The MgtC virulence factor of Salmonella enterica serovar Typhimurium activates Na(+),K(+)-ATPase

The mgtC gene of Salmonella enterica serovar Typhimurium encodes a membrane protein of unknown function that is important for full virulence in the mouse. Since mgtC is part of an operon with mgtB which encodes a Mg(2+)-transporting P-type ATPase, MgtC was hypothesized to function in ion transport, possibly in Mg(2+) transport. Consequently, MgtC was expressed in Xenopus laevis oocytes, and its effect on ion transport was evaluated using ion selective electrodes. Oocytes expressing MgtC did not exhibit altered currents or membrane potentials in response to changes in extracellular H(+), Mg(2+), or Ca(2+), thus ruling out a previously postulated function as a Mg(2+)/H(+) antiporter. However, addition of extracellular K(+) markedly hyperpolarized membrane potential instead of the expected depolarization. Addition of ouabain to block the oocyte Na(+),K(+)-ATPase completely prevented hyperpolarization and restored the normal K(+)-induced depolarization response. These results suggested that the Na(+),K(+)-ATPase was constitutively activated in the presence of MgtC resulting in a membrane potential largely dependent on Na(+),K(+)-ATPase. Consistent with the involvement of Na(+),K(+)-ATPase, oocytes expressing MgtC exhibited an increased rate of (86)Rb(+) uptake and had increased intracellular free [K(+)] and decreased free [Na(+)] and ATP. The free concentrations of Mg(2+) and Ca(2+) and cytosolic pH were unchanged, although the total intracellular Ca(2+) content was slightly elevated. These results suggest that the serovar Typhimurium MgtC protein may be involved in regulating membrane potential but does not directly transport Mg(2+) or another ion.     

Yeast SMF1 mediates H(+)-coupled iron uptake with concomitant uncoupled cation currents

Yeast membrane proteins SMF1, SMF2, and SMF3 are homologues of the DCT1 metal ion transporter family. Their functional characteristics and the implications of these characteristics in vivo have not yet been reported. Here we show that SMF1 expressed in Xenopus oocytes mediates H(+)-dependent Fe(2+) transport and uncoupled Na(+) flux. SMF1-mediated Fe(2+) transport exhibited saturation kinetics (K(m) = 2.2 microM), whereas the Na(+) flux did not, although both processes were electrogenic. SMF1 is also permeable to Li(+), Rb(+), K(+), and Ca(2+), which likely share the same uncoupled pathway. SMF2 (but not SMF3) mediated significant increases in both Fe(2+) and Na(+) transport compared with control oocytes. These data are consistent with the concept that uptake of divalent metal ions by SMF1 and SMF2 is essential to yeast cell growth. Na(+) inhibited metal ion uptake mediated by SMF1 and SMF2 expressed in oocytes. Consistent with this, we found that increased sensitivity of yeast to EGTA in the high Na(+) medium is due to inhibition of SMF1- and SMF2-mediated metal ion transport by uncoupled Na(+) pathway. Interestingly, DCT1 also mediates Fe(2+)-activated uncoupled currents. We propose that uncoupled ion permeabilities in metal ion transporters protect cells from metal ion overload.     

Ion selectivity and current saturation in inward-rectifier K+ channels

We investigated the features of the inward-rectifier K channel Kir1.1 (ROMK) that underlie the saturation of currents through these channels as a function of permeant ion concentration. We compared values of maximal currents and apparent K(m) for three permeant ions: K(+), Rb(+), and NH(4)(+). Compared with K(+) (i(max) = 4.6 pA and K(m) = 10 mM at -100 mV), Rb(+) had a lower permeability, a lower i(max) (1.8 pA), and a higher K(m) (26 mM). For NH(4)(+), the permeability was reduced more with smaller changes in i(max) (3.7 pA) and K(m) (16 mM). We assessed the role of a site near the outer mouth of channel in the saturation process. This site could be occupied by either permeant ions or low-affinity blocking ions such as Na(+), Li(+), Mg(2+), and Ca(2+) with similar voltage dependence (apparent valence, 0.15-0.20). It prefers Mg(2+) over Ca(2+) and has a monovalent cation selectivity, based on the ability to displace Mg(2+), of K(+) > Li(+) ～ Na(+) > Rb(+) ～ NH(4)(+). Conversely, in the presence of Mg(2+), the K(m) for K(+) conductance was substantially increased. The ability of Mg(2+) to block the channels was reduced when four negatively charged amino acids in the extracellular domain of the channel were mutated to neutral residues. The apparent K(m) for K(+) conduction was unchanged by these mutations under control conditions but became sensitive to the presence of external negative charges when residual divalent cations were chelated with EDTA. The results suggest that a binding site in the outer mouth of the pore controls current saturation. Permeability is more affected by interactions with other sites within the selectivity filter. Most features of permeation (and block) could be simulated by a five-state kinetic model of ion movement through the channel.     

