Comparison of monovalent and divalent ion distributions around a DNA duplex with molecular dynamics simulation and a Poisson‐Boltzmann approach

The ion atmosphere created by monovalent (Na⁺) or divalent (Mg²⁺) cations surrounding a B‐form DNA duplex were examined using atomistic molecular dynamics (MD) simulations and the nonlinear Poisson‐Boltzmann (PB) equation. The ion distributions predicted by the two methods were compared using plots of radial and two‐dimensional cation concentrations and by calculating the total number of cations and net solution charge surrounding the DNA. Na⁺ ion distributions near the DNA were more diffuse in PB calculations than in corresponding MD simulations, with PB calculations predicting lower concentrations near DNA groove sites and phosphate groups and a higher concentration in the region between these locations. Other than this difference, the Na⁺ distributions generated by the two methods largely agreed, as both predicted similar locations of high Na⁺ concentration and nearly identical values of the number of cations and the net solution charge at all distances from the DNA. In contrast, there was greater disagreement between the two methods for Mg²⁺ cation concentration profiles, as both the locations and magnitudes of peaks in Mg²⁺ concentration were different. Despite experimental and simulation observations that Mg²⁺ typically maintains its first solvation shell when interacting with nucleic acids, modeling Mg²⁺ as an unsolvated ion during PB calculations improved the agreement of the Mg²⁺ ion atmosphere predicted by the two methods and allowed for values of the number of bound ions and net solution charge surrounding the DNA from PB calculations that approached the values observed in MD simulations. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 834–848, 2014.     

Effects of Inhibitors of Protein and Nucleic Acid Synthesis on the Development of Ion Uptake Mechanisms in Beetroot Slices (Beta vulgaris)

Mechanisms for the uptake of K⁺, Na⁺ and Cl^- develop sequentially in thin slices of beetroot tissue washed under aerobic conditions. Actinomycin D inhibited or prevented the development of K⁺, Na⁺ and Cl^- uptake mechanisms when added to freshly cut slices, but had no effect on net ion uptake when added after the development of the ion uptake mechanisms. The use of puromycin as a specific inhibitor of protein synthesis was unsatisfactory as it caused leakage of pigments and excessive loss of ions from the disks. Cycloheximide prevented the development of ion uptake mechanisms when added at the start of the experiment, but when added after the development of ion uptake mechanisms its inhibitory effect did not become apparent until after a certain time interval which varied from 3 hours for Cl^- to 25 hours for K⁺ uptake in the same experiment. p-Fluorophenylalanine caused an appreciable shortening of the time required for the development of Na⁺ and K⁺ uptake capabilities, while it completely prevented the development of a Cl^- uptake mechanism. p-Fluorophenylalanine-induced early uptake of Na⁺ and K⁺, however, was followed by periods of net leakage of these ions. It is suggested that the development of ion uptake mechanisms depends on the production of m-RNA, which appears to be relatively stable after its synthesis. The synthesis and decay characteristics of specific proteins required for the ion uptake mechanisms appear to differ for each ion species.     

Effects of tricyclohexylhydroxytin on the kinetics of adenosine triphosphatase system and protection by thiol reagents

Tricyclohexylhydroxytin, commonly known as Plictran® inhibited Na⁺, K⁺ -ATPase activity of rat brain synaptosomes in a concentration-dependent manner with median inhibitory concentration (IC-50) of 2 μM. Both K⁺ -stimulated para-nitrophenylphosphatase and [3-H]-ouabain binding to synaptosomes were also inhibited by Plictran with IC-50 values of 11 and 30 μM, respectively. Altered pH and Na⁺, K⁺ -ATPase activity curves demonstrated comparable inhibition in buffered neutral and alkaline pH ranges, and no inhibition was observed in acidic pH. The inhibition of Na⁺, K⁺ -ATPase was independent of temperature. Kinetic studies of substrate (ATP) activation of Na⁺, K⁺ -ATPase indicated uncompetitive inhibition. Results also showed noncompetitive inhibition for p-nitrophenylphosphate and uncompetitive inhibition for K⁺ activations of p-nitrophenylphosphatase. Preincubation of synaptosomes with dithiothreitol, a sulfhydryl (SH) agent, resulted in the complete protection of Plictran inhibition of Na⁺, K⁺ -ATPase, K⁺ -para-nitrophenylphosphatase, and [3-H]-ouabain binding. The protection was specific and concentration dependent since cysteine and glutathione did not afford protection. These results indicate that Plictran inhibited Na⁺, K⁺ -ATPase by interacting with dephosphorylation of the enzyme-phosphoryl complex and exerted a similar effect to that of SH-blocking agents.     

