Flavobacterium meningosepticum peptide:N-glycosidase: influence of ionic strength on enzymatic activity.

Flavobacterium meningosepticum peptide:N-glycosidase-mediated deglycosylation of N-linked glycan strands of glycoproteins has been found to be strongly influenced by the ionic strength of the assay medium. By use of a modification of a previously published assay procedure for quantitative analysis of glycan release we have been able to improve reproducibility and thus to compare the extent of deglycosylation achieved under a variety of conditions of ionic strength. We have observed that enzyme activity is adversely affected by high ionic strength buffers such as those recommended for deglycosylation of various glycoproteins and recommend the use of low ionic strength buffers for routine use.     

Thermodynamics of the interaction of sodium-n-dodecyl sulphate with Aspergillus niger catalase in high ionic strength aqueous solutions

The thermodynamic parameters for the interaction between sodium n-dodecyl sulphate (SDS) and Aspergillus niger catalase in aqueous solution at pH 3.2 and 6.4 have been measured by microcalorimetry and equilibrium dialysis over a range of ionic strength from 0.05 to 0.2 at 25 degrees C. Binding isotherms have been interpreted in terms of theoretical models (Hill equation and Wyman binding potential). The Gibbs energies of interaction become increasingly negative with increase in ionic strength and the entropies of interaction become increasingly positive. The ionic strength dependence of the Gibbs energies are much greater than predicted by the Debye-Hückel limiting law indicating a strongly ionic strength dependent hydrophobic contribution to the interactions.     

Influence of ionic strength on Hoechst 33258 binding with DNA

The binding of Hoechst 33258 with DNA at various ionic strengths of solution and different ligand concentrations has been investigated. Existence of more than one type of interactions of Hoechst 33258 with DNA has been revealed, which were very sensitive to the ionic strength. Hoechst 33258 doesn't show specificity to AT sequences of DNA at low ionic strength. High affinity binding mode becomes obvious at high ionic strength. The values of binding constants and binding site sizes for revealed strong and weak interactions have been determined.     

Ionic Strength-Dependent Physicochemical Factors in Cytochrome c3 Regulating the Electron Transfer Rate

The effect of ionic strength on the macroscopic and microscopic redox potentials and the heme environment of cytochrome c₃ from Desulfovibrio vulgaris Miyazaki F have been investigated by NMR and electrochemical methods. The redox potentials of this tetraheme protein are found to be ionic strength-dependent. Especially, the microscopic redox potentials of hemes 2 and 3 at the fourth reduction step increase significantly with increasing ionic strength, which is in contradiction to the theoretical expectation. The coordinated imidazole proton signals are unaffected by ionic strength. However, the methyl and propionate proton signals of hemes 1 and 4 showed significant ionic strength dependencies that are distinct from those for hemes 2 and 3. This heme classification is the same as that found in the ionic strength dependencies of the microscopic redox potentials at the fourth reduction step. Furthermore, the effect of ionic strength on the electrostatic potentials at the heme irons has been examined on the theoretical basis. The electrostatic potential at heme 4 changes up to 1 M ionic strength, which was not expected from the observations reported on cytochromes so far. These results are discussed in connection with the reported anomalous ionic strength dependency of the reduction rate of cytochrome c₃.     

Transient homodimer interactions studied using the electron self-exchange reaction

Transient homodimer protein interactions have been investigated by analyzing the influence of ionic strength (NaCl) on the electron self-exchange (the bimolecular reaction whereby the two oxidation states of a redox protein interconvert) rate constant (k(ese)) of four plastocyanins. The k(ese) values for the plastocyanins from spinach, Dryopteris crassirhizoma (a fern), and the green alga Ulva pertusa, which possess acidic patches of varying size and locations, increase 190-, 29-, and 21-fold, respectively, at elevated ionic strength (I = 2.03 M). In contrast, the k(ese) for the almost neutral cyanobacterial plastocyanin from Anabaena variabilis exhibits very little dependence on ionic strength. The temperature dependence of the k(ese) for spinach plastocyanin (I = 0.28 M) provides evidence for poor packing at the homodimer interface. Representative structures of the transient homodimers involved in electron self-exchange, which are consistent with fits of the ionic strength dependence of k(ese) to van Leeuwen theory, have been obtained from protein modeling and docking simulations. The Coulombic energy of the docked homodimers follows the order spinach > D. crassirhizoma > U. pertusa > A. variabilis, which matches that of the overall influence of ionic strength on k(ese). Analysis of the homodimer structures indicates that poor packing and high planarity are features of the interface that favor transient interactions. The physiologically relevant Mg2+ ion has a much more pronounced influence on the k(ese) of spinach plastocyanin, which along with the known properties of the thylakoid lumen suggests a biological role for electron self-exchange.     