Molecular dynamics simulation of cation-phospholipid clustering in phospholipid bilayers: possible role in stalk formation during membrane fusion

In this study, we performed all-atom long-timescale molecular dynamics simulations of phospholipid bilayers incorporating three different proportions of negatively charged lipids in the presence of K(+), Mg(2+), and Ca(2+) ions to systemically determine how membrane properties are affected by cations and lipid compositions. Our simulations revealed that the binding affinity of Ca(2+) ions with lipids is significantly stronger than that of K(+) and Mg(2+) ions, regardless of the composition of the lipid bilayer. The binding of Ca(2+) ions to the lipids resulted in bilayers having smaller lateral areas, greater thicknesses, greater order, and slower rotation of their lipid head groups, relative to those of corresponding K(+)- and Mg(2+)-containing systems. The Ca(2+) ions bind preferentially to the phosphate groups of the lipids. The complexes formed between the cations and the lipids further assembled to form various multiple-cation-centered clusters in the presence of anionic lipids and at higher ionic strength-most notably for Ca(2+). The formation of cation-lipid complexes and clusters dehydrated and neutralized the anionic lipids, creating a more-hydrophobic environment suitable for membrane aggregation. We propose that the formation of Ca(2+)-phospholipid clusters across apposed lipid bilayers can work as a "cation glue" to adhere apposed membranes together, providing an adequate configuration for stalk formation during membrane fusion.     

D443 of the N domain of Na+,K+-ATPase interacts with the ATP-Mg2+ complex, possibly via a second Mg2+ ion

This paper provides evidence for an interaction of D443 in the N domain of Na(+),K(+)-ATPase with a Mg(2+) ion. Wild-type, D443N/A/C and S445A mutants of porcine Na(+),K(+)-ATPase (alpha1beta1) have been expressed in Pichia pastoris. By comparison with wild-type, D443N reduces the turn-over rate by about 40%. Binding affinity of ATP, measured directly, was not affected by D443N, D443A, or D443C mutations. AMP-PNP-Fe(2+)-catalyzed oxidative cleavage of Na(+),K(+)-ATPase produces two characteristic fragments, at (708)VNDS (P domain) and near (440)VAGDA (N domain), respectively. In the D443N and D443A mutants, both cleavages are suppressed, indicating an interaction between the residues with AMP-PNP-Fe(2+) bound. Previous work suggested that with ATP-Fe(2+) bound the N and P domains come into proximity, both D710 and D443 making contact with a single Fe(2+) (or Mg(2+)) ion. However, the crystal structure of Ca(2+)-ATPase with bound AMP-PCP and Mg(2+) confirm the involvement of D703 (D710) but show that E439 (D443) is too far to make contact with the Mg(2+). By contrast, in the crystal structure with bound ADP, AlF(4), and Mg(2+), representing the E(1)-P conformation, two Mg(2+) ions were observed. Significantly, ADP-Fe(2+)-mediated oxidative cleavage of renal Na,K-ATPase produces the fragment near (440)VAGDA (N domain), while the cleavage at (708)VNDS (P domain) is almost completely absent. The results are explained economically by the hypothesis that ATP is bound with two Mg(2+) (Fe(2+)) ions, a "catalytic" Mg(2+) interacting with D710 via the gamma phosphate and a "structural" Mg(2+) interacting with D443 via the alpha and beta phosphates and a water molecule, respectively.     

