Interactions of bromide, iodide, and fluoride with the pathways of chloride transport and diffusion in human neutrophils

Isolated human neutrophils possess three distinct pathways by which Cl- crosses the plasma membrane of steady state cells: anion exchange, active transport, and electrodiffusion. The purpose of the present work was to investigate the selectivity of each of these separate processes with respect to other external halide ions. (a) The bulk of total anion movements represents transport through an electrically silent anion-exchange mechanism that is insensitive to disulfonic stilbenes, but which can be competitively inhibited by alpha-cyano-4-hydroxycinnamate (CHC; Ki approximately 0.3 mM). The affinity of the external translocation site of the carrier for each of the different anions was determined (i) from substrate competition between Cl- and either Br-, F-, or I-, (ii) from trans stimulation of 36Cl- efflux as a function of the external concentrations of these anions, (iii) from changes in the apparent Ki for CHC depending on the nature of the replacement anion in the bathing medium, and (iv) from activation of 82Br- and 125I- influxes by their respective ions. Each was bound and transported at roughly similar rates (Vmax values all 1.0-1.4 meq/liter cell water.min); the order of decreasing affinities is Cl- greater than Br- greater than F- greater than I- (true Km values of 5, 9, 23, and 44 mM, respectively). These anions undergo 1:1 countertransport for internal Cl-. (b) There is a minor component of total Cl- influx that constitutes an active inward transport system for the intracellular accumulation of Cl- [( Cl-]i approximately 80 meq/liter cell water), fourfold higher than expected for passive distribution. This uptake is sensitive to intracellular ATP depletion by 2-deoxy-D-glucose and can be inhibited by furosemide, ethacrynic acid, and CHC, which also blocks anion exchange. This active Cl- uptake process binds and transports other members of the halide series in the sequence Cl- greater than Br- greater than I- greater than F- (Km values of 5, 8, 15, and 41 mM, respectively). (c) Electrodiffusive fluxes are small. CHC-resistant 82Br- and 125I- influxes behave as passive leak fluxes through low-conductance ion channels: they are nonsaturable and strongly voltage dependent. These anions permeate the putative Cl- channel in the sequence I- greater than Br- greater than Cl- with relative permeability ratios of 2.2:1.4:1, respectively, where PCl approximately 5 X 10(-9) cm/s.     

Comparative study of tyrosinases from different sources: relationship between halide inhibition and the enzyme active site

The inhibition of tyrosinases from frog epidermis (Rana esculenta ridibunda), mushroom (Agaricus bisporus) and Harding-Passey mouse melanoma by halides is compared. In all cases, the inhibition is pH dependent, increasing when the pH decreases. The order of inhibition is I- greater than Br- greater than Cl- much greater than F- for frog epidermis tyrosinase, F- greater than I- greater than Cl- greater than Br- for mushroom tyrosinase and F- greater than Cl- much greater than Br- greater than I- for the mouse melanoma enzyme. These results are discussed in terms of the active site accessibility to exogenous ligands. The activation energies of the enzyme-catalysed L-dopa oxidation were also calculated, being the values 6.86, 17.01 and 20.25 kcal/mol for frog epidermis, mushroom and Harding-Passey mouse melanoma, respectively. A relationship between these values and the evolutionary adaptation of these enzymes is proposed.     