Identification of Electric-Field-Dependent Steps in the Na+,K+-Pump Cycle

The charge-transporting activity of the Na⁺,K⁺-ATPase depends on its surrounding electric field. To isolate which steps of the enzyme’s reaction cycle involve charge movement, we have investigated the response of the voltage-sensitive fluorescent probe RH421 to interaction of the protein with BTEA (benzyltriethylammonium), which binds from the extracellular medium to the Na⁺,K⁺-ATPase’s transport sites in competition with Na⁺ and K⁺, but is not occluded within the protein. We find that only the occludable ions Na⁺, K⁺, Rb⁺, and Cs⁺ cause a drop in RH421 fluorescence. We conclude that RH421 detects intramembrane electric field strength changes arising from charge transport associated with conformational changes occluding the transported ions within the protein, not the electric fields of the bound ions themselves. This appears at first to conflict with electrophysiological studies suggesting extracellular Na⁺ or K⁺ binding in a high field access channel is a major electrogenic reaction of the Na⁺,K⁺-ATPase. All results can be explained consistently if ion occlusion involves local deformations in the lipid membrane surrounding the protein occurring simultaneously with conformational changes necessary for ion occlusion. The most likely origin of the RH421 fluorescence response is a change in membrane dipole potential caused by membrane deformation.     

Identification of Electric-Field-Dependent Steps in the Na,K-Pump Cycle

The charge-transporting activity of the Na⁺,K⁺-ATPase depends on its surrounding electric field. To isolate which steps of the enzyme’s reaction cycle involve charge movement, we have investigated the response of the voltage-sensitive fluorescent probe RH421 to interaction of the protein with BTEA (benzyltriethylammonium), which binds from the extracellular medium to the Na⁺,K⁺-ATPase’s transport sites in competition with Na⁺ and K⁺, but is not occluded within the protein. We find that only the occludable ions Na⁺, K⁺, Rb⁺, and Cs⁺ cause a drop in RH421 fluorescence. We conclude that RH421 detects intramembrane electric field strength changes arising from charge transport associated with conformational changes occluding the transported ions within the protein, not the electric fields of the bound ions themselves. This appears at first to conflict with electrophysiological studies suggesting extracellular Na⁺ or K⁺ binding in a high field access channel is a major electrogenic reaction of the Na⁺,K⁺-ATPase. All results can be explained consistently if ion occlusion involves local deformations in the lipid membrane surrounding the protein occurring simultaneously with conformational changes necessary for ion occlusion. The most likely origin of the RH421 fluorescence response is a change in membrane dipole potential caused by membrane deformation.     

The effect of ultraviolet light on the sodium and potassium composition of resting yeast cells

The Na⁺ and K⁺ content of non-metabolizing yeast cells was determined before and after monochromatic ultraviolet (UV) irradiation. UV facilitated the uptake of Na⁺ into and the loss of K⁺ from the cells (net ion flux); the effect is greatest for the shortest wavelength employed (239 mµ) and is partly dependent upon the presence of oxygen. The UV effect on net ion flux persists for at least 90 minutes during which tests were made and it occurs following dosages which are without measurable effect on colony formation. The UV effect on net ion flux is decreased by acidity and promoted by alkalinity. Addition of calcium ions in sufficient amount prevents the usual net ion flux changes observed in irradiated yeast. Increase in concentration gradient between the inside and the outside of the cell increases the net ion flux of irradiated yeast, Na⁺ uptake leading K⁺ loss in all cases. UV appears to act by disorganizing the constituents of the cell surface, permitting K⁺ to leave the cell in exchange for Na⁺. At low intensities of UV this ionic exchange approaches equivalence, but at higher intensities more Na⁺ is taken up than K⁺ is lost. Some evidence suggests that the Na⁺ in excess over that exchanged for K⁺ is adsorbed to charged groups produced by the photochemical effect of UV on the cell surface.     

