Translocation of polysialic acid across model membranes: kinetic analysis and dynamic studies.

Transmembrane translocation of polyion homopolymers takes place in the case of polyanionic polysialic acid (polySia), polyanionic polynucleotides and polycationic polypeptides. The purpose of this work was to determine the role of membrane electrical parameters on the kinetics of polyion translocation, the influence of polysialic acid on ion adsorption on positively charged membrane surface and the dynamics of the phospholipid hydrocarbon chains and choline group by using 1H-NMR. The analysis of polyion translocation was performed by using the electrical equivalent circuit of the membrane for the initial membrane potential equal to zero. The changes in polysialic acid flux was up to 75% after 1 ms in comparison with the zero-time flux. Both a decrease of membrane conductance and an increase of polyion chain length resulted in the diminution of this effect. An increase of praseodymium ions adsorption to positively charged liposomes and an increase of the rate of segmental movement of the -CH2 and -CH3 groups, and the choline headgrup of lipid molecules, was observed in the presence of polySia. The results show that the direction of the vectorial polyion translocation depends both on the membrane electrical properties and the degree of polymerization of the polymer, and that polysialic acid can modulate the degree of ion adsorption and the dynamics of membrane lipids.     

Site binding as a local equilibrium. Mn2+ binding to poly(acrylic acid) as a function of the degree of ionization

Mn2+ binding to poly(acrylic acid) at different degrees of ionization, alpha, has been studied from the frequency dependence of the water protons' relaxation rates T1(-1) and T2(-1). Site binding is treated as an equilibrium with the concentration of free ions at the immediate vicinity (CIV) of the polyion. The CIV is calculated as the solution of the Poisson-Boltzmann equation at the surface of the cylindrical polyion. A single value of K is shown to fit the results at all values of alpha. The amount of site binding is higher than the total amount of condensed divalent counterions predicted for a finite polyion concentration in the presence of monovalent counterions by Manning's theory.     

Transference numbers, polyion mobilities, and charge fractions in aqueous solutions of lithium, sodium, and potassium dextransulfate

Results of Hittorf type transference number measurements are reported for aqueous solutions of lithium, sodium, and potassium dextransulfate (DS) in the concentration range 0.008-0.09 moles of sulfate groups per liter. The results for the polyion transference number are combined with equivalent conductance measurements reported earlier to calculate the polyion equivalent conductance, and the polyion charge fraction based on Wall's method. Our results show that the transference number of this high charge density polyion is larger than unity over the entire concentration range studied, and decreases monotonously with increasing concentration. The calculated charge fraction f of the polyion increases with increasing concentration, and at any concentration/decreases in the order LiDS > NaDS > KDS. A comparison between the conclusions derived from these transport experiments and from both mean and single ion activity measurements in dextransulfate solutions shows the considerable uncertainties involved in either of these methods, and emphasizes the need for the application of a variety of techniques, including spectroscopic techniques, to determine differential ion binding by polyions.     

Electromigration of polyion homopolymers across biomembranes: a biophysical model

The analysis of polyion transmembrane translocation was performed using membrane electrical equivalent circuit. The dependence of polyion flux across membranes on time, membrane electrical conductance, membrane electrical capacitance, degree of polymerization, water solution conductance and applied transmembrane potential is discussed. The changes in polyion flux were up to 88% after 1 ms. Both the increase of polyion chain length and the decrease of membrane conductance resulted in the diminution of this effect. Inversion of flux direction was observed as a result of external potential changes. Reversal curves, representing the values of considered parameters for zero-flux were also shown. The replacement of a polyanion by a polycation of the same chain length resulted in the same shape of the surface plot but with opposite orientation. The analysis describes the effect of transmembrane potential on the translocation rate of polyanionic polysialic acid and polynucleotides, and polycationic peptides across membranes.     

Ultrasonic study of the kinetics of counterion site binding in aqueous solutions of polyelectrolytes

