Effects of preparation method on reversible conformational changes induced by neutral salts in spin-labeled erythrocyte membranes

Addition of “chaotropic” neutral salts to spin-labeled erythrocyte membranes produced changes in membrane conformation proportional to the known activities of the salts: KSCN > CaCl₂ ≥ KNO₃ ? NaCl. The effects had a threshold of about 0.10 m and increased through 0.60 m for all salts. Above 0.6 m other changes due to dielectric effects and/or protein loss began.The chaotropic effects were reversible on removal of the chaotropic ion, for human (or bovine) membranes prepared with a buffer ionic strength of 0.02. For membranes prepared with a buffer ionic strength of 0.005 the changes were irreversible. Exposure to salt concentration greater than 1 m also produced irreversible changes, probably due to solubilization. The results appear to show that the water-soluble membrane proteins are essential to maintain the “native” membrane conformation.     

Glucosylglycerol-phosphate synthase: target for ion-mediated regulation of osmolyte synthesis in the cyanobacterium Synechocystis sp. strain PCC 6803

The response of cyanobacteria to a changing osmotic environment includes the accumulation of organic osmolytes such as glucosylglycerol. The activation of the enzymes involved in glucosylglycerol synthesis [glucosylglycerol-phosphate synthase (GGPS) and glucosylglycerol-phosphate phosphatase (GGPP)] in Synechocystis sp. strain PCC 6803 by various salts and salt concentrations was investigated in vitro. GGPS seemed to be the target for salt-mediated regulation of glucosylglycerol synthesis in vitro. GGPS activation was dependent on the concentration of NaCl, and a sigmoidal plot was obtained. Sensitivity to NaCl was markedly enhanced by low Mg⁺² concentrations (optimal at 4 mM), but Mg²⁺ was not absolutely necessary for the Na⁺ stimulation. As in the case of NaCl, other salts (including MgCl₂) stimulated GGPS. The relative order of GGPS activation in the presence of chloride by the cations at constant ionic strength was Li⁺ > Na⁺ > K⁺, Mg²⁺ Mn²⁺. No absolute dependence on ionic strength was observed in Mg²⁺/Na⁺-exchange experiments. The degree of activation by ions at various concentrations was positively related to the increasing destabilizing properties of the cations according to the Hofmeister rule, where chaotropic cations are most efficient. Cations were responsible for activation since chaotropic anions counteracted the activating effect of cations. Received: 10 August 1998 / Accepted: 11 November 1998     

Effects of chaotropic agents versus detergents on dihydroorotate dehydrogenase.

As chaotropic salts are generally believed to affect water structure in a manner which increases lipophilicity of water, they may seem to be capable of substituting for detergents in the solubilization of particulate enzyme. Although solubilization either by detergents or by chaotropic salts has been demonstrated with several membrane proteins, the effects these agents have on the properties and activity of an enzyme may be quite different. This is illustrated by the effects on mammalian mitochondrial dihydroorotate dehydrogenase. Stability of the solubilized enzymic activity is dependnet on the presence of a detergent and maximum enzymic activity is observed at the critical micelle concentration of the detergent. Addition of low concentrations of various anions of the chaotropic series further enhances activity while higher concentrations of these anions, although increasing solubility of the enzyme, irreversibly inhibit catalysis.     

The Function of the Na+-Driven Flagellum of Vibrio cholerae Is Determined by Osmolality and pH

Vibrio cholerae is motile by its polar flagellum, which is driven by a Na⁺-conducting motor. The stators of the motor, composed of four PomA and two PomB subunits, provide access for Na⁺ to the torque-generating unit of the motor. To characterize the Na⁺ pathway formed by the PomAB complex, we studied the influence of chloride salts (chaotropic, Na⁺, and K⁺) and pH on the motility of V. cholerae. Motility decreased at elevated pH but increased if a chaotropic chloride salt was added, which rules out a direct Na⁺ and H⁺ competition in the process of binding to the conserved PomB D23 residue. Cells expressing the PomB S26A/T or D42N variants lost motility at low Na⁺ concentrations but regained motility in the presence of 170 mM chloride. Both PomA and PomB were modified by N,N′-dicyclohexylcarbodiimide (DCCD), indicating the presence of protonated carboxyl groups in the hydrophobic regions of the two proteins. Na⁺ did not protect PomA and PomB from this modification. Our study shows that both osmolality and pH have an influence on the function of the flagellum from V. cholerae. We propose that D23, S26, and D42 of PomB are part of an ion-conducting pathway formed by the PomAB stator complex.     

