Salt effects on peptide conformers: a dielectric study of tuftsin.

Four 1-ns molecular dynamics computer simulations of tuftsin, Thr-Lys-Pro-Arg, are analyzed: (1) cis tuftsin in water, (2) trans tuftsin in water, (3) cis tuftsin in 1 M NaCl, and (4) trans tuftsin in 1 M NaCl. Independently of the salt concentration, the trans conformer has a higher dielectric constant than the cis conformer because the former exhibits a more widely distributed charge distribution in space. Independently of the peptide conformation, the presence of salt reduces the dielectric constants of both the peptide and the solvating water molecules because ions, on binding, restrict the motion of other atoms. In contrast to the dielectric constants, neither the peptide conformation nor the salt concentration shows a significant influence on the dielectric relaxation time of water molecules.     

Regulation of cell volume and ion concentrations in a Halobacterium.

Changes in cell volume and ion content of a Halobacterium species are described in terms of the NaCl concentration (0.5--3.5M) and pH(4-8) of the suspending medium. Cell volume, per unit content of protein of bacteria in stationary phase cultures, rose as the [NaCl] of the growth medium was increased. Logarithmic-phase bacteria shrank as the pH fell from 7 to 5.5. These changes are characteristic of bacteria with a moderate or rapid rate of O2 consumption. Starving (i.e. nonmetabolizing) bacteria, on the other hand, did not change in size within the above ranges of [NaCl] and pH. At lower values, however, such bacteria swelled and eventually lysed. Effects of low pH on cell ions are compared in metabolizing and starving bacteria, and it is shown that changes in the state of the cell K are correlated with movements of cell Na. It appears that the cell K is used to maintain cell [Na] below the NaCl concentration of the medium. The results are explained in terms of a model involving interactions between polyelectrolytes, salts and water in the concentrated cytoplasm of these halophilic organisms.     

Regulation of intracellular osmotic pressure during the initial stages of salt stress in a salt-tolerant yeast, Zygosaccharomyces rouxii.

The accumulation of glycerol and inorganic ions as it related to osmotic pressure, and the regulation of intracellular osmotic pressure in a salt-tolerant yeast, Zygosaccharomyces rouxii, were examined for several hours after salt stress. Intracellular contents of glycerol increased for up to 6 h in media supplemented with 1 M and 2 M NaCl and did not increase in medium containing 3 M NaCl. Intracellular contents of Na+ and Cl- reached a maximum value within 1 and 3 h, respectively, in all NaCl-containing media and increases were proportional to the concentration of NaCl in the medium. As glycerol was accumulated in cells, the intracellular contents of Na+ and Cl- gradually decreased in media containing 1 M and 2 M NaCl. After salt stress, cell volume decreased within 1 h and the original volume was re-established for 3 to 6 h in media with 1 M and 2 M NaCl but not in medium with 3 M NaCl. Intracellular concentrations of solutes, which were calculated from the total contents of glycerol and inorganic ions and the cell volume, became almost equivalent to the external osmotic pressure within 1 h after salt stress. Experiments using various inhibitors showed that a large amount of ATP was required not only for the synthesis and accumulation of glycerol but also for the exclusion of Na+ and Cl- from cells under salt-stressed conditions.     

微囊藻碳酸酐酶活性在不同环境因素下的调节与适应

测定了3种微囊藻水华中的优势种类,即铜锈微囊藻（Microcystis aetlzginosa Kutz．）,绿色微囊藻（Microcystis viridis（A．Br．）Lemm）,惠氏微囊藻（Microcystis wesenbergii（Kom．）Kom．）,以及微囊藻573（Microcystis sp．573）的碳酸酐酶活性;研究了无机碳、pH、温度、光强、NIP比等环境因素和外源葡萄糖对铜锈微囊藻碳酸酐酶活性的影响,发现微囊藻碳酸酐酶活性受环境中碳酸氢根浓度的调节,故推断碳酸氢根是铜锈微囊藻利用的主要无机碳形式;相比添加葡萄糖进行混合营养培养的细胞,无外源葡萄糖和暗饥饿培养的微囊藻细胞会产生高约6倍的碳酸酐酶活性;光强的改变也会影响碳酸酐酶的活性。     