Stoicheiometrical proton and potassium ion movements accompanying the absorption of amino acids by the yeast Saccharomyces carlsbergensis

1. Proton uptake into the yeast Saccharomyces carlsbergensis, was studied at pH4.5-5.5 in the presence of both antimycin and 2-deoxyglucose to inhibit energy metabolism. Previous work had shown that the cells then absorbed about 20nmol of glycine or l-phenylalanine against a considerable amino acid concentration gradient. The addition of the amino acid immediately stimulated the rate of uptake of protons two- to three-fold. About 2 extra equivalents of H(+) accompanied a given amount of the amino acids into the yeast preparations exposed to the metabolic inhibitors for 2-4min and about 1.2 equivalents after 20min exposure. 2. Analogous observations were made during serial additions of glycine, l-phenylalanine, l-leucine and l-lysine to preparations lacking the metabolic inhibitors and deficient in substrates needed for energy metabolism. In fresh cellular preparations the influx of glycine was then closely coupled to a stimulated flow of 2.1 equiv. of H(+) into the yeast. A similar number of K(+) ions left the cells. About 30% of the extra protons was subsequently ejected from the yeast. Deoxyglucose and antimycin together inhibited the ejection of protons. When the yeast had been fed with glucose energy metabolism was stimulated and almost as many protons as were absorbed with the amino acid were apparently ejected again. 3. Yeast preparations containing Na(+), instead of K(+), as the principal cation absorbed about 1 extra equivalent of H(+) after the addition of phenylalanine, glycine or leucine. This response was not observed in the presence of both deoxyglucose and antimycin. 4. The observations show that H(+) and, in certain circumstances, K(+) are co-substrates in the transport of the amino acids into the yeast. An analogy is drawn with the roles of Na(+) and K(+) as co-substrates in certain mammalian systems. The results lead to various models relating the physical flow of the co-substrate ions on the amino acid carrier to the transduction of chemical energy in an associated ion pump forming part of the mechanism for transporting amino acids into the yeast.     

K+ transport by the OsHKT2;4 transporter from rice with atypical Na+ transport properties and competition in permeation of K+ over Mg2+ and Ca2+ ions

Members of class II of the HKT transporters, which have thus far only been isolated from grasses, were found to mediate Na(+)-K(+) cotransport and at high Na(+) concentrations preferred Na(+)-selective transport, depending on the ionic conditions. But the physiological functions of this K(+)-transporting class II of HKT transporters remain unknown in plants, with the exception of the unique class II Na(+) transporter OsHKT2;1. The genetically tractable rice (Oryza sativa; background Nipponbare) possesses two predicted K(+)-transporting class II HKT transporter genes, OsHKT2;3 and OsHKT2;4. In this study, we have characterized the ion selectivity of the class II rice HKT transporter OsHKT2;4 in yeast and Xenopus laevis oocytes. OsHKT2;4 rescued the growth defect of a K(+) uptake-deficient yeast mutant. Green fluorescent protein-OsHKT2;4 is targeted to the plasma membrane in transgenic plant cells. OsHKT2;4-expressing oocytes exhibited strong K(+) permeability. Interestingly, however, K(+) influx in OsHKT2;4-expressing oocytes did not require stimulation by extracellular Na(+), in contrast to other class II HKT transporters. Furthermore, OsHKT2;4-mediated currents exhibited permeabilities to both Mg(2+) and Ca(2+) in the absence of competing K(+) ions. Comparative analyses of Ca(2+) and Mg(2+) permeabilities in several HKT transporters, including Arabidopsis thaliana HKT1;1 (AtHKT1;1), Triticum aestivum HKT2;1 (TaHKT2;1), OsHKT2;1, OsHKT2;2, and OsHKT2;4, revealed that only OsHKT2;4 and to a lesser degree TaHKT2;1 mediate Mg(2+) transport. Interestingly, cation competition analyses demonstrate that the selectivity of both of these class II HKT transporters for K(+) is dominant over divalent cations, suggesting that Mg(2+) and Ca(2+) transport via OsHKT2;4 may be small and would depend on competing K(+) concentrations in plants.     

Altering the intermediate in the equilibrium folding of unmodified yeast tRNAPhe with monovalent and divalent cations