Enthalpy and entropy changes for the intercalation of small molecules to DNA. II. Ethidium and propidium fluoride

Enthalpy changes (ΔH_B) for the binding of ethidium (a monocation) and propidium (a dication) to calf thymus DNA have been determined calorimetrically in piperazine-N, N′-bis(2-ethanesulfonic acid) buffer with the fluoride ion as the counterion. Heats of dilution for the fluoride salts of ethidium and propidium were substantially less than the corresponding values found for other halide salts of these cations. At a Na⁺ ion concentrations of 0.019, ΔH_B = ?8.3 and ?7.9 ± 0.3 kcal mol^?1 for ethidium and propidium, respectively. For these two cations, just as was observed for the naphthalene monoimide (monocation) and diimide (dication) [H. P. Hopkins, K. A. Stevenson, and W. D. Wilson, (1986) J. Sol. Chem. 15 , 563–579], ΔH_B is within the same experimental error for both cations. Apparently, charge–charge interactions in DNA–cation complexes produce only small changes in the enthalpy for the system. In the concentration range 0.019–0.207, the ΔH_B values for propidium did not depend appreciably on the Na⁺ ion concentration, and a similar pattern was shown to exist for ethidium. When these results were combined with ΔG_B values for the binding of these cations to DNA, we found the variation of ΔS_B with Na⁺ ion concentration to be remarkably close to the predictions of modern polyelectrolyte theory, i.e., propidium binding to DNA causes approximately twice as many Na⁺ ions to be released into the bulk solution as does the binding of ethidium. The much stronger binding of propidium, relative to ethidium, at low ionic strengths is thus seen to be primarily due to entropic effects.     

Density functional study of isoguanine tetrad and pentad sandwich complexes with alkali metal ions

Isoguanine tetraplexes and pentaplexes contain two or more stacked polyads with intercalating metal ions. We report here the results of a density functional study of sandwiched isoguanine tetrad and pentad complexes consisting of two polyads with Na⁺, K⁺ and Rb⁺ ions at the B3LYP level. In comparison to single polyad metal ion complexes, there is a trend towards increased non-planarity of the polyads in the sandwich complexes. In general, the pentad sandwiches have relatively planar polyad structures, whereas the tetrad complexes contain highly non-planar polyad building blocks. As in other sandwich complexes and in metal ion complexes with single polyads, the metal ion-base interaction energy plays an essential role. In iG sandwich structures, this interaction energy is slightly larger than in the corresponding guanine sandwich complexes. Because the base–base interaction energy is even more increased in passing from guanine to isoguanine, the isoguanine sandwiches are thus far the only examples where the base–base interaction energy is larger than the base–metal ion interaction energy. Stacking interactions have been studied in smaller models consisting of two bases, retaining the geometry from the complete complex structures. From the data obtained at the B3LYP and BH&;H levels and with Møller-Plesset perturbation theory, one can conclude that the B3LYP method overestimates the repulsion in stacked base dimers. For the complexes studied in this work, this is only of minor importance because the direct inter-tetrad or inter-pentad interaction is supplemented by a strong metal ion-base interaction. Using a microsolvation model, the metal ion preference K⁺≈Rb⁺?>?Na⁺ is found for tetrad complexes. On the other hand, for pentads the ordering is Rb⁺?>?K⁺?>?Na⁺. In the latter case experimental data are available that agree with this prediction.

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Figure Structures of isoguanine pentad complexes with Rb⁺ at different symmetries.

