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1.
A novel and sensitive biosensor based on aptamer and pyrene-labeled fluorescent probes for the determination of K+ was developed. The aptamer was used as a molecular recognition element and a partially complementary oligonucleotide with the aptamer was labeled by pyrene moieties at both ends to transduce the binding event of K+ with aptamer. In the presence of K+, the complementary oligonucleotides were displaced from aptamers, which was accompanied by excimer fluorescence of pyrenes because the self-hairpin structure of the complementary oligonucleotide brought pyrene moieties into close proximity. However, it gave only monomer emission in the absence of K+. Under optimum conditions, the relative fluorescence intensity of pyrene was proportional to the concentration of K+ in the range of 6.0 × 10−4 to 2.0 × 10−2 M. A detection limit of 4.0 × 10−4 M was achieved. Moreover, this method was able to detect K+ with high selectivity in the presence of Na+, , Mg2+, and Ca2+ ions of biological fluids. In brief, the assay may have great potential applications, especially in a biological environment because of its simplicity, sensitivity, and specificity. 相似文献
2.
Many organic anions bind free Ca2+, the total concentration of which must be adjusted in experimental solutions. Because published values for the apparent dissociation constant (Kapp) describing the Ca2+ affinity of short chain fatty acids (SCFAs) and gluconate are highly variable, Ca2+ electrodes coupled to either a 3 M KCl or a Na+ selective electrode were used to redetermine Kapp. All solutions contained 130 mM Na+, whereas the concentration of the studied anion was varied from 15 to 120 mM, replacing Cl− that was decreased concomitantly to maintain osmolarity. This induces changes in the liquid junction potential (LJP) at the 3 M KCl reference electrode, leading to a systematic underestimation of Kapp if left uncorrected. Because the Na+ concentration in all solutions was constant, a Na+ electrode was used to directly measure the changes in the LJP at the 3 M KCl reference, which were under 5 mV but twice those predicted by the Henderson equation. Determination of Kapp either after correction for these LJP changes or via direct reference to a Na+ electrode showed that SCFAs do not bind Ca2+ and that the Kapp for the binding of Ca2+ to gluconate at pH 7.4, ionic strength 0.15 M, and 23 °C was 52.7 mM. 相似文献
3.
4.
Christine PeineltHans-Jürgen Apell 《Biophysical journal》2002,82(1):170-181
Electrochromic styryl dyes were used to investigate mutually antagonistic effects of Ca2+ and H+ on binding of the other ion in the E1 and P-E2 states of the SR Ca-ATPase. On the cytoplasmic side of the protein in the absence of Mg2+ a strictly competitive binding sequence, H2E1?HE1?E1?CaE1?Ca2E1, was found with two Ca2+ ions bound cooperatively. The apparent equilibrium dissociation constants were in the order of K1/2(2 Ca) = 34 nM, K1/2(H) = 1 nM and K1/2(H2) = 1.32 μM. Up to 2 Mg2+ ions were also able to enter the binding sites electrogenically and to compete with the transported substrate ions (K1/2(Mg) = 165 μM, K1/2(Mg2) = 7.4 mM). In the P-E2 state, with binding sites facing the lumen of the sarcoplasmatic reticulum, the measured concentration dependence of Ca2+ and H+ binding could be described satisfactorily only with a branched reaction scheme in which a mixed state, P-E2CaH, exists. From numerical simulations, equilibrium dissociation constants could be determined for Ca2+ (0.4 mM and 25 mM) and H+ (2 μM and 10 μM). These simulations reproduced all observed antagonistic concentration dependences. The comparison of the dielectric ion binding in the E1 and P-E2 conformations indicates that the transition between both conformations is accompanied by a shift of their (dielectric) position. 相似文献
5.