The isothermal equilibrium folding of the unmodified yeast tRNA(Phe) is studied as a function of Na(+), Mg(2+), and urea concentration with hydroxyl radical protection, circular dichroism, and diethyl pyrocarbonate (DEPC) modification. These assays indicate that this tRNA folds in Na(+) alone. Similar to folding in Mg(2+), folding in Na(+) can be described by two transitions, unfolded-to-intermediate-to-native. The I-to-N transition has a Na(+) midpoint of approximately 0.5 M and a Hill constant of approximately 4. Unexpectedly, the urea m-value, the dependence of free energy on urea concentration, for the I-to-N transition is significantly smaller in Na(+) than in Mg(2+), 0.4 versus 1.7 kcal mol(-1) M(-1), indicating that more structure is formed in the Mg(2+)-induced transition. DEPC modification indicates that the I state in Na(+)-induced folding contains all four helices of tRNA and the I-to-N transition primarily corresponds to the formation of the tertiary structure. In contrast, the intermediate in Mg(2+)-induced folding contains only three helices, and the I-to-N transition corresponds to the formation of the acceptor stem plus tertiary structure. The cation dependence of the intermediates arises from the differences in the stability of the acceptor stem and the tertiary structure. The acceptor stem is stable at a lower Na(+) concentration than required for the tertiary structure formation. The relative stability is reversed in Mg(2+) so that the acceptor stem and the tertiary structure form simultaneously in the I-to-N transition. These results demonstrate that formation of the RNA secondary structure can be independent or coupled to the formation of the tertiary structure depending on their relative stability in monovalent and divalent ions.     

Influence of cations on growth of thermophilic Geobacillus spp. and Anoxybacillus flavithermus in planktonic culture

Free ions of Na(+), K(+), Ca(2+), and Mg(2+) influenced the optical density of planktonic cultures of thermophilic bacilli. Anoxybacillus flavithermus E16 and Geobacillus sp. strain F75 (milk powder manufacturing plant isolates) and A. flavithermus DSM 2641 and G. thermoleovorans DSM 5366 were studied. Ca(2+) and Mg(2+) were associated with increases in optical density more so than Na(+) and K(+). Overall, it appeared that Ca(2+) and/or Mg(2+) was required for the production of protein in thermophilic bacilli, as shown by results obtained with A. flavithermus E16, which was selected for further study.     

Selective Fe2+-catalyzed oxidative cleavage of gastric H+,K+-ATPase: implications for the energy transduction mechanism of P-type cation pumps

In the presence of ascorbate/H(2)O(2), Fe(2+) ions or the ATP-Fe(2+) complex catalyze selective cleavage of the alpha subunit of gastric H(+),K(+)-ATPase. The electrophoretic mobilities of the fragments and dependence of the cleavage patterns on E(1) and E(2) conformational states are essentially identical to those described previously for renal Na(+),K(+)-ATPase. The cleavage pattern of H(+),K(+)-ATPase by Fe(2+) ions is consistent with the existence of two Fe(2+) sites: site 1 within highly conserved sequences in the P and A domains, and site 2 at the cytoplasmic entrance to trans-membrane segments M3 and M1. The change in the pattern of cleavage catalyzed by Fe(2+) or the ATP-Fe(2+) complex induced by different ligands provides evidence for large conformational movements of the N, P, and A cytoplasmic domains of the enzyme. The results are consistent with the Ca(2+)-ATPase crystal structure (Protein Data Bank identification code; Toyoshima, C., Nakasako, M., Nomura, H., and Ogawa, H. (2000) Nature 405, 647-655), an E(1)Ca(2+) conformation, and a theoretical model of Ca(2+)-ATPase in an E(2) conformation (Protein Data Bank identification code ). Thus, it can be presumed that the movements of N, P, and A cytoplasmic domains, associated with the E(1) <--> E(2) transitions, are similar in all P-type ATPases. Fe(2+)-catalyzed cleavage patterns also reveal sequences involved in phosphate, Mg(2+), and ATP binding, which have not yet been shown in crystal structures, as well as changes which occur in E(1) <--> E(2) transitions, and subconformations induced by H(+),K(+)-ATPase-specific ligands such as SCH28080.     

Alcohol dehydrogenase activity and electron transport in living yeast

1. The excretion of H(+) ions, with practically equivalent uptake of K(+) ions (from 0.1m-potassium chloride), occurs during the aerobic oxidation of ethanol. 2. Acetaldehyde and acetic acid formed at the same time are quantitatively equal to the amount of ethanol oxidized. 3. A slow uptake of K(+) ions occurs during the oxidation of acetaldehyde and a more rapid uptake during the oxidation of d-glyceraldehyde 3-phosphate. 4. The anaerobic reduction of methylene blue is studied, and the inhibitory effect of K(+) and other inorganic cations on the system demonstrated. 5. The cation requirement for equal inhibitory effect is parallel with the reciprocals of the transport affinities for the ;physiological K-carrier' (as taken from Conway & Duggan, 1958). 6. The cation inhibition of methylene blue reduction is reversed by treatment of the yeast with Teepol or by freezing-and-thawing. 7. Azide is shown to inhibit the reduction of methylene blue with intact cells. The inhibition is partially reversed by Teepol treatment and completely by freezing-and-thawing.     

