHi but DIDS (1 mM) treatment, HCOS-free condition, 1 mM ouabain, 50 mM K+, and 2 mM BaCl, failed to change pHi. Hydrogen peroxide (1 mM) acidified pHi but no change occurred with 50 wM. Trabecular cells possess an Na+/H+ exchanger similar to that in other cell types.
The Na+-dependence of alkaliphily in Bacillus
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2001
A Na cycle plays a central role in the remarkable capacity of aerobic, extremely alkaliphilic Bacillus species for pH homeostasis. The capacity for pH homeostasis, in turn, appears to set the upper pH limit for growth. One limb of the alkaliphile Na cycle consists of Na /H antiporters that achieve net H accumulation that is coupled to Na efflux. The major antiporter on which pH homeostasis depends is thought to be the Mrp(Sha)-encoded antiporter, first identified from a partial clone in Bacillus halodurans C-125. Mrp(Sha) may function as a complex. While this antiporter is capable of secondary antiport energized by an imposed or respiration-generated protonmotive force, the possibility of a primary mode has not been excluded. In Bacillus pseudofirmus OF4, at least two additional antiporters, including NhaC, have supporting roles in pH homeostasis. Some of these additional antiporters may be especially important for antiport at low [Na ] or at near-neutral pH. The second limb of the Na cycle facilitates Na re-entry via Na /solute symporters and, perhaps, the ion channel associated with the Na -dependent flagellar motor. The process of pH homeostasis is also enhanced, perhaps especially during transitions to high pH, by different arrays of secondary cell wall polymers in the two alkaliphilic Bacillus species studied most intensively. The mechanisms whereby alkaliphiles handle the challenge of Na stress at very elevated [Na ] are just beginning to be identified, and a hypothesis has been advanced to explain the finding that B. pseudofirmus OF4 requires a higher [Na ] for growth at near-neutral pH than at very alkaline pH values. ß 2001 Elsevier Science B.V. All rights reserved.
Microbiology (Reading, England), 2001
Two genes in the Escherichia coli genome, b4065 (yjcE) and b1191 (ycgO), are similar to genes encoding eukaryotic Na+/H+ exchangers. Mutants were constructed in which yjcE (GRN11), ycgO (GRF55) or both (GRD22) were inactivated. There was no change in respiration-driven Na+ efflux in any of the mutants when grown in media containing 50-500 mM Na+. The only striking finding was that growth of GRF55 was impaired at low osmolarity. In complex low-salt medium, GRF55 grew at a wild-type rate for three to four generations but then stopped; the growth was partially recovered after a pause, the length of which was dependent on salt concentration. Measurement of cytoplasmic alkali cations showed that an abrupt loss of about one-half of the intracellular K+ preceded the pause. When grown in low-salt medium with only 20 mM added Na+, GRF55 also lost the ability to maintain a sodium concentration gradient. However, this phenomenon appears to be a secondary effect of the ycgO deletion. The double...
… of bioenergetics and …, 2003
is a model organism for investigation of adaptation of photosynthetic organisms to extreme environmental conditions: the cell functions in this cyanobacterium are optimized to high pH and high concentration (150-250 mM) of Na + . However, the mechanism of the possible fine-tuning of the photosynthetic functions to these extreme conditions and/or the regulation of the cellular environment to optimize the photosynthetic functions is poorly understood. In this work we investigated the effect of Na-ions on different photosynthetic activities: linear electron transport reactions (measured by means of polarography and spectrophotometry), the activity of photosystem II (PS II) (thermoluminescence and chlorophyll a fluorescence induction), and redox turnover of the cytochrome b 6 f complex (flash photolysis); and measured the changes of the intracellular pH (9-aminoacridine fluorescence). It was found that sodium deprivation of cells in the dark at pH 10 inhibited, within 40 min, all measured photosynthetic reactions, and led to an alkalinization of the intracellular pH, which rose from the physiological value of about 8.3-9.6. These were partially and totally restored by readdition of Na-ions at 2.5-25 mM and about 200 mM, respectively. The intracellular pH and the photosynthetic functions were also sensitive to monensin, an exogenous Na + /H + exchanger, which collapses both proton and sodium gradients across the cytoplasmic membrane. These observations explain the strict Na + -dependency of the photosynthetic electron transport at high extracellular pH, provide experimental evidence on the alkalization of the intracellular environment, and support the hypothesized role of an Na + /H + antiport through the plasma membrane in pH homeostasis . J. Phycol. 32, 608-613). Further, we show that (i) the specific site of inactivation of the photosynthetic electron transport at alkaline pH is to be found at the water splitting enzyme; (ii) in contrast to earlier reports, the inactivation occurs in the dark and, for short periods, without detectable damage in the photosynthetic apparatus; and (iii) in contrast to high pH, Na + dependency in the neutral pH range is shown not to originate from PSII, but from the acceptor side of PSI. These data permit us to conclude that the intracellular environment rather than the machinery of the photosynthetic electron transport is adjusted to the extreme conditions of high pH and high Na + concentration.