High Ionic Strength Conditions Research Articles

The multisubunit acetyl-CoA carboxylase is strongly associated with the chloroplast envelope through non-ionic interactions to the carboxyltransferase subunits

The committed step for de novo fatty acid biosynthesis is the carboxylation of acetyl-CoA catalyzed by acetyl-CoA carboxylase (ACCase). Plastidial ACCase from most plants is a multisubunit complex composed of multiple copies of four different polypeptides, biotin carboxyl carrier protein (BCCP), biotin carboxylase (BC), and carboxyltransferase (α-CT and β-CT). Immunoblot analyses revealed these four proteins were mostly (69% of total) associated with a 17,000 g insoluble fraction from lysed pea chloroplasts. Under the same conditions only 8% of ribulose-1,5-bisphosphate carboxylase was associated with this insoluble fraction. BCCP and biotin carboxylase BC subunits freely dissociated from 17 kg insoluble fractions under high ionic strength conditions, whereas α-CT and β-CT subunits remained tightly associated. Both CT subunits were highly enriched in envelope versus stroma and thylakoid preparations whereas BC and BCCP subunits were predominantly stromal-localized due to partial dissociation. Rapid solubilization of intact chloroplasts with Triton X-100 followed by centrifugation at 30 kg resulted in a pellet that was up to 8-fold enriched in ACCase activity and 21-fold enriched in BC activity. Triton-insoluble 30 kg pellets were reduced in lipid and chlorophyll content but enriched in chloroplast DNA due to the isolation of nucleoid particles. However, ACCase was not directly associated with nucleoids since enzymatic digestion of DNA or RNA had no effect on the association with Triton-insoluble matter. The amount of Triton-insoluble ACCase was similar in chloroplasts isolated from dark- or light-adaptated leaves suggesting transitory starch granules were also not involved in this association. It is proposed that ACCase is associated with envelope membranes through interactions with an unidentified integral membrane protein.

Oscillatory Peroxodisulfate Reduction on Pt and Au Electrodes under High Ionic Strength Conditions, Caused by the Catalytic Effect of Adsorbed OH

Four electrochemical oscillations of different types (named oscillations α, β, γ, and δ), which are different from previously reported Frumkin-type oscillations in low ionic strength electrolytes, appear for peroxodisulfate (S2O82-) reduction on Pt and Au electrodes under high ionic strength conditions. Impedance measurements for oscillation γ that appears in a region of the most positive potentials have shown that it is classified as hidden negative differential resistance (hidden-NDR or HNDR) oscillators. Detailed electrochemical measurements have suggested that the NDR arises from a decrease (with a negative potential shift) in the surface coverage of adsorbed OH, which acts as a catalyst for the dissociative adsorption of S2O82- (the first step of its reduction). The NDR is hidden by the desorption (with the negative potential shift) of adsorbed sulfate (SO42-) produced by the dissociative adsorption and reduction of S2O82-, which works as a site-blocking agent. Mathematical simulation based on this m...

Settling velocity of a sodium montmorillonite floc under high ionic strength

The settling velocity of a single floc of sodium montmorillonite coagulated under a condition of high ionic strength was analyzed as a function of floc diameter to determine the fractal dimension of the floc. The obtained values of the fractal dimension were found to be approximately 2.0 irrespective of ionic strength and pH values. This result clearly demonstrated that the difference of the viscosity of coagulated montmorillonite suspension against ionic strength earlier reported is not affected by the difference of the fractal structure of a floc but by that of the attractive force between individual clay particles in a floc.

Characterization of Hydride Transfer to Flavin Adenine Dinucleotide in Neuronal Nitric Oxide Synthase Reductase Domain: Geometric Relationship between the Nicotinamide and Isoalloxazine Rings