Hofmeister effects on activity and stability of alkaline phosphatase

We have studied the effects on alkaline phosphatase of adding high concentrations (normally 1.0 M) of simple salts. It is necessary to allow for significant effects of salts on the extinction coefficient of the reaction product, and on the apparent pH of the buffer. Both activity and stability of the enzyme correlate well with the Hofmeister series in terms of the salt's kosmotropic/chaotropic properties, which are assessed by the Jones–Dole viscosity B coefficients (B₊ for cations and B₋ for anions). The catalytic activity or V_max/K_m of the enzyme showed a bell-shaped relationship with the (B₋ − B₊) values of the salts present, being optimal with salts (such as NaCl, KCl, and KNO₃) where the anion and cation have similar kosmotropic/chaotropic properties. This effect is believed to be enzyme-specific and relates to the impact of both cations and anions on the enzyme's surface pH, active site, and catalytic mechanism. Anions play a more predominant role than cations in affecting enzyme stability. The rate of irreversible thermal inactivation is strongly reduced by addition of kosmotropic anions like SO₄²⁻ (half-life increased from 8 to 580 min at 60 °C). This effect is general and the mechanism probably involves the ability of the ions to affect the water solvation layer around the enzyme molecule and to interact with both the surface and internal structure of the enzyme.     

Reversibility of nucleoside diphosphate kinase solubilization from the surface of the outer mitochondrial membrane

It was found that in medium with low ionic strength nucleoside diphosphate kinase (NDPK) solubilization from the outer membrane of liver mitochondria could be partially reversed by the addition of 3.3 mM MgCl₂. Complete rebinding of the enzyme after the addition of MgCl₂ was observed when the mitochondrial washing and storage medium contained leupeptin, an inhibitor of cathepsins. It was demonstrated that leupeptin and another inhibitor of cysteine proteinases, E-64, do not influence the rate of NDPK solubilization as well as its solubilized and membrane-associated activity. We conclude that NDPK becomes sensitive to proteolysis only after its solubilization; proteolysis does not affect the part of the enzyme molecule that is responsible for catalysis. After solubilization of NDPK in the absence of leupeptin, cathepsins damage sites of its binding on the membranes. The rate of the enzyme solubilization is dependent on the pH of the storage medium (pH 6.0–8.0); it decreases with increase in pH. It was shown that in the medium with high ionic strength, MgCl₂ does not reverse pH-dependent NDPK solubilization, but solubilization could be reversed by increase in medium pH in the presence of E-64 and BSA. The physiological importance of these results is discussed. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 5, pp. 710–720.     

Purification and properties of adenosinetriphosphatase from Zea mays seedling microsomes

A simple method was developed for selective solubilization of membrane ATPase from etiolated corn seedlings using 0.01% Triton X100 and 0.01% deoxycholate containing 200 mM KI. An 81-fold enriched enzyme preparation, with specific activity of 133 μmol Pi/mg protein/hr, was obtained. The enzyme stored in 25 mM Tris-HCl buffer (pH 7.5) at 4° showed rapid loss of activity. The enzyme was stabilized by 1 mM EDTA with addition of 1.2 mM Mg²⁺°. Mg²⁺ and Ca²⁺ (1.2 mM) increased enzymatic activity by 12 and 10.8% respectively, whereas Na⁺ and K⁺ brought about a 20% increase in ATP-hydrolysis. The effect of combined mono- and di-valent ions was neither synergistic nor additive. Ouabain exerted no effect on enzyme activity. The enzyme showed two pH optima (6.0 and 7.5) in the presence of Na⁺ and K⁺, and one optimum at pH 6.5 in the absence of these ions. On polyacrylamide gel the enzyme was resolved into two protein bands, both exhibiting ATPase activity. It is suggested that the soluble enzyme from the microsomal fraction of corn seedlings contains two ATP-hydrolyzing enzymes, one of them being stimulated by Na⁺ and K⁺ ions.     