     

Isolation and characterization of a novel Rhodococcus strain with switchable carbonyl reductase and para-acetylphenol hydroxylase activities

In the search for an effective biocatalyst for the reduction of acetophenones with unprotected hydroxy group on the benzene ring, a microorganism, which reduced para-acetylphenol to S-(?)-1-(para-hydroxyphenyl)ethanol under anaerobic conditions, was isolated from soil samples and the 16S rDNA study showed that it was phylogenetically affiliated with species of the genus Rhodococcus and was most similar to Rhodococcus pyridinivorans. Unexpectedly, this strain also hydroxylated para-acetylphenol to give 4-acetylcatechol in presence of oxygen, possessing para-acetylphenol hydroxylase activity. While the reduction of para-acetylphenol had an optimal reaction pH at 7 and a broad optimal temperature range (35–45 °C), the hydroxylation reached the maximum conversion at the pH range of 7–8 and 35 °C. This study identified for the first time a Rhodococcus strain with para-acetylphenol hydroxylase activity, which also contains highly enantioselective carbonyl reductase activity with potential applications for the asymmetric reduction of these less-explored but important ketones such as α-aminoacetophenone, 3′-hydroxyacetophenone and 4′-hydroxyacetophenone. The para-acetylphenol hydroxylase and carbonyl reductase activity are switchable by the reaction conditions.     

Active and passive Na+ fluxes across the basolateral membrane of rabbit urinary bladder

Summary The apical membrane of rabbit urinary bladder can be functionally removed by application of nystatin at high concentration if the mucosal surface of the tissue is bathed in a saline which mimics intracellular ion concentrations. Under these conditions, the tissue is as far as the movement of univalent ions no more than a sheet of basolateral membrane with some tight junctional membrane in parallel. In this manner the Na⁺ concentration at the inner surface of the basolateral membrane can be varied by altering the concentration in the mucosal bulk solution. When this was done both mucosal-to-serosal²²Na flux and net change in basolateral current were measured. The flux and the current could be further divided into the components of each that were either blocked by ouabain or insensitive to ouabain. Ouabain-insensitive mucosal-to-serosal Na⁺ flux was a linear function of mucosal Na⁺ concentration. Ouabain-sensitive Na⁺ flux and ouabain-sensitive, Na⁺-induced current both display a saturating relationship which cannot be accounted for by the presence of unstirred layers. If the interaction of Na⁺ with the basolateral transport process is assumed to involve the interaction of some number of Na⁺ ions,n, with a maximal flux,M _max, then the data can be fit by assuming 3.2 equivalent sites for interaction and a value forM _max of 287.8pm cm^–2 sec^–1 with an intracellular Na concentration of 2.0mm Na⁺ at half-maximal saturation. By comparing these values with the ouabain-sensitive, Na⁺-induced current, we calculate a Na⁺ to K⁺ coupling ratio of 1.40±0.07 for the transport process.     

Interaction of phospholipids (Lysophosphatidylethanolamines) with water and sodium cation

We have performed detailed ab initio SCF calculations on the intermolecular interaction energies for one Na⁺ ion and one water molecule with two molecular fragments, one exemplifying a phospholipid (PL) head (PLHD) and the other, a phospholipid tail (PLTL). A 6-12-1 atom-atom pair potential for the interaction of a Na⁺ ion and water with a lysophosphatidyl-ethanolamine (LPEA) was derived from these results by a fitting procedure. This fitted potential was used to obtain isoenergy maps that provide energy profiles of the Na⁺ ion and the water around the phospholipids. The interaction of the Na⁺ ion with PL, as well as the interaction of water with the PL, can be visualized from these maps, which, as expected, show regions of hydrophilicity and hydrophobicity for the water and indicate a very strong binding site for the Na⁺ ion on the phosphate. It appears to be a stationary site that would limit the Na⁺ ion mobility. This binding site is located near the double-bonded oxygen atom of the phosphate group; its binding energy for Na⁺ is 67 kcal/mol. On the other hand the NH⁺ group of PLHD ahows strong electrostatic repulsion of Na⁺ while interacting with water with a binding energy of 13 kcal/mol. This potential energy well region for water is separated from another of similar depth near the phosphate by a barrier and both regions are expected to act as binding sites for water.     

