Role of a conserved salt bridge between the PAS core and the N-terminal domain in the activation of the photoreceptor photoactive yellow protein

The effect of ionic strength on the conformational equilibrium between the I(2) intermediate and the signaling state I(2)' of the photoreceptor PYP and on the rate of recovery to the dark state were investigated by time-resolved absorption and fluorescence spectroscopy. With increasing salt concentration up to approximately 600 mM, the recovery rate k(3) decreases and the I(2)/I(2)' equilibrium (K) shifts in the direction of I(2)'. At higher ionic strength both effects reverse. Experiments with mono-(KCl, NaBr) and divalent (MgCl(2), MgSO(4)) salts show that the low salt effect depends on the ionic strength and not on the cation or anion species. These observations can be described over the entire ionic strength range by considering the activity coefficients of an interdomain salt bridge. At low ionic strength the activity coefficient decreases due to counterion screening whereas at high ionic strength binding of water by the salt leads to an increase in the activity coefficient. From the initial slopes of the plots of log k(3) and log K versus the square root of the ionic strength, the product of the charges of the interacting groups was found to be -1.3 +/- 0.2, suggesting a monovalent ion pair. The conserved salt bridge K110/E12 connecting the beta-sheet of the PAS core and the N-terminal domain is a prime candidate for this ion pair. To test this hypothesis, the mutants K110A and E12A were prepared. In K110A the salt dependence of the I(2)/I(2)' equilibrium was eliminated and of the recovery rate was greatly reduced below approximately 600 mM. Moreover, at low salt the recovery rate was six times slower than in wild-type. In E12A significant salt dependence remained, which is attributed to the formation of a novel salt bridge between K110 and E9. At high salt reversal occurs in both mutants suggesting that salting out stabilizes the more compact I(2) structure. However, chaotropic anions like SCN shift the I(2)/I(2)' equilibrium toward the partially unfolded I(2)' form. The salt linkage K110/E12 stabilizes the photoreceptor in the inactive state in the dark and is broken in the light-induced formation of the signaling state, allowing the N-terminal domain to detach from the beta-scaffold PAS core.     

Salt effects on the bacteriophage T7-II structure and activity changes

Optically detected thermal stability and biological activity of phage T7 has been compared as the function of the ionic composition and strength of the buffers. The ionic strength range was studied between 20-140 mmol/1. In Tris buffer containing only monovalent ions the biological activity of the phages decreases abruptly below 50 mmol/1 ionic strength. Structural studies show a logarithmic dependence between the ionic strength and the intraphage DNA stability and no significant change in the thermal stability of the whole phage. Mg2+ and Ca2+ ions at low concentration (1 mmol/1) given into a Tris buffer of 20 mmol/1 original ionic strength highly stabilize the biological activity, which stabilization is also to be seen in the intraphage DNA and also in the whole phage thermal denaturation process.     

Heterogeneity of leghemoglobin from yellow lupine nodules

A possibility is demonstrated to separate summary lupine leghemoglobins (which are salted out within 55--90% of ammonium sulphate saturation) into Lb I and Lb II components by means of ionic exchange chromatography on DEAE-cellulose. Lb I is eluted at lower ionic strength buffer than LbII, and differs from the latter in the form and the size of crystals. Both components have the same electrophoretic mobility and contain N-terminal glycine. LbII and LbI precipitate under gradual salting out within 55--75% and 78--90% of saturation respectively.     

Purification of a novel 30,000 Da calcium-binding protein from bovine cerebellum

A novel very acidic calcium-binding protein (CaBP) was purified from bovine cerebellum, using 45Ca autoradiography as a marker, through a preparative procedure involving salting out with a very high concentration of ammonium sulfate, DE52 column chromatography, RNAase treatment, and HPLC gel filtration. This protein showed a molecular weight of 30,0000 dalton (Da) on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and of 120,000 on in gel filtration chromatography analysis under physiological ionic strength. The calcium binding activity of this 30,000 Da CaBP was monitored on the basis of calcium-dependent changes in tyrosine fluorescence (Kd = 3.0 microM).     

Effect of Ionic Strength on the Kinetics of Trypsin and Alpha Chymotrypsin

The kinetic effects resulting from changes in the medium ionic strength on reactions involving trypsin or α-chymotrypsin are different. The reaction rate increases continuously as the ionic strength increases with α-chymotrypsin. With trypsin, the rate increases at low ionic strengths but as the ionic strength further increases a gradual inhibitory effect is observed. The effects produced by different salts of various valence types (from uni-univalent to uni-trivalent or tri-univalent) are essentially the same, and they are a function of the square root of the ionic strength. The quantitative differences among the various salts may be accounted for on the basis of individual properties of the ions, such as the size of the hydrated ion, "association," etc. The effects of salts on the enzymic reactions described herein are amenable to the same electrostatic treatment applicable to non-enzymatic reactions. By applying Brönsted's basic kinetic concepts and the Debye-Hückel law of electrolyte activity, it appears that the salt effects are mainly due to changes in the dissociation of ionizable groups. This appears to be a general method for analyzing the effect of inorganic ions on enzymic reactions.     