Competitive inhibition of liver glucokinase by its regulatory protein

The regulatory protein of rat liver glucokinase (hexokinase IV or D) behaved as a fully competitive inhibitor of this enzyme when glucose was the variable substrate, i.e. it increased the half-saturating concentration of glucose as a linear function of its concentration without affecting V (velocity at infinite concentration of substrate). The inhibition by the regulatory protein and that by palmitoyl-CoA were synergistic with that by N-acetyl-glucosamine, indicating that the two former inhibitors bind to a site distinct from the catalytic site. In contrast, the effects of the regulatory protein and palmitoyl-CoA were competitive with each other, indicating that these two inhibitors bind to the same site. The regulatory protein exerted a non-competitive inhibition with respect to Mg-ATP at concentrations of this nucleotide less than 0.5 mM. At higher concentrations, the latter antagonized the inhibition by the regulatory protein partly by decreasing the apparent affinity for fructose 6-phosphate. The following anions inhibited glucokinase non-competitively with respect to glucose: Pi, sulfate, I-, Br-, No3-, Cl-, F- and acetate. Pi and sulfate, at concentrations in the millimolar range, decreased the inhibition by the regulatory protein by competing with fructose 6-phosphate. Monovalent anions also antagonized the inhibition by the regulatory protein with the following order of potency: I- greater than Br- greater than NO3- greater than Cl- greater than F- greater than acetate and their effect was non-competitive with respect to fructose 6-phosphate. Glucokinase from Buffo marinus and pig liver were, like the rat liver enzyme, inhibited by the regulatory protein, as well as by palmitoyl-CoA at micromolar concentrations. In contrast, neither compound inhibited hexokinases from rat brain, beef heart or yeast, or the low-Km specific glucokinase from Bacillus stearothermophilus.     

Lysis of Streptococcus mutans by hen egg white lysozyme and inorganic sodium salts.

Streptococcus mutans BHT was grown in a synthetic medium containing radioactive thymidine to monitor deoxyribonucleic acid release. Kinetic experiments demonstrated that although lysozyme alone could not liberate deoxyribonucleic acid, cellular deoxyribonucleic acid was liberated from lysozyme-treated cells by addition of low concentrations of inorganic sodium salts. When the salts were tested for their ability to dislodge cell-bound tritiated lysozyme, the extent of the initial release of enzyme by individual anions correlated with the anion potency for deoxyribonucleic acid liberation (SCN- greater than ClO4- greater than I- greater than Br- greater than NO3- greater than Cl- greater than F-), although the total amount of lysozyme dislodged did not correspond directly with cell lysis. Differences in the effectiveness of anions (SCN-, HCO3-, Cl- and F-) in potentiating cell lysis could be enhanced or minimized by varying the lysozyme, anion, and bacterial cell concentrations. As the anion concentration was increased for each enzyme concentration and cell concentration, the lysis increased, in some cases markedly, until maximum levels of released deoxyribonucleic acid were attained. The maximum levels of lysis of SCN- and HCO3- were similar and were greater than those for Cl- and F-. In addition, the maximum levels were observed to increase for each of the anions as the concentration of lysozyme increased.     

Modulation of activity of NADH oxidase from Thermus thermophilus through change in flexibility in the enzyme active site induced by Hofmeister series anions.

The conformational dynamics of NADH oxidase from Thermus thermophilus was modulated by the Hofmeister series of anions (H2PO4-, SO42-, CH3COO-, Cl-, Br-, I-, ClO4-, SCN-) in the concentration range 0-3 M. Both chaotropic and kosmotropic anions, at high concentration, inhibit the enzyme by different mechanisms. Chaotropic anions increase the apparent Michaelis constant and decrease the activation barrier of the reaction. Kosmotropic anions have the opposite effect. Anions from the middle of the Hofmeister series do not significantly affect the enzyme activity even at high concentration. We detected no significant changes in ellipticity of the aromatic region in the presence of the anions studied. There is a decreased Stern-Volmer quenching constant for FAD fluorescence quenching in the presence of kosmotropic anions and an increased quenching constant in the presence of chaotropic anions. All of this indicates that active site flexibility is important in the function of the enzyme. The data demonstrate that both the high rigidity of the active site in the presence of kosmotropic anions, and its high flexibility in the presence of chaotropic anions have a decelerating effect on enzyme activity. The Hofmeister series of anions proved to be suitable agents for altering enzyme activity through changes in flexibility of the polypeptide chain, with potential importance in modulating extremozyme activity at room temperature.     

Anion binding properties of human serum albumin from halide ion quadrupole relaxation.