Robert D. GrayJonathan B. Chaires 《Biophysical chemistry》2011,159(1):205-209
Formation of DNA quadruplexes requires monovalent cation binding. To characterize the cation binding stoichiometry and linkage between binding and folding, we carried out KCl titrations of Tel22 (d[A(GGGTTA)3]), a model of the human telomere sequence, using a fluorescent indicator to determine [K+]free and circular dichroism to assess the extent of folding. At [K+]free = 5 mM (sufficient for > 95% folding), the apparent binding stoichiometry is 3K+/Tel22; at [K+]free = 20 mM, it increased to 8-10K+/Tel22. Thermodynamic analysis shows that at [K+]free = 5 mM, K+ binding contributes approximately − 4.9 kcal/mol for folding Tel22. The overall folding free energy is − 2.4 kcal/mol, indicating that there are energetically unfavorable contributions to folding. Thus, quadruplex folding is driven almost entirely by the energy of cation binding with little or no contribution from other weak molecular interactions. 相似文献
6.
Lucena MN Garçon DP Mantelatto FL Pinto MR McNamara JC Leone FA 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2012,161(4):380-391
We examine hemolymph ion regulation and the kinetic properties of a gill microsomal (Na+, K+)-ATPase from the intertidal hermit crab, Clibanarius vittatus, acclimated to 45‰ salinity for 10 days. Hemolymph osmolality is hypo-regulated (1102.5 ± 22.1 mOsm kg−1 H2O) at 45‰ but elevated compared to fresh-caught crabs (801.0 ± 40.1 mOsm kg−1 H2O). Hemolymph [Na+] (323.0 ± 2.5 mmol L−1) and [Mg2+] (34.6 ± 1.0 mmol L−1) are hypo-regulated while [Ca2+] (22.5 ± 0.7 mmol L−1) is hyper-regulated; [K+] is hyper-regulated in fresh-caught crabs (17.4 ± 0.5 mmol L−1) but hypo-regulated (6.2 ± 0.7 mmol L−1) at 45‰. Protein expression patterns are altered in the 45‰-acclimated crabs, although Western blot analyses reveal just a single immunoreactive band, suggesting a single (Na+, K+)-ATPase α-subunit isoform, distributed in different density membrane fractions. A high-affinity (Vm = 46.5 ± 3.5 U mg−1; K0.5 = 7.07 ± 0.01 μmol L−1) and a low-affinity ATP binding site (Vm = 108.1 ± 2.5 U mg−1; K0.5 = 0.11 ± 0.3 mmol L−1), both obeying cooperative kinetics, were disclosed. Modulation of (Na+, K+)-ATPase activity by Mg2+, K+ and NH4+ also exhibits site-site interactions, but modulation by Na+ shows Michaelis-Menten kinetics. (Na+, K+)-ATPase activity is synergistically stimulated up to 45% by NH4+ plus K+. Enzyme catalytic efficiency for variable [K+] and fixed [NH4+] is 10-fold greater than for variable [NH4+] and fixed [K+]. Ouabain inhibited ≈80% of total ATPase activity (KI = 464.7 ± 23.2 μmol L−1), suggesting that ATPases other than (Na+, K+)-ATPase are present. While (Na+, K+)-ATPase activities are similar in fresh-caught (around 142 nmol Pi min−1 mg−1) and 45‰-acclimated crabs (around 154 nmol Pi min−1 mg−1), ATP affinity decreases 110-fold and Na+ and K+ affinities increase 2-3-fold in 45‰-acclimated crabs. 相似文献
7.
Mitochondrial volume regulation depends on K+ movement across the inner membrane and a mitochondrial Ca2+-dependent K+ channel (mitoKCa) reportedly contributes to mitochondrial K+ uniporter activity. Here we utilize a novel KCa channel activator, NS11021, to examine the role of mitoKCa in regulating mitochondrial function by measuring K+ flux, membrane potential (ΔΨm), light scattering, and respiration in guinea pig heart mitochondria. K+ uptake and the influence of anions were assessed in mitochondria loaded with the K+ sensor PBFI by adding either the chloride (KCl), acetate (KAc), or phosphate (KH2PO4) salts of K+ to energized mitochondria in a sucrose-based medium. K+ fluxes saturated at ∼ 10 mM for each salt, attaining maximal rates of 172 ± 17, 54 ± 2.4, and 33 ± 3.8 nmol K+/min/mg in KCl, KAc, or KH2PO4, respectively. NS11021 (50 nM) increased the maximal K+ uptake rate by 2.5-fold in the presence of KH2PO4 or KAc and increased mitochondrial volume, with little effect on ΔΨm. In KCl, NS11021 increased K+ uptake by only 30% and did not increase volume. The effects of NS11021 on K+ uptake were inhibited by the KCa toxins charybdotoxin (200 nM) or paxilline (1 μM). Fifty nanomolar of NS11021 increased the mitochondrial respiratory control ratio (RCR) in KH2PO4, but not in KCl; however, above 1 μM, NS11021 decreased RCR and depolarized ΔΨm. A control compound lacking KCa activator properties did not increase K+ uptake or volume but had similar nonspecific (toxin-insensitive) effects at high concentrations. The results indicate that activating K+ flux through mitoKCa mediates a beneficial effect on energetics that depends on mitochondrial swelling with maintained ΔΨm. 相似文献
8.