Steric selectivity in Na channels arising from protein polarization and mobile side chains

Monte Carlo simulations of equilibrium selectivity of Na channels with a DEKA locus are performed over a range of radius R and protein dielectric coefficient epsilon(p). Selectivity arises from the balance of electrostatic forces and steric repulsion by excluded volume of ions and side chains of the channel protein in the highly concentrated and charged (approximately 30 M) selectivity filter resembling an ionic liquid. Ions and structural side chains are described as mobile charged hard spheres that assume positions of minimal free energy. Water is a dielectric continuum. Size selectivity (ratio of Na+ occupancy to K+ occupancy) and charge selectivity (Na+ to Ca2+) are computed in concentrations as low as 10(-5) M Ca2+. In general, small R reduces ion occupancy and favors Na+ over K+ because of steric repulsion. Small epsilon(p) increases occupancy and favors Na+ over Ca2+ because protein polarization amplifies the pore's net charge. Size selectivity depends on R and is independent of epsilon(p); charge selectivity depends on both R and epsilon(p). Thus, small R and epsilon(p) make an efficient Na channel that excludes K+ and Ca2+ while maximizing Na+ occupancy. Selectivity properties depend on interactions that cannot be described by qualitative or verbal models or by quantitative models with a fixed free energy landscape.     

An extreme halophilic enzyme active at low salt in reversed micelles.

Possible biotechnological applications of extreme halophilic enzymes are strongly determined by their high salt requirement of around 4 M NaCl. Consequently, the use of these in organic media seemed to be unlikely. However, we have succeeded in dissolving a halophilic enzyme, p-nitrophenylphosphate phosphatase from the archaeon Halobacterium salinarum, in an organic medium by creating a reverse micellar system with very low salt concentration. The enzyme retained its catalytic properties in reversed micelles made with an anionic surfactant (dioctyl sodium sulphosuccinate) or with a cationic surfactant (hexadecyltrimethylammonium bromide) in cyclohexane plus 1-butanol as co-surfactant. The dependence of the rate of hydrolysis of p-nitrophenylphosphate phosphate on the molar water/surfactant ratio (w(0) value) showed a bell-shaped curve for each surfactant system. Kinetic parameters were determined in each system. The enzymatic reaction appeared to follow Michaelis-Menten kinetics with the anionic surfactant only. The kinetic behaviour was determined at different concentrations of Mn(2+) in reversed micelles of dioctyl sodium sulphosuccinate as surfactant.     

General continuum theory for multiion channel. II. Application to acetylcholine channel.

The general theory (Levitt, D. G. 1990. Biophys. J. 59:271-277) is applied to a model channel that resembles the acetylcholine receptor channel (ACH). The model incorporates the known features of the ACH geometry and fixed charge locations. The channel has a wide mouth facing the outer solution, tapering to a narrow region facing the interior of the cell. Rings of fixed negative charge are placed at the two surfaces where the bilayer begins, corresponding to the known charges at the ends of the M2 segment. It is assumed that the forces acting on the ion are electrostatic: ion-channel wall, ion-ion, Born image and applied voltage. Analytical expressions for these forces are derived that take account of the low dielectric lipid region. In addition, there is a local hard sphere repulsive force that prevents ions from piling up on each other in regions of the channel with a high fixed charge density. A classical continuum theory is used to obtain an expression for the diffusion coefficient in the channel. The model can mimic the major qualitative and, in many cases, quantitative experimental features of the ACH channel: current-voltage relation, conductance versus concentration and interaction between monovalent and divalent ions. The model calculations were also compared with the site directed mutagenesis experiments of Imoto, K., C. Busch, B. Sakmann, M. Mishina, T. Konno, J. Nakai, H. Bujo, Y. Mori, K. Fukuda, and S. Numa. (1988. Nature (Lond.). 335:645-648) in which the charge at the ends of the channel was systematically varied.     

