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1.
Na+ strongly promoted HCO3− transport in Anabaena variabilis. The effect was highly specific to this cation. Kinetic analysis indicated a progressive decrease in the Km (HCO3−) of the transport system with increasing Na+ concentration. Vmax was also affected. We raise the possibility that the transport is a Na+-HCO3− symport; alternatively, that a Na+-H+ antiport (or Na+-OH+ symport) system mediates the efflux of the OH− ions derived from the entering HCO3− ions, and that this antiport can rate-limit HCO3− influx. 相似文献
2.
Sodium-dependence of glycylglycine (Gly-Gly) influx and stimulation of Na+ transport by Gly-Gly were studied in everted sacs, sheet preparations and brush-border membrane vesicles isolated from guinea-pig ileum. Gly-Gly influx was found to be independent of the presence of Na+, while Na+ transport was stimulated by Gly-Gly as evidenced by increases in transmural potential difference (PDt), short-circuit current (Isc) and Na+ influx. The change in PDt (ΔPDt) induced by Gly-Gly was a saturable function of Gly-Gly concentration, showing a Michaelis-Menten type relationship. The half-saturation concentration for Gly-Gly estimated from the electrical data was nearly identical with that estimated from influx data. At a constant Gly-Gly concentration the relationship between Isc and Na+ concentration was sigmoid, and the Hill coefficient was 1.5. Kinetic analysis according to Garay Garrahan indicates that each Gly-Gly carrier has two equivalent non-interacting binding sites for Na+, and that translocation of Na+ occurs when the two Na+ sites on the carrier loaded with Gly-Gly are occupied by Na+. However, our results indicate that the resultant Na+ flow is not capable of stimulating Gly-Gly translocation. 相似文献
3.
Inga H?nelt Sonja Jensen Dorith Wunnicke Dirk Jan Slotboom 《The Journal of biological chemistry》2015,290(26):15962-15972
GltPh from Pyrococcus horikoshii is a homotrimeric Na+-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters that take up the neurotransmitter glutamate. Each protomer in GltPh consists of a trimerization domain involved in subunit interactions and a transport domain containing the substrate binding site. Here, we have studied the dynamics of Na+ and aspartate binding to GltPh. Tryptophan fluorescence measurements on the fully active single tryptophan mutant F273W revealed that Na+ binds with low affinity to the apoprotein (Kd 120 mm), with a particularly low kon value (5.1 m−1s−1). At least two sodium ions bind before aspartate. The binding of Na+ requires a very high activation energy (Ea 106.8 kJ mol−1) and consequently has a large Q10 value of 4.5, indicative of substantial conformational changes before or after the initial binding event. The apparent affinity for aspartate binding depended on the Na+ concentration present. Binding of aspartate was not observed in the absence of Na+, whereas in the presence of high Na+ concentrations (above the Kd for Na+) the dissociation constants for aspartate were in the nanomolar range, and the aspartate binding was fast (kon of 1.4 × 105
m−1s−1), with low Ea and Q10 values (42.6 kJ mol−1 and 1.8, respectively). We conclude that Na+ binding is most likely the rate-limiting step for substrate binding. 相似文献
4.
T. I. Ivanova A. O. Sherstobitov G. P. Gusev 《Journal of Evolutionary Biochemistry and Physiology》2007,43(6):557-563
To activate Na+/H+ exchange, intracellular pH (pHi) of erythrocytes of the river lamprey Lampetra fluviatilis were changed from 6 and 8 using nigericin. The Na+/H+ exchanger activity was estimated from the values of amiloride-sensitive components of Na+ (22Na) inflow or of H+ outflow from erythrocytes. Kinetic parameters of the carrier functioning were determined by using Hill equation. Dependence of Na+ and H+ transport on pHi value is described by hyperbolic function with the Hill coefficient value (n) close to 1. Maximal rate of ion transport was within the limits of 9–10 mmol/l cells/min, and the H+ concentration producing the exchanger 50% activation amounted to 0.6–1.0 μM. Stimulation of H+ outcome from acidified erythrocytes (pHi 5.9) with increase of H+ concentration in the incubation medium is described by Hill equation with n value of 1.6. Concentration Na+ for the semimaximal stimulation of H+ outcome amounted to 10 mM. The obtained results indicate the presence in lamprey erythrocytes of only binding site for H+ from the cytoplasm side and the presence of positive cooperativity in Na+-binding from the extracellular side of the Na+/H+ exchanger. Na+ efflux from cells in the Na+-free medium did not change at a 10-fold increase of H+ concentration in the incubation medium. The presented data indicate differences of kinetic properties of the lamprey erythrocyte Na+/H+ exchanger and of this carrier isoforms in mammalian cells. In intact erythrocytes the dependence of the amiloride-sensitive Na+ inflow on its concentration in the medium is described by Hill equitation with n 1.6. The Na+ concentration producing the 50% transport activation amounted to 39 mM and was essentially higher as compared with that in acidified erythrocytes. These data confirm conception of the presence of two amiloride-sensitive pathways of Na+ transport in lamprey erythrocytes. 相似文献
5.