To assess whether foliar application of K+S as potassium sulfate (K2SO4) could alleviate the adverse effects of salt on sunflower (Helianthus annuus L. cv. SF-187) plants, a greenhouse experiment was conducted. There were two NaCl levels (0 and 150 mM) applied to the growth medium and six levels of K+S as K2SO4 (NS (no spray), WS (spray of water+0.1% Tween 20 solution), 0.5% K+0.21% S, 1.0% K+0.41% S, 1.5% K+0.62% S, and 2.0% K+0.82% S in 0.1% Tween-20 solution) applied two times foliarly to non-stressed and salt-stressed sunflower plants. Salt stress markedly repressed the growth, yield, photosynthetic pigments, water relations and photosynthetic attributes, quantum yield (Fv/Fm), leaf and root K+, Mg2+, P, Ca2+, N as well as K+/Na+ ratios, while it enhanced the cell membrane permeability, and leaf and root Na+ and Cl− concentrations. Foliar application of potassium sulfate significantly improved growth, achene yield, photosynthetic and transpiration rates, stomatal conductance, water use efficiency, leaf turgor and enhanced shoot and leaf K+ of the salt-stressed sunflower plants, but it did not improve leaf and root Na+, Cl−, Mg2+, P, Ca2+, N as well as K+/Na+ ratios. The most effective dose of K+S for improving growth and achene yield was found to be 1.5% K+0.62% S and 1% K+0.41% S, respectively. Improvement in growth of sunflower plants due to exogenously applied K2SO4 was found to be linked to enhanced photosynthetic capacity, water use efficiency, leaf turgor and relative water content. 相似文献
9.
The electrochemical potential differences
for potassium, between excised barley (Hordeum vulgare L.) roots and external media containing 0.05 mM KCl+0.5 mM CaSO4, were determined over a 4-h period during which initially low-K+ roots accumulated K+ by pretreatment in 50 mM KCl plus 0.5 mM CaCl2. This pretreatment resulted in increased internal [K+], decreased K+ influx (as measured from 0.05 mM KCl+0.5 mM CaSO4) and decreased values of
. These observations indicate that the decline of K+ influx associated with increased internal K+ concentration cannot be accounted for by passive adjustment to the electrochemical gradient for this ion. 相似文献
10.
Potassium fluxes across the blood-brain barrier of the cockroach Periplaneta americana were measured using the scanning ion-selective microelectrode technique. In salines containing 15 mM or 25 mM K+, an efflux of K+ from the ganglia of isolated nerve cords was counterbalanced by an influx across the connectives. Metabolic inhibition with CN− resulted in an increase in K+ efflux across both the ganglia and the connectives. Depletion of K+ by chilling the nerve cords in K+-free saline was associated with subsequent K+ influx across the connectives in K+-replete saline at room temperature. There were dramatic increases in K+ efflux across both ganglia and connectives when the nerve cords were exposed to the pore-forming antibiotic amphotericin B. K+ fluxes across the ventral nerve cord were also altered when paracellular leakage was augmented by transient exposure to 3 M urea. K+ efflux was reduced by the K+ channel blockers Ba2+ and tetraethylammonium or by exposure to Ca2+-free saline and K+ efflux from the ganglia was increased by addition of ouabain to the bathing saline. The results provide direct support for a model proposing that K+ is cycled through a current loop between the ganglia and the connectives and that both the Na+/K+-ATPase and K+ channels are implicated in extracellular K+ homeostasis within the central nervous system. 相似文献
11.