The nuclear magnetic quadrupole relaxation enhancement of 35Cl-, 81Br-, and 12I- anions on binding to human serum albumin has been studied under conditions of variable protein and anion concentration and also in the presence of simple inorganic, amphiphilic, and complex anions which compete with the halide ions for the protein anion binding sites. Two classes of anion binding sites with greatly different binding constans were identified. Experiments at variable halide ion concentration were employed to determin the Cl- and I- binding constants. By means of 35 Cl nuclear magnetic resonance (NMR) the relative affinity for different anions was determined by competition experiments for both the strong and the weak anion binding sites. Anion binding follows the sequence SO42- smaller than F- smaller than CH3COO- smaller than Ci- smaller Br- smaller than NO3- smaller than I- smaller than ClO4- smaller than SCN- smaller than Pt(CN)42- smaller than Au(CN)2- smaller than CH3(CH2)11OSO3- for the high affinity sites, and the sequence SO42- congruent to F- congruent to Cl- smaller CH3COO- smaller than NO3- smaller than Br- smaller than I- smaller than ClO4- smaller than SCN- for the low affinity sites. These series are nearly identical with the well-known lyotropic series. Consequently, those effects of anions on proteins described by the lyotropic series can be correlated with the affinities of the anions for binding to the protein. The data suggest that the physical nature of the interaction is the same for both types of biding sites, and that the differences in affinity between different binding sites must be explained in terms of tertiary structure. Analogous experiments performed using 127I- quadrupole relaxation gave results very similar to those obtained with 35Cl-. A comparison between the Cl-, Br- and I- ions revealed that, as a result of the increasing affinity for the weak anion binding sites in the series Cl- smaller than Br- smaller than I-, Cl- is much more useful as a probe for the specific anion binding sites than the other two halide ions. The findings with human serum albumin in this and other respects are probably of general relevance in studies of protein-anion interactions. In addition to competition experiments, the magnitude of the relaxation rate is also discussed. Line broadening not related to anion binding to the protein is found to be small. A comparison of transverse and longitudinal 35Cl relaxation rates gives a value for the quadrupole coupling constant of the high affinity sites in good agreement with a calculated coupling constant assuming anion binding to arginine.     

Anion regulation of active transport of protons across the membrane of the synaptic vesicles of the brain

The dependence of active transport of H+ on the presence of anions in synaptic vesicle membranes from rat brain was studied. The H+ transport was measured by monitoring the acidification of the vesicles with a permeant weak base-acridine orange. The fluorescence changes in the latter were proportional to the magnitude of artificially imposed pH gradients (delta pH). The ATP-dependent generation of delta pH was completely dependent on the presence of a permeant anion, was maximal at 150 mM Cl- and was inhibited, when the medium osmolarity was further increased by sucrose or KCl. At 150 mM only Br-, similar to Cl-, behaved as permeant anions, whereas I- was effective only at low (5-20 mM) concentrations. The anions--SCN-, ClO4-, HSO3- and I-(10-20 mM) as well as 4-acetamido-4'-isothiocyanatostilbene-2.2'-disulfonate (K0.5 = 14 microM) blocked the ATP-dependent generation of delta pH observed in the presence of Cl-, while other anions tested (F-, phosphate, bicarbonate, some organic anions) were virtually without effect and did not support the H+ transport. The dependence of the rate and extent of H+ accumulation on Cl- concentration was sigmoidal with a Hill coefficient of 2.8 and a Km value of 85-90 mM. The effects of anions point to the presence in the membrane of synaptic vesicles of an anion (chloride) channel whose conductance can regulate the H+ transport by switching it from an electrogenic to an electroneutral (coupled entry of H+ and Cl-) mode of operation.     