Modulation and adaptation of carbonic anhydrase activity in Microcystis spp. under different environmental factors

The carbonic anhydrase (CA) activities were determined in three cyanobacterial species, namely Microcystis aeruginosa Kütz., Microcystis viridis (A.Br.) Lemm, and Microcystis wesenbergii (Kom.) Kom, which were dominant in a lake (Dianchi Lake) subject to major blooms. In more detailed experiments on M. aeruginosa, the effects of inorganic carbon, pH, temperature, nitrogen/phosphorus ratio, glucose, and light intensity on CA activity were also investigated. Because of the relatively alkaline pH value of the culture media for the optimum growth of algal cells, bicarbonate ions were the main form of exogenous inorganic carbon. The results showed that the CA activity of M. aeruginosa was influenced dramatically by the concentration of bicarbonate. Consequently, it was suggested that bicarbonate ions were the main form of exogenous inorganic carbon that M. aeruginosa could utilize. Cultures grown in the dark exhibited CA activity six times higher than that of cells cultured mixotrophically with the addition of glucose. Features of eutrophic water bodies promoted an increase in CA activity, and the resulting higher CA activity would accelerate the utilization of inorganic carbon and favor the growth and blooming of Microcystis spp. in eutrophic lakes. Although the experiments were carried out under controlled experimental conditions, they could provide some basic data that would prove useful for the control of cyanobacterial blooms in nature.     

Adsorption to dipalmitoylphosphatidylcholine membranes in gel and fluid state: pentachlorophenolate, dipicrylamine, and tetraphenylborate.

We measured the dependence of electrophoretic mobility of dipalmitoylphosphatidylcholine (DPPC) vesicles on the aqueous concentration of negatively charged ions of pentachlorophenol (PCP), dipicrylamine (DPA), and tetraphenylborate (TPhB). The objective was to determine how the physical state of hydrocarbon chains of lipids affects adsorption of lipophilic ions. The studies were done at 25 and 42 degrees C to determine adsorption properties of DPPC membrane in the gel and fluid state, respectively. From the analysis of zeta-potential isotherms in terms of Langmuir-Stern-Grahame model we obtained the association constant, K, the area of the adsorption site, Ps, and the linear partition coefficient, beta. Results: K, (x 10(4)M-1): K(gel): PCP (0.49 +/- 0.28), DPA (25 +/- 10), TPhB (31 +/- 10); K(fluid): PCP (4.5 +/- 0.9), DPA (74 +/- 21), TPhB (59 +/- 14); Ps, (nm2): Ps(gel): PCP (5.4 +/- 2.3), DPA (5.9 +/- 2), TPhB (5.0 +/- 1.7); Ps(fluid): PCP (4.5 +/- 0.4), DPA (5.2 +/- 0.4), TPhB (4.1 +/- 0.2); beta, (x 10(-5) m): beta(gel): PCP (0.15 +/- 0.09), DPA (7.1 +/- 0.3), TPhB (10 +/- 7); beta(fluid): PCP (1.7 +/- 0.3), DPA (24 +/- 7), TPhB (24 +/- 6). It was interesting to find that the adsorption site area for PCP, DPA, and TPhB were very similar for both the gel and fluid membranes; also, the areas were independent of the size and molecular structure of the adsorbing species. Using a simple discrete charge model the adsorption site areas for all species were consistent with a dielectric constant of 8-10 and with an ion adsorption depth of 0.4-0.6 nm below the water/dielectric interface. The delta delta G0 = delta G0(gel) - delta G0(fluid) was found to be about twice as large for PCP than for DPA and TPhB. This indicates that PCP will be significantly more adsorbed in the fluid and disordered regions of biomembranes, whereas the distribution of DPA and TPhB is expected to be relatively more even.     

Halotolerance of Methanobacterium thermoautotrophicum delta H and Marburg.