Biological membranes composed of lipids and proteins are in contact with electrolytes like aqueous NaCl solutions. Based on molecular dynamics studies it is widely believed that Na+ ions specifically bind to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes, whereas Cl− ions stay in solution. Here, we present a careful comparison of recent data from electrophoresis and isothermal titration calorimetry experiments as well as molecular dynamics simulations suggesting that in fact both ions show very similar affinities. The corresponding binding constants are 0.44(±0.05) M−1 for Na+ and 0.40(±0.04) M−1 for Cl− ions. This is highlighted by our observation that a widely used simulation setup showing asymmetric affinities of Na+ and Cl− for POPC bilayers overestimates the effect of NaCl on the electrophoretic mobility of a POPC membrane by an order of magnitude. Implications for previous simulation results on the effect of NaCl on polarization of interfacial water, transmembrane potentials, and mechanisms for ion transport through bilayers are discussed. Our findings suggest that a range of published simulations results on the interaction of NaCl with phosphocholine bilayers have to be reconsidered and revised and that force field refinements are necessary for reliable simulation studies of membranes at physiological conditions on a molecular level. 相似文献
6.
Alvaro Garcia Natasha A.S. Fry Keyvan Karimi Chia-chi Liu Hans-Jürgen Apell Helge H. Rasmussen Ronald J. Clarke 《Biophysical journal》2013
Whole-cell patch-clamp measurements of the current, Ip, produced by the Na+,K+-ATPase across the plasma membrane of rabbit cardiac myocytes show an increase in Ip over the extracellular Na+ concentration range 0–50 mM. This is not predicted by the classical Albers-Post scheme of the Na+,K+-ATPase mechanism, where extracellular Na+ should act as a competitive inhibitor of extracellular K+ binding, which is necessary for the stimulation of enzyme dephosphorylation and the pumping of K+ ions into the cytoplasm. The increase in Ip is consistent with Na+ binding to an extracellular allosteric site, independent of the ion transport sites, and an increase in turnover via an acceleration of the rate-determining release of K+ to the cytoplasm, E2(K+)2 → E1 + 2K+. At normal physiological concentrations of extracellular Na+ of 140 mM, it is to be expected that binding of Na+ to the allosteric site would be nearly saturated. Its purpose would seem to be simply to optimize the enzyme’s ion pumping rate under its normal physiological conditions. Based on published crystal structures, a possible location of the allosteric site is within a cleft between the α- and β-subunits of the enzyme. 相似文献
7.