EFFECT OF SALTS ON THE PHYSICAL AND KINETIC PROPERTIES OF HUMAN PLACENTAL CHOLINE ACETYLTRANSFERASE

Abstract— The effects of salt on the properties of human placental choline acetyltransferase have been examined. Increases in enzyme activity, thermal denaturation and susceptibility to proteolysis can be related to increases in ionic strength, rather than to specific salt effects. Increased ionic strength increases the maximal velocity (K_m) of the reaction, with no change in the kinetic parameter V_max/K_m (choline). The pH-K_m profile, measured over the range of 6.5–8.0, indicates the requirement of a dissociated acidic residue whose pKa is below 7.5 at high ionic strength, and a protonated residue whose pKa is above 7.5 at low ionic strength. It is proposed that the conformation of the enzyme is different at high ionic strength and at low ionic strength, and that these different conformational states of the enzyme result in different rate-determining steps of the reaction.     

Application of high performance liquid chromatography to the analysis of cytoplasmic and nuclear oestrogen receptors

Soluble and nuclear preparations of fresh human endometrium have been analysed by HPLC on a gel permeation column. With high ionic strength eluants recoveries of receptor binding were less than 40% while low ionic strength eluants gave recoveries of 70% or greater. Non-specific binding by endometrial and plasma proteins was largely lost at both high and low ionic strength. Soluble binding components saturable by diethylstilboestrol (DES) were resolved into two components differing in heat lability and response to variations in ionic strength. Molybdate appears to alter the receptor protein rather than the activity of inactivating enzymes. At low ionic strength nuclear binding components saturable by DES could be resolved into two components.     

Proton-induced phase separation in phosphatidylserine/phosphatidylcholine membranes

Effects of ph and ionic strength on phosphatidylserine/phosphatidylcholine mixed membranes prepared on Millipore filter pore surfaces have been studied using spin-labeled phosphatidylcholine. Lowering pH at constant ionic strength and lowering ionic strength at constant pH caused a lateral reorganization of the membrane. The trigger was protonation of the serine carboxyl group which caused solidification of phosphatidylserine molecules in the membrane, leaving a fluid phase consisting mainly of phosphatidylcholine. The appearent pK for the proton-induced phase separation was measured in a wide range of salt concentrations. The ionic strength dependence was satisfactorily explained based on the electrostatic free energy of proton in the field of membrane surface potential. The Gouy-Chapman theory gave a good approximation for the surface potential. The surface pK of phosphatidylserine and phosphatidic acid vesicles was directly measured in various salt concentrations by 31P-NMR and the results confirmed validity of the Gouy-Chapman-type analysis. The lateral reorganization was triggered by electrostatic interaction but the bulk of the stabilization energy for the structural changes would be the gains in intermolecular van der Waals energy due to closer packing of phosphatidylserine on solidification.     

Effect of ionic strength on the regulatory properties of 2-oxoglutarate dehydrogenase complex.

The effects of changing ionic strength on the activity of the 2-oxoglutarate dehydrogenase complex from pig kidney cortex were explored. This enzyme complex is found to be influenced in many ways by the ionic strength of the reaction medium. The enzyme shows an optimum activity at 0.1 M ionic strength. Increase in ionic strength from 0.1 M to 0.2 M resulted in a decrease of S0.5 for 2-oxoglutarate, and in an increase of S0.5 for NAD. Changes in ionic strength over the range of 0.05-0.2 M have little, if any, effect on S0.5 for CoA. The Hill coefficient for 2-oxoglutarate and NAD at 0.2 M ionic strength was 1.0, whereas at 0.05 M ionic strength it was 0.85 and 1.2 for 2-oxoglutarate and NAD, respectively. At 0.05 M ionic strength the pH optimum of the enzyme ranges between 7.4-7.6, but at 0.15 M ionic strength the pH optimum shifts to 7.8. The magnitude of inhibition of enzyme activity by ATP is not influenced by changes in ionic strength in the absence of calcium. However, in the presence of Ca2+, increases in ionic strength lower the inhibitory effects of ATP. The Si0.5 for ATP in both presence and absence of Ca2+ was not affected by changes in ionic strength in the range of 0.1-0.2 M. In contrast, the Sa0.5 for ADP in the absence of Ca2+ decreases as ionic strength increases. In the presence of calcium and 0.2 M ionic strength ADP has no effect on 2-oxoglutarate dehydrogenase complex activity.     

