Effect of lipid phase transition on the binding of anions to dimyristoylphosphatidylcholine liposomes

Temperature dependence of the electrophoretic mobility of multilamellar liposomes prepared from dimyristoylphosphatidylcholine was measured in the presence of salts with different anions in aqueous solutions. It was established that specific binding of anions to liposome surface induced a pronounced zeta potential (electrostatic potential at the hydrodynamic plane of shear). A combination of Langmuir, Gouy-Chapman, and Boltzmann equations was used to describe the dependence of the zeta potential on the concentration of anions. The values of binding constants (K) and maximum numbers of binding sites per unit area (σ_max) were determined by this method. The sequence for anion affinities to liposome surface was found to be as follows: trinitrophenol >ClO₄⁻ >I⁻ >SCN⁻ >Br⁻ >NO₃⁻ >Cl⁻ SO₄²⁻. A sharp increase in the negative zeta potential was detected at the temperature of phase transition of the lipid from the gel to liquid-crystalline state. It was found that the parameter K did not change at lipid phase transition and the shifts in zeta potential might be due to alterations of σ_max. The binding sites were considered as defects in the package of lipid molecules in membranes.     

Specific cation modulation of anion transport across the human erythrocyte membrane

The specific modulation by three cations, Ca²⁺, Mg²⁺, and tetracaine of the equilibrium exchange of SO₄²⁻ across the erythrocyte membrane was investigated. While external calcium had no effect on SO₄²⁻ exchange, internal calcium, and external calcium in the presence of 10 μM A23187 were found to be potent inhibitors of the exchange reaction. The apparent inhibition constants (K₁) for Ca²⁺ were calculated to be 6.1 μM and 5 μM for the above two conditions, respectively.Unlike Ca²⁺, Mg²⁺ was shown to be a weak activator of SO₄²⁻ exchange with an apparent dissociation constant of 3.6 μM. Competition experiments demonstrated that the Ca²⁺ and Mg²⁺ sites associated with anion transport are distinct and noninteracting.Tetracaine, a cation at neutral pH, was also found to be an inhibitor of SO₄²⁻ exchange with an apparent K₁ of 0.8 mM. Although tetracaine was observed to displace calcium from non-specific sites on the erythrocyte membrane, it showed no effect on the apparent inhibition constant of Ca²⁺ for SO₄²⁻ exchange. Thus, the Ca²⁺ and tetracaine sites also appear to be independent. The difficulty of situating three mutually independent sites on a single subunit protein, i.e., band 3, is considered.Using the experimental data obtained from five individuals, the concentration of free calcium in the red cell cytoplasm was calculated to range from 0.2 to 0.7 μM. This concentration was sufficient to reduce SO₄²⁻ exchange only 3–8%. It was concluded that calcium inhibition of anion exchange, and, hence, impairment of CO₂ transport, may be physiologically significant only in senescent cells and in certain types of anemia where calcium concentrations are significantly increased.     

Partitioning of small organic molecules in aqueous biphasic systems

Aqueous biphasic systems (ABS) are suitable for the separation of small organic molecules in industrial and environmental applications and thus, it is important to correlate partitioning behavior of model organic solutes with their structure in order to develop predictive models. The partitioning behavior of five, uncharged, substituted benzenes (benzene, toluene, chlorobenzene, 1,4-dichlorobenzene and 1,2,4-trichlorobenzene) were studied in ABS prepared from stock solutions of 40% (w/w) PEG-2000 and increasing concentrations of four water-structuring salts (K₃PO₄, K₂CO₃, (NH₄)₂SO₄ and NaOH). For a given solute and a defined concentration of salt, the partition coefficients increase as the ΔG_hyd value of the salt anion becomes more negative (e.g., D_benzene increases in the order OH⁻<SO₄²⁻<CO₃²⁻<PO₄³⁻). In a given salt, the distribution ratios increase in the order benzene<toluene<chlorobenzene<1,4-dichlorobenzene<1,2,4-trichlorobenzene. The partitioning behavior of the solutes in PEG–salt ABS was found to be strongly correlated with their partitioning coefficients in 1-octanol–water biphasic systems.     