H. Koepsell 《The Journal of membrane biology》1978,44(1):85-102
Summary Antibodies which were raised against highly purified membrane-bound (Na+–K+)-ATPase from the outer medulla of rat kidneys inhibit the (Na+–K+)-ATPase activity up to 95%. The antibody inhibition is reversible. The time course of enzyme inhibition and reactivation is biphasic in semilogarithmic plots.In the purified membrane-bound (Na+–K+)-ATPase negative cooperativity was observed (a) for the ATP dependence of the (Na+–K+)-ATPase activity (n=0.86), (b) for the ATP binding to the enzyme (n=0.58), and (c) for the ouabain inhibition of the (Na+–K+)-ATPase activity (n=0.77). By measuring the Na+ dependence of the (Na+–K+-ATPase reaction, a positive homotropic cooperativity (n=1.67) was found.As reactivation of the antibody-inhibited enzyme proceeds very slowly (t
0.5=5.2hr), it was possible to measure characteristics of the antibody-(Na+–K+)-ATPase complex: The antibodies exerted similar effects on the ATP dependence of the (Na+–K+)-ATPase reaction and on the ATP binding of the enzyme.V
max of the (Na+–K+)-ATPase reaction and the number of ATP binding sites were reduced whileK
0.5 ATP for the (Na+–K+)-ATPase activity and for the ATP binding were increased by the antibodies. The Hill coefficients for the ATP binding and for the ATP dependence of the enzyme activity were not significantly altered by the antibodies. The antibodies increased theK
0.5 value for the Na+ stimulation of the (Na+–K+)-ATPase activity, but they did not alter the homotropic interactions between the Na+-binding sites. The negative cooperativity which was observed for the ouabain inhibition of the (Na+–K+)-ATPase activity was abolished by the antibodies.The data are tentatively explained by the following model: The antibodies bind to the (Na+–K+)-ATPase from the inner membrane side, reduce the ATP binding symmetrically at the ATP binding sites and reduce thereby also the (Na+–K+)-ATPase activity of the enzyme. The antibodies may inhibit the ATP binding by a direct interaction or by means of a conformational change at the ATP binding sites. This may possibly also lead to the alteration of the Na+ dependence of the (Na+–K+)-ATPase activity and to the observed alteration of the dose response to the ouabain inhibition. 相似文献
8.
Olaf Sparre Andersen 《The Journal of general physiology》1998,111(1):1-18
The voltage dependence of the rat renal type II Na+/Pi cotransporter (NaPi-2) was investigated by expressing NaPi-2 in Xenopus laevis oocytes and applying the two-electrode voltage clamp. In the steady state, superfusion with inorganic phosphate (Pi) induced inward currents (Ip) in the presence of 96 mM Na+ over the potential range −140 ≤ V ≤ +40 mV. With Pi as the variable substrate, the apparent affinity constant (K
m
Pi) was strongly dependent on Na+, increasing sixfold for a twofold reduction in external Na+. K
m
Pi increased with depolarizing voltage and was more sensitive to voltage at reduced Na+. The Hill coefficient was close to unity and the predicted maximum Ip (Ipmax) was 40% smaller at 50 mM Na+. With Na+ as the variable substrate, K
m
Na was weakly dependent on both Pi and voltage, the Hill coefficient was close to 3 and Ipmax was independent of Pi at −50 mV. The competitive inhibitor phosphonoformic acid suppressed the steady state holding current in a Na+-dependent manner, indicating the existence of uncoupled Na+ slippage. Voltage steps induced pre–steady state relaxations typical for Na+-coupled cotransporters. NaPi-2-dependent relaxations were quantitated by a single, voltage-dependent exponential. At 96 mM Na+, a Boltzmann function was fit to the steady state charge distribution (Q-V) to give a midpoint voltage (V0.5) in the range −20 to −50 mV and an apparent valency of ∼0.5 e−. V0.5 became more negative as Na+ was reduced. Pi suppressed relaxations in a dose-dependent manner, but had little effect on their voltage dependence. Reducing external pH shifted V0.5 to depolarizing potentials and suppressed relaxations in the absence of Na+, suggesting that protons interact with the unloaded carrier. These findings were incorporated into an ordered kinetic model whereby Na+ is the first and last substrate to bind, and the observed voltage dependence arises from the unloaded carrier and first Na+ binding step. 相似文献
9.