Tony Christopeit 《Analytical biochemistry》2009,391(1):39-22
The interactions between Ca2+ and C-reactive protein (CRP) have been characterized using a surface plasmon resonance (SPR) biosensor. The protein was immobilized on a sensor chip, and increasing concentrations of Ca2+ or phosphocholine were injected. Binding of Ca2+ induced a 10-fold higher signal than expected from the molecular weight of Ca2+. It was interpreted to result from the conformational change that occurs on binding of Ca2+. Two sites with different characteristics were distinguished: a high-affinity site with KD = 0.03 mM and a low-affinity site with KD = 5.45 mM. The pH dependencies of the two Ca2+ interactions were different and enabled the assignment of the different sites in the three-dimensional structure of CRP. There was no evidence for cooperativity in the phosphocholine interaction, which had KD = 5 μM at 10 mM Ca2+. SPR biosensors can clearly detect and quantify the binding of very small molecules or ions to immobilized proteins despite the theoretically very low signals expected on binding, provided that significant conformational changes are involved. Both the interactions and the conformational changes can be characterized. The data have important implications for the understanding of the function of CRP and suggest that Ca2+ is an efficient regulator under physiological conditions. 相似文献
12.
Aim of the present study was to investigate the effects of two key environmental factors of estuarine ecosystems, salinity and hypoxia, on the physiological attributes in reed plants (Phragmites australis (Cav.) Trin. ex Steudel). Growth, leaf gas exchange, water (and ion) relations, and osmotic adjustment were determined in hydroponically grown plants exposed to hypoxia at varying NaCl-salinity concentrations (0, 50, 100, and 200 mM). Plants grew well under hypoxia treatment with standard nutrient solution without added salt and at NaCl concentrations up to 100 mM. Reed plants were able to produce and allocate phytomass to all their organs even at the highest salt level (200 mM NaCl). In plants subjected to hypoxia at various water potentials no clear relationships were found between growth and photosynthetic parameters except for gs, whereas growth displayed a highly significant correlation with plant–water relations. A and gs of reed plants treated with hypoxia at varying water potential of nutrient solutions were positively correlated and the former variable also had a strong positive relationship with E. Leaf Ψw and Ψπ followed a similar trend and declined significantly as water potential of watering solutions was lowered. Highly significant positive correlations were identified between leaf Ψw and photosynthetic parameters. At all NaCl concentrations, the increase in total inorganic ions resulted from increased Na+ and Cl− while K+, Ca2+, and Mg2+ concentrations decreased with increasing osmolality of nutrient solutions. Common reed has an efficient mechanism of Na+ exclusion from the leaves and exhibited a high leaf K+/Na+ selectivity ratio over a wide range of salinities under hypoxia treatment. In Phragmites australis grown in 200 mM NaCl, K+ contributed 17% toΨπ, whereas Na+ and Cl− accounted for only 11% and 6%, respectively. At the same NaCl concentration, the estimated contribution of proline to Ψπ was less than 0.2%. Changes in leaf turgor occurred with a combined effect of salinity and hypoxia, suggesting that reed plants could adjust their water status sufficiently. 相似文献
13.