Role of the choline exchanger in Na(+)-independent Mg(2+) efflux from rat erythrocytes

Two types of Na(+)-independent Mg(2+) efflux exist in erythrocytes: (1) Mg(2+) efflux in sucrose medium and (2) Mg(2+) efflux in high Cl(-) media such as KCl-, LiCl- or choline Cl-medium. The mechanism of Na(+)-independent Mg(2+) efflux in choline Cl medium was investigated in this study. Non-selective transport by the following transport mechanisms has been excluded: K(+),Cl(-)- and Na(+),K(+),Cl(-)-symport, Na(+)/H(+)-, Na(+)/Mg(2+)-, Na(+)/Ca(2+)- and K(+)(Na(+))/H(+) antiport, Ca(2+)-activated K(+) channel and Mg(2+) leak flux. We suggest that, in choline Cl medium, Na(+)-independent Mg(2+) efflux can be performed by non-selective transport via the choline exchanger. This was supported through inhibition of Mg(2+) efflux by hemicholinum-3 (HC-3), dodecyltrimethylammonium bromide (DoTMA) and cinchona alkaloids, which are inhibitors of the choline exchanger. Increasing concentrations of HC-3 inhibited the efflux of choline and efflux of Mg(2+) to the same degree. The K(d) value for inhibition of [(14)C]choline efflux and for inhibition of Mg(2+) efflux by HC-3 were the same within the experimental error. Inhibition of choline efflux and of Mg(2+) efflux in choline medium occurred as follows: quinine>cinchonine>HC-3>DoTMA. Mg(2+) efflux was reduced to the same degree by these inhibitors as was the [(14)C]choline efflux.     

Effects of lower alcohols on potassium transport and microsomal adenosine-triphosphatase activity of rat cerebral cortex

1. Slices of rat cerebral cortex, incubated anaerobically at 37 degrees , lost K(+) from an initial concentration of 102m-equiv./kg. to a concentration of 57m-equiv./kg. after 10min. On subsequent aerobic incubation they regained K(+) rapidly at a rate that varied with the K(+) concentration of the medium. 2. Lower aliphatic alcohols, present at equal thermodynamic activity, produced approximately equal degrees of inhibition of K(+) uptake during the aerobic incubation. This inhibition was reduced by an increase in K(+) content of the medium. Ethanol did not affect the rate of K(+) loss during anaerobic incubation. 3. Li(+), in concentrations of 1-10mm, also inhibited K(+) uptake by brain-cortex slices, the degree of inhibition varying with the Li(+) concentration. Ouabain also inhibited K(+) uptake. 4. The same series of alcohols, at equal thermodynamic activity, produced comparable degrees of inhibition of Na(+),K(+),Mg(2+)-stimulated adenosine-triphosphatase activity in brain microsomes. 5. It is suggested that inhibition of cation transport is an important, but not a primary, mechanism in the production of central nervous depression by alcohols and other substances.     

Quantification of Mg2+ extrusion and cytosolic Mg2+-buffering in Xenopus oocytes

Intracellular Mg(2+) buffering and Mg(2+) extrusion were investigated in Xenopus laevis oocytes. Mg(2+) or EDTA were pressure injected and the resulting changes in the intracellular Mg(2+) concentration were measured simultaneously with Mg(2+)-selective microelectrodes. In the presence of extracellular Na(+), injected Mg(2+) was extruded from the oocytes with an estimated v(max) and K(M) of 74 pmol cm(-2)s(-1) and 1.28 mM, respectively. To investigate genuine cytosolic Mg(2+) buffering, measurements were carried out in the nominal absence of extracellular Na(+) to block Mg(2+) extrusion, and during the application of CCCP (inhibiting mitochondrial uptake). Under these conditions, Mg(2+) buffering calculated after both MgCl(2) and EDTA injections could be described by a buffer equivalent with a concentration of 9.8mM and an apparent dissociation constant, K(d-app), of 0.6mM together with an [ATP](i) of 0.9 mM with a K(d-app) 0.12 mM. Xenopus oocytes thus possess highly efficient mechanisms to maintain their intracellular Mg(2+) concentration.     

