Direct visualization of changes in deacylated Na(+) gellan polymer morphology during the sol-gel transition

Changes in gellan polymer morphology during the sol-gel transition were directly visualized by transmission electron microscopy and a model incorporating these changes and existing physical data is proposed. Our observations suggest that the most thermodynamically stable conformations of gellan polymers in solution, in the absence of added cations, are the double helix and double-helical duplexes. We have demonstrated two forms of lateral aggregation of gellan helices in the presence of Ca(2+) and K(+) ions. One type forms junction zones that lead to network formation and gelation, while the second type leads to the formation of isolated fibers of aggregated helices and inhibition of gelation. The proposed model of gellan gelation is based on these observations where thermoreversibility, gel strength, and endothermic transitions of gellan gels can be explained.     

Effects of Potassium Chloride and Sodium Chloride on the Thermal Properties of Gellan Gum Gels

The exothermic and endothermic peaks in cooling and heating curves of differential scanning calorimetry (DSC) for gellan gum gels without and with potassium chloride and sodium chloride were analyzed. The gelling and melting temperatures shifted to higher temperatures with increasing gellan and salt concentration in the concentration range of gellan from 0.3 to 2.0% (w/w). The exothermic and endothermic enthalpy increased with increasing gellan and salt concentrations. Cooling DSC curves showed one exothermic peak for samples with salts and at low gellan concentration. Heating DSC curves showed many peaks for all samples except 0.3% (w/w) gellan gum gels. The sol-gel transition of samples was examined numerically by using a zipper model approach. The introduction of cations increases the number of junction zones or zippers and decreases the rotational freedom of parallel links. This makes the structure of junction zones more heat resistant, and increases the elastic modulus of the gel.     

Effect of divalent metal cations on the dimerization of OXA-10 and -14 class D beta-lactamases from Pseudomonas aeruginosa

The factors influencing the oligomerization state of OXA-10 and OXA-14 class D beta-lactamases in solution have been investigated. Both enzymes were found to exist as an equilibrium mixture of a monomer and dimer, with a K(d) close to 40 microM. The dimeric form was stabilized by divalent metal cations. The ability of different metal ions to stabilize the dimer was in the following order: Cd(2+) > Cu(2+) > Zn(2+) > Co(2+) > Ni(2+) > Mn(2+) > Ca(2+) > Mg(2+). The apparent K(d)s describing the binding of Zn(2+) and Cd(2+) cations to the OXA-10 dimer were 7.8 and 5.7 microM, respectively. The metal ions had a profound effect on the thermal stability of the protein complex observed by differential scanning calorimetry. The enzyme showed a sharp transition with a T(m) of 58.7 degrees C in the absence of divalent cations, and an equally sharp transition with a T(m) of 78.4 degrees C in the presence of a saturating concentration of the divalent cation. The thermal transition observed at intermediate concentrations of divalent metal ions was rather broad and lies between these two extremes of temperature. The equilibrium between the monomer and dimer is dependent on pH, and the optimum for the formation of the dimer shifted from pH 6.0 in the absence of divalent cations to pH 7.5 at saturating concentrations. The beta-lactamase activity increased approximately 2-fold in the presence of saturating concentrations of zinc and cadmium ions. Reaction with beta-lactams caused a shift in the equilibrium toward monomer formation, and thus an apparent inactivation, but the divalent cations protected against this effect.     

Structural transition from the random coil to quadruplex of AG(3)(T(2)AG(3))(3) induced by Zn(2+)

Structures of G-quadruplex DNAs can be typically stabilized by monovalent cations such as K(+), Na(+). Some divalent and trivalent cations, such as Sr(2+), Pb(2+), Tb(3+) and Eu(3+), can also induce the formation of G-quadruplex DNA. Here we show that Zn(2+) can induce the human telomeric sequence AG(3)(T(2)AG(3))(3) to fold the G-quadruplex structure by UV absorbance difference spectra and circular dichroism (CD) spectroscopy. At micromolar concentrations, the Zn(2+)-induced changes in the UV absorbance difference spectra and CD spectra are the characteristics of antiparallel G-quadruplexes although the long wavelength CD maximum is around 285 nm rather than the typical value of 295 nm. The binding stoichometry of Zn(2+) per one AG(3)(T(2)AG(3))(3) molecule is four. To our knowledge, the structural transition of human telomeric sequence induced by Zn(2+) was observed for the first time.     