Light-scattering investigations of the subunit dissociation of human hemoglobin A. Effects of various neutral salts

The effectiveness of various salts of the Hofmeister series as dissociating agents for human hemoglobin A tetramers has been investigated by light-scattering molecular-weight measurements. Dissociation of hemoglobin to half-molecules of alpha beta dimers follows the order of the series dictated predominantly by the sequence of the anions F- less than Cl- less than Br- less than ClO4- less than SCN-, I-, with the cations Na+ and K+ having relatively little effect on the observed dissociation. The use of equations derived for predicting the effects of dissociating reagents on the structure of subunit proteins [Herskovits, T. T., and Ibanez, V. S. (1976), Biochemistry 15, 5715] together with Setschenow constants based on the model amino acid data of Nandi and Robinson were found to give a satisfactory account of the dissociation behavior observed with many of the salts, giving reasonable estimates of the number of amino acids that form the smaller contact area of the alpha beta subunits of hemoglobin shown by the Perutz crystallographic model. The analysis of the dissociation data also extends the utility of the Setschenow constants tested for the characterization of the dissociation behavior of other subunit proteins.     

Halide permeation through 10 pS and 20 pS anion channels in human airway epithelial cells.

Halide permeability sequences were obtained from reversal potential measurements of single-channel currents through 10 pS and 20 pS anion channels in human airway epithelial cells. The sequences obtained were Cl- greater than I- greater than Br- greater than or equal to F- for the 10 pS channel and Cl- greater than I- greater than or equal to Br- greater than or equal to F- for the 20 pS channel. However, the permeability differences were not large, the greatest being 0.66 for the ratio of fluoride to chloride permeability in the 20 pS channel. Single-channel currents were also measured with solutions of constant halide concentration but varying ratios of chloride to fluoride ions. An anomalous mole fraction effect was observed for the 20 pS channel but not for the 10 pS channel, suggesting that the former is a multi-ion channel. Comparison of the halide permeability sequences of these two channels with those of whole-cell currents in other epithelial cells does not support their involvement in any of the known whole-cell epithelial currents.     

Lateral conductance parallel to membrane surfaces: effects of anesthetics and electrolytes at pre-transition.

The effects of dilute salts and anesthetics were studied on the impedance dispersion in the dipalmitoylphosphatidylcholine (DPPC) liposomes. Below the pre-transition temperature, the apparent activation energy for conductance in DPPC-H2O without salts was equivalent to pure water, 18.2 kJ mol-1. This suggests that the mobile ions (H3O+ and OH-) interact negligibly with the lipid surface below the pre-transition temperature. At pre-transition temperature, the apparent activation energy of the conductance decreased by the increase in the DPPC concentrations. The effects of various salts (LiCl, NaCl, KCl, KBr, and KI) on the apparent activation energy of the conductance were studied. Changes in anions, but not in cations, affected the activation energy. The order of the effect was Cl- less than Br- less than I-. Cations appear to be highly immobilized by hydrogen bonding to the phosphate moiety of DPPC. The smaller the ionic radius, the more ions are fixed on the surface at the expense of the free-moving species. The apparent activation energy of the transfer of ions at the vesicle surface was estimated from the temperature-dependence of the dielectric constant, and was 61.0 kJ mol-1 in the absence of electrolytes. In the presence of electrolytes, the order of the activation energy was F- greater than Cl- greater than Br- greater than I-. When the ionic radius is smaller, these anions interact with the hydration layer at the vesicle surface and the ionic transfer may become sluggish. In the absence of electrolytes, the apparent activation energy of the dielectric constant decreased by the increase in halothane concentrations. In the presence of electrolytes, however, the addition of halothane increased the apparent activation energy. We propose that the adsorption of halothane on the vesicle surface produces two effects: (1) destruction of the hydration shell, and (2) increase in the binding of electrolytes to the vesicle surface. In the absence of electrolytes, the first effect predominates and the apparent activation energy is decreased. In the presence of electrolytes, the latter effect predominates and the apparent activation energy is increased.     

Properties of single chloride channels in primary neuronal cultures of Drosophila

An anion channel from Drosophila neurons had the selectivity sequence: NO3- (1.97) greater than Br- (1.12) approximately equal to I- (1.03) approximately equal to Cl- (1) greater than F- (0.32) much greater than glutamate (less than 0.02) as estimated by the permeability ratio based on the reversal potential measurement. 4-Acetoamido-4'-isothiocyanostilbene-2,2'-disulfonic acid applied internally at 10 microM blocked the channel. We suggest that the chloride channel identified here may provide a pathway for Cl- in the resting membrane of Drosophila neurons.     