Effect of the filamentous structure of myosin on the actomyosin ATPase activity

The ATPase activities of acto-heavy meromyosin and of acto-myosin minifilaments have been compared under the same conditions at low ATP (0.1 mM) and at several KC1 concentrations. The activities, which are strongly salt-dependent in both systems, have been found to be similar at high ionic strength (about 0.16 M) but different at lower ionic strength (0.06-0.07 M). Under this last condition, the catalytic constants kcat and Km are lower for acto-myosin minifilaments than for acto-heavy meromyosin ATPase. In addition, at low ionic strength, any decrease in the concentration of any of the ionic species (ATP, citrate, etc.) induces an increase in the interaction strength between myosin and actin filaments, as revealed by the Km changes. The presence of the troponintropomyosin complex and of Ca2+ also enhances the strength of this interaction. On the other hand, the occurrence of particular interactions between F-actin and myosin minifilaments is further substantiated by the phenomenon of superprecipitation which occurs when the ATP concentration decreases. The favourable effect of the organized structure of the myosin minifilaments on the ATPase activity of actomyosin is discussed.     

Electrophoresis of absorbed RNA

The electrophoretic mobilities of adsorbed yeast ribonucleic acid have been measured as functions of pH, ionic strength, and biopolymer concentration and the results so obtained have been critically compared with those for adsorbed DNA. Like DNA, ribonucleic acid has also been found to reverse the positive charge of alumina owing to its adsorption on the solid-liquid interface. The mobilities of adsorbed RNA have been found to be less than those of adsorbed DNA under identical conditions. The observed mobilities of adsorbed heat- and alkali-denatured RNA are significantly less than those of adsorbed native RNA at a given pH and ionic strength of the medium. The electrophoretic mobilities as observed also show the evidence of RNA adsorption on the negatively charged surface of Dowex-50 resin, but practically no adsorption of RNA on the negatively charged glass surface has been predicted.     

ELECTROPHORETIC STUDIES ON HUMAN RED BLOOD CELLS

1. The electrophoretic mobility of unhemolyzed human red cells has been determined as a function of ionic strength at approximately constant pH in isotonic mixtures of glucose solution and saline-phosphate buffer solution. 2. Above an ionic strength of about 0.02 the cells behave as particles with a smooth surface of large radius of curvature. Below an ionic strength of about 0.02, changes of the surface occur, probably involving a decrease of charge density and perhaps connected with injury of the surface. 3. The mobility as a function of pH at an ionic strength of 0.172 has been determined for human red cells, for the lipid extract of the cells, and for the stroma protein of the cells. The isoelectric points of cells, lipid, and protein have been found to be about 1.7, 2.6, and 4.7 respectively. 4. The pH-mobility data lead to the conclusion that a red cell surface is composed largely of lipid and dominated by strong acid groups, possibly the phosphoric acid groups of cephalin molecules.     

Nuclear magnetic resonance studies on the solution conformation of histone IV fragments obtained by cyanogen bromide cleavage.

Two histone IV fragments obtained by cleavage at Met-84 by cyanogen bromide have been examined by proton magnetic resonance (PMR) spectroscopy as a function of temperature, peptide concentration, ionic strength, and pD. Sedimentation and gel electrophoresis studies on these peptides have also been carried out. The 220-MHz PMR spectrum of the N-peptide in both the high- and low-field regions was shown to be almost identical with that calculated for an extended coil N-peptide monomer. The calculated random coil and experimental C-peptide spectra, on the other hand, differ in many respects. Evidence was obtained for the presence of rigid secondary structure in the C-peptide. In addition, the Val, Leu, Ile CH3 resonance displays a prominent high-field satellite band which shifts downfield with increasing temperature. Sedimentation studies on the N-peptide reveal the formation of extremely large, remarkably homogeneous aggregates at ionic strengths larger than or equal to 0.01. The C-peptide, on the other hand, does not appear to form aggregates of sufficient size to be detectable in velocity sedimentation studies of a few hours duration. The relative area changes which have previously been noted in the PMR spectrum of histone IV with increasing ionic strength were also observed for the N-peptide but not the C-peptide. Interpretation of these relative area changes has been made in terms of the amino acid sequence of histone IV, and an effort was made to identify that segment of the polypeptide which undergoes secondary structural change with increasing ionic strength.     