Methanobacterium thermoautotrophicum delta H and Marburg were adapted to grow in medium containing up to 0.65 M NaCl. From 0.01 to 0.5 M NaCl, there was a lag before cell growth which increased with increasing external NaCl. The effect of NaCl on methane production was not significant once the cells began to grow. Intracellular solutes were monitored by nuclear magnetic resonance (NMR) spectroscopy as a function of osmotic stress. In the delta H strain, the major intracellular small organic solutes, cyclic-2,3-diphosphoglycerate and glutamate, increased at most twofold between 0.01 and 0.4 M NaCl and decreased when the external NaCl was 0.5 M. M. thermoautotrophicum Marburg similarly showed a decrease in solute (cyclic-2,3-diphosphoglycerate, 1,3,4,6-tetracarboxyhexane, and L-alpha-glutamate) concentrations for cells grown in medium containing > 0.5 M NaCl. At 0.65 M NaCl, a new organic solute, which was visible in only trace amounts at the lower NaCl concentrations, became the dominant solute. Intracellular potassium in the delta H strain, detected by atomic absorption and 39K NMR, was roughly constant between 0.01 and 0.4 M and then decreased as the external NaCl increased further. The high intracellular K+ was balanced by the negative charges of the organic osmolytes. At the higher external salt concentrations, it is suggested that Na+ and possibly Cl- ions are internalized to provide osmotic balance. A striking difference of strain Marburg from strain delta H was that yeast extract facilitated growth in high-NaCl-containing medium. The yeast extract supplied only trace NMR-detectable solutes (e.g., betaine) but had a large effect on endogenous glutamate levels, which were significantly decreased. Exogenous choline and glycine, instead of yeast extract, also aided growth in NaCl-containing media. Both solutes were internalized with the choline converted to betaine; the contribution to osmotic balance of these species was 20 to 25% of the total small-molecule pool. These results indicate that M. thermoautotrophicum shows little changes in its internal solutes over a wide range of external NaCl. Furthermore, they illustrate the considerable differences in physiology in the delta H and Marburg strains of this organism.     

Some properties of the citrate synthase from the extreme halophile, Halobacterium cutirubrum.

1. Citrate synthase [citrate oxaloacetate-lyase (CoA-acetylating), EC 4.1.3.7] was purified about 400-fold from the extreme halophile, Halobacterium cutirubrum, by a method involving (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and hydroxyapatite and gel filtration on Sephadex G-200. 2. The purified enzyme was best activated by high concentrations of KCl (3M); the chlorides of other cations and K+ salts of other anions (Br-, NO3-, SCN-) were less effective than KCl as activators. The enzyme was best stabilized by high concentrations of NaCl or KCl. Cold-lability was found in the presence of 3M-KCl, but not in the presence of NaCl at concentrations up to 5M. The results suggest that both the shielding of negative charges on the enzyme molecule and the stabilization of hydrophobic bonds by high KCl concentrations were required for maximum activity of the enzyme. 3. The double-reciprocal plots for acetyl-CoA or oxaloacetate at several concentrations of the co-substrate intersected at the abscissa in the presence of either KCl or NaCl, at either 1 or 3M. The Km for oxaloacetate increased about fivefold with the salt concentration, from 1 to 3M.     

The effect of ouabain on the guinea pig ileum longitudinal smooth muscle: 2. Intracellular levels of Ca, Na, K, and Mg during the ouabain response and the dependence of the response on extracellular Ca.

Isotonic Tris-HCl containing 10 mM LaCl3 at 4 degrees C effectively removed extracellular ions in 30 min while preventing loss of intracellular ions. Intracellular Ca and Na increased during the contraction in the presence of 10 mM ouabain and then decreased during relaxation. Intracellular Na increased again during the latter part of the relaxation phase when K loss became apparent. Mg levels remained essentially constant. Ouabain responses were rapidly lost in Ca-free medium indicating that they were dependent on extracellular Ca. A 5.5-fold increase in the normal levels of extracellular K did not reduce the contraction to a submaximal dose of ouabain. A full phasic response to high K (60 mM) was observed after a 10-min exposure of the tissue to ouabain, at which time the ouabain response had returned to basal tension. The contraction to ouabain appears to be dissociated from inhibition of the Na,K-ATPase at the K site. The changes in intracellular ions indicated that ouabain contracted the muscle by increasing the plasma membrane permeability to Ca and Na and later decreased the K and Na concentration gradients, probably by inhibition of the Na,K-ATPase.     