Effect of abiotic factors on the antibacterial activity of chitosan against waterborne pathogens

To assess the adaptability of chitosan (from agricultural waste) as a natural disinfectant, its antibacterial activity against bacteria associated with waterborne diseases was investigated by varying such abiotic conditions, as pH and ionic strength and by adding different amounts of acid solvent, metal ions, and EDTA. Two major waterborne pathogens, Escherichia coli and Staphylococcus aureus, were examined. Results showed that organic acids with low carbon number were better solvents for chitosan than were inorganic acids. The effect of pH below 6 on the antibacterial activity of chitosan was significant. The antibacterial activity of chitosan increased with ionic strength but decreased with the addition of metal ions. The addition of Zn(2+) ions inhibited the antibacterial activity of chitosan the most, while the addition of Mg(2+) ions inhibited the antibacterial activity of chitosan the least. This was due to the chelating capacity of chitosan toward metal ions. The antibacterial activity of chitosan against E. coli was enhanced by EDTA. However, the antibacterial activity of chitosan against S. aureus was partially suppressed by EDTA. The antibacterial activity of chitosan was also dependent on its charges and solubility. The antibacterial mechanism of chitosan has currently been hypothesized as being related to surface interference. The results show that the chitosan is a potential bactericide under various environmental conditions.     

Effect of the irradiation conditions on the radiation inactivation of subtilisin-72

A comparative study was made of gamma-inactivation of subtilisin-72 solutions in 5 X 10(-3) M acetate buffer and 0.1 M NaCl in the presence and absence of Ca2+ ions. It was shown that the acetate buffer had a protective action, and the influence of Ca2+ ions depended on the ionic strength of the solution. In general, Ca2+ ions exerted a stabilizing effect irrespective of the subtilisin concentration in the acetate buffer, but this effect competed with the destabilizing influence of the ionic strength increased by Ca2+ ions.     

Effect of dipolar ions on the entropy-driven polymerization of tobacco mosaic virus protein

The effect of the dipolar ions, glycine, glycylglycine, and glycylglycylglycine on the polymerization of tobacco mosaic virus (TMV) protein has been studied by the methods of light scattering and ultracentrifugation. All three dipolar ions promote polymerization. The major reaction in the early stage is transition from the 4 S to the 20 S state. As in the absence of dipolar ions, the polymerization is enhanced by an increase in temperature; it is endothermic and therefore entropy-driven. The effect of the dipolar ions can be understood in terms of their action as salting-out agents; they increase the activity coefficient of TMV A protein, the 4 S material, and thus shift the equilibrium toward the 20 S state. The salting-out constants, K, for the reaction in 0.10 ionic strength phosphate buffer at pH 6.7 was found by the light scattering method to be 1.6 for glycine, 2.5 for glycylglycine, and 2.5 for glycylglycylglycine. A value of 2.7 was obtained by the ultracentrifugation method for glycylglycine in phosphate buffer at 0.1 ionic strength and pH 6.8 at 10 degrees C. For both glycine and glycylglycine, K increases when the ionic strength of the phosphate buffer is decreased. This result suggests that electrolytes decrease the activity coefficient of the dipolar ions, a salting-in phenomenon. However, the salting-in constants evaluated from these results are substantially higher than those previously determined by solubility measurements. The effect of glycine and glycylglycine on polymerization was studied at pH values between 6.2 and 6.8. The effectiveness of both dipolar ions is approximately 50% greater at pH 6.8 than at pH 6.2. The variation of the extent of polymerization with pH in the presence of the dipolar ions is consistent with the interpretation that approximately one hydrogen ion is bound for half of the polypeptide units in the polymerized A protein.     