Based on the similarity in both structure and function of the reductase domain of neuronal nitric oxide synthase (nNOSred) to that of NADPH-cytochrome P450 reductase (CPR), we determined whether the characteristics of hydride transfer from NADPH to flavin adenine dinucleotide (FAD) were similar for both proteins. Secondly, we questioned whether hydride transfer from NADPH to either nNOSred or holo-nNOS was rate limiting for reactions catalyzed by these two proteins. Utilizing 500 MHz proton NMR and deuterated substrate, we determined that the stereospecificity of hydride transfer from NADPH and the conformation of the nicotinamide ring around the glycosidic bond were similar between CPR and nNOSred. Specifically, nNOSred abstracts the A-side hydrogen from NADPH, and the nicotinamide ring is in the anti conformation. We determined that the rate of hydride transfer to FAD appears to become partially rate limiting only for exceptionally good electron acceptors such as cytochrome c. Hydride transfer is not rate limiting for NO· production under any conditions used in this study. Interestingly, the deuterium isotope effect was decreased in the cytochrome c reductase assay with both nNOS and nNOSred when the assays were conducted in high ionic strength buffer, suggesting an increase in the rate of hydride transfer to FAD. These results are in stark contrast to results obtained with CPR (D. S. Sem and C. B. Kasper, 1995, Biochemistry 34, 3391–3398) whereby hydride transfer is partially rate limiting at high, but not at low, ionic strength. The seemingly opposite results in deuterium isotope effect observed with CPR and nNOSred, under conditions of high and low ionic strength, suggest differences in structure and/or regulation of these important flavoproteins.

Capillary electrochromatography analysis of hormonal cyclic and linear peptides.

The retention behavior of linear and cyclic peptides has been studied by capillary electrochromatography (CEC) with a variety of different n-alkyl silica reversed-phase sorbents and also with mixed-mode phases containing both strong cation-exchange (sulfonic acid) and n-alkyl groups bonded onto the silica surface, using eluents ranging from pH 2.0 to pH 5.0. Depending upon the amino acid sequence, electrochromatographic retention of the peptides was strongly affected by the composition of the eluent, its pH value, and the choice of sorbent packed into the capillaries. The dominant separation processes operating with these charged analytes could be modulated inter alia by the content of organic modifier, acetonitrile, in the eluent, with peptide resolution predominantly arising from electrophoretic migration processes at high acetonitrile content. As the concentration of acetonitrile was decreased, chromatographic retention processes became more pronounced. With the n-alkyl silica CEC columns used in this study, silanophilic interactions between the sorbents and the charged peptides could be suppressed by increasing the molarity of the buffer and by adjusting the pH of the eluent to lower values. On the other hand, electrostatic interactions between basic peptides and the surface of strong cation-exchanger, mixed-mode materials can be suppressed at low pH values by using higher ionic strength conditions in the eluent. Different selectivity behavior was achieved with desmopressin and the other peptides with Spherisorb C18/SCX and Hypersil mixed-mode materials when an identical eluent composition of 60% (v/v) acetonitrile with 7.6 mM triethylammonium phosphate, pH 3.0, was used. These findings confirm that the surface charge density of the sorbent fulfills an important role in the modulation of peptide selectivity in CEC. These studies also confirm that the dependency of the logarithm of the CEC retention coefficients, i.e., log Kcec, of a peptide separated with n-octadecyl silica sorbents under CEC conditions, on the volume fraction, psi, of the organic solvent modifier, acetonitrile, within the range of 0.20 < or = psi < or = 0.60, can be approximated by a linear relationship. Moreover, these studies show that the selectivity differences of peptides separated by CEC with nonpolar sorbents in packed capillary systems can be discussed in terms of semiempirical dependencies that link peptide retention behavior with their molecular descriptor properties, e.g., their hydrophobicity, surface charge anisotropy, surface area, molecular mass and intrinsic charge, and thus to their corresponding linear free energy relationships.

Characterization of peptides released from rabbit skeletal muscle troponin-T by μ-calpain under conditions of low temperature and high ionic strength

Rabbit skeletal muscle troponin-T (200 μg ml −1) was incubated with μ-calpain (2 μl ml −1) under conditions of low temperature and high ionic strength for 180 min at 4°C and the peptides released analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Troponin-T was hydrolyzed rapidly with the simultaneous appearance of eight peptides with masses of less than 14 up to 26 kDa. Two peptides produced by 10 min of incubation were electroblotted and analysis of these peptides by N-terminal sequencing and mass spectrometry showed that the principal cleavage sites of μ-calpain on troponin-T were at Thr 45–Ala 46, Leu 69–Met 70, Glu 220–Lys 221 and Asn 231–Val 232. The peptides present in insufficient quantity for electroblotting were isolated by reverse-phase high performance liquid chromatography (RP-HPLC). Cleavage sites were also identified at Met 151–Gly 152, Asn 188–Ile 189, Lys 223–Arg 224, Arg 233–Ala 234 and Ala 240–Lys 241. In general, μ-calpain cleaved bonds containing one hydrophobic amino acid residue and mainly towards the C-terminus of troponin-T.