Rapid purification of commercial gellan gum to highly soluble and gellable monovalent cation salts

Commercial gellan gum contains divalent cation contaminants (mainly Ca²⁺ and Mg²⁺) in levels sufficient to neutralize over one-third of its car☐yl groups. Consequently, in order to dissolve gellan gum in water, the mixtures must be heated to more than 90°C. This has prevented applications of gellan gum to such uses as the immobilization of viable enzymes and cells in beads. A rapid two-step method is described here to purify commercial gellan gum to the monovalent cation salts in an overall yield of 85%, through the intermediate free acid form. The gellan monovalent cation salts were highly soluble at temperatures as low as 5°C, and readily gelled upon exposure to solutions of divalent cations. Laboratory-scale preparations of 100 g of gellan monovalent cation salts were readily achieved in a day.     

The reinforcement of gellan gel network by the immersion into salt solution

The effect of immersion into salt solutions on rheological properties of gellan gels was investigated. The storage Young's modulus of gellan gels increased with time during the immersion into salt solutions. The increase of the storage Young's modulus can not be explained solely by change in the concentration of gellan. The ellipticity at 202 nm decreased by the immersion, suggesting the formation and aggregation of gellan helices. It was considered that during immersion cations penetrated into gellan gels to induce the formation and aggregation of gellan helices in gels, resulting in reinforcement of the gel network.     

Effect of pH on the conformation of gellan chains in aqueous systems

The optical rotation and fluorescence anisotropy for gellan aqueous systems were measured at pH 4, 7, and 10 to elucidate the effect of pH on the conformation of gellan chains. The optical rotation study suggests that pH affects the conformation of helical gellan chains and their aggregation behavior but the coil-helix transition temperature. By comparing the chain mobility estimated from the fluorescence anisotropy between different pH conditions, it has been revealed that the degree of expansion of random-coiled gellan chains varies with pH. These results indicate that the effect of pH is explained by the change in the anionic nature of gellan chains rather than in the shielding effect of hydrogen ions surrounding gellan chains as a cation species.     

Kinetics and thermodynamics of thermal denaturation in acyl carrier protein.

The denaturation of Escherichia coli acyl carrier protein (ACP) in buffers containing both monovalent and divalent cations was followed by variable-temperature NMR and differential scanning calorimetry. Both high concentrations of monovalent salts (Na+) and moderate concentrations of divalent salts (Ca2+) raise the denaturation temperature, but calorimetry indicates that a significant increase in the enthalpy of denaturation is obtained only with the addition of a divalent salt. NMR experiments in both low ionic strength monovalent buffers and low ionic strength monovalent buffers containing calcium ions show exchange between native and denatured forms to be slow on the NMR time scale. However, in high ionic strength monovalent buffers, where the temperature of denaturation is elevated as it is in the presence of Ca2+, the transition is fast on the NMR time scale. These results suggest that monovalent and divalent cations may act to stabilize ACP in different ways. Monovalent ions may nonspecifically balance the intrinsic negative charge of this protein in a way that is similar for native, denatured, and intermediate forms. Divalent cations provide stability by binding to specific sites present only in the native state.     

螺旋脂质体的研究——Ⅰ.含有心磷脂的两相体系形成螺旋脂质体的条件

用CL(心磷脂)与DMPC(二肉豆蔻酰磷脂酰胆碱)或DPPC(二棕榈酰磷脂酰胆碱)所组成的两组体系制备脂质体,可形成少量管状脂质体.加Ca~(2+)或其它二价阳离子后可形成单股或双股螺旋.对产生这类螺旋脂质体的各种条件进行了研究.     