Susan Meier Neslihan N. Tavraz Katharina L. Dürr Thomas Friedrich 《The Journal of general physiology》2010,135(2):115-134
The Na+/K+-ATPase mediates electrogenic transport by exporting three Na+ ions in exchange for two K+ ions across the cell membrane per adenosine triphosphate molecule. The location of two Rb+ ions in the crystal structures of the Na+/K+-ATPase has defined two “common” cation binding sites, I and II, which accommodate Na+ or K+ ions during transport. The configuration of site III is still unknown, but the crystal structure has suggested a critical role of the carboxy-terminal KETYY motif for the formation of this “unique” Na+ binding site. Our two-electrode voltage clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human Na+/K+-ATPase α2 subunit decreases the affinity for extracellular and intracellular Na+, in agreement with previous biochemical studies. Apparently, the ΔYY deletion changes Na+ affinity at site III but leaves the common sites unaffected, whereas the more extensive ΔKETYY deletion affects the unique site and the common sites as well. In the absence of extracellular K+, the ΔYY construct mediated ouabain-sensitive, hyperpolarization-activated inward currents, which were Na+ dependent and increased with acidification. Furthermore, the voltage dependence of rate constants from transient currents under Na+/Na+ exchange conditions was reversed, and the amounts of charge transported upon voltage pulses from a certain holding potential to hyperpolarizing potentials and back were unequal. These findings are incompatible with a reversible and exclusively extracellular Na+ release/binding mechanism. In analogy to the mechanism proposed for the H+ leak currents of the wild-type Na+/K+-ATPase, we suggest that the ΔYY deletion lowers the energy barrier for the intracellular Na+ occlusion reaction, thus destabilizing the Na+-occluded state and enabling inward leak currents. The leakage currents are prevented by aromatic amino acids at the carboxy terminus. Thus, the carboxy terminus of the Na+/K+-ATPase α subunit represents a structural and functional relay between Na+ binding site III and the intracellular cation occlusion gate. 相似文献
10.
Ionic Regulation for Cytokinin-dependent Betacyanin Synthesis in Amaranthus Seedlings 总被引:8,自引:7,他引:1
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Elliott DC 《Plant physiology》1979,63(2):264-268
Potassium ions at low concentrations stimulate cytokinin-dependent betacyanin synthesis in Amaranthus tricolor seedlings more than other alkali metal ions when tested as the chloride salts. The sequence of relative stimulation is K+ > Rb+ > (Na+ = Li+). Calcium and Mg2+ ions are inhibitory at concentrations > 1 millimolar when tested as chlorides. Anions also have an effect on the degree of alkali metal stimulation in the order PO43− > NO3− > Cl−. The high activity of phosphate may be partly due to its chelating effect on inhibitory Ca2+ ions, or to effects on K+ uptake. A mixture of Na+ and K+ in the presence of phosphate is more effective than either cation alone. This result may be due either to effects on tyrosine transport or on the potassium uptake system. Phytochrome-dependent betacyanin synthesis shows the same stimulation by Na+ plus K+. The effect of a number of inhibitors of transport systems on betacyanin accumulation is reported. The possible role of the ionic environment of cells in their metabolic regulation is discussed, particularly in relation to cytokinin action. 相似文献
11.
Fusao Kawai Takashi Kurahashi Akimichi Kaneko 《The Journal of general physiology》1997,109(2):265-272
Effects of odorants on voltage-gated ionic channels were investigated in isolated newt olfactory receptor cells by using the whole cell version of the patch–clamp technique. Under voltage clamp, membrane depolarization to voltages between −90 mV and +40 mV from a holding potential (Vh) of −100 mV generated time-
and voltage-dependent current responses; a rapidly (< 15 ms) decaying initial inward current and a late outward
current. When odorants (1 mM amyl acetate, 1 mM acetophenone, and 1 mM limonene) were applied to the recorded cell, the voltage-gated currents were significantly reduced. The dose-suppression relations of amyl acetate
for individual current components (Na+ current: INa, T-type Ca2+ current: ICa,T, L-type Ca2+ current: ICa,L, delayed
rectifier K+ current: IKv and Ca2+-activated K+ current: IK(Ca)) could be fitted by the Hill equation. Half-blocking
concentrations for each current were 0.11 mM (INa), 0.15 mM (ICa,T), 0.14 mM (ICa,L), 1.7 mM (IKv), and 0.17 mM
(IK(Ca)), and Hill coefficient was 1.4 (INa), 1.0 (ICa,T), 1.1 (ICa,L), 1.0 (IKv), and 1.1 (IK(Ca)), suggesting that the inward current is affected more strongly than the outward current. The activation curve of INa was not changed significantly by amyl acetate, while the inactivation curve was shifted to negative voltages; half-activation voltages were
−53 mV at control, −66 mV at 0.01 mM, and −84 mV at 0.1 mM. These phenomena are similar to the suppressive
effects of local anesthetics (lidocaine and benzocaine) on INa in various preparations, suggesting that both types of
suppression are caused by the same mechanism. The nonselective blockage of ionic channels observed here is
consistent with the previous notion that the suppression of the transduction current by odorants is due to the direst blockage of transduction channels. 相似文献
12.