ROLE OF INORGANIC IONS IN CONTROLLING SEDIMENTATION RATE OF A MARINE CENTRIC DIATOM DITYLUM BRIGHTWELLI1,2

The settling rates and intracellular levels of K⁺, Na⁺, Cl^-, Mg²⁺ and Ca²⁺ were measured in Ditylum bright-welli (West) Grunow, grown axenically in an enriched seawater medium at 20 C at 4,000 lx on an 8:16 LD schedule. Cells at the end of the dark period have high Na⁺ (118 mM), low K⁺ (64 mM) and low Cl^- (117 mM) relative to levels at the end of the light period when K⁺ (126 mM) and Cl^- (154 mM) are high and Na⁺ (101 mM) is low. There is no significant change in Mg²⁺ (16–18 mM) or Ca²⁺ (3–4 mM) with time. The net result of the ion changes during the light period is to increase cell density by about 3.4 mg ml^-1. This change can account for the increase in settling rate of ca. 0.3 day^-1 during the same interval. The density of the cell contents, calculated from observed ion concentrations, is 15–18 mg.ml^-1 less than that of the seawater medium. The ion and settling rate changes are light-dependent and do not persist in the dark or under constant light (ca. 850 lx), but cells do exhibit a free-running circadian rhythm in cell division under continuous dim illumination. The cell vacuole expands during the light period and contracts during the dark, apparently in response to the net ion fluxes. D. brightwelli appears to regulate its density by active ion selectivity accompanied by trans-vacuolar water movement.     

Enniatin B and Valinomycin as Ion Carriers: An Empirical Force Field Analysis

Abstract

The alkali-ion binding properties of two natural depsipeptide ion carriers, enniatin B (EnB) and valinomycin (VM), are examined and compared by the empirical force field method. While VM has been shown to bind preferentially K⁺, Rb⁺, and Cs⁺ over Na⁺ in most solvents, EnB is considerably less specific.

We find that EnB forms two kinds of complexes, internal and external. In internal complexes, the ion binds to all six carbonyl oxygens, while in external ones, only three oxygens, preferentially those of the D-hydroxy-isovaleryl residues, are bound. The size of the internal cavity is best suited for Na⁺, while K⁺ and Rb⁺ squeeze in asymmetrically by distorting the molecule, and Cs⁺ not at all. External binding is much less specific. Since internal complexes possess much higher strain energies than external ones, the latter may be at least as stable as the former, even in fairly non-polar solvents.

VM is calculated to bind only internally, and with much less strain energy than EnB. The size of its internal cavity is well suited for binding the ions K⁺, Rb⁺, and Cs⁺, but is too big for Na⁺. The difference between the binding energies of Na⁺ and K⁺ is much smaller than that between the corresponding hydration enthalpies, thus explaining the binding preference for the latter ion.     

Voltage-dependent processes in the electroneutral amino acid exchanger ASCT2

Neutral amino acid exchange by the alanine serine cysteine transporter (ASCT)2 was reported to be electroneutral and coupled to the cotransport of one Na⁺ ion. The cotransported sodium ion carries positive charge. Therefore, it is possible that amino acid exchange is voltage dependent. However, little information is available on the electrical properties of the ASCT2 amino acid transport process. Here, we have used a combination of experimental and computational approaches to determine the details of the amino acid exchange mechanism of ASCT2. The [Na⁺] dependence of ASCT2-associated currents indicates that the Na⁺/amino acid stoichiometry is at least 2:1, with at least one sodium ion binding to the amino acid–free apo form of the transporter. When the substrate and two Na⁺ ions are bound, the valence of the transport domain is +0.81. Consistently, voltage steps applied to ASCT2 in the fully loaded configuration elicit transient currents that decay on a millisecond time scale. Alanine concentration jumps at the extracellular side of the membrane are followed by inwardly directed transient currents, indicative of translocation of net positive charge during exchange. Molecular dynamics simulations are consistent with these results and point to a sequential binding process in which one or two modulatory Na⁺ ions bind with high affinity to the empty transporter, followed by binding of the amino acid substrate and the subsequent binding of a final Na⁺ ion. Overall, our results are consistent with voltage-dependent amino acid exchange occurring on a millisecond time scale, the kinetics of which we predict with simulations. Despite some differences, transport mechanism and interaction with Na⁺ appear to be highly conserved between ASCT2 and the other members of the solute carrier 1 family, which transport acidic amino acids.     