Interactions of neomycin and calcium in synaptosomal membranes and polyphosphoinositide monolayers

Neomycin and related aminoglycosidic antibiotics displace calcium from synaptosomes of guinea pig cerebral cortex and from preparations of phosphatidylinositol diphosphate. At low drug concentrations, inhibition of synaptosomal calcium binding is competitive (K_i = 3·10⁻⁵M), at high concentrations it is non-competitive (K_i = 4·10^−4M). Monomolecular films of phosphatidylinositol diphosphate are contracted by low concentrations of neomycin in the subphase, and are expanded at high concentrations. This expansion persists even at the collapse pressure indicating a strong interaction between the drug and the lipid.     

Coordinated cations in dipicolinato complexes of divalent metal ions

Several salts of alkali, alkaline earth metal and organic ammonium cations of a complex anion [ML₂]²⁻ {Where L = dipicolinato dianion, M = copper(II), nickel(II) and zinc(II)} are prepared. The coordination effect of [ML₂]²⁻ with the cations such as sodium, potassium, calcium, magnesium, and organic cations namely diammonium cation of 1,5-pentanediamine, diammonium cation of 1,8-octyldiamine, mono ammonium cation of 4-aminobenzylamine are studied by determining their X-ray crystal structures. Depending on the nature of cations, four different types of structures are obtained. When calcium is the cation a polymeric structure with calcium ions bridging the [ML₂]²⁻ is observed. The salts having sodium and potassium cations form polymeric chain like structures by oxo and aqua bridges. In the case of magnesium, the hydrated form of magnesium cations coordinates to [ML₂]²⁻. The organic ammonium salts of [ML₂]²⁻ have the structural features of conventional ionic complexes. These salts easily exchange cations. The organic ammonium salts of [ML₂]²⁻ decomposes to give the corresponding metal oxides at relatively low temperature range 300-450 °C.     

Identification and characterization of beta1-adrenergic receptors in rat parotid membranes

&#x02022; Beta-adrenergic receptor identification and properties are probed in rat parotid membranes utilizing the high affinity β-adrenergic antagonist(−)-[³H]dihydroalprenolol.

&#x02022; The binding of (−)-[³H]dihydroalprenolol to membrane preparations of parotid is rapid, equilibrium being reached in 5 min. Strict stereospecificity is observed, (−)-propanolol being 100 times more potent than (+)-propranolol in competing with (−)-[³H]dihydroalprenolol for binding sites. Beta-adrenergic agonists compete for the binding sites with (−)-[³H]dihydroalprenolol with the same characteristics, i.e., much higher concentrations of the (+)-stereoisomers than the (−)-stereoisomers are required to produce 50% inhibition, the range varies from 14-fold for epinephrine to 300-fold for isoproterenol. Among the (−)-stereoisomers, the relative potency of inhibitory action is (−)-propranolol > (−)-isoproterenol > (−)-epinephrine ≡ (−)-norepinephrine. (−)-Isoproterenol is about 20 times as potent as norepinephrine, the least potent agonist among all the catecholamine (−)-stereoisomers.

&#x02022; The binding of (−)-[³H]dihydroalprenolol is saturable, with a maximum number of binding sites equalling 450 fmol/mg protein and a dissociation constant of 7.9 nM. The Scatchard plots show no significant curvilinear character. Hill plots consistently give a Hill coefficient close to unity (0.92–1.05). Both pieces of evidence suggest a single-component system with no significant cooperativity.

&#x02022; Dissociation kinetics study after the method of De Metys et al. (1973) Biochem. Biophys. Res. Commun. 155, 154, indicates a lack of site-to-site interactions among the binding sites. The rate of dissociation of bound (−)-[³H]dihydroalprenolol is the same in the presence and absence of 1 · 10⁻⁵ M (±)-alprenolol.

&#x02022; Based on the binding of (−)-[³H]dihydroalprenolol, it is concluded that the beta-adrenergic receptors can be identified in rat parotid and that these binding sites display β₁ character. Results of the study indicate a one-component system with no observable site-to-site interactions.