Osmo and ionic regulation of black tiger prawn (Penaeus monodon Fabricius 1798) juveniles exposed to K(+) deficient inland saline water at different salinities

An 11-day trial was conducted to investigate the osmoregulatory capacity (OC) and regulation of K(+), Na(+), Ca(2+) and Mg(2+) of Penaeus monodon juveniles when exposed to K(+) deficient inland saline water (ISW) of four different salinities (5, 15, 25 and 35 ppt). The survival of juveniles showed a positive linear relationship (R(2) ranging from 0.72 to 0.98) with salinity. At the end of the trial, juveniles were able to survive only in 5 ppt of ISW and showed no changes in OC when transferred from ocean water (OW) to ISW. Further, the OC of juveniles in 5 ppt of ISW was significantly different (P<0.05) from the OC of juveniles exposed to 15, 25 and 35 ppt and exhibited strong serum K(+), Na(+), Ca(2+) and Mg(2+) regulation monitored over 16 h. In contrast, at 35 ppt, significant decrease (P<0.05) in serum K(+) and Mg(2+) concentrations and accumulation of serum Na(+) concentration occurred after 16 h of exposure to ISW. At higher salinity, an increase in serum Na(+) concentration leads to an increase in the serum osmolality of the juveniles, which in turn causes decrease in the OC of the juveniles. The results of this study suggest that K(+) deficiency in ISW has a negative effect on survival, OC and the ability of P. monodon juveniles to regulate serum Na(+), K(+), Ca(2+) and Mg(2+) concentrations. These effects are compounded as salinity increases.     

Phenylene-ethynylene trication as an efficient fluorescent signal transducer in an aptasensor for potassium ion

A tricationic phenylene-ethynylene (N(3+)) fluorophore is investigated as a fluorescent transducer in homogeneous aptasensing system for potassium ion (K(+)) assay in aqueous media. The enhancement of the fluorescent signal of N(3+) by three K(+) aptamers consisting of 12, 15, and 21 nucleotides are observed and used for the determination of N(3+)-aptamer binding affinities. The binding affinities increase with the length of the aptameric oligonucleotides and are proven to be important to the sensitivity and selectivity of the aptasensors. The enhanced fluorescent signal of each N(3+)-aptamer solution is selectively quenched by K(+) due to the ability of K(+) in stabilizing the G-quadruplex structure of the aptamer. Among three aptamers, the 15-base aptamer provides optimal sensitivity and selectivity over other ions such as Li(+), Na(+), NH(4)(+), Mg(2+), Ca(2+) and Sr(2+). The sensing system shows the detection limit of 1 μM of K(+) in clean buffered solution and 30 μM of K(+) in the solution containing 4800-fold excess of Na(+), with wide linear dynamic ranges of micro- to millimolar concentration. This label-free fluorescence aptasensor is conveniently and effectively applicable for analysis of K(+) in urine samples.     

Effect of metal ions on the activity of green crab (Scylla serrata) alkaline phosphatase

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. The present paper deals with the study of the effect of some kinds of metal ions on the enzyme. The positive monovalent alkali metal ions (Li(+), Na(+) and K(+)) have no effect on the enzyme; positive bivalent alkaline-earth metal ions (Mg(2+), Ca(2+) and Ba(2+)) and transition metal ions (Mn(2+), Co(2+), Ni(2+) and Cd(2+)) activate the enzyme; heavy metal ions (Hg(2+), Ag(+), Bi(2+), Cu(2+) and Zn(2+)) inhibit the enzyme. The activation of magnesium ion on the enzyme appears to be a partial noncompetitive type. The kinetic model has been set up and a new plot to determine the activation constant of Mg(2+) was put forward. From the plot, we can easily determine the activation constant (K(a)) value and the activation ratio of Mg(2+) on the enzyme. The inhibition effects of Cu(2+) and Hg(2+) on the enzyme are of noncompetitive type. The inhibition constants have been determined. The inhibition effect of Hg(2+) is stronger than that of Cu(2+).     