Lupus Anticoagulant-Like Activity Observed in a Dimeric λ Protein Produced by Myeloma Cells

We report here a lupus anticoagulant (LA)-like activity observed in a 45-year-old man with Bence-Jones protein (BJP) lambda-type multiple myeloma. This patient showed no clinical symptoms of thrombosis or bleeding diathesis. Laboratory examination on admission showed mild anemia, prolongation of activated partial thromboplastin time (APTT) (APTT, 56.2 seconds; control, 29.1 seconds), normal prothrombin time, normal thrombin time, and massive proteinuria (2.3 g/d). The mix test with normal plasma showed the presence of circulating anticoagulant. Based on the assumption that the lambda-type BJP may have been responsible for the prolongation of APTT, we purified the BJP from the patient's urine using column works. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting showed that the purified protein was a 48-kd homodimer of immunoglobulin lambda-chains. Addition of the purified dimeric lambda-type BJP to the normal plasma prolonged both APTT and dilute Russell's viper venom time (DRVVT) in a dose-dependent manner, and the negatively charged phospholipid-dependent prothrombinase activity was significantly inhibited in the presence of this protein. Furthermore, both the prolongation of DRVVT and the inhibition of the prothrombinase activity were almost completely abrogated under the condition of high ionic strength. These findings collectively suggest that the dimeric lambda-type BJP showed LA-like activity via the mechanism of ionic charge.

Affinity purification of Csk protein tyrosine kinase based on its catalytic requirement for divalent metal cations.

Protein tyrosine kinase Csk requires two Mg2+ ions for activity: one magnesium is part of the ATP-Mg complex, and the second free Mg2+ ion is required as an essential activator. Zn2+ can bind to this site to replace Mg2+, which inhibits Csk kinase activity. The binding is reversible and removal of Zn2+ results in an active Csk apoenzyme. In this communication, we report that this tight binding can be used as a mechanism for affinity purification of Csk. When bacterial cell lysate containing overexpressed GST-Csk was applied to a column of Zn2+-iminodiacetic acid immobilized to agarose, Csk was specifically retained by the column. Since the binding of Csk to Zn2+ is not affected by up to 200 mM NaCl, high ionic strength conditions were used in the purification procedure, minimizing nonspecific binding due to ionic interactions. Washing the column with 200 mM NaCl and 50 mM imidazole removed virtually all other proteins from the column while Csk remained bound. The retained Csk enzyme was eluted with 1 M imidazole. The 1 M imidazole-eluted fraction contained pure Csk that had a specific activity similar to the enzyme purified by a glutathione-agarose affinity column.

Sorption mechanism of U(VI) on a reference montmorillonite: Binding to the internal and external surfaces

Batch type experiments of U(VI) sorption on a reference montmorillonite(SWy-2) were carried out over wide ranges of pH, ionic strength, and totalU(VI) concentration. The influences of these factors on the sorption behaviorof U(VI) were analyzed to gain a macroscopic understanding of the sorptionmechanism. The sorption of U(VI) on montmorillonite showed a distinct dependencyon ionic strength. When it was low (0.01 or 0.001M), almost all of the totalU(VI) was sorbed over the whole pH range studied, therefore, the dependencyon pH was not clear. But the sorption of U(VI) on montmorillonite showed asorption pH edge in the high ionic strength condition (0.1M), like those onother clay minerals, kaolinite and chlorite. A mechanistic model was establishedby considering the mineral structure of montmorillonite together with ourprevious EPR result, which successfully explained the U(VI) sorption on montmorilloniteover the whole range of experimental conditions. The model describes the U(VI)sorption on montmorillonite as simultaneous and competitive reactions of ionexchange and surface complexation, whose relative contribution to the totalsorption depends on pH and ionic strength. At low ionic strength and low pHconditions, ion exchange was the dominant mechanism for U(VI) sorption onmontmorillonite. At high ionic strength and high pH conditions, surface complexationwas the dominant