Paloma Olvera-Caltzontzin Guadalupe Delgado Carmen Aceves Brenda Anguiano 《Molecular medicine (Cambridge, Mass.)》2013,19(1):409-416
Iodine supplementation exerts antitumor effects in several types of cancer. Iodide (I−) and iodine (I2) reduce cell proliferation and induce apoptosis in human prostate cancer cells (LNCaP and DU-145). Both chemical species decrease tumor growth in athymic mice xenografted with DU-145 cells. The aim of this study was to analyze the uptake and effects of iodine in a preclinical model of prostate cancer (transgenic adenocarcinoma of the mouse prostate [TRAMP] mice/SV40-TAG antigens), which develops cancer by 12 wks of age. 125I− and 125I2 uptake was analyzed in prostates from wild-type and TRAMP mice of 12 and 24 wks in the presence of perchlorate (inhibitor of the Na+/I− symporter [NIS]). NIS expression was quantified by quantitative polymerase chain reaction (qPCR). Mice (6 wks old) were supplemented with 0.125 mg I− plus 0.062 mg I2/mouse/day for 12 or 24 wks. The weight of the genitourinary tract (GUT), the number of acini with lesions, cell proliferation (levels of proliferating cell nuclear antigen [PCNA] by immunohistochemistry), p53 and p21 expression (by qPCR) and apoptosis (relative amount of nucleosomes by enzyme-linked immunosorbent assay) were evaluated. In both age-groups, normal and tumoral prostates take up both forms of iodine, but only I− uptake was blocked by perchlorate. Iodine supplementation prevented the overexpression of NIS in the TRAMP mice, but had no effect on the GUT weight, cell phenotype, proliferation or apoptosis. In TRAMP mice, iodine increased p53 expression but had no effect on p21 (a p53-dependent gene). Our data corroborate NIS involvement in I− uptake and support the notion that another transporter mediates I2 uptake. Iodine did not prevent cancer progression. This result could be explained by a strong inactivation of the p53 pathway by TAG antigens. 相似文献
13.
The Na+ requirement for photosynthesis and its relationship to dissolved inorganic carbon (DIC) concentration and Li+ concentration was examined in air-grown cells of the cyanobacterium Synechococcus leopoliensis UTEX 625 at pH 8. Analysis of the rate of photosynthesis (O2 evolution) as a function of Na+ concentration, at fixed DIC concentration, revealed two distinct regions to the response curve, for which half-saturation values for Na+ (K½[Na+]) were calculated. The value of both the low and the high K½(Na+) was dependent upon extracellular DIC concentration. The low K½(Na+) decreased from 1000 micromolar at 5 micromolar DIC to 200 micromolar at 140 micromolar DIC whereas over the same DIC concentration range the high K½(Na+) decreased from 10 millimolar to 1 millimolar. The most significant increases in photosynthesis occurred in the 1 to 20 millimolar range. A fraction of total photosynthesis, however, was independent of added Na+ and this fraction increased with increased DIC concentration. A number of factors were identified as contributing to the complexity of interaction between Na+ and DIC concentration in the photosynthesis of Synechococcus. First, as revealed by transport studies and mass spectrometry, both CO2 and HCO3− transport contributed to the intracellular supply of DIC and hence to photosynthesis. Second, both the CO2 and HCO3− transport systems required Na+, directly or indirectly, for full activity. However, micromolar levels of Na+ were required for CO2 transport while millimolar levels were required for HCO3− transport. These levels corresponded to those found for the low and high K½(Na+) for photosynthesis. Third, the contribution of each transport system to intracellular DIC was dependent on extracellular DIC concentration, where the contribution from CO2 transport increased with increased DIC concentration relative to HCO3− transport. This change was reflected in a decrease in the Na+ concentration required for maximum photosynthesis, in accord with the lower Na+-requirement for CO2 transport. Lithium competitively inhibited Na+-stimulated photosynthesis by blocking the cells' ability to form an intracellular DIC pool through Na+-dependent HCO3− transport. Lithium had little effect on CO2 transport and only a small effect on the size of the pool it generated. Thus, CO2 transport did not require a functional HCO3− transport system for full activity. Based on these observations and the differential requirement for Na+ in the CO2 and HCO3− transport system, it was proposed that CO2 and HCO3− were transported across the membrane by different transport systems. 相似文献
14.