Homeostasis of Sodium and Potassium Ions in Erythrocytes of the Lamprey Lampetra fluviatilis: Effect of Ion Transport and Metabolic Blockers, and Ionophores

After incubation of lamprey Lampetra fluviatilis erythrocytes in the standard medium for 90–120 min, intracellular Na⁺ and K⁺ content remained unchanged (28.7 ± 1.1 and 66.3 ± 1.5 mmol/l cells, respectively, n = 33). The erythrocyte ion content also did not change after treatment of the cells with ion transport inhibitors, Ba^{2 +} and amiloride. Addition of 0.1 mM ouabain to the incubation medium led to a decrease of K⁺ content by 8.4 ± 1.2 and to an increase of Na⁺ content by 2.4 ± 0.8 mmol/l/2 h. Similar reciprocal changes in the cellular ion composition were observed after treatment of the erythrocytes by oxidative metabolism inhibitors (rotenone and CCCP—carbonyl cyanide m-chlorophenyl-hydrazone). The metabolic blockers produced more significant ion composition changes in comparison with ouabain. An increase of intracellular Na⁺ content under effect of CCCP was completely inhibited by amiloride. It can be suggested that inhibition of oxidative metabolism is accompanied by a cell acidification and Na⁺/H⁺ exchange activation. Erythrocyte acidification by a K⁺/H⁺ ionophore led to a rapid cellular Na⁺ accumulation, which indicates the presence of a Na⁺/H⁺ exchanger with high activity. The K⁺ ionophore valinomycin produced a relatively small K⁺ loss from the lamprey erythrocytes to indicate a low anion conductance of the cells. The data obtained indicate an important role of oxidative metabolism in the monovalent ion homeostasis in the lamprey red blood cells.     

Molecular dynamics simulation study for diffusion of Na+ ion in water-filled carbon nanotubes at 25°C

We present results of molecular dynamics simulations for diffusion of Na⁺ ion in water-filled carbon nanotubes (CNTs) at 25°C using the extended simple point charge water potential. Simulation results indicate the general trend that the diffusion coefficients of Na⁺ ion and water molecule in CNTs decrease with an increase in water density and are larger than those in the bulk solution. The average potential energies of ion–water and water–water, the radial distribution functions, the hydration numbers and the residence times of the hydrated water molecules are discussed. The classical solventberg picture describes Na⁺ ion in water adequately for systems with the small values of diffusion coefficients.     

Activity and Stoichiometry of Na+:HCO− 3 Cotransport in Immortalized Renal Proximal Tubule Cells

The proximal tubule Na⁺-HCO⁻ ₃ cotransporter is located in the basolateral plasma membrane and moves Na⁺, HCO⁻ ₃, and net negative charge together out of the cell. The presence of charge transport implies that at least two HCO⁻ ₃ anions are transported for each Na⁺ cation. The actual ratio is of physiological interest because it determines direction of net transport at a given membrane potential. To determine this ratio, a thermodynamic approach was employed that depends on measuring charge flux through the cotransporter under defined ion and electrical gradients across the basolateral plasma membrane. Cells from an immortalized rat proximal tubule line were grown as confluent monolayer on porous substrate and their luminal plasma membrane was permeabilized with amphotericin B. The electrical properties of these monolayers were measured in a Ussing chamber, and ion flux through the cotransporter was achieved by applying Na⁺ or HCO⁻ ₃ concentration gradients across the basolateral plasma membrane. Charge flux through the cotransporter was identified as difference current due to the reversible inhibitor dinitro-stilbene disulfonate. The cotransporter activity was Cl⁻ independent; its conductance ranged between 0.12 and 0.23 mS/cm² and was voltage independent between −60 and +40 mV. Reversal potentials obtained from current-voltage relations in the presence of Na⁺ gradients were fitted to the thermodynamic equivalent of the Nernst equation for coupled ion transport. The fit yielded a cotransport ratio of 3HCO⁻ ₃:1Na⁺. Received: 19 January 1996/Revised: 24 April 1996     