Influence of monovalent cation identity on parvalbumin divalent ion-binding properties

Rat alpha- and beta-parvalbumins have distinct monovalent cation-binding properties [Henzl et al. (2000) Biochemistry 39, 5859-5867]. Beta binds two Na(+) or one K(+), and alpha binds one Na(+) and no K(+). Ca(2+) abolishes these binding events, suggesting that the monovalent ions occupy the EF-hand motifs. This study compares alpha and beta divalent ion affinities in Na(+) and K(+) solutions. Solvent cation identity seriously affects alpha. In Hepes-buffered NaCl, at 5 degrees C, the macroscopic Ca(2+)-binding constants are 2.6 x 10(8) and 6.4 x 10(7) M(-1) and the Mg(2+) constants, 1.8 x 10(4) and 4.3 x 10(3) M(-1). In Hepes-buffered KCl, the Ca(2+) values increase to 2.9 x 10(9) and 6.6 x 10(8) M(-1) and the Mg(2+) values to 2.2 x 10(5) and 3.7 x 10(4) M(-1). Monte Carlo simulation of alpha binding data-employing site-specific constants and explicitly considering Na(+) binding-yields a K(Na) of 630 M(-1) and indicates that divalent ion-binding is positively cooperative. NMR data suggest that the lone Na(+) ion occupies the CD loop. Solvent cation identity has a smaller impact on beta. In Na(+), the Ca(2+) constants for the EF and CD sites are 2.3 x 10(7) and 1.5 x 10(6) M(-1), respectively; the Mg(2+) constants are 9.2 x 10(3) and 1.7 x 10(2) M(-1). In K(+), these values shift to 3.1 x 10(7) and 3.8 x 10(6) M(-1) and the latter to 1.4 x 10(4) and 2.9 x 10(2) M(-1). These data suggest that parvalbumin divalent ion affinity, particularly that of rat alpha, can be significantly attenuated by increased intracellular Na(+) levels.     

Potassium ion-stimulated and sodium ion-dependent adenosine diphosphate–adenosine triphosphate exchange activity in a kidney microsomal fraction

1. K(+) did not affect the Mg(2+)-dependent transphosphorylation but markedly increased the Na(+)-stimulated ADP-ATP exchange rate mediated by a microsomal fraction from guinea-pig kidney. 2. Rb(+), Cs(+), NH(4) (+) and Li(+) were equally effective in stimulating the Na(+)-dependent ADP-ATP exchange activity. 3. Treatment of the microsomal fraction with N-ethylmaleimide or increased concentrations of Mg(2+) prevented stimulation of the Na(+)-dependent exchange reaction by K(+). 4. Ouabain (2.5mum) inhibited ATP hydrolysis by 33% but did not decrease the K(+)-stimulated Na(+)-dependent ADP-ATP exchange rate. 5. A possible mechanism for stimulation of exchange activity by K(+) is discussed.     

Effect of divalent cations on ion fluxes and leaf photochemistry in salinized barley leaves

Photosynthetic characteristics, leaf ionic content, and net fluxes of Na(+), K(+), and Cl(-) were studied in barley (Hordeum vulgare L) plants grown hydroponically at various Na/Ca ratios. Five weeks of moderate (50 mM) or high (100 mM) NaCl stress caused a significant decline in chlorophyll content, chlorophyll fluorescence characteristics, and stomatal conductance (g(s)) in plant leaves grown at low calcium level. Supplemental Ca(2+) enabled normal photochemical efficiency of PSII (F(v)/F(m) around 0.83), restored chlorophyll content to 80-90% of control, but had a much smaller (50% of control) effect on g(s). In experiments on excised leaves, not only Ca(2+), but also other divalent cations (in particular, Ba(2+) and Mg(2+)), significantly ameliorated the otherwise toxic effect of NaCl on leaf photochemistry, thus attributing potential targets for such amelioration to leaf tissues. To study the underlying ionic mechanisms of this process, the MIFE technique was used to measure the kinetics of net Na(+), K(+), and Cl(-) fluxes from salinized barley leaf mesophyll in response to physiological concentrations of Ca(2+), Ba(2+), Mg(2+), and Zn(2+). Addition of 20 mM Na(+) as NaCl or Na(2)SO(4) to the bath caused significant uptake of Na(+) and efflux of K(+). These effects were reversed by adding 1 mM divalent cations to the bath solution, with the relative efficiency Ba(2+)>Zn(2+)=Ca(2+)>Mg(2+). Effect of divalent cations on Na(+) efflux was transient, while their application caused a prolonged shift towards K(+) uptake. This suggests that, in addition to their known ability to block non-selective cation channels (NSCC) responsible for Na(+) entry, divalent cations also control the activity or gating properties of K(+) transporters at the mesophyll cell plasma membrane, thereby assisting in maintaining the high K/Na ratio required for optimal leaf photosynthesis.