Bovine brain phosphatidylinositol transfer protein. Effects of pH, ionic strength and lipid composition on transfer activity

Phosphatidylinositol and phosphatidylcholine are transferred between bilayer membranes in the presence of a specific phosphatidylinositol transfer protein isolated from bovine brain. The effects of pH, ionic strength and lipid composition on the rate of transfer of these phospholipids between small unilamellar vesicles have been investigated. At low ionic strength, phosphatidylinositol transfer between vesicles prepared from phosphatidylcholine and 5 mol% phosphatidylinositol was maximal at about pH 5 and moderately dependent on hydrogen ion concentration in more alkaline regions. A similar dependence on pH was noted for phosphatidylcholine transfer between membranes containing phosphatidylcholine or mixtures of phosphatidylcholine and 5 mol% phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, phosphatidylethanolamine or stearylamine. The rate of transfer between anionic vesicles was somewhat higher than that between neutral or cationic vesicles. At higher ionic strength the transfer reactions in neutral and alkaline regions were less sensitive to pH. Phospholipid transfers between vesicles containing 5 mol% of anionic lipid increased sharply as ionic strength decreased below 0.1. In contrast, phosphatidylcholine transfer between membranes which contained only zwitterionic phospholipids or 5 mol% stearylamine was unaffected by variations of ionic strength. Irrespective of the lipid composition of membranes, pH affected both the apparent Km and Vmax, while ionic strength generally affected the apparent Vmax. These results indicate a significant role of electrostatic interactions in the phospholipid transfer catalyzed by phosphatidylinositol transfer protein.     

Effects of ionic strength,serum protein and surface charge on membrane movements and vesicle production in heated erythrocytes

Morphological changes and fragmentation of human erythrocytes heated at various rates through the spectrin inactivation temperature have been examined by cinephotomicroscopy. Most cells heated in 0.20 ionic strength buffered saline developed a wavy disturbance along the cell rim when heated. Vesicles developed from the crests of the growing waves within 0.3 s of the initiation of a wave when the heating rate was 1°C/s. At an ionic strength of 0.02, only 48% of the cells developed a wave outline. The average number of waves per cell was half that at 0.2 ionic strength. When the cell surface charge was reduced by neuraminidase treatment, only 12% of the cells fragmented. Bovine serum albumin or homologous plasma also reduced fragmentation. The dependence of the wave growth on ionic strength and surface charge was broadly consistent with theoretical predictions for the growth of a displacement instability on a low interfacial tension interface. Attention has been paid to the importance of bending energy in the development of the wave. Where wave development was suppressed, the morphological changes due to heating appeared to involve membrane internalization in the region of the cell dimple.     

Higher-order structures of chromatin in solution.

Neutron scatter studies have been made on gently prepared chicken erythrocyte chromatin over a range of ionic strength. At low ionic strength the mass per unit length of the '10 nm nucleofilament corresponds to one nucleosome per 8--12 nm and a DNA packing ratio of between 6 and 9. From the contrast dependence of the cross-section radius of gyration of the nucleofilament the following parameters have been obtained; RgDNA' the cross-section radius of gyration (Rg) when DNA dominates the scatter; RgP, the cross-section Rg when protein dominates the scatter; Rc, the cross-section Rg at infinite contrast and alpha, the constant which describes the dependence of the cross-section Rg on contrast variation. From our understanding of the structure of the core particle, various arrangement of core particles in the nucleofilament have been tested. In models consistent with the above parameters the core particles are arranged edge-to-edge or with the faces of the core particles inclined to within 20 degrees to the axis of the nucleofilament. With increase of ionic strength the transition to the second-order chromatin structure has been followed. This gave the interesting result that above 20 microM NaCL or 0.4 mM MgCL2 the cross-section Rg increases abruptly to about 9 nm with a packing ratio of 0.2 nucleosome/mn and with further increase of ionic strength the Rg increases to 9.5 nm while the packing ratio increases threefold to 0.6 nucleosome/nm. This suggests a family of supercoils of nucleosomes which contract with increasing ionic strength. In its most contracted form the diameter of the hydrated supercoil has been found from the radial distribution function to be 34 nm. Models for the arrangements of core particles in the 34-nm supercoil are discussed.     