The effect of chaotropic anions on the activation and the activity of spinach chloroplast fructose-1,6-bisphosphatase

The effect of chaotropic anions was studied on processes that constitute the chloroplast fructose-1,6-bisphosphatase reaction, i.e. enzyme activation and catalysis. The specific activity of chloroplast fructose-1,6-bisphosphatase was enhanced by preincubation with dithiothreitol, fructose 1,6-bisphosphate, Ca2+, and a chaotropic anion. When chaotropes were ranked in the order of increasing concentrations required for maximal activation they followed a lyotropic (Hofmeister) series: SCN- less than Cl3C-COO- less than ClO4- less than I- less than Br- less than Cl- less than SO4(2-). On the contrary, salts inhibited the catalytic step. The stimulation of chloroplast fructose-1,6-bisphosphatase by chaotropic anions arose from a decrease of the activation kinetic constants of both fructose 1,6-bisphosphate and Ca2+; on the other hand, in catalysis neutral salts caused a decrease of kcat because the S0.5 for both fructose 1,6-bisphosphate and Mg2+ remained unaltered. The molecular weight of chloroplast fructose-1,6-bisphosphatase did not change after the activation by incubation with dithiothreitol, fructose 1,6-bisphosphate, Ca2+, and a chaotrope; consequently, the action of these modulators altered the conformation of the enzyme. Modification in the relative position of aromatic residues of chloroplast fructose-1,6-bisphosphatase was detected by UV differential spectroscopy. In addition, the concerted action of modulators made the enzyme more sensitive to (a) trypsin attack and (b) S-carboxymethylation by iodoacetamide. These results provide a new insight on the mechanism of light-mediated regulation of chloroplast fructose-1,6-bisphosphatase; concurrently to the action of a sugar bisphosphate, a bivalent cation, and a reductant, modifications of hydrophobic interactions in the structure of chloroplast fructose-1,6-bisphosphatase play a crucial role in the enhancement of the specific activity.     

Kinetic study of the interaction between frog epidermis tyrosinase and chloride

The effect of halide ions on frog epidermis tyrosinase has been characterized with the trypsin-activated enzyme. At pH 7, the order of inhibition is I- greater than Br- greater than Cl- greater than F-. Chloride, the most extensively studied halide, shows a competitive pattern with respect to the substrate, L-DOPA. Inhibition is strongly pH-dependent, with a pKa of 6.12 for the responsible protonatable group. Other kinetic constants are also calculated using a novel approach. The mechanism of interaction between chloride and the enzyme is discussed, and a model is proposed in which chloride interferes the tyrosinase activity by displacing a catalytically important ligand, probably a histidine residue of the side-chain, from the copper at the enzyme-active site.     

Anion effects on the reaction of lecithin-cholesterol acyltransferase with discoidal complexes of phosphatidylcholines . apolipoprotein A-I . cholesterol

Discoidal complexes of phosphatidylcholine (PC) . apolipoprotein A-I . cholesterol were prepared with egg PC, palmitoyloleoylPC, dipalmitoylPC, or dimyristoylPC, and were used as substrates of purified lecithin-cholesterol acyltransferase to investigate the effects of neutral salts on the enzymatic reaction. Sodium fluoride, chloride and bromide concentrations up to 1 M, did not affect the properties of the substrate particles, but caused marked and distinct changes in the activity of the enzyme with the various PC particles. The effects of salts were largely due to the anions, which followed the order of the lyotropic series in their inactivating capacity: F- less than Cl- less than Br- less than NO3- less than I- less than SCN-. Sodium salts (F-, Cl-, and Br-) produced a very large increase in the pH optimum of the enzymatic reaction (7.4 to at least 8.5) essentially obliterating the ionization of a functional group with pK of 8.1. The kinetics of the enzymatic reaction revealed major differences among the PC particles, and different responses of their kinetic parameters with increasing salt concentrations. The conclusions reached in this work are the following: (1) The relative reactivity of PC substrates, in discoidal particles, with lecithin-cholesterol acyltransferase depends strongly on the concentration and type of salts in the medium. (2) Anions (in lyotropic series) rather than cations affect the enzymatic reaction. (3) There are functional groups with pK of 8.1 which are affected markedly in their ionization behavior by anion binding. (4) The active site of lecithin-cholesterol acyltransferase and its interaction with anions are affected by the exact nature of the PC-apolipoprotein interface.     