Bruce Felts Akula Bala Pramod Walter Sandtner Nathan Burbach Simon Bulling Harald H. Sitte L. Keith Henry 《The Journal of biological chemistry》2014,289(3):1825-1840
Neurotransmitter transporters of the SLC6 family of proteins, including the human serotonin transporter (hSERT), utilize Na+, Cl−, and K+ gradients to induce conformational changes necessary for substrate translocation. Dysregulation of ion movement through monoamine transporters has been shown to impact neuronal firing potentials and could play a role in pathophysiologies, such as depression and anxiety. Despite multiple crystal structures of prokaryotic and eukaryotic SLC transporters indicating the location of both (or one) conserved Na+-binding sites (termed Na1 and Na2), much remains uncertain in regard to the movements and contributions of these cation-binding sites in the transport process. In this study, we utilize the unique properties of a mutation of hSERT at a single, highly conserved asparagine on TM1 (Asn-101) to provide several lines of evidence demonstrating mechanistically distinct roles for Na1 and Na2. Mutations at Asn-101 alter the cation dependence of the transporter, allowing Ca2+ (but not other cations) to functionally replace Na+ for driving transport and promoting 5-hydroxytryptamine (5-HT)-dependent conformational changes. Furthermore, in two-electrode voltage clamp studies in Xenopus oocytes, both Ca2+ and Na+ illicit 5-HT-induced currents in the Asn-101 mutants and reveal that, although Ca2+ promotes substrate-induced current, it does not appear to be the charge carrier during 5-HT transport. These findings, in addition to functional evaluation of Na1 and Na2 site mutants, reveal separate roles for Na1 and Na2 and provide insight into initiation of the translocation process as well as a mechanism whereby the reported SERT stoichiometry can be obtained despite the presence of two putative Na+-binding sites. 相似文献
15.
To investigate Na+ binding to the ion-binding sites presented on the cytoplasmic side of the Na,K-ATPase, equilibrium Na+-titration experiments were performed using two fluorescent dyes, RH421 and FITC, to detect protein-specific actions. Fluorescence
changes upon addition of Na+ in the presence of various Mg2+ concentrations were similar and could be fitted with a Hill function. The half-saturating concentrations and Hill coefficients
determined were almost identical. As RH421 responds to binding of a Na+ ion to the third neutral site whereas FITC monitors conformational changes in the ATP-binding site or its environment, this
result implies that electrogenic binding of the third Na+ ion is the trigger for a structural rearrangement of the ATP-binding moiety. This enables enzyme phosphorylation, which is
accompanied by a fast occlusion of the Na+ ions and followed by the conformational transition E1/E2 of the protein. The coordinated action both at the ion and the nucleotide binding sites allows for the first time a detailed
formulation of the mechanism of enzyme phosphorylation that occurs only when three Na+ ions are bound.
Received: 8 October 1998/Revised: 29 December 1998 相似文献
16.