The excess ultrasonic absorption due to counterion binding has been studied as a function of frequency for a series of polysalts in the range 1–150 MHz. All the relaxation spectra can be represented by a relaxation equation with two relaxation terms. The relaxation frequencies appear concentration independent and the relaxation amplitudes seem proportional to concentration. The low frequency relaxation process appears to depend mainly on the nature of the counterion while the high frequency relaxation process seems to be mostly dependent on the nature of the polyion. These results are quite similar to those obtained in ultrasonic studies of ion-pairing in solutions of divalent sulfates. The kinetic model used for the quantitative analysis of these results has been modified for polysalts through introducing the concept of“counterion condensation”. In this modified model the excess absorption is assigned to the perturbation by the ultrasonic waves of the equilibria between the three states of hydration of ths complex formed by a counterion and that part of the polyion where it is bound. Analytical expressions of the relaxation amplitudes have been derived using classical procedures for this modified kinetic model. In the case of cobalt-polyphosphate (Co-PP), the ultrasonic data together with the results of NMR measurements on either Co²⁺ or Co-PP have been used for the evaluation of the volume changes, the rate constants and the fractions of counterions in the three states of hydration involved in the binding equilibria. The volume changes obtained in this manner depend only slightly on the method of calculation and appear to be consistent with volume changes for outer-sphere and inner-sphere complex formation. These results are discussed.     

Structure-activity relationship for extracellular block of K+ channels by tetraalkylammonium ions

External tetraalkylammonium ion binding to potassium channels is studied using microscopic molecular modelling methods and the experimental structure of the KcsA channel. Relative binding free energies of the KcsA complexes with Me4N+, Et4N+, and n-Pr4N+ are calculated with the molecular dynamics free energy perturbation approach together with automated ligand docking. The four-fold symmetry of the entrance cavity formed by the Tyr82 residues is found to provide stronger binding for the D2d than for the S4 conformation of the ligands. In agreement with experiment the Et4N+ blocker shows several kcal/mol better binding than the other tetraalkylammonium ions.     

Estimation of bound quaternary ammoniumion to a rod-like polyion with a large alkyl residue

The amount of quaternary ammonium ion (Bu4N+), which is believed not to be bound to general carboxylpolyelectrolytes, bound to poly(iso BVE-co-MA) was estimated by conductivity measurements. In the region of the density of polyion charge in which the polyions are thought to take a free drain'ng conformation, it has been confirmed that the activity coefficient of Bu4N+ ions is less than 0.5 in the presence of a small amount of Bu4NCl, showing that the force between the counterions and the polyion is probably due to the hydrophobic interaction. Moreover, from the electrophoretic mobility Up of the polyion observed from the data of conductivity, it has been ascertained that Up of this polyion is two times larger than PAA, and the behavior of the quantity e/xiP with changing degree of ionization corresponds to that of the viscosity.     

Raman spectroscopic studies on the interaction between counterion and polyion

The interaction between alkali metal ions and the polyacrylate ion was investigated by means of Raman spectroscopy. in comparison with the Raman spectra of propionate salts. The Raman bands due to the metal-oxygen bond were not apparent and no significant difference was observed among the Raman spectra of several univalent salts of polyacrylate. except in the case of the lithium salt. The apparent degree of dissociation of lithium polyacrylate, as determined from the relative intensity of a specific band characteristic of the carboxylate ion, was lower than those of the other alkali metal salts. It is concluded from the Raman data that the electrostatic interaction between counterions and a polyion is not specifically modified by forces of a nonionic nature. Moreover, it is pointed out that the local conformation of polyacrylate changes gradually with the degree of neutralization, but that the change is not like a conformational transition between globular and random coil forms.     

Ion-binding and conformation of poly-L-methionine S-methylsulfonium salts in added salt systems

In order to study the type of ion binding and the conformation of several salts of poly-L -methionine S-methylsulfonium hydroxide, the viscosity, conductance, counterion activity, and optical rotatory dispersion of the polysalts were measured in systems with a small amount of added salt. It was shown that the ion binding of chloride and bromide salts was of a diffuse binding type due only to the electrostatic potential of the polyion, and that both polysalts underwent no conformational transition by the addition of a simple salt, NaCl for chloride salt and NaBr for bromide salt, and retained a random coil conformation. Iodide and thiocyanate salts showed a conformational change, probably from the random coil into the β form, with increasing concentrations of NaI and NaSCN, respectively. On the other hand, perchlorate salt existed in the α-helix conformation in part even in pure aqueous solution, and the fraction of α-helix increased on the addition of NaClO₄. On considering several possible situations, it is suggested that there is a specific and strong interaction between the polyion and the small counterion in iodide, thiocyanate, and perchlorate salts.     

Crystal structure of the alpha1beta1 integrin I-domain: insights into integrin I-domain function.