Ionic selectivity of volume-sensitive currents in human epithelial cells.

The ion selectivity of swelling-activated Cl- currents has been investigated in three different human epithelial cell lines, two derived from the airway epithelium (9HTEo- and CFNPE9o-) and one from a colon carcinoma (T84). The relative permeability of volume-sensitive currents with respect to Cl- is: I- (1.19) greater than NO3- (1.07) approximately Br-(1.05) greater than Cl-(1.0) greater than F-(0.5) approximately HCO3-(0.48) greater than isethionate(0.28) greater than aspartate (0.14) approximately gluconate(0.13) approximately SO4(2-)(0.12). This type of ion selectivity is similar to that described for depolarization-activated outwardly rectifying Cl- channels found in epithelial cells.     

Influence of anions and cations on the dipole potential of phosphatidylcholine vesicles: a basis for the Hofmeister effect

Anions and cations have long been recognized to be capable of modifying the functioning of various membrane-related physiological processes. Here, a fluorescent ratio method using the styrylpyridinium dyes, RH421 and di-8-ANEPPS, was applied to determine the effect of a range of anions and cations on the intramembrane dipole potential of dimyristoylphosphatidylcholine vesicles. It was found that certain anions cause a decrease in the dipole potential. This could be explained by binding within the membrane, in support of a hypothesis originally put forward by A. L. Hodgkin and P. Horowicz [1960, J. Physiol. (Lond.) 153:404-412.] The effectiveness of the anions in reducing the dipole potential was found to be ClO4- > SCN- > I- > NO3- > Br- > Cl- > F- > SO42-. This order could be modeled by a partitioning of ions between the membrane and the aqueous phase, which is controlled predominantly by the Gibbs free energy of hydration. Cations were also found to be capable of reducing the dipole potential, although much less efficiently than can anions. The effects of the cations was found to be trivalent > divalent > monovalent. The cation effects were attributed to binding to a specific polar site on the surface of the membrane. The results presented provide a molecular basis for the interpretation of the Hofmeister effect of lyotropic anions on ion transport proteins.     

Activating anions that replace Cl- in the O2-evolving complex of photosystem II slow the kinetics of the terminal step in water oxidation and destabilize the S2 and S3 states

Photosystem II, the multisubunit protein complex that oxidizes water to O2, requires the inorganic cofactors Ca2+ and Cl- to exhibit optimal activity. Chloride can be replaced functionally by a small number of anionic cofactors (Br-, NO3-, NO2-, I-), but among these anions, only Br- is capable of restoring rates of oxygen evolution comparable to those observed with Cl-. UV absorption difference spectroscopy was utilized in the experiments described here as a probe to monitor donor side reactions in photosystem II in the presence of Cl- or surrogate anions. The rate of the final step of the water oxidation cycle was found to depend on the activating anion bound at the Cl- site, but the kinetics of this step did not limit the light-saturated rate of oxygen evolution. Instead, the lower oxygen evolution rates supported by surrogate anions appeared to be correlated with an instability of the higher oxidation states of the oxygen-evolving complex that was induced by addition of these anions. Reduction of these states takes place not only with I- but also with NO2- and to a lesser extent even with NO3- and Br- and is not related to the ability of these anions to bind at the Cl- binding site. Rather, it appears that these anions can attack higher oxidation states of the oxygen evolving complex from a second site that is not shielded by the extrinsic 17 and 23 kDa polypeptides and cause a one-electron reduction. The decrease of the oxygen evolution rate may result from accumulated damage to the reaction center protein by the one-electron oxidation product of the anion.     