Potassium stimulation of corn root plasmalemma ATPase : I. Hydrolytic activity of native vesicles and purified enzyme

Potassium stimulation of the plasmalemma (Zea mays L. var Mona) was studied by using a constant ionic strength to prevent indirect stimulation by the electrostatic effect of K⁺ salts. The transmembrane electrochemical H⁺ gradient was eliminated by using gramicidin. In these conditions, K⁺ stimulation was attributable to a direct effect of the cation on plasmalemma proteins. We used both native vesicles isolated on a sucrose cushion, and solubilized and purified ATPase from phase-partitioned plasmalemma, according to the method of T. Nagao, W. Sasakawa, and T. Sugiyama ([1987] Plant Cell Physiol 28: 1181-1186). The purified enzyme had a high specific activity (15 micromoles per minute per milligram protein), but was only about 20% stimulated by K⁺. In both preparations, potassium (in the range around 1 millimolar) specifically decreased two-fold the vanadate inhibition constant, and increased the maximum rate of ATP hydrolysis. In plasmalemma vesicles, the Eadie-Scatchard graph of the K⁺-dependent ATPase activity as a function of K⁺ concentration was linear only at constant ionic strength. The purified ATPase preparation appeared as two closely spaced bands in the 100 kilodalton region with isoelectric point about 6.5. Nevertheless, this biochemical heterogeneity seems unlikely to be related to K⁺ stimulation, since K⁺ modified neither the pH optimum of the activity (pH 6.5) nor the monophasic kinetics of the vanadate inhibition, in both native plasmalemma and purified enzyme preparation.     

Extracellular charge adsorption influences intracellular electrochemical homeostasis in amphibian skeletal muscle

The membrane potential measured by intracellular electrodes, E_m, is the sum of the transmembrane potential difference (E₁) between inner and outer cell membrane surfaces and a smaller potential difference (E₂) between a volume containing fixed charges on or near the outer membrane surface and the bulk extracellular space. This study investigates the influence of E₂ upon transmembrane ion fluxes, and hence cellular electrochemical homeostasis, using an integrative approach that combines computational and experimental methods. First, analytic equations were developed to calculate the influence of charges constrained within a three-dimensional glycocalyceal matrix enveloping the cell membrane outer surface upon local electrical potentials and ion concentrations. Electron microscopy confirmed predictions of these equations that extracellular charge adsorption influences glycocalyceal volume. Second, the novel analytic glycocalyx formulation was incorporated into the charge-difference cellular model of Fraser and Huang to simulate the influence of extracellular fixed charges upon intracellular ionic homeostasis. Experimental measurements of E_m supported the resulting predictions that an increased magnitude of extracellular fixed charge increases net transmembrane ionic leak currents, resulting in either a compensatory increase in Na⁺/K⁺-ATPase activity, or, in cells with reduced Na⁺/K⁺-ATPase activity, a partial dissipation of transmembrane ionic gradients and depolarization of E_m.     

The influence of the thylakoid membrane surface properties on the distribution of ions in chloroplasts

Thylakoid membranes isolated from peas have been subjected to ionic analyses using the technique of neutron activation. This has allowed the analyses of K⁺, Na⁺, Mg²⁺, Ca²⁺ and Cl^? to be measured simultaneously on the same sample. By varying the ionic composition of the suspending medium it has been shown that these chloroplast membranes have no obvious chemical specificity for the inorganic cations studied and that the major controlling factor is the electrostatic neutralization of the surface negative charges. In agreement with the Gouy-Chapman theory and for the conditions used, divalent cations were preferentially attracted to the membrane surface. This finding, together with the ionic analysis of the unwashed thylakoids and of isolated intact chloroplasts, indicated that the major physiological surface cation is Mg²⁺ and that K⁺ is probably the main inorganic cation of the stroma. This conclusion is discussed in terms of counterion movement in response to light induced proton pumping at the thylakoid membrane.     