The alpha1beta1 integrin is a major cell surface receptor for collagen. Ligand binding is mediated, in part, through a 200 amino acid inserted 'I'-domain contained in the extracellular part of the integrin alpha chain. Integrin I-domains contain a divalent cation binding (MIDAS) site and require cations to interact with integrin ligands. We have determined the crystal structure of recombinant I-domain from the rat alpha1beta1 integrin at 2.2 A resolution in the absence of divalent cations. The alpha1 I-domain adopts the dinucleotide binding fold that is characteristic of all I-domain structures that have been solved to date and has a structure very similar to that of the closely related alpha2beta1 I-domain which also mediates collagen binding. A unique feature of the alpha1 I-domain crystal structure is that the MIDAS site is occupied by an arginine side chain from another I-domain molecule in the crystal, in place of a metal ion. This interaction supports a proposed model for ligand-induced displacement of metal ions. Circular dichroism spectra determined in the presence of Ca2+, Mg2+ and Mn2+ indicate that no changes in the structure of the I-domain occur upon metal ion binding in solution. Metal ion binding induces small changes in UV absorption spectra, indicating a change in the polarity of the MIDAS site environment.     

Conductometric analysis of the competition between monovalent and divalent counterions in their interaction with polyelectrolytes

A procedure is described for the analysis of the conductivity of solutions of anionic polyelectrolytes in which both mono- and divalent counterions are present. The method is based on analysis of the relation between the overall conductivity of the system and the conductivity of the individual monovalent cations which are only electrostatically (non-specifically) bound. The system is described in terms of the two-state approach, implying that the counterions are considered to be either fully bound to the polyion or completely free. The potentialities of the proposed method are explored by studying solutions of alkali polyacrylates with and without added zinc nitrate at several alkali nitrate concentrations. The results give a picture of the composition of the counterionic atmosphere around the polyion in systems with both mono- and divalent counterions present. To a certain degree, the divalent ion Zn(II) was found to be bound quantitatively by the polyion. The composition of the counterionic atmosphere around the polyion was largely independent of alkali nitrate concentration when the latter was present in not too large an excess with respect to both Zn(II) and the charged monomers.     

Postnatal changes in Na,K-ATPase isoform expression in rat cardiac ventricle. Conservation of biphasic ouabain affinity.

The cardiac glycoside sensitivity of the rat heart changes during postnatal maturation and in response to certain pathological conditions. The Na,K-ATPase is thought to be the receptor for cardiac glycosides, and there are three isozymes of its catalytic (alpha) subunit with different cardiac glycoside affinities: alpha 1 (low affinity) and alpha 2 and alpha 3 (high affinity). We examined the developmental expression of the alpha subunit isozymes in rat ventricular membrane preparations by immunoblotting with isozyme-specific antibodies. The alpha 1 isozyme was present throughout all stages of maturation. A developmental switch from alpha 3 to alpha 2 occurred between 14 and 21 days after birth. Measurements of [3H]ouabain binding and inhibition of Na,K-ATPase activity indicated that alpha 2 and alpha 3 should make equivalent contributions to ion pump capacity; in both neonatal natal and adult preparations, ouabain interacted with a single class of high-affinity binding sites (KD = 15 or 40 nM, respectively; Bmax = 4-5 pmol/mg protein), and at low concentrations produced a similar degree of Na,K-ATPase inhibition (25%). The results indicate that the developmental difference in cardiac glycoside sensitivity cannot be explained by quantitative differences in the proportion of high-affinity isozymes of the Na,K-ATPase. The switch from alpha 3 to alpha 2 coincides with other major changes in cardiac electrophysiology and calcium metabolism.     

Acknowledgement

In the series of tetraalkylammonium ions from tetramethylammonium to tetra-n-heptylammonium, tetra-n-pentylammonium ion was a potent protector of both murine L1210 leukemia and bone marrow progenitor cells against mechlorethamine cytotoxicity. It was also a non-competitive inhibitor of choline uptake. Phosphonium analogs of the tetraalkylammonium ions were equal to their corresponding tetraalkylammonium ions in their protection against mechlorethamine cytotoxicity and in their inhibition of choline uptake. Treatment of tumor bearing L1210 leukemia mice with the combination of tetra-n-pentylammonium ion and mechlorethamine resulted in no major improvement in survival of the tumor-bearing mice compared to mechlorethamine treatment alone.     

Quasielastic light scattering by biopolymers. VI. Diffusion of mononucleosomes and oligonucleosomes in the presence of static and sinusoidal electric fields

Quasielastic light scattering and electrophoretic light scattering studies were carried out on mononucleosome and oligonucleosome systems. The electrophoretic light scattering experiments employed static and sinusoidal electric fields. Data are presented that suggest at least two relaxation modes. It is proposed that the small amplitude sinusoidal field effectively polarizes the ion atmosphere about the polyion, thus leading to an induced dipole moment that varies sinusoidally in time. This model is, in essence, an extension of the current interpretation of low-frequency dielectric dispersion data on DNA as being due to fluctuations of counterions along the polyion.     