Role of CFTR and anion exchanger in bicarbonate fluxes in C127 cell lines

C127 cell lines transfected with wtCFTR, ΔF508CFTR or vector were employed to determine HCO⁻₃ fluxes in the presence or absence of functional CFTR, using the pH-sensitive dye BCECF. Both cytosolic alkalinization and acidification were due to activity of anion exchanger and were similar in the three cell lines, indicating that expression of CFTR did not influence anion exchanger activity. In C127wt cells only, cAMP elevating agents significantly stimulated HCO⁻₃ fluxes, insensitive to the inhibitor of anion exchanger 4,4′-diisothiocyanate dihydrostilbene-2,2′-disulfonic acid, suggesting that activated CFTR directly mediates both HCO⁻₃ influx and efflux and therefore can contribute to intracellular and extracellular pH regulation.     

Effects of addition of calcium and magnesium salts on ammonia volatilization during manure decomposition

Ammonia volatilization during aerobic decomposition of poultry manure was significantly reduced through additions of calcium and magnesium salts. The percentage reduction in ammonia loss decreased during the 48 day decomposition period from 85–100% in the first 2–3 weeks, to 23–52% at the end of the experiment. The maximum amount of ammonia which was retained (i.e. maximum reduction in ammonia loss) through addition of the chloride salts of Mg²⁺ or Ca²⁺ was independent of the type of cation. However, CaCl₂ released some of the ammonia initially retained as production of CO₂ and NH₃ from the manure decreased after 3 weeks of decomposition, whereas both MgCl₂ and MgSO₄ did not release any of the initially retained ammonia over the 7 week incubation period. Over the entire incubation period MgCl₂ therefore retained more ammonia than CaCl₂. Magnesium sulphate was considerably less effective in retaining ammonia than either chloride salts.     

Modulation of human myometrial PGE2 receptor by GTP characterization of receptor subtype

We studied PGE₂ specific binding sites in human myometrial microsomes prepared from uterine specimens obtained by hysterectomy (women between 38 and 55 years of age). Competition experiments showed that the potency order for various prostaglandins (PGs) was : PGE₂ ≥ PGE₁ PGF_2α > Iloprost ≥ Carbacyclin ZK 110841 (PGD₂ analogue). These relative affinities indicated that the receptor was of the EP type.In kinetic experiments GTP, GppNHp and GTPγS increased the rate of PGE₂ binding (steady state was reached more rapidly in the presence of nucleotides) but maximal specific binding was not significantly different. Complete dissociation could not be obtained, even in the presence of GTP. Only 50% of maximal binding was readily dissociable. The dissociation rate was 4.56.10⁻⁴ sec⁻¹ (half time of about 660 sec) and in the presence of GTP analogues it was slightly increased (k−1 = 7.16 10⁻⁴ sec⁻¹ half time 420 sec.). Scatchard analysis of saturation curves showed an increase in ligand receptor affinity in the presence of GTP or nucleotide analogues: the Kd shifted from 9.66 ± 2.8.10⁻⁹ M to 4.96 ± 1.25.10⁻⁹M, but the number of binding sites did not change significantly (310 ± 37 to 350 ± 17 fmol/mgP). The effect of GTP was observed at a concentration of 5.10⁻⁴M. GppNHp and GTPγS were effective at 1.10⁻⁵M. Pretreatment of myometrial membranes with pertussis or cholera toxins had no effect on PGE₂ binding to membrane sites. Our conclusion is that GTP induced conversion of a population of low affinity sites into a population of higher affinity sites. This effect of guanine nucleotides was described in adipocytes and kidney medulla.Competition studies with PGE₂ analogues (sulprostone, 17-phenyl-ω-trinor PGE₂, M&B 28,767, misoprostol, butaprost) showed that this receptor mediates a contractile response and is probably an EP₃ subtype.     

The effect of pressure on the photochemistry of tris(bipyridyl)chromium(III) ion

The quantum yield for the photoaquation of Cr(bpy)₃³⁺ in basic medium decreases with increasing pressure, with an apparent volume of activation of 3.8 ± 1.0 ml mol⁻¹. From this value and that associated with the phosphorescence lifetime, the volumes of activation for non-radiative decay to the ground state and for formation of photoproduct are derived as −1.6 and +2.9 ml mol⁻¹, respectively. The latter value is consistent with either an associative process with water entering from pockets between the ligands or a dissociative process involving one or both bonds to a bipyridyl ligand.     