Impact of air ions of both polarity on evaporation of certain organic and inorganic liquids

Air ions of both polarity, produced by corona electrodes, were used to evaporate to dryness liquid samples of ethyl alcohol (EA), water (W), and carbon tetrachloride (CTC). Drying times were determined with a beta-ray gauge. Ion exposed samples of EA, W, and CTC dried, respectively, 2.3, 3.2, and 5.4 times faster than the corresponding control samples when exposed simultaneously to 0.94×10¹² positive and 1.83×10¹² negative air ions cm^–2s^–1 under the same laboratory conditions. Drying by corona discharge could be explained by three different mechanisms. Electric wind caused by the ionic drag is proposed as the principal driving force for the observed enhancement of evaporation. The decrease in free energy of a dielectric in the presence of an electric field compared to its absence may have increased the escaping tendency of the molecules of the treated liquids. The turbulence in the liquids created by the rotational effect on the dielectric molecules by the electric field may also be a factor in further enhancing the mass transfer rates from the samples.     

Photochemically induced charge separation occurring in bacteriorhodopsin. Detection by time-resolved dielectric loss.

Time-resolved dielectric loss (TRDL) measurements are reported for the photochemical excitation of bacteriorhodopsin (bR) in solid films of Halobacterium halobium purple membranes. These measurements provide an independent confirmation for the existence of an important component of charge separation in these membranes after photochemical excitation. The separation of charge is detected by the absorption of microwave energy by the multilayer films of purple membranes in a microwave cavity during flash photolysis experiments. The TRDL method has the advantage of being sensitive to charge separation occurring in both oriented and unoriented films of purple membranes. One disadvantage is that the water content of the samples must be minimized, however, there is some absorbed water present in our electrodeposited solid film samples. To the best of our knowledge, TRDL measurements have not been reported previously for photochemical charge separation in biological membranes. It is significant that an early decay component of TRDL in the 20-microseconds time domain corresponds to the relaxation of the negative charge displacement photocurrent in oriented samples of purple membranes. In addition, a component of charge separation persists during the first several hundred microseconds of the bR photocycle.     

Adsorption equilibria between liposome membrane formed of phosphatidylcholine and aqueous sodium chloride solution as a function of pH

The effect has been studied of the adsorption of ions (H(+), Na(+), OH(-), Cl(-)) which are present in solution upon the electric charge of the liposome membrane formed of phosphatidylcholine (PC). The surface charge density of the membrane was determined as a function of pH and electrolyte concentration from electrophoretic mobility measurements. The measurements were carried out by the laser-Doppler microelectrophoresis method. A four-equilibria model has been proposed to describe the phenomena occurring on the membrane surface. The equilibria in which the adsorption of other ions on the liposome membrane surface was involved were assumed to exist beside the equilibria in which the H(+) and OH(-) ions were engaged. The idea was confirmed by mathematical calculations. Association constants of the liposome membrane surface with ions of solution (K(AH), K(ANa), K(BOH), K(BCl)) were determined. The proposed model has been proved to be correct by comparing the resulting theoretic charge variation curves of the lecithin membrane with the experimental data.     

The relation between sodium ion content and efflux of labelled sodium ions from yeast

1. The activity of the Na(+) pump in an Na(+)-rich yeast was compared with that in an Na(+)-rich frog sartorius muscle, and found to be very similar to it over the first hour if both were immersed in fluid containing 104mm-Na(+) plus 10mm-K(+). 2. The efflux of labelled Na(+) from an Na(+)-rich yeast into an Na(+)-free medium was investigated. In this Na(+)-free medium, Li(+) or choline replaced the Na(+), and the efflux-content curves obtained with either of these ions were very similar. The curves were sigmoid, reaching or approaching a saturation at the higher internal Na(+) concentrations. 3. The curves obtained with yeast resembled those similarly obtained with frog sartorius muscle by Keynes & Swan (1959), Mullins & Frumento (1963), Harris (1965) and Keynes (1965). The slope of the plot of the logarithm of the Na(+) efflux against the logarithm of the Na(+) concentration in the cells reached its highest value at an internal Na(+) concentration of 15m-equiv./kg. (27m-equiv./l. of cell water). 4. The effect of external K(+) concentration on the efflux-content relationship was examined. An increased K(+) concentration was found to increase the Na(+) efflux by raising the saturation value, which is similar to observations made by Harris (1965) with frog muscle. 5. The effect of increasing the external carbon dioxide concentration was investigated. No effect on the slope of the plot of the logarithm of the Na(+) efflux against the logarithm of the Na(+) content was noticed even when the yeast suspension was equilibrated with 100% carbon dioxide. There was, however, a decrease in the amount of Na(+) efflux on equilibrating the solution with carbon dioxide.     