Na-Independent HCO(3) Transport and Accumulation in the Cyanobacterium Synechococcus UTEX 625
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The active transport and intracellular accumulation of HCO3− by air-grown cells of the cyanobacterium Synechococcus UTEX 625 (PCC 6301) was strongly promoted by 25 millimolar Na+.Na+-dependent HCO3− accumulation also resulted in a characteristic enhancement in the rate of photosynthetic O2 evolution and CO2 fixation. However, when Synechococcus was grown in standing culture, high rates of HCO3− transport and photosynthesis were observed in the absence of added Na+. The internal HCO3− pool reached levels up to 50 millimolar, and an accumulation ratio as high as 970 was observed. Sodium enhanced HCO3− transport and accumulation in standing culture cells by about 25 to 30% compared with the five- to eightfold enhancement observed with air-grown cells. The ability of standing culture cells to utilize HCO3− from the medium in the absence of Na+ was lost within 16 hours after transfer to air-grown culture and was reacquired during subsequent growth in standing culture. Studies using a mass spectrometer indicated that standing culture cells were also capable of active CO2 transport involving a high-affinity transport system which was reversibly inhibited by H2S, as in the case for air-grown cells. The data are interpreted to indicate that Synechococcus possesses a constitutive CO2 transport system, whereas Na+-dependent and Na+-independent HCO3− transport are inducible, depending upon the conditions of growth. Intracellular accumulation of HCO3− was always accompanied by a quenching of chlorophyll a fluorescence which was independent of CO2 fixation. The extent of fluorescence quenching was highly dependent upon the size of the internal pool of HCO3− + CO2. The pattern of fluorescence quenching observed in response to added HCO3− and Na+ in air-grown and standing culture cells was highly characteristic for Na+-dependent and Na+-independent HCO3− accumulation. It was concluded that measurements of fluorescence quenching provide an indirect means for following HCO3− transport and the dynamics of intracellular HCO3− accumulation and dissipation. 相似文献
17.
Mark O. Bevensee Michael Apkon Walter F. Boron 《The Journal of general physiology》1997,110(4):467-483
In the preceding paper (Bevensee, M.O., R.A. Weed, and W.F. Boron. 1997. J. Gen. Physiol. 110: 453–465.), we showed that a Na+-driven influx of HCO3
− causes the increase in intracellular pH (pHi) observed when astrocytes cultured from rat hippocampus are exposed to 5% CO2/17 mM HCO3
−. In the present study, we used the pH-sensitive fluorescent indicator 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and the perforated patch-clamp technique to determine whether this transporter is a Na+-driven Cl-HCO3 exchanger, an electrogenic Na/HCO3 cotransporter, or an electroneutral Na/HCO3 cotransporter. To determine if the transporter is a Na+-driven Cl-HCO3 exchanger, we depleted the cells of intracellular Cl− by incubating them in a Cl−-free solution for an average of ∼11 min. We verified the depletion with the Cl−-sensitive dye N-(6-methoxyquinolyl)acetoethyl ester (MQAE). In Cl−-depleted cells, the pHi still increases after one or more exposures to CO2/HCO3
−. Furthermore, the pHi decrease elicited by external Na+ removal does not require external Cl−. Therefore, the transporter cannot be a Na+-driven Cl-HCO3 exchanger. To determine if the transporter is an electrogenic Na/ HCO3 cotransporter, we measured pHi and plasma membrane voltage (Vm) while removing external Na+, in the presence/absence of CO2/HCO3
− and in the presence/absence of 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS). The CO2/HCO3
− solutions contained 20% CO2 and 68 mM HCO3
−, pH 7.3, to maximize the HCO3
− flux. In pHi experiments, removing external Na+ in the presence of CO2/HCO3
− elicited an equivalent HCO3
− efflux of 281 μM s−1. The HCO3
− influx elicited by returning external Na+ was inhibited 63% by DIDS, so that the predicted DIDS-sensitive Vm change was 3.3 mV. Indeed, we found that removing external Na+ elicited a DIDS-sensitive depolarization that was 2.6 mV larger in the presence than in the absence of CO2/ HCO3
−. Thus, the Na/HCO3 cotransporter is electrogenic. Because a cotransporter with a Na+:HCO3
− stoichiometry of 1:3 or higher would predict a net HCO3
− efflux, rather than the required influx, we conclude that rat hippocampal astrocytes have an electrogenic Na/HCO3 cotransporter with a stoichiometry of 1:2. 相似文献
18.