Sol–Gel transition of alginate solution by the addition of various divalent cations: A rheological study

Several rheological properties of aqueous alginate solution have been investigated in the absence and presence of four divalent cations: Ca, Cu, Mn, and Co. The concentration dependence of viscosity in alginate solution without addition of divalent cations shows a similar behavior to that found for other polysaccharides and synthetic polymers. We have found that for Ca(II) -added systems, sol-gel transition curves for different alginate concentrations can be superimposed over the concentration range of this study by using a normalized parameter. Shear-thinning behavior has been observed for all the alginate solutions by addition of four divalent cations over sol-gel transition process. The values of exponent b in the power-law relation $ {\eta \propto \dot \gamma }$ were found in the range of ?0.3 ～ ?0.6. It is suggested that a considerable portion of divalent cations remains in a cross-linking state even under shear flow. The results obtained from thixotropy measurements indicate the existence of a major framework formed in the gel-like and well-gelled samples, which can be maintained until shear rate (or shear stress) reaches a threshold value. © 1994 John Wiley & Sons, Inc.     

Purification and characterization of arginine kinase from locust

L-Arginine kinase (AK; ATP:L-arginine N-phosphotransferase; EC 2.7.3.3) catalyzes the reversible transphosphorylation between N-phospho-L-arginine (PArg) and ATP thus buffering cellular ATP levels. AK was purified from the leg muscle of the locust Migratoria manilensis by Sephacryl S-200 HR gel filtration chromatography and DEAE Sepharose CL-6B fast flow anion exchange chromatography to an apparent homogeneity with a recovery of 80%. The enzyme behaved as monomeric protein with molecular mass of about 40 kD, and had a pH and temperature optimum of 8.6 and 30 degrees C, respectively, and a pI of about 6.3. The Michaelis constants for synthesis of PArg are 0.936 and 1.290 mM for L-arginine and ATP, respectively and k(cat)/K(m)(Arg) 174. The activity of AK required divalent cations such as Mg(2+) and Mn(2+). In the presence of Cu(2+) and Zn(2+), AK activity was greatly inhibited. The intrinsic protein fluorescence emission maximum at 330 nm using the excitation wavelength at 295 nm suggested that tryptophan residues are below the surface of the protein and not exposed to solvent.     

Conformational changes in surface structures of isolated connexin 26 gap junctions

Gap junction channels mediate communication between adjacent cells. Using atomic force microscopy (AFM), we have imaged conformational changes of the cytoplasmic and extracellular surfaces of native connexin 26 gap junction plaques. The cytoplasmic domains of the gap junction surface, imaged at submolecular resolution, form a hexameric pore protruding from the membrane bilayer. Exhibiting an intrinsic flexibility, these cytoplasmic domains, comprising the C-terminal connexin end, reversibly collapse by increasing the forces applied to the AFM stylus. The extracellular connexon surface was imaged after dissection of the gap junction with the AFM stylus. Upon injection of Ca(2+) into the buffer solution, the extracellular channel entrance reduced its diameter from 1.5 to 0.6 nm, a conformational change that is fully reversible and specific among the divalent cations tested. Ca(2+) had a profound effect on the cytoplasmic surface also, inducing the formation of microdomains. Consequently, the plaque height increased by 0.6 nm to 18 nm. This suggests that calcium ions induce conformational changes affecting the structure of both the hemichannels and the intact channels forming cell-cell contacts.     

A structural basis for Mg2+ homeostasis and the CorA translocation cycle

We describe the CorA Mg(2+) transporter homologue from Thermotoga maritima in complex with 12 divalent cations at 3.7 A resolution. One metal is found near the universally conserved GMN motif, apparently stabilized within the transmembrane region. This portion of the selectivity filter might discriminate between the size and preferred coordination geometry of hydrated substrates. CorA may further achieve specificity by requiring the sequential dehydration of substrates along the length of its approximately 55 A long pore. Ten metal sites identified within the cytoplasmic funnel domain are linked to long extensions of the pore helices and regulate the transport status of CorA. We have characterized this region as an intrinsic divalent cation sensor and provide evidence that it functions as a Mg(2+)-specific homeostatic molecular switch. A proteolytic protection assay, biophysical data, and comparison to a soluble domain structure from Archaeoglobus fulgidus have revealed the potential reaction coordinate for this diverse family of transport proteins.     