Orthophosphate anion enhances the stability and activity of endoxylanase from Bacillus sp

Endoxylanase, for which the optimum temperature is 60 degrees C (optimum pH 7), is labile to heat. Because the isoelectric point (pI) value of this xylanase is 10.6, the net charge of this enzyme is positive at pH 7. Thus, ions are likely to influence its enzyme structure and the thermal stability of endoxylanase may improve. Among the various ions tested, orthophosphate anion (HPO(4)(2-)) was found to significantly improve not only the stability but the activity of xylanase. When K(2)HPO(4) concentration was increased from 50 mM to 1.2 M, the T(m )value of xylanase was increased from 60.0 degrees C to 74.5 degrees C. The affinity of xylanase on xylan also increased along with K(2)HPO(4) concentration. Thus, the xylanase activity at 0.6 M K(2)HPO(4) was 2.3-fold higher than that at 50 mM K(2)HPO(4), and 120.2-fold higher than that in 40 mM MOPS buffer. This enhanced activity in the presence of K(2)HPO(4 )probably takes place because the orthophosphate anion affects the binding and catalytic residues of endoxylanase.     

Surface-enhanced Raman spectroscopy of biopolymers: membrane proteins, bacteriorhodopsin and rhodopsin adsorbed on silver electrodes and silver hydrosols

Surface-enhanced Raman (SER) spectra of purple membranes of Halobacterium halobium and photoreceptor disks of the rod outer segments adsorbed on silver hydrosols were analysed. It has been shown that the intensity of SER spectra of bacterial and visual rhodopsins increases 5 X 10(4) times at adsorption. Concentration relationship of the signal intensity of SER spectra has the maximum at bacteriorhodopsin concentration about 2 X 10(-7) M. It has been shown that adsorption on silver hydrosol leads to fixation of light-induced photochemical transformations in bacterial and visual rhodopsins. Adsorption on the "smooth" electrodes at the potential of the zero charge of silver does not affect the photocycle of bacteriorhodopsin. An increase or decrease of the electrode potential relative to the zero charge point of silver leads to the accumulation of kinetic intermediate K610 and a decrease of the concentration of the form BRh570. It has been shown that on the "smooth" electrode primarily the long-range component of the SER mechanism is realized. Bands corresponding to the vibrations of the atom groups directly contacting with the metal are mainly intensified after redox cycle which increases the concentration of chemosorption centres. A conclusion is drawn that the method of SER spectroscopy of biomolecules adsorbed on "smooth" electrodes, permits obtaining information similar to that obtained from the analysis of Raman spectra of unadsorbed molecules, but at concentrations by two orders less. Adsorption on the electrodes treated with the help of redox cycle permits to obtain highly oriented preparations and to study topography of biopolymers in water solutions and suspensions.     

A potential anti-oxidant protein in a ferrous iron-oxidizing Sulfolobus species

Abstract Eight species of halophilic Archaea were tested for the presence of isocitrate lyase activity. High activities (up to 100 nmol min⁻¹ mg protein⁻¹) were detected in Haloferax mediterranei and Haloferax volcanii when grown in medium containing acetate as the principal carbon source. Little activity was found in representatives of the genera Halobacterium and Haloarcula . Isocitrate lyase from Haloferax mediterranei required high potassium chloride concentrations, optimal activity being found at 1.5–3 M potassium chloride and pH 7.0. Replacement of potassium chloride by sodium chloride resulted in much lower activities. Sulfhydryl compounds (cysteine, glutathione) were not stimulatory. In other properties (stimulation by magnesium ions, sensitivity to different inhibitors) the enzyme resembled isocitrate lyases from representatives of the Bacteria and Eucarya.