Hydration Potential of Lysozyme: Protein Dehydration Using a Single Microparticle Technique

For biological molecules in aqueous solution, the hydration pressure as a function of distance from the molecular surface represents a very short-range repulsive pressure that limits atom-atom contact, opposing the attractive van der Waals pressure. Whereas the separation distance for molecules that easily arrange into ordered arrays (e.g., lipids, DNA, collagen fibers) can be determined from x-ray diffraction, many globular proteins are not as easily structured. Using a new micropipette technique, spherical, glassified protein microbeads can be made that allow determination of protein hydration as a function of the water activity (a_w) in a surrounding medium (decanol). By adjusting a_w of the dehydration medium, the final protein concentration of the solid microbead is controlled, and ranges from 700 to 1150 mg/mL. By controlling a_w (and thus the osmotic pressure) around lysozyme, the repulsive pressure was determined as a function of distance between each globular, ellipsoid protein. For separation distances, d, between 2.5 and 9 Å, the repulsive decay length was 1.7 Å and the pressure extrapolated to d = 0 was 2.2 × 10⁸ N/m², indicating that the hydration pressure for lysozyme is similar to other biological interfaces such as phospholipid bilayers.     

Ion fluxes and abscisic Acid-induced proline accumulation in barley leaf segments

The increase in proline induced by ABA, a process stimulated by NaCl or KCl in barley leaves, did not occur when Na⁺ (or K⁺) was present in the external medium as the gluconate salt, namely with an anion unable to permeate the plasma membrane. However, proline increase was restored, to different extents, by the addition of various chloride salts but not by ammonium chloride. Moreover, it was shown that the stimulation of the process by NaCl (or KCl) was variously affected by the presence of different salts; all the ammonium salts (10 millimolar NH₄⁺ concentration) inhibited this stimulation almost completely. Inhibition by NH₄⁺ was accompanied by a decreased Na⁺ influx (−40%). Also, in the case of Na-gluconate, Na⁺ uptake was reduced and the addition of Cl⁻ as the calcium or magnesium salt (but not as ammonium salt) restored both the ion influxes and the increase in proline typical of NaCl treatments. Both 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS), an anion transport inhibitor, and tetraethylammonium chloride (TEA), a K⁺ channels-blocking agent, caused, as well as with a reduction of ion influxes, an inhibition of the proline accumulation. The inhibition was practically total with 1 millimolar DIDS and about 80% with 20 millimolar TEA. A possible role of ion influxes in the process leading to the increase in proline induced by ABA is proposed.     

Thyroid hormone: Aldosterone antagonism in cultured epithelial cells

Thyroid hormone (T₃) has been demonstrated to inhibit the action of aldosterone on sodium transport in toad urinary bladder and rat kidney. We have exammined the effect of T₃ on aldosterone action and specific nuclear binding in cultured epithelial cells derived from toad urinary bladder. In cell line TB6-C, addition of 5·10⁻⁸ M T₃ to culture media for up to 3 days results in no change in short-circuit current or transepithelial resistance. This concentration of T₃ completely inhibits the maximal increase in short-circuit current in response to 1·10⁻⁷ M aldosterone. The inhibition can be demonstrated with 18 h preincubation or with simultaneous addition of T₃ and aldosterone. The half-maximal concentration for the inhibition of the aldosterone effect is approx. 5·10⁻⁹ M T₃. T₃ has no effect on cyclic AMP-stimulated short-circuit current in these cells. The effect of T₃ on nuclear binding of [³H]aldosterone was examined using a filtration assay with data analysis by at least-squares curve-fitting program. Best fit was obtained with a model for two binding sites. The dissociation constants for the binding were K′_d1 = (0.82 ± 0.36)·10⁻¹⁰ M and K′_d2 = (3.2±0.60)·10⁻⁸ M.The half-maximal concentration for aldosterone-stimulated sodium transport in these cells is approx. 1·10⁻⁸ M. Analysis of nuclear aldosterone binding in cells preincubated for 18 h with 5·10⁻⁸ M T₃ showed a K′_d1 = (0.15 ± 0.10)·10⁻¹⁰ M and K′_d2 = (3.5 ± 0.10)·10⁻⁸ M. We conclude that T₃ i action of aldosterone on sodium transport at a site after receptor binding in the nucleus.     