Activation of Glucose-6-phosphate Dehydrogenase Promotes Acute Hypoxic Pulmonary Artery Contraction

Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to a decrease in airway O₂ tension, but the underlying mechanism is incompletely understood. We studied the contribution of glucose-6-phosphate dehydrogenase (Glc-6-PD), an important regulator of NADPH redox and production of reactive oxygen species, to the development of HPV. We found that hypoxia (95% N₂, 5% CO₂) increased contraction of bovine pulmonary artery (PA) precontracted with KCl or serotonin. Depletion of extracellular glucose reduced NADPH, NADH, and HPV, substantiating the idea that glucose metabolism and Glc-6-PD play roles in the response of PA to hypoxia. Our data also show that inhibition of glycolysis and mitochondrial respiration (indicated by an increase in NAD⁺ and decrease in the ATP-to-ADP ratio) by hypoxia, or by inhibitors of pyruvate dehydrogenase or electron transport chain complexes I or III, increased generation of reactive oxygen species, which in turn activated Glc-6-PD. Inhibition of Glc-6-PD decreased Ca²⁺ sensitivity to the myofilaments and diminished Ca²⁺-independent and -dependent myosin light chain phosphorylation otherwise increased by hypoxia. Silencing Glc-6-PD expression in PA using a targeted small interfering RNA abolished HPV and decreased extracellular Ca²⁺-dependent PA contraction increased by hypoxia. Similarly, Glc-6-PD expression and activity were significantly reduced in lungs from Glc-6-PD^mut(−/−) mice, and there was a corresponding reduction in HPV. Finally, regression analysis relating Glc-6-PD activity and the NADPH-to-NADP⁺ ratio to the HPV response clearly indicated a positive linear relationship between Glc-6-PD activity and HPV. Based on these findings, we propose that Glc-6-PD and NADPH redox are crucially involved in the mechanism of HPV and, in turn, may play a key role in increasing pulmonary arterial pressure, which is involved in the development of pulmonary hypertension.     

Salt-induced pH changes in spinach chloroplast suspension. Changes in surface potential and surface pH of thylakoid membranes

A pH decrease in chloroplast suspension in media of low salt concentration was observed when a salt was added at pH values higher than 4.4, while at lower pH values a pH increase was observed. The salt-induced pH changes depended on the valence and concentration of cations of added salts at neutral pH values (higher than 4.4) and on those of anions at acidic pH values (lower than 4.4). The order of effectiveness was trivalent > divalent > monovalent. The pH value change by salt addition was affected by the presence of ionic detergents depending on the sign of their charges. These characteristics agreed with those expected from the Gouy-Chapman theory on diffuse electrical double layers. The results were interpreted in terms of the changes in surface potential, surface pH and the ionization of surface groups which result in the release (or binding) of H⁺ to (or from) the outer medium.The analysis of the data of KCl-induced pH change suggests that the change in the surface charge density of thylakoid membranes depends mainly on the ionization of carboxyl groups, which is determined by the surface pH. When the carboxyl groups are fully dissociated, the surface charge density reaches ?1.0 ± 0.1 · 10^?3 elementary charge/square Å.Dependence of the estimated surface potential on the bulk pH was similar to that of electrophoretic mobility of thylakoid membrane vesicles.     

Taurine: a preventive agent of the acute ethanol depletive action on the isolated human amniotic membrane

Summary The preventive effect of taurine towards the acute ethanol reduction action was studied on the ionic transfer through the isolated human amniotic membrane. Taurine increased 3 components of the ionic transfer expressed by the conductance measurements (Na⁺ and K⁺ paracellular conductances through the intercellular spaces and coupling cell factor between 2 adjacent epithelial cells, expressed by a voltage ratio). These components were decreased by ethanol. Electrophysiological studies (conductance and voltage measurements) indicated that the addition of taurine (0.1–1 mM) before ethanol (0.4 g/l) hindered the decrease action of ethanol on the Na⁺ and K⁺ paracellular conductances and on the coupling cell factor. These data indicated a common target between taurine and ethanol: the membraneous phospholipids, particularly the distribution of the external fixed charges. The preventive action of taurineversus ethanol, on the human amniotic membrane, was exerted on the polar groups of phospholipids, hindering the incorporation of ethanol molecules.     