Cells of Nicotiana tabacum L. var Wisconsin 38 adapted to NaCl (up to 428 millimolar) which have undergone extensive osmotic adjustment accumulated Na+ and Cl− as principal solutes for this adjustment. Although the intracellular concentrations of Na+ and Cl− correlated well with the level of adaptation, these ions apparently did not contribute to the osmotic adjustment which occurred during a culture growth cycle, because the concentrations of Na+ and Cl− did not increase during the period of most active osmotic adjustment. The average intracellular concentrations of soluble sugars and total free amino acids increased as a function of the level of adaptation; however, the levels of these solutes did not approach those observed for Na+ and Cl−. The concentration of proline was positively correlated with cell osmotic potential, accumulating to an average concentration of 129 millimolar in cells adapted to 428 millimolar NaCl and representing about 80% of the total free amino acid pool as compared to an average of 0.29 millimolar and about 4% of the pool in unadapted cells. These results indicate that although Na+ and Cl− are principal components of osmotic adjustment, organic solutes also may make significant contributions. 相似文献
19.
The physiological ligands for Na,K-ATPase (the Na,K-pump) are ions, and electrostatic forces, that could be revealed by their ionic strength dependence, are therefore expected to be important for their reaction with the enzyme. We found that the affinities for ADP3−, eosin2−, p-nitrophenylphosphate, and Vmax for Na,K-ATPase and K+-activated p-nitrophenylphosphatase activity, were all decreased by increasing salt concentration and by specific anions. Equilibrium binding of ADP was measured at 0–0.5 M of NaCl, Na2SO4, and NaNO3 and in 0.1 M Na-acetate, NaSCN, and NaClO4. The apparent affinity for ADP decreased up to 30 times. At equal ionic strength, I, the ranking of the salt effect was NaCl ≈ Na2SO4 ≈ Na-acetate < NaNO3 < NaSCN < NaClO4. We treated the influence of NaCl and Na2SO4 on K
diss for E·ADP as a “pure” ionic strength effect. It is quantitatively simulated by a model where the binding site and ADP are point charges, and where their activity coefficients are related to I by the limiting law of Debye and Hückel. The estimated net charge at the binding site of the enzyme was about +1. Eosin binding followed the same model. The NO3
− effect was compatible with competitive binding of NO3
− and ADP in addition to the general I-effect. K
diss for E·NO3 was ∼32 mM. Analysis of Vmax/K
m for Na,K-ATPase and K+-p-nitrophenylphosphatase activity shows that electrostatic forces are important for the binding of p-nitrophenylphosphate but not for the catalytic effect of ATP on the low affinity site. The net charge at the p-nitrophenylphosphate-binding site was also about +1. The results reported here indicate that the reversible interactions between ions and Na,K-ATPase can be grouped according to either simple Debye-Hückel behavior or to specific anion or cation interactions with the enzyme. 相似文献
20.
A new simple procedure has been developed for the purification of plasma membranes from rabbit kidney microsomes which yields a three- to fourfold increase in the specific activity of Na+-K+-adenosine triphosphatase (ATPase). The procedure differs from previous methods with deoxycholate or other detergents and does not change the molecular activity of the ATPase. The K+-dependent p-nitrophenylphosphatase activity of the native Na+-K+-ATPase is controlled more effectively by Mg2+ in the presence of K+ at concentrations higher than that of Mg2+, and by K+ in the presence of Mg2+ at concentrations higher than that of K+. The enzyme in its Mg2+-regulating state, which shows K+-saturation curves with a Hill coefficient of 1, is less sensitive to ouabain (I0.5 = 90 μM) and corresponds to the enzyme conformation reported previously which is inhibited by the concurrent presence of Na+ and ATP or of Na+ and oligomycin (I0.5 is the midpoint of the saturation curve). The enzyme in its K+-regulating state, which shows K+-saturation curves with a Hill coefficient of 2, is more sensitive to ouabain inhibition (I05 = 8 μM) and corresponds to the enzyme conformation which is stimulated by the concurrent presence of Na+ and ATP or of Na+ and oligomycin. There appear to be two conformations of the enzyme that are regulated by Mg2+ binding on the inhibitory sites of the enzyme. 相似文献