The interaction of sodium alginate with univalent cations

The M/G ratio, dyad and triad frequencies in the sodium alginate chain, were determined from ¹³C-nmr spectra. The interactions of sodium alginate in solution with the univalent cations K⁺ ion and Na⁺ ion have been investigated by viscometry and membrane osmometry. The dependencies of intrinsic viscosity, Huggins constant, and second virial coefficient on ionic strength were observed, and the maximums in reduced viscosity were obtained in low KCl and NaCl concentrations, respectively. These show that the electroviscous effects play an important role in polyelectrolyte solution, and the effect of the Na⁺ ion on aqueous solution of sodium alginate is greater than the K⁺ ion. The experimental observations are interpreted in terms of ion-pair formation with carboxyl groups of mannuronate and isolated guluronate residues and cooperation “egg-box” binding between polyguluronate chain sequence. The difference of interaction between univalent cations and alginate chains in solution is attributed to the ability of their binding with the polyion, which depends on the properties of ions itself. © 1998 John Wiley & Sons, Inc. Biopoly 46: 395–402, 1998     

NMR-based localization of ions involved in salting out of hen egg white lysozyme

NaCl-induced aggregation of hen egg white lysozyme (HEWL) was monitored by NMR spectroscopy. Small, but significant, changes induced by salt addition in TOCSY spectra were attributed to the effect of local reorganization of protein backbone upon ion binding. Salt-induced variations in HN and H alpha chemical shifts were mapped on the HEWL 3D structure which allowed the construction of a scheme of the spatial localization of potential ion binding sites. It was found that in a 0.5 M NaCl solution six chloride anions and at least one sodium cation are bound to preferred sites on the HEWL surface.     

Flash chronopotentiometric sensing of the polyions protamine and heparin at ion-selective membranes

We report here on a highly sensitive and rapid detection technique, multipulse flash chronopotentiometry, for the anticoagulant polyion heparin and its antidote protamine. The technique is based on a localized titration of the polyions at the surface of an appropriately formulated polymeric ion-selective membrane devoid of ion exchange properties to prohibit spontaneous extraction processes. A defined ion flux from the sample side to the membrane is induced electrochemically by applying a current pulse of appropriate amplitude and sign. The resulting depletion of the measured ions at the membrane surface gives rise to a characteristic limiting current or transition time and is observed as an inflection point in the resulting chronopotentiogram. The limiting current and the square root of the transition time are linear functions of the concentration of the polyion and yield sensitive and rapid analytical information attractive for clinical diagnostics applications. The polyion protamine is detected in 10-fold diluted blood samples in a matter of seconds via a cathodic current pulse. The utility of the technique for monitoring heparin/protamine titrations in physiological saline solutions is demonstrated.     

Interaction of Calcium Ion with Bovine Caseins

In order to clarify the interaction of calcium ion with casein, the volume change associated with the interaction was measured by dilatometric procedures. When CaCl₂ was added to the casein solutions at neutral pH, a volume increase occurred and reached a constant saturated value of about 700 ml per 10⁶ g protein with increasing CaCl₂ concentrations for whole-, α_s- and β-casein solutions, but there was no volume change for κ-casein solution. On the other hand, the binding of calcium ion to the casein fractions was determined by a gel filtration procedure at pH 6.0 to 9.0. The number of Ca²⁺ ions bound to the caseins increased with the CaCl₂ concentration and pH value, and the relative order of binding capacities for the caseins was: α_s-casein > whole-casein > β-casein > κ-casein.

It was found that the volume changes obtained by the dilatometry were smaller than the calculated volume increases based on the assumption that these are caused by the binding of Ca²⁺ ion to the caseins. Therefore it is necessary to introduce another factor which reduces the volume increase due to the Ca²⁺ ion binding in order to reasonably explain the measured volume changes. At present it is presumed that there occurs the unfolding of peptide chain of casein molecule on Ca²⁺ ion binding, which has been known to decrease the volume of the protein solution.     

Theory of the delocalized binding of Mg(II) to DNA: Preliminary analysis for low binding levels

A simple theoretical equation for the binding of Mg²⁺ to DNA in the presence of excess l : l salt is derived from a model that does not specify discrete binding sites but rather allows the associated metal ions to move freely near the surface of the DNA polyion. Use of a numerical value for the free volume, determined uniquely, in a separate communication, by a free energy minimization, leads to predicted values for the Mg²⁺binding constant that are in essential agreement with measured values taken from the literature.