The effect of temperature on DNA structural transitions under the action of Cu2+ and Ca2+ ions in aqueous solutions

The work examines the structural transitions of DNA under the action of Cu2+ and Ca2+ ions in aqueous solution at temperatures of 29 and 45 degrees C by ir spectroscopy. Upon binding to the divalent ions studied, DNA transits into the compact state both at 29 and 45 degrees C. In the compact state DNA remains in B-form limits. The compaction process is of high positive cooperativity. As temperature increases the divalent metal ion concentration required to induce DNA compaction decreases in the case of Cu(2+)-induced compaction and increases in the case of Ca(2+)-induced compaction. It is suggested that the mechanism of the temperature effect on DNA compaction in the presence of Cu2+ ions possessing higher affinity for DNA bases differs from that of the temperature influence on Ca(2+)-induced DNA compaction. In the case of copper ions the determining factor is the increase of binding constants of the Cu2+ ions interacting with the denatured parts formed on DNA while in the case of calcium ions it is the decreased screening action of counterions upon the increase of their hydration with temperature. The efficiency of divalent metal ions studied in inducing DNA compaction depends on hydration of counterions. DNA compaction occurs in a narrow interval of Cu2+ concentrations. As the Cu2+ ion concentration increases, DNA compaction is replaced with Cu(2+)-induced DNA aggregation. At elevated temperatures Cu(2+)-induced DNA compaction could acquire a phase transition character.     

A Fourier transform infrared spectroscopic study of the interaction of alkaline earth cations with the negatively charged phospholipid 1, 2-dimyristoyl-sn-glycero-3-phosphoglycerol

The interaction of aqueous phospholipid dispersions of negatively charged 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol, sodium salt (DMPG) with the divalent cations Mg(2+), Ca(2+) and Sr(2+) at equimolar ratios in 100 mM NaCl at pH 7 was investigated by Fourier transform infrared spectroscopy. The binding of the three cations induces a crystalline-like gel phase with highly ordered and rigid all-trans acyl chains. These features are observed after storage below room temperature for 24 h. When the gel phase is heated after prolonged incubation at low temperature phase transitions into the liquid crystalline phase are observed at 58 degrees C for the DMPG:Sr(2+), 65 degrees C for the DMPG:Mg(2+), and 80 degrees C for the DMPG:Ca(2+) complex. By subsequent cooling from temperatures above T(m) these complexes retain the features of a liquid crystalline phase with disordered acyl chains until a metastable gel phase is formed at temperatures between 38 and 32 degrees C. This phase is characterized by predominantly all-trans acyl chains, arranged in a loosely packed hexagonal or distorted hexagonal subcell lattice. Reheating the DMPG:Sr(2+) samples after a storage time of 2 h at 4 degrees C results in the transition of the metastable gel to the liquid crystalline phase at 35 degrees C. This phase transition into the liquid crystalline state at 35 degrees C is also observed for the Mg(2+) complex. However, for DMPG:Mg(2+) at higher temperatures, a partial recrystallization of the acyl chains occurs and the high temperature phase transition at 65 degrees C is also detected. In contrast, DMPG:Ca(2+) exhibits only the phase transition at 80 degrees C from the crystalline gel into the fluid state upon reheating. Below 20 degrees C, the rate of conversion from the metastable gel to a thermodynamically stable, crystalline-like gel phase decreases in the order Ca(2+)&z. Gt;Mg(2+)>Sr(2+). This conversion into the crystalline gel phase is accompanied by a complete dehydration of the phosphate groups in DMPG:Mg(2+) and by a reorientation of the polar lipid head groups in DMPG:Ca(2+) and in DMPG:Sr(2+). The primary binding sites of the cations are the PO(2)(-) groups of the phosphodiester moiety. Our infrared spectroscopic results suggest a deep penetration of the divalent cations into the polar head group region of DMPG bilayers, whereby the ester carbonyl groups, located in the interfacial region of the bilayers, are indirectly affected by strong hydrogen bonding of immobilized water molecules. In the liquid crystalline phase, the interaction of all three cations with DMPG is weak, but still observable in the infrared spectra of the DMPG:Ca(2+) complex by a slight ordering effect induced in the acyl chains, when compared to pure DMPG liposomes.     