Virus Transport through Saturated Sand Columns as Affected by Different Buffer Solutions

AbstractTo accurately examine the mechanisms that control virus retention and transport in porous media, it is essential to perform experiments under environmentally relevant conditions. Laboratory studies of virus transport are generally conducted using buffered solutions. However, there are no standards for the concentration and composition of the solution, making comparisons between different studies difficult. Our objective was to systematically investigate the effects of the ionic strength and composition of buffer solutions on adsorption of viruses during transport through saturated sand columns. Two phosphate buffered saline solutions (PBS and PBS2) and one artificial ground water (AGW) were used and experiments were conducted under saturated, steady state flow conditions. Bromide tracer and bacteriophages φX174 and MS‐2 were introduced into saturated sand columns as step inputs and their concentrations in the outflow samples were determined. Change in ionic strength did not affect the behavior of φX174, whereas MS‐2 was largely removed in the high ionic strength PBS buffer (0.16 M) but moved through the columns in the low ionic strength PBS2 and AGW buffers (0.002 2M). An additional experiment conducted under an ionic strength gradient (from 0.002 to 0.2 M) using PBS indicated that the outflow concentration of MS‐2 decreased slowly from an ionic strength of ∼0.03 M, reached a minimum at an apparent critical value of ∼0.1 M, and then increased gradually. This study also suggests that metals or metal oxides on sand surfaces can cause significant virus removal under high ionic strength conditions.

Structural and kinetic characterization of NADP-dependent, non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from celery leaves

NADP-dependent, non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9) from celery leaves was purified over 1200-fold to a specific activity of 35 units/mg protein, and its kinetic, regulatory and structural properties were characterized. The purified enzyme exhibited a homotetrameric structure with a subunit molecular mass of 54 kDa. A high specificity of the enzyme for the substrates NADP + ( K m=7 μM) and d-glyceraldehyde-3-phosphate ( K m=127 μM) was observed. Maximal activity was determined at pH 8.5. The purified enzyme was highly unstable, requiring the addition of NADP + or conditions of high ionic strength in the medium. A hysteretic behavior, with a lag phase of minutes, was observed during activity measurement of the enzyme preincubated in the absence of substrates. The lag was inversely proportional to the protein concentration during preincubation. The hysteretic parameters were affected by the substrates, KCl and mannitol among other compounds. Distinctively, incubation with NADP + produced a near twofold activation of the enzyme. Results suggest that in alditol producing plants the enzyme plays a key role in the synthesis and partitioning of photoassimilates.

Calcium- and magnesium-dependent aggregation of legume seed storage proteins.

The solubility characteristics and sedimentation behavior of total or individual globulins from legume seeds [Lupinus albus L., Pisum sativum L., and Glycine max (L.) Merr.] were investigated. The typical insolubility of globulins detected during their extraction seems to be due to the presence of a low molecular weight factor(s) in the seed extract. The solubility of the purified globulins decreases with increasing concentrations of calcium and/or magnesium, but not of other cations, showing minimum values at concentrations that vary with the particular globulin considered. Ultracentrifugation analyses revealed that the Ca(2+)- and/or Mg(2+)-induced insolubilization of the globulins involves the formation of high-order aggregates of molecules of the same or of different globulins. These macromolecular structures are dissociated under conditions of high ionic strength, suggesting the involvement of electrostatic interactions in the aggregation process. The degree of association relies heavily on the amount of Ca(2+) and/or Mg(2+) available, on the presence of chelating agents for these divalent cations, and on the ionic strength of the surrounding medium. The possible physiological significance of the findings is discussed.