Non-aqueous capillary electrophoresis chiral separations with sulfated β-cyclodextrin

This paper reports the application of an anionic cyclodextrin (CD), sulfated β-cyclodextrin with a degree of substitution of four (β-CD-(SO₄⁻)₄, in chiral separations of pharmaceutical enantiomers by non-aqueous capillary electrophoresis (NACE). Upon complexation with the anionic CD, electrophoretic mobilities of the basic enantiomers decreased, however, both separation selectivity and resolution were enhanced. The advantage of NACE chiral separations over the aqueous CE with the charged CD is that higher electric field strength and higher ionic strength could be applied due to the characteristics of the solvent formamide. The higher ionic strength leads to stacking of peaks and reduces the electrodispersion caused by the mobility mismatch between β-CD-(SO₄⁻)₄–analyte complexes and the co-ions in the running buffer. As a result, better peak shapes and higher separation efficiency were obtained. Comparing with NACE chiral separations with neutral CDs, lower concentration of β-CD-(SO₄⁻)₄ was needed due to the fact that the electrostatic attraction caused stronger binding between β-CD-(SO₄⁻)₄ and the enantiomers. The effects of the experimental parameters, such as concentration of the CD, apparent pH (pH*), degree of substitutions of the CDs, percentage of water in mixed solvent systems, and type of solvents were also studied.     

Enantioselective acylation of (RS)-phenylethylamine catalysed by lipases

The enzymatic acylation of (RS)-phenylethylamine with different acyl donors catalysed by lipases, was studied in organic solvents with different hydrophobicities and in mixtures with ionic liquids ((ILs); [BMIm][BF₄], [BMIm][SCN], [BMIm][Cl] and [BMIm][PF₆]). Using lipases from Candida antarctica B (CAL-B) and from Aspergillus niger higher conversion degrees and E-values were obtained with ethyl acetate as the acyl donor. When CAL-B was used as the biocatalyst, in a two-phase system formed by [BMIm][X]/dichloromethane or [BMIm][X]/chloroform, the selectivity was better than that obtained in pure organic solvents. The selectivity was found to be related to individual anions in ILs. In this reaction, the ion effectiveness in enhancing the enzyme selectivity followed the series: Cl⁻ > SCN⁻ > BF₄⁻ > PF₆⁻ in mixtures with dichloromethane, and PF₆⁻ > BF₄⁻ > SCN⁻ > Cl⁻ in mixtures with chloroform.     

Effect of ions on phospholipid layer structure as indicated by Raman Spectroscopy

Various anions and cations are found to induce changes in the layered structure of phosphatidylcholine-water systems as indicated by Raman Spectroscopy. From the ratio of Raman intensities, , it is inferred that dispositive ions decrease the proportion of gauche character in the hydrocarbon chains, with the relative influence being: Ba²⁺ < Mg²⁺ < Ca²⁺ ≈ Cd²⁺. Unipositive ions (Li⁺, K⁺ and Na⁺) produce no observed changes in the Raman spectrum of the lecithin dispersion. The proportion of gauche character of the hydrocarbon chains is found to be nearly independent of the anion for: Br⁻, Cl⁻, acetate⁻, I⁻, ClO₄⁻, CNS⁻ and SO₄²⁻. Dispersions prepared with a solution of KI + I₂ produced Raman spectra in which the 1089 cm⁻¹ peak, which is characteristic of random lipid chains, was greatly intensified, presumably because of the presence of I₃⁻ which is known to penetrate the lipid lamellae. The observed trends are discussed.     