Stability and solvation of alkali metal ion complexes with dibenzo-30-crown-10

Stability constants for the 1:1 complexes of dibenzo-30-crown-10 (DB30C10) with alkali metal ions have been determined at 25 °C in nitromethane and water by conductometry and capillary electrophoresis, respectively. Transfer activity coefficients of DB30C10 and its complexes from nitromethane to S (S = water, acetonitrile, propylene carbonate, methanol, and N,N-dimethylformamide) have been determined at 25 °C to evaluate the solvation properties. The stability constant in the poorly solvating solvent, nitromethane, decreases with increasing metal ion size, Na⁺ > K⁺ > Rb⁺ > Cs⁺, reflecting the intrinsic selectivity governed by electrostatic interaction between the metal ion and the ether oxygen atoms. It is also suggested that a part of the ether oxygen atoms does not bind to the metal ion in the Na(DB30C10)⁺ complex. The aqueous stability constant varies as Na⁺ ? K⁺ ≈ Rb⁺ ≈ Cs⁺; this selectivity pattern is similar to that in acetonitrile, propylene carbonate, and methanol. The complex stability in water is very low compared to that in the nonaqueous solvents, owing to hydrogen bonding of water to the oxygen atoms of the free crown ether. The transfer activity coefficient values show that DB30C10 shields all the metal ions effectively from the solvents and lead to the conclusion that the complexation selectivity in S receives a significant contribution from the solvation of the free metal ions. The Na(DB30C10)⁺ complex has specific interaction with water, causing much lower K⁺/Na⁺ selectivity in H₂O than in MeOH.     

Kinetics of K+-p-Nitrophenyl Phosphatase Stimulation by a Brain Soluble Fraction

We have already described the separation of two brain soluble fractions by Sephadex G-50, one of which stimulates (peak I) and the other inhibits (peak II) Na⁺, K⁺-ATPase and K⁺-p-nitrophenylphosphatase (K⁺-p-NPPase) activities. Here we examine the features of synaptosomal membrane p-NPPase activity in the presence and absence of brain peak I. It was observed that stimulation of Mg²⁺, K⁺-p-NPPase activity by peak I was concentration dependent, The ability of peak I to stimulate p-NPPase activity was lost by heat treatment followed by brief centrifugation. Pure serum albumin also stimulated enzyme activity. K⁺-p-NPPase stimulation by peak I proved dependent on K⁺ concentration but independent of Mg²⁺ and substrate p-nitrophenylphosphate concentrations. Since our determinations were performed in a non-phosphorylating condition reflecting the Na⁺, K⁺-ATPase Na⁺ site, it is suggested that peak I may stimulate the Na⁺-dependent enzyme phosphorylation known to take place from the internal cytoplasmic side.     

Oxygen binding to cobalt(II) proto-, deutero- and meso-porphyrin IX dimethyl ester complexes in organic solvents

The binding of oxygen to cobalt(II) meso, deutero- and proto-porphyrin IX dimethyl esters complexed with pyridine or 2-methylimidazole was investigated at −10°–−60°C in toluene or DMF solution, and the thermodynamic data related with the binding were presented. The oxygen affinity of cobalt meso-porphyrin complex was larger by the factor of 2.0–1.4 than those of the other complexes where oxygen affinities were not explained by a simple electron-withdrawing capability of 2,4-substituents of the porphyrin ring. The oxygen binding property was, generally, dependent on the solvent, suggesting that the solvation affects appreciably the oxygen binding to the complexes.The oxygen affinities of cobalt porphyrin complexes in various organic solvents were compared with those of their apomyoglobin complexes. The differences of oxygen affinities between both systems decreased with increasing the size of 2,4-substituents, and it was in the following order on 2,4-substituted porphyrins: Deutero ⪢ Proto ⪢ Meso. It was suggested that the 2,4-substitutent effect on the oxygen affinity of cobalt myoglobin complexes was not only caused by the direct electronic effect on the central cobalt atom, but also controlled by the stereochemical interaction between apomyoglobin and the porphyrin.