Spermidine and Spermine Interfere with in vitro BAPNA-mediated Proteolytic Activity in Organogenic Tobacco Thin Layers

Summary The effect of different concentrations of putrescine, spermidine and spermine on proteolytic activity was tested in tobacco thin layers cultured for 15 days under vegetative bud-inducing conditions (indoleacetic acid, IAA plus benzyladenine, BA). Two artificial substrates were used for the proteolytic assay: azocasein and BAPNA. With the former substrate, both under low or high ionic strength conditions, exogenous polyamines had no effect, independently of the polyamine or the concentration used. With the latter substrate, spermidine and spermine at the highest concentrations strongly reduced protease activity, confirming that these long, flexible molecules affect biological activities. This behaviour is discussed in relation to pH, ionic strength, and number of charged Rroupsllength of the molecule.

Investigation of static structure factor in dense suspensions by use of multiply scattered light

Near-infrared, frequency-domain photon migration measurements of phase shift are used to derive accurate values of isotropic scattering coefficients in concentrated, interacting suspensions of aqueous polystyrene microspheres with volume concentrations ranging from 1% to 45% by solids and mean diameters ranging from 135 to 500 nm. Under conditions of high ionic strength, the isotropic scattering coefficient can be quantitatively predicted by the Percus-Yevick model for hard-sphere interactions and Mie theory. In addition, the attractive interactions between scatterers arising from the addition of soluble poly(ethylene glycol) with molecular weights of 100 and 600 K cause hindered scattering. The increases in static structure and decreases in isotropic scattering coefficient agree with that predicted by Mie theory and the depletion interaction model developed by Asakura and Oosawa [J. Chem. Phys. 22, 1255 (1954)]. These results demonstrate the success of monitoring interaction between particles by use of multiple-scattered light and the necessity of incorporating models for these interactions when predicting scattering of dense, concentrated suspensions.

Role of reducing co-factor in cerulenin-insensitivity of 6-hydroxymellein synthase in carrot cell extract.

The activity of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme of carrot, was not inhibited by cerulenin in the presence of NADPH. However, cerulenin showed a marked inhibitory activity to the synthase if the reducing co-factor was omitted from the assay mixture. The synthase was also sensitive to the antibiotic even in the presence of NADPH when the acyl condensation site and the reducing domain at the reaction center of the enzyme were dissociated under the high ionic strength condition. In addition, the synthase activity was appreciably inhibited when NADH was employed instead of NADPH. These observations strongly suggest that a phosphate group attached to 2'-position of adenosyl moiety of NADPH molecule plays an important role in the apparent insensitivity of 6-hydroxymellein synthase toward cerulenin.

Alkaline unfolding and salt-induced folding of bovine liver catalase at high pH.

We have studied the alkaline unfolding of bovine liver catalase and its dependence on ionic strength by enzymic activity measurements and a combination of optical methods like circular dichroism, fluorescence and absorption spectroscopies. Under conditions of high pH (11.5) and low ionic strength, the native tetrameric enzyme dissociates into monomers with complete loss of enzymic activity and a significant loss of alpha-helical content. Increase in ionic strength by addition of salts like potassium chloride and sodium sulphate resulted in folding of alkaline-unfolded enzyme by association of monomers to tetramer but with significantly different structural properties compared to native enzyme. The salt-induced tetrameric intermediate is characterized by a significant exposure of the buried hydrophobic clusters and significantly reduced alpha-helical content compared to the native enzyme. The refolding/reconstitution studies showed that the salt-induced partially folded tetrameric intermediate shows significantly higher efficiency of refolding/reconstitution as compared to alkaline-denatured catalase in the absence of salts. These studies suggest that folding of multimeric enzymes proceeds probably through the hydrophobic collapse of partially folded multimeric intermediate with exposed hydrophobic clusters.