Characterization of GABAA receptor-mediated 36chloride uptake in rat brain synaptoneurosomes

gamma-Aminobutyric acid (GABA) receptor-mediated 36chloride (36Cl-) uptake was measured in synaptoneurosomes from rat brain. GABA and GABA agonists stimulated 36Cl- uptake in a concentration-dependent manner with the following order of potency: Muscimol greater than GABA greater than piperidine-4-sulfonic acid (P4S)greater than 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP) = 3-aminopropanesulfonic acid (3APS) much greater than taurine. Both P4S and 3APS behaved as partial agonists, while the GABAB agonist, baclofen, was ineffective. The response to muscimol was inhibited by bicuculline and picrotoxin in a mixed competitive/non-competitive manner. Other inhibitors of GABA receptor-opened channels or non-neuronal anion channels such as penicillin, picrate, furosemide and disulfonic acid stilbenes also inhibited the response to muscimol. A regional variation in muscimol-stimulated 36Cl- uptake was observed; the largest responses were observed in the cerebral cortex, cerebellum and hippocampus, moderate responses were obtained in the striatum and hypothalamus and the smallest response was observed in the pons-medulla. GABA receptor-mediated 36Cl- uptake was also dependent on the anion present in the media. The muscimol response varied in media containing the following anions: Br- greater than Cl- greater than or equal to NO3- greater than I- greater than or equal to SCN- much greater than C3H5OO- greater than or equal to ClO4- greater than F-, consistent with the relative anion permeability through GABA receptor-gated anion channels and the enhancement of convulsant binding to the GABA receptor-gated Cl- channel.     

The systematic characterization by aqueous column chromatography of solutes which affect protein stability

We have systematically characterized, by aqueous column chromatography on a size exclusion cross-linked dextran gel (Sephadex G-10), 12 solutes, 11 of which are known to affect protein stability. Six are chaotropes (water structure breakers) and destabilize proteins, while five are polar kosmotropes (polar water structure makers) and stabilize proteins. Analysis of the chromatographic behavior of these neutral (ethylene glycol, urea), positively charged (Tris, guanidine, as the hydrochloride salts) and negatively charged (SO2-4, HPO2-4, F-, Cl-, Br-, Cl3CCO-2, I-, SCN-, as the sodium salts, in order of elution) solutes at pH 7 as a function of sample concentration (up to 0.6 M), supporting electrolyte, and temperature yields four conclusions, based largely on the behavior of the anions. Chaotropes adsorb to the gel according to their position in the Hofmeister series, with the most chaotropic species adsorbing most strongly. ++Chaotropes adsorb to the gel less strongly in the presence of chaotropes (a salting in effect) and more strongly in the presence of polar kosmotropes (a salting out effect). Polar kosmotropes do not adsorb to the gel, and are sieved through the gel according to their position in the Hofmeister series, with the most kosmotropic species having the largest relative hydrodynamic radii. The hydrodynamic radii of polar kosmotropes is increased by chaotropes and decreased by polar kosmotropes. These results suggest that a chaotrope interacts with the first layer of immediately adjacent water molecules somewhat less strongly than would bulk water in its place; a polar kosmotrope, more strongly.     

Effect of picrotoxin on the Cl-activated ATPase microsomal fraction of bream brain (Abramis brama L.)

It is found that picrotoxine in range concentrations 0.1-10 microM stimulates the basal Mg(2+)-ATPase from microsomal fraction of fish bream (Abramis brama L.), however decreases activating effect of 10(-5) M GABA on the enzyme. The stimulative effect of picrotoxine dependants on duration of preincubation with microsomes. It was established that basal Mg(2+)-ATP-ase activity was activated by anions (Cl- > Br- > I-). The activated effect of anions on the Mg(2+)-ATP-ase is decreased in the presence 1 microM picrotoxine. It was shown that in the dependence on concentration of the Mg(2+)-ATP (0.2 or 1 mM) in the incubation medium the picrotoxine serves as on activator or inhibitor of the enzyme activity. It is supposed that picrotoxine allosterially influences on the enzyme by the receptor-dependent way.