Binding of the Mn(III) complex of meso-tetrakis (4-N-methyl-pyridiniumyl) porphyrin to DNA. Effect of ionic strength

Interactions of the water-soluble Mn(III) complex of meso-tetrakis (4-N-methyl-pyridiniumyl) porphyrin (Mn(III)TMPyP) with DNA in aqueous solutions at low (0.01 M) and high (0.2 M) ionic strengths have been studied by optical absorption, resonance light scattering (RLS) and 1H NMR spectroscopies. Optical absorption and RLS measurements have demonstrated that in DNA solutions at low ionic strength the Mn(III)TMPyP form aggregates, which are decomposed at DNA excess. At high ionic strength the aggregation was not observed. We explain this effect by assuming that upon increase in ionic strength, Mn(III) TMPyP dislocates from the DNA sites, which produces better conditions for the porphyrin aggregation, to sites where the aggregation is hindered. The 1H NMR data demonstrated that the aggregation observed at low ionic strength reduces the paramagnetism of Mn(III)TMPyP. This phenomenon was not observed at the high ionic strength in the absence of aggregation.     

Studies on the binding of FMN by apoflavodoxin from Peptostreptococcus elsdenii, pH and NaCl concentration dependence.

1. The pH and ionic strength dependence of the interaction of FMN with apoflavodoxin has been studied by fluorometry in the pH region 2-5, at 22 degrees C. 2. The rate constant of dissociation and the dissociation constant were experimentally determined; the rate constants of association were claculated at a given pH value. These constants depend on the ionic strength. The plots of these constants against the square root of the ionic strength are straight. 3. Our data have been interpreted in terms of the Br?nsted theory, which relates chemical reaction rates to ionic strength. The data indicate that the apoenzyme reaches its maximum net positive charge at pH 2.0-2.6. The calculated net charge in this pH region is between 11 and 12 and is in agreement with the theoretical value of 12 as deduced from the primary structure of the protein. The isoelectric point of the holoenzyme is about 4. 4. The rate constant of association extrapolated to zero ionic strength is 3.2-10(5)M-1-s-1 and is pH-independent. 5. The rate constant of dissociation and the dissociation constant extrapolated to zero ionic strength depend on the pH. The results are explained by assuming that there are two protein ionizations with a pK value of 3.4; these ionizing groups are possibly close to the FMN binding site.     

Cytochrome c: ion binding and redox properties. Studies on ferri and ferro forms of horse, bovine, and tuna cytochrome c

The ion binding properties of horse, bovine, and tuna cytochrome c (both oxidized and reduced) have been measured using a combination of ultrafiltration, neutron activation, and ion chromatography. The ions investigated were chloride, phosphate, and Tris-cacodylate. Ion chromatography and neutron activation analysis techniques were employed to determine the concentration of free anions. Binding constants are obtained from modified Scatchard plots (in the range of 10-2000 M-1). The redox potentials for cytochrome c at different ionic strengths, pH 7.0, have been determined. In this paper we report the ionic strength and ion binding effects on the redox properties of horse, bovine, and tuna cytochrome c. Potential versus ionic strength dependence for horse, bovine, and tuna cytochrome c from the experimental data were compared with a theoretical model.     

Transient kinetics of electron transfer reactions of flavodoxin: ionic strength dependence of semiquinone oxidation by cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic acid and computer modeling of reaction complexes

Electron transfer reactions between Clostridum pasteurianum flavodoxin semiquinone and various oxidants [horse heart cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic [horse heart cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic acid (EDTA)] have been studied as a function of ionic strength by using stopped-flow spectrophotometry. The cytochrome c reaction is complicated by the existence of two cytochrome species which react at different rates and whose relative concentrations are ionic strength dependent. Only the faster of these two reactions is considered here. At low ionic strength, complex formation between cytochrome c and flavodoxin is indicated by a leveling off of the pseudo-first-order rate constant at high cytochrome c concentration. This is not observed for either ferricyanide or ferric EDTA. For cytochrome c, the rate and association constants for complex formation were found to increase with decreasing ionic strength, consistent with negative charges on flavodoxin interacting with the positively charged cytochrome electron transfer site. Both ferricyanide and ferric EDTA are negatively charged oxidants, and the rate data respond to ionic strength changes as would be predicted for reactants of the same charge sign. These results demonstrate that electrostatic interactions involving negatively charged groups are important in orienting flavodoxin with respect to oxidants during electron transfer. We have also carried out computer modeling studies of putative complexes of flavodoxin with cytochrome c and ferricyanide, which relate their structural properties to both the observed kinetic behavior and some more general features of physiological electron transfer processes. The results of this study are consistent with the ionic strength behavior described above.