Formation of Stable and Monodispersive Polyion Complex Micelles in Aqueous Medium from Poly(L-lysine) And Poly(Ethylene Glycol)-Poly(Aspartic Acid) Block Copolymer

In this study, the formation of polyion complex micelles from a pair of poly(L-lysine) homopolymers (P(Lys)) and poly(ethylene glycol)-poly(aspartic acid) block copolymers (PEG-P(Asp)) with varying chain length was demonstrated in aqueous medium. There exists the lower critical chain length in the charged segments of both P(Lys) and PEG-P(Asp) to form stable polyion complex micelles in nanometric scale. The scaled average characteristic line width (ΓTK2) was independent on the detection angles for all combinations, suggesting that the formed polyion complex micelles may have a spherical shape. Furthermore, the transitional diffusion coefficient (DT) had no concentration dependence, indicating the micelle system was free from secondary aggregates (the cluster of micelles). It is of interest that the micellar size was almost constant (ca. 50 nm) regardless of the change in the chain length of the charged segments. Size distribution was extremely narrow, and the values of variance μ2/Γ 2) were always less than 0.1. Laser-Doppler electrophoresis measurements revealed that the polyion complex micelles were electrically neutral, suggesting that the PEG corona surrounding the polyion complex core may contribute to their stable dispersion in an aqueous medium through steric repulsion of the tethered hydrophilic chain, in this case, PEG. This system was considerably stable against the change in ionic strength, and it maintained a constant diameter in the region below 0.4 M NaCl. However, they dissociated under high ionic strength condition as 0.6 M NaCl. The system may have potential utility to include charged peptides and nucleotides in the core, delivering these biologically useful substances into a target site in the body.

Interaction between botulinum neurotoxin type a and ganglioside: ganglioside inactivates the neurotoxin and quenches its tryptophan fluorescence

This study found that ganglioside quenched the tryptophan fluorescence of botulinum neurotoxin type A (BoNT A), accompanied by the inactivation of the toxin under low ionic strength conditions. This finding suggests that the ganglioside-binding site of BoNT A contains tryptophan residues. The quantum yield (a conformation parameter) in BoNT A under high ionic strength conditions differed from that under low ionic strength. This observation indicates that high ionic strength may alter the conformation of BoNT A, resulting in failure of the interaction between BoNT A and ganglioside.

Nanofiltration of Natural Organic Matter: pH and Ionic Strength Effects

Studies were conducted to examine the effect of solution chemistry, defined here as pH and ionic strength, on the permeability of negatively charged polymeric nanofiltration membranes. Water permeation through the membrane was demonstrated to decrease at conditions of low pH and high ionic strength in the absence of organic macromolecules. The reduction in membrane permeability was attributed to a compaction of the membrane matrix resulting from charge neutralization at the membrane surface and electric double layer compression. An uncharged model organic macromolecule (polyethylene glycol) was used to quantify the effects of solution chemistry on membrane compaction and solute rejection capabilities of the charged membrane. Studies of membrane permeability and rejection were then repeated with solutions containing natural organic matter (NOM), enabling concurrent evaluation of the effects of electric double layer compression as well as changes in both membrane structure and the apparent macromolecular size of charged NOM macromolecules.

Comparison of the Salt-Dependent Self-Association of Brain and Erythroid Spectrin

The self-association of ovine brain spectrin in 0.1-1.5 M NaCl (pH 7.5) was studied using sedimentation velocity and sedimentation equilibrium techniques. Brain spectrin is tetrameric at sedimentation equilibrium at a 0.13 M ionic strength at 18-37 degrees C and at ionic strengths of up to 0.33 M at 20 degrees C. At ionic strengths greater than 0.33 M at 20 degrees C, the brain spectrin tetramer is destabilized, resulting in both dissociation to dimers and indefinite association to higher oligomers, in a manner similar to that seen with erythroid spectrin. The equilibrium constants describing all steps in the association involving the addition of dimers are around 15-fold higher for brain spectrin than for erythroid spectrin, at ionic strengths of > or = 0.43 M. We propose that the stronger association of brain spectrin compared to that of erythroid spectrin is due to a relative inability of brain spectrin to form closed dimers. Sedimentation velocity analysis confirms that brain spectrin readily forms open dimers and that the association of open dimers is not kinetically trapped even at 2 degrees C. Our results suggest that the destabilization of spectrin tetramers in high-ionic strength conditions is due to increased independent movement of the alpha and beta subunits resulting from disruption of electrostatic interactions. The greater stability of brain spectrin oligomers relative to those of erythroid spectrin is due to stronger nonelectrostatic interactions which stabilize the rigidity of the individual subunits and thereby increase the conformational strain associated with dimer closure.