The thermal denaturation of nonpolymerizable alpha alpha-tropomyosin and its segments as a function of ionic strength

Nonpolymerizable tropomyosin (NPTm) is found to unfold thermally at high ionic strength almost exactly as the parent protein, but it does not aggregate at low ionic strength. Thus, NPTm can be used as a tropomyosin surrogate whose coiled-coil structural stability can be probed by varying the ionic strength. Studies of NPTm by CD show that increasing ionic strength stabilizes the coiled-coil structure. CD spectra over a wide range of helix content, obtained by varying either temperature or ionic strength, show an isodichroic point at 203 nm, suggesting a local, residue-level, two-state model. At given temperature, such a local helix in equilibrium random equilibrium suggests ln [phi h/(1-phi h)] = A1 + A2In, wherein phi h is the fraction helix, and A1, A2, and n are constants. In the low ionic strength region, theoretical limiting laws for ionic strength mediated charge-charge, dipole-dipole, and apolar-apolar (salting out) interactions give, respectively, n = 0.5, 1.0, and 1.0. Our experimental values for 40 degrees C, where the data span a wide range of helix content, show n = 1.0, suggesting that ionic strength stabilizes either by reducing dipole-dipole repulsions or by enhancing hydrophobic interactions, both probably interhelix in nature. Two segments of tropomyosin, 11Tm127 and 142Tm281, neither of which aggregate at low ionic strength, give results similar to those for NPTm, i.e., n = 0.96 and 0.84, respectively.     

Some aspects of the copper(II)-DNA interaction

The Cu(II) ion interaction with calf-thymus DNA was studied by means of differential pulse polarography and sweep voltammetry as well as chromatography and viscosimetry. Most of the complexes formed at high ionic strength (0.2 M) and lower Cu(II) concentrations are of a nondenaturing nature. Their formation has but a minor effect on unwinding process of the DNA double helix. The excess of Cu(II) (P = 5) leads, however, to distinct denaturation of the DNA structure. Metal ions have little effect on the denaturation induced by the polarographic reduction of DNA on the mercury electrode. This conclusion is consistent with the character of the polarographic process and with the fact that Cu(II) ions are not very effective in the interaction with AT pairs. Cupric ions have no renaturing ability towards thermally denatured DNA at 0.2 M ionic strength but distinct renaturation was observed at low ionic strength (0.05 M).     

Experimental conditions affecting ribonucleic acid polymerase in isolated rat liver nuclei. Effect of nucleoside triphosphate concentration, temperature, ammonium sulphate and heparin

1. The conditions affecting the activity of RNA polymerase in isolated rat liver nuclei were studied with Mg(2+) or Mn(2+) as activating ions. 2. The enzyme assayed with Mg(2+) and at low ionic strength is saturated by a lower concentration of nucleotide substrates than if assayed with Mn(2+) at low ionic strength or with either ion at high ionic strength. 3. At low and at high ionic strength the incorporation of AMP is affected in a similar way by variations in the temperature of incubation. Preincubation at 37 degrees impairs the AMP incorporation. 4. Heparin stimulates the RNA polymerase activity in the presence of Mn(2+). 5. Both ammonium sulphate and heparin ;restart' the reaction if added after 15min., the effect being more marked with ammonium sulphate than with heparin, and also more marked in the presence of Mn(2+) than of Mg(2+). 6. alpha-Amanitin abolishes the effect of ammonium sulphate and of heparin.     

Effect of glycerol on the interactions and solubility of bovine pancreatic trypsin inhibitor

The effects of additives used to stabilize protein structure during crystallization on protein solution phase behavior are poorly understood. Here we investigate the effect of glycerol and ionic strength on the solubility and strength of interactions of the bovine pancreatic trypsin inhibitor. These two variables are found to have opposite effects on the intermolecular forces; attractions increase with [NaCl], whereas repulsions increase with glycerol concentration. These changes are mirrored in bovine pancreatic trypsin inhibitor solubility where the typical salting out behavior for NaCl is observed with higher solubility found in buffers containing glycerol. The increased repulsions induced by glycerol can be explained by a number of possible mechanisms, all of which require small changes in the protein or the solvent in its immediate vicinity. Bovine pancreatic trypsin inhibitor follows the same general phase behavior as other globular macromolecules where a robust correlation between protein solution second virial coefficient and solubility has been developed. This study extends previous reports of this correlation to solution conditions involving nonelectrolyte additives.     

Sorting out molecules reacting with acetylcholinesterase by enzyme encapsulation in liposome

Enzymes are considered as providential molecules for biosensor design because of their sensitivity and the high specificity of the reactions they catalyse. However, their active sites often display low selectivity, a lot of molecules may enter and interfere with catalysis. These molecules may be either competitive inhibitors, activators or molecules which change the physico-chemical environment of the enzyme (pH, ionic strength). They produce the "matrix effect" that lowers the reliability of biosensors. We show here that encapsulation of enzymes in liposomes inserts a barrier between the enzyme and the external environment and protects the enzyme in a stable nano-environment for an optimal activity. This barrier sorts out the molecules that could react with the enzyme according to their hydrophobicity. Acetylcholinesterase is used to detect organophosphorous and carbamate insecticide residues but several molecules (reversible inhibitors, pH and ionic strength modifiers) generate matrix effects in free conditions. These perturbations were completely ineffective following enzyme encapsulation.     