Alvarez-Pizarro JC Gomes-Filho E Prisco JT Grossi-de-Sá MF de Oliveira-Neto OB 《Journal of plant physiology》2011,168(14):1617-1626
The effect of external inorganic nitrogen and K+ content on K+ uptake from low-K+ solutions and plasma membrane (PM) H+-ATPase activity of sorghum roots was studied. Plants were grown for 15 days in full-nutrient solutions containing 0.2 or 1.4 mM K+ and inorganic nitrogen as NO3-, NO3-/NH4+ or NH4+ and then starved of K+ for 24, 48 and 72 h. NH4+ in full nutrient solution significantly affected the uptake efficiency and accumulation of K+, and this effect was less pronounced at the high K+ concentration. In contrast, the translocation rate of K+ to the shoot was not altered. Depletion assays showed that plants grown with NH4+ more efficiently depleted the external K+ and reached higher initial rates of low-K+ uptake than plants grown with NO3-. One possible influence of K+ content of shoot, but not of roots, on K+ uptake was evidenced. Enhanced K+-uptake capacity was correlated with the induction of H+ extrusion by PM H+-ATPase. In plants grown in high K+ solutions, the increase in the active H+ gradient was associated with an increase of the PM H+-ATPase protein concentration. In contrast, in plants grown in solutions containing 0.2 mM K+, only the initial rate of H+-pumping and ATP hydrolysis were affected. Under these conditions, two specific isoforms of PM H+-ATPase were detected, independent of the nitrogen source and deficiency period. No change in enzyme activity was observed in NO3--grown plants. The results suggest that K+ homeostasis in NH4+-grown sorghum plants may be regulated by a high capacity for K+ uptake, which is dependent upon the H+-pumping activity of PM H+-ATPase. 相似文献
14.
The oxidation of thiocyanate by iron(V) (Fe(V)) was studied as a function of pH in alkaline solutions by a premix pulse radiolysis technique. The rates decrease with an increase in pH. The rate law for the oxidation of SCN− by Fe(V) was obtained as −d[Fe(V)]/dt = k10{[H+]2/([H+]2 + K2[H+] + K2K3)}[Fe(V)][SCN−], where k10 = 5.72 ± 0.19 × 106 M−1 s−1, pK2 = 7.2, and pK3 = 10.1. The reaction precedes via a two-electron oxidation, which converts Fe(V) to Fe(III). Thiocyanate reacts approximately 103× faster with iron(V) than does with iron(VI). 相似文献
15.
Different sensitivity of Phragmites australis and Glyceria maxima to high availability of ammonium-N
The ability to cope with NH4+-N was studied in the littoral helophytes Phragmites australis and Glyceria maxima, species commonly occupying fertile habitats rich in NH4+ and often used in artificial wetlands. In the present study, Glyceria growth rate was reduced by 16% at 179 μM NH4+-N, and the biomass production was reduced by 47% at 3700 μM NH4+-N compared to NO3−-N. Similar responses were not found in Phragmites. The amounts (mg g−1 dry wt) of starch and total non-structural carbohydrates (TNC) in rhizomes were significantly lower in NH4+ (8.9; 12.2 starch; 20.1; 41.9 TNC) compared to NO3− treated plants (28.0; 15.6 starch; 58.5; 56.3 TNC) in Phragmites and Glyceria, respectively. In addition, Glyceria showed lower amounts (mg g−1 dry wt) of soluble sugars, TNC, K+, and Mg2+ in roots under NH4+ (5.6; 14.3; 20.6; 1.9) compared to NO3− nutrition (11.6; 19.9; 37.9; 2.9, for soluble sugars, TNC, K+, and Mg2+, respectively), while root internal levels of NH4+ and Ca2+ (0.29; 4.6 mg g−1 dry wt, mean of both treatments) were only slightly affected. In Phragmites, no changes in soluble sugars, TNC, Ca2+, K+, and Mg2+ contents of roots (7.3; 14.9; 5.1; 17.3; 2.6 mg g−1 dry wt, means of both treatments) were found in response to treatments. The results, therefore, indicate a more pronounced tolerance towards high NH4+ supply in Phragmites compared to Glyceria, although the former may be susceptible to starch exhaustion in NH4+-N nutrition. In contrast, Glyceria's ability to colonize fertile habitats rich in NH4+ is probably related to the avoidance strategy due to shallow rooting or to the previously described ability to cope with high NH4+ levels when P availability is high and NO3− is also provided. 相似文献
16.
A glucose-tolerant β-glucosidase was purified to homogeneity from prune (Prunus domestica) seeds by successive ammonium sulfate precipitation, hydrophobic interaction chromatography and anion-exchange chromatography. The molecular mass of the enzyme was estimated to be 61 kDa by SDS-PAGE and 54 kDa by gel permeation chromatography. The enzyme has a pI of 5.0 by isoelectric focusing and an optimum activity at pH 5.5 and 55 °C. It is stable at temperatures up to 45 °C and in a broad pH range. Its activity was completely inhibited by 5 mM of Ag+ and Hg2+. The enzyme hydrolyzed both p-nitrophenyl β-d-glucopyranoside with a Km of 3.09 mM and a Vmax of 122.1 μmol/min mg and p-nitrophenyl β-d-fucopyranoside with a Km of 1.65 mM and a Vmax of 217.6 μmol/min mg, while cellobiose was not a substrate. Glucono-δ-lactone and glucose competitively inhibited the enzyme with Ki values of 0.033 and 468 mM, respectively. 相似文献
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18.