Ion exchange properties of isolated tomato fruit cuticular membrane: Exchange capacity,nature of fixed charges and cation selectivity

Summary Isolated tomato fruit cuticular membrane, free of extractable materials, was titrated potentiometrically using various bases. Three dissociable groups were observed in the pH ranges 3–6 (0.2 meq g^-1), 6–9 (0.3 meq g^-1) and 9–12 (0.55 meq g^-1). The first group was tentatively assigned to-COOH groups of pectic materials and protein embedded in the membrane, the second to nonesterified-COOH groups of the cutin polymer and the third to phenolic-OH groups, such as non-extractable flavenoids present in the membrane, and to a small amount of-NH ₃ ⁺ groups of proteins. The cuticular membrane exhibited a behavior typical of highly cross-linked, high-capacity ion exchange resins of the weak-acid type. Ion exchange capacity increased with increasing pH and neutral salt concentration. At constant pH and salt concentration, the exchange capacity increased with increasing counter ion valence and decreasing crystal radius, e.g. [tris (ethylenediamine) Co]³⁺Ca²⁺>Ba²⁺>Li⁺>Na⁺>Rb⁺>N(CH₃) ₄ ⁺ . The cutin polymer exhibited a pronounced selectivity for Ca²⁺ over Na⁺ which increased with increasing neutralization of fixed charges. The large trivalent [Co(en)₃]³⁺ was preferred only at low equivalent ionic fractions in the polymer. These results are discussed in relation to the structure and function of cuticular membranes.     

Possible existence of ion pairs at the mouths of ion channels

An electrostatic calculation suggests that when an ion is bound near the mouth of a channel penetrating a low-dielectric membrane, a counter ion may form an ion pair with this ion. The tendency towards ion-pair formation is remarkably enhanced at channel mouths by forces (image forces) arising from the charges induced on the boundaries between different dielectrics. The binding constant for the formation of ion-pairs of monovalent ions is estimated under the assumption that local interactions between the counter ion and the channel wall are negligibly small. It is of the order of 1–10 molal^?1 or more for the binding of a Cl^? (F^?) counter ion to an $\text{Na}{}^{\mathrm{+}}$ ($\text{Li}{}^{\mathrm{+}}$) ion if appropriate conditions are fulfilled. The binding constant depends on the position of the binding site, the dimensions and geometries of the channel and channel mouth, and the state of ion loading of the channel, as well as the ionic species. The present results also indicate that when cation (anion) channels have anionic (cationic) groups as integrant parts of their channel walls, interactions between these charged groups and permeant ions are markedly enhanced by the image forces.     

Studies on the quaternary structure of class I major histocompatibility complex antigens. Effect of different agents on the interaction between subunits

The effect of temperature, urea, guanidine HCl, ionic and nonionic detergents, organic solvents, chaotropic salts, pH, and divalent cations has been investigated on purified human histocompatibility antigens solubilized by papain (HLApap) or solubilized by sodium cholate (HLAchol). HLApap and HLAchol are fairly stable proteins to agents acting predominantly on hydrogen bonds (temperature, urea) or hydrophobic forces (ionic and nonionic detergents). However, agents which affect ionic interactions (pH, salts, divalent cations) dissociate the molecules into subunits. A single binding site for beta 2-microglobulin with an affinity constant of 1.0 X 10(7) M-1 was found for the alpha chain of HLAchol. The dissociated subunits can be separated by affinity chromatography on Sepharose-rabbit IgG anti-human beta 2-microglobulin and reassociate in vitro when incubated under the appropriate conditions. The results point toward an important role of ionic interactions between subunits in the stabilization of the quaternary structure of HLA.     

Analysis of the Properties of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Insecticidal Toxins Using a Potential-sensitive Fluorescent Probe

A potential-sensitive fluorescent probe, 3,3-dipropylthiadicarbocyanine iodide, was used to analyze, at pH 7.5 and 10.5, the effects of Bacillus thuringiensis toxins on the membrane potential generated by the efflux of K⁺ ions from brush border membrane vesicles purified from the midgut of the tobacco hornworm, Manduca sexta. Fluorescence levels were strongly influenced by the pH and ionic strength of the media. Therefore, characterization of the effects of the toxins was conducted at constant pH and ionic strength. Under these conditions, the toxins had little effect on the fluorescence levels measured in the presence or absence of ionic gradients, indicating that the ionic selectivity of their pores is similar to that of the intact membrane. Valinomycin greatly increased the potential generated by the diffusion of K⁺ ions although membrane permeability to the other ions used to maintain the ionic strength constant also influenced fluorescence levels. In the presence of valinomycin, active toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1C and Cry1E) efficiently depolarized the membrane at pH 7.5 and 10.5.     