Progress towards a DNA-based model system for the study of electron spin-spin interactions

A DNA-based model system is described for studying electron spin-spin interactions between a paramagnetic metal ion and a nitroxide spin label. The modified base deoxythymidine-EDTA (dT-EDTA) chelates the divalent or trivalent metal ion and produces a new feature in the circular dichroism (CD) spectra that makes it possible to monitor local DNA melting. Based on the results of optical and electron paramagnetic resonance (EPR) experiments, we find that the terminus of the DNA duplex that incorporates dT-EDTA and the spin-label melts at a higher temperature than the rest of the DNA duplex. EPR microwave progressive power saturation experiments performed at 77 K are consistent with the specific binding of Dy(III) at the EDTA site and an intramolecular dipole-dipole interaction between the nitroxide spin-label and the chelated Dy(III). This model system should be suitable for studying the relaxation properties of metal ions by saturation-recovery EPR.     

Effect of divalent cations on antiparallel G-quartet structure of d(G4T4G4)

The thermodynamic parameters of an antiparallel G-quartet formation of d(G4T4G4) with 1 mM divalent cation (Mg(2+), Ca(2+), Mn(2+), Co(2+), and Zn(2+)) were obtained. The thermodynamic parameters showed that the divalent cation destabilizes the antiparallel G-quartet of d(G4T4G4) in the following order: Zn(2+)>Co(2+)>Mn(2+)>Mg(2+)>Ca(2+). In addition, a higher concentration of a divalent cation induced a transition from an antiparallel to a parallel G-quartet structure. These results indicate that these divalent cations are a good tool for regulating the G-quartet structures.     

Effect of competing self-structure on triplex formation with purine-rich oligodeoxynucleotides containing GA repeats.

Competition between triplex formation with double-stranded DNA and oligonucleotide self-association was investigated in 23mer GA and GT oligonucleotides containing d(GA)5 or d(GT)5 repeats. Whereas triplex formation with GT oligonucleotides was diminished when temperature increased from 4 to 37 degrees C, triplex formation with GA oligonucleotides was enhanced when temperature increased within the same range due to the presence of competing intermolecular GA oligonucleotide self-structure. This self-structure was determined to be a homoduplex stabilized by the internal GA repeats. UV spectroscopy of these homoduplexes demonstrated a single sharp transition with rapid kinetics (Tm = 38.5-43.5 degrees C over strand concentrations of 0.5-4 microM, respectively, with transition enthalpy, delta H = -89 +/- 7 kcal/mol) in 10 mM MgCl2, 100 mM NaCl, pH 7.0. Homoduplex formation was strongly stabilized by multivalent cations (spermine > Mg2+ = Ca2+) and destabilized by low concentrations of monovalent cations (K+ = Li+ = Na+) in the presence of divalent cations. However, unlike GA or GT oligonucleotide-containing triplexes, the homoduplex formed even in the absence of multivalent cations, stabilized by only moderate concentrations of monovalent cations (Li+ > Na+ > K+). Through the development of multiple equilibrium states and the resulting depletion of free oligonucleotide, it was found that the presence of competing self-structure could decrease triplex formation under a variety of experimental conditions.     

Thermodynamic parameters associated with the binding of adrenalin and norephedrine to heparin

Pulse radiolysis has been used to determine the thermodynamic parameters (delta G', delta H' and delta S') governing the binding of adrenalin and norephedrine to heparin. These complexes were completely dissociated by increasing concentrations of inorganic salts. Lower concentrations of divalent cations (e.g. Ca2+) than of monovalent cations (e.g. Na+) were necessary to effect dissociation of the complex. For each interaction an increase in drug binding occurred as the temperature was increased from ambient. However, a transition temperature was observed (48 degrees C) above which the drug was progressively released as the temperature was increased. These observations are discussed in terms of conformational changes induced in the polymer below and above its melting temperature.