Proper assembly of RNA into catalytically active three-dimensional structures requires multiple tertiary binding interactions, individual characterization of which is crucial to a detailed understanding of global RNA folding. This work focuses on single-molecule fluorescence studies of freely diffusing RNA constructs that isolate the GAAA tetraloop-receptor tertiary interaction. Freely diffusing conformational dynamics are explored as a function of Mg2+ and Na+ concentration, both of which promote facile docking, but with 500-fold different affinities. Systematic shifts in mean fluorescence resonance energy transfer efficiency values and line widths with increasing [Na+] are observed for the undocked species and can be interpreted with a Debye model in terms of electrostatic relaxation and increased flexibility in the RNA. Furthermore, we identify a 34 ± 2% fraction of freely diffusing RNA constructs remaining undocked even at saturating [Mg2+] levels, which agrees quantitatively with the 32 ± 1% fraction previously reported for immobilized constructs. This verifies that the kinetic heterogeneity observed in the docking rates is not the result of surface tethering. Finally, the KD value and Hill coefficient for [Mg2+]-dependent docking decrease significantly for [Na+] = 25 mM vs. 125 mM, indicating Mg2+ and Na+ synergy in the RNA folding process. 相似文献
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20.
The effects of fluphenazine (FLU) on the noradrenaline (NA) induced cAMP-synthesis in intact rat retinae were studied as a function of extracellular K+- and Ca2+-ions. Thus NA-induced cAMP levels were measured after incubating intact rat retinae with 50 μM NA in the presence or absence of FLU and in the presence of 1 or 10 mM theophylline. Results were: (1) Experimental condition a: standard NA-responses were measured after incubating retinae at 0.75 mM Ca2+, at 10 mM theophylline, at 10 μM FLU and at 2 and 0 mM K+. FLU does not affect the NA-response at 2 mM K+ significantly; however, it inhibits the NA-response at 0 mM K+ in this condition. (2) Experimental condition b: NA-responses were measured after incubating retinae at 0.125 mM Ca2+, 10 mM theophylline, 10 μM FLU and at 2 and 0 mM K+. At 2 mM K+ FLU replaces a Ca2+ function probably connected with the synthesis part of the NA-cAMP system and NA-responses in this low Ca2+ condition are consequently enhanced by FLU; however, FLU inhibits the NA-response at 0 mM K+ in this condition. (3) Experimental condition c: NA-responses were measured after incubating retinae at 0.75 mM Ca2+, 1 mM theophylline, 10 μM FLU and at 2 and 0 mM K+. At 2 mM K+ FLU enhances the NA-response by further inhibition of the degradation part of the NA-cAMP system; FLU inhibits the NA-response at 0 mM K+ in this condition. (4) The inhibitions of the NA-responses by FLU at 0 mM K+ in all three conditions a, b and c showed an apparent Km of 1 μM. (5) Low concentrations of K+ (0.4–0.8 mM) maintain the property of FLU to enhance the NA-responses at condition b (0.125 mM Ca2+) and at condition c (1 mM theophylline). Results suggest that the activation of NA-receptor coupled adenylate cyclases (NA-AC-ases) by NA, resulting in activation of phosphodiesterase activity by the NA-elevated cAMP-levels, is sustained by (a) membraneous factor(s) connected to the NA-receptor. This (these) factor(s) is (are) switched off in the absence of K+. Evidence has been presented, that Ca2+ and FLU do not have access to this intramembraneous factor-enzyme activating moiety of the NA-cAMP system at 0 mM K+. Between 0.4 and 0.8 mM K+ the factor-enzyme-NA-receptor complex is still intact. 相似文献