Functional domains of the gastric HK ATPase

The gastric H⁺ + K⁺ ATPase is a member of the phosphorylating class of transport ATPase. Based on sequence homologies and CHO content, there may be ab subunit associated with the catalytic subunit of the H⁺ + K⁺ ATPase. Its function, if present, is unknown. The pump catalyzes a stoichiometric exchange of H⁺ for K⁺, but is also able to transport Na⁺ in the forward direction. This suggests that the transport step involves hydronium rather than protons. The initial binding site is likely to contain a histidine residue to account for the high affinity of the cellular site. The extracellular site probably lacks this histidine, so that a low affinity for hydronium allows release into a solution of pH 0.8. Labelling with positively charge, luminally reactive reagents that block ATPase and pump activity has shown that a region containing H5 and H6 and the intervening luminal loop is involved in necessary conformational changes for normal pump activity. The calculated structure of this loop shows the presence of ana helical,b turn, andb strand sector, with negative charges close to the membrane domain. This sector provides a possible site of interaction of drugs with the H⁺ + K⁺ ATPase, and may be part of the K⁺ pathway in the enzyme.Emory University, Atlanta, Georgia.     

Tension in Skinned Frog Muscle Fibers in Solutions of Varying Ionic Strength and Neutral Salt Composition

The maximal calcium-activated isometric tension produced by a skinned frog single muscle fiber falls off as the ionic strength of the solution bathing this fiber is elevated declining to zero near 0.5 M as the ionic strength is varied using KCl. When other neutral salts are used, the tension always declines at high ionic strength, but there is some difference between the various neutral salts used. The anions and cations can be ordered in terms of their ability to inhibit the maximal calcium-activated tension. The order of increasing inhibition of tension (decreasing tension) at high ionic strength for anions is propionate^- SO₄^-- < Cl^- < Br^-. The order of increasing inhibition of calcium-activated tension for cations is K⁺ Na⁺ TMA⁺ < TEA⁺ < TPrA⁺ < TBuA⁺. The decline of maximal calcium-activated isometric tension with elevated salt concentration (ionic strength) can quantitatively explain the decline of isometric tetanic tension of a frog muscle fiber bathed in a hypertonic solution if one assumes that the internal ionic strength of a muscle fiber in normal Ringer's solution is 0.14–0.17 M. There is an increase in the base-line tension of a skinned muscle fiber bathed in a relaxing solution (no added calcium and 3 mM EGTA) of low ionic strength. This tension, which has no correlate in the intact fiber in hypotonic solutions, appears to be a noncalcium-activated tension and correlates more with a declining ionic strength than with small changes in [MgATP], [Mg], pH buffer, or [EGTA]. It is dependent upon the specific neutral salts used with cations being ordered in increasing inhibition of this noncalcium-activated tension (decreasing tension) as TPrA⁺ < TMA⁺ < K⁺ Na⁺. Measurements of potentials inside these skinned muscle fibers bathed in relaxing solutions produced occasional small positive values (<6 mV) which were not significantly different from zero.     

Extraction of horseradish peroxidase and its conjugates formed by surface-active agent micelles

The author studied peculiarities of the extraction of horseradish peroxidase (HRP) and its conjugates with 3 and 7 molecules of progesterone (PROG) from the aqueous solution into heptane and chloroform containing reversed micelles of surfactants. Micelles of cetyltrimethylammonium bromide, Aerosol OT, and Triton X-45 protect the enzyme from denaturation in the biphasic system. The enzyme is readily extracted from the aqueous phase in the organic medium containing reversed micelles of surfactants at low values of pH. The addition of PEG-6000 (5%) to the aqueous phase enhances the enzyme solubilization at pH 8.6-9.0. The enzyme solubilization significantly increases, when surfactants with unlike charges are used. Inorganic salts decrease the specific solubilization of the enzyme. The HRP modification with progesterone has a weak effect on the enzyme solubilization with reversed micelles.