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.     

Electrostatic effects on the kinetics of oxidation-reduction reactions of c-type cytochromes.

The kinetics of the oxidation-reduction reactions between horse heart cytochrome c, Euglena gracilis cytochrome c552, and ions (ascorbate, ferricyanide, and ferrocyanide) was investigated as a function of ionic strength at pH 7, 25 degrees C. The ionic strength was varied between 0.002 and 0.02 M. Data were analyzed according to four different functions of ionic strength. Results showed that the Kirkwood-Tanford smeared charge model holds well for the calculation of the activity coefficients and that the whole charges of these proteins are reflected in the rates of their reactions. Chemical modifications or changes in the pH that altered the charge of the proteins affected the primary salt effects as predicted by the smeared charge model.     

Effect of ionic strength on the organization and dynamics of membrane-bound melittin

Melittin, a cationic hemolytic peptide, is intrinsically fluorescent due to the presence of a single functionally important tryptophan residue. We have previously shown that the sole tryptophan of melittin is localized in a motionally restricted environment in the membrane interface. We have monitored the effect of ionic strength on the organization and dynamics of membrane-bound melittin utilizing fluorescence and circular dichroism (CD) spectroscopic approaches. Our results show that red edge excitation shift (REES) of melittin bound to membranes is sensitive to the change in ionic strength of the medium. This could be attributed to a change in the immediate environment around melittin tryptophan with increasing ionic strength due to differential solvation of ions. Interestingly, the rotational mobility of melittin does not appear to be affected with change in ionic strength. In addition, fluorescence parameters such as lifetime and acrylamide quenching of melittin indicate an increase in water penetration in the membrane interface upon increasing ionic strength. Our results suggest that the solvent dynamics and water penetration in the interfacial region of the membranes are significantly affected at physiologically relevant ionic strength. These results assume significance in the overall context of the influence of ionic strength in the organization and dynamics of membrane proteins and membrane-active peptides.     

Role of ions in the regulation of porcine lactate dehydrogenase.

Different ions affect the H4 and M4 isoenzymes of porcine lactate dehydrogenase (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) in the same way, inhibiting the enzyme at low pyruvate concentrations, whereas at high pyruvate concentrations, the activities were enhanced. The inhibition was competitive with regard to pyruvate and NADH. The enhancement of the enzyme activity at high pyruvate concentration is due to the increase in the Km value for pyruvate, implying that higher substrate concentrations are needed to obtain substrate inhibition. Sulphate behaved differently from the other ions. It inhibited in a noncompetitive manner with regard to pyruvate and did not activate the enzyme at high pryvuate concentration. The effect of ions increased with the size of the anion. The ionic strength was of less importance.     

Phosphate Uptake and Root Electrical Potential

It is shown that the potential difference between the rootsof five plant species and their external solution is relatedto the ionic strength of the solution in a logarithmic fashionas predicted by diffuse double-layer theory. The slope of thestraight-line relationship indicates a change of 48 mV for atenfold change in the ionic strength of the solution. At lowphosphate concentrations the rate of uptake is linearly relatedto the square root of the ionic strength of the solution. Thisis in agreement with the effect of ionic strength on surfacepotential and in turn with the effect of potential on surfaceconcentration of adsorbed ions.     

Surface charge measurements on Micrococcus lysodeikticus and the catalytic implications for lysozyme

Electrophoresis measurements on Micrococcus lysodeikticus have shown that the net surface charge density on the cell wall is constant at around -1.5 microC/cm2 for the pH range 4-8. This result has enabled a quantitative analysis to be made of how the electrostatic field associated with the negatively charged cell wall influences the ionic strength and pH dependency of the lytic activity of lysozyme towards M. lysodeikticus. A dominant effect is the creation of a local pH gradient at the cell wall, and at high ionic strengths the lytic activity is found to be controlled by an electrostatic force of attraction between the lysozyme molecule and the cell wall. As the ionic strength of the supporting electrolyte is decreased, however, an electrostatic force of repulsion becomes dominant and is associated with a negative charge carried by the lysozyme molecule, which could possibly be the ionized Asp-52 residue at the active site. This is considered to arise from the fact that at low ionic strengths the fine details of the heterogeneous charge distribution on the cell wall and lysozyme molecule are only partially screened by counter ions.