Temperature dependence of anion transport in the human red blood cell

Arrhenius plots of chloride and bromide transport yield two regions with different activation energies (E_a). Below 15 or 25°C (for Cl⁻ and Br⁻, respectively), E_a is about 32.5 kcal/mol; above these temperatures, about 22.5 kcal/mol (Brahm, J. (1977) J. Gen. Physiol. 70, 283–306). For the temperature dependence of SO₄²⁻ transport up to 37°C, no such break could be observed. We were able to show that the temperature coefficient for the rate of SO₄²⁻ transport is higher than that for the rate of denaturation of the band 3 protein (as measured by NMR) or the destruction of the permeability barrier in the red cell membrane. It was possible, therefore, to extend the range of flux measurements up to 60°C and to show that, even for the slowly permeating SO₄²⁻ in the Arrhenius plot, there appears a break, which is located somewhere between 30 and 37°C and where E_a changes from 32.5 to 24.1 kcal/mol. At the break, the turnover number is approx. 6.9 ions/band 3 per s. Using ³⁵Cl⁻-NMR (Falke, Pace and Chan (1984) J. Biol. Chem. 259, 6472–6480), we also determined the temperature dependence of Cl⁻-binding. We found no significant change over the entire range from 0 to 57°C, regardless of whether the measurements were performed in the absence or presence of competing SO₄²⁻. We conclude that the enthalpy changes associated with Cl⁻-or SO₄²⁻-binding are negligible as compared to the E_a values observed. It was possible, therefore, to calculate the thermodynamic parameters defined by transition-state theory for the transition of the anion-loaded transport protein to the activated state for Cl⁻, Br⁻ and SO₄²⁻ below and above the temperatures at which the breaks in the Arrhenius plots are seen. We found in both regions a high positive activation entropy, resulting in a low free enthalpy of activation. Thus the internal energy required for carrying the complex between anion and transport protein over the rate-limiting energy barrier is largely compensated for by an increase of randomness in the protein and/or its aqueous environment.     

Effect of inorganic salts on hemochromogen aggregation

The absorbance and difference absorbance spectra of pyridine-hemochromogen at different concentrations showed the presence of two different types of pyridine-hemochromogens, one unstable form (compound III) at low concentration and a more stable form (compound II) at higher concentrations, suggesting that there is an interaction between hemochromogen molecules at high concentration. In the presence of inorganic salts, the unstable compound III is converted to stable compound II. The effect of various inorganic ions on the formation of compound II has been studied. The order of increasing effectiveness of the anions on compound II formation was HPO₄²⁻SO₄²⁻F⁻Cl⁻Br⁻NO₃⁻SCN⁻ and that of cation was Li⁺Na⁺K⁺. The results are discussed on the basis of the effect of these ions on the structure of water. It appears compound II is an aggregate of compound III and the aggregation is due to hydrophobic interaction.     

Characterization of Anion Effects on the Nitrate-Sensitive ATP-Dependent Proton Pumping Activity of Soybean (Glycine max L.) Seedling Root Microsomes

The ATP-dependent proton-pumping activity of soybean (Glycine max L.) root microsomes is predominantly nitrate sensitive and presumably derived from the tonoplast. We used microsomes to characterize anion effects on proton pumping of the tonoplast vesicles using two distinctly different techniques.

Preincubation of the vesicles with nitrate caused inhibition of proton pumping and ATPase activity, with similar concentration dependence. Fluoride, which preferentially inhibits the plasma membrane ATPase, inhibited ATPase activity strongly at concentrations which did not affect proton pumping activity.

Addition of potassium salts, after a steady-state pH gradient is established in the absence of such salts, caused an increased pH gradient which was due to alleviation of Δ Ψ and subsequent increased influx of H⁺ into these vesicles. This anion-induced increase in the pH gradient could be used as a measure of the relative anion permeabilities, which were of the order Br⁻ = NO₃⁻ > Cl⁻ SO₄²⁻. Phosphate and fluoride caused no increase in the pH gradient. Since the concentration dependence of KCl- and KNO₃-induced quenching exhibited a saturable component, and since H⁺ uptake was increased by only certain anions, the data suggest that there may be a relatively specific anion channel associated with tonoplast-derived vesicles.

     

Effects of prostacyclin on coronary circulation, heart rate and myocardial contractile force in isolated hearts of guinea PIG and rabbit — Comparison with prostaglandin E2

Infusions of prostacyclin (PGI₂) (3 × 10⁻¹⁰ − 3 × 10⁻⁷M) into the coronary circulation of isolated hearts from guinea pigs or rabbits resulted in a concentration-dependent decrease in the coronary perfusion pressure (CPP). There was a slight decrease in left ventricular systolic pressure in the heart of the rabbit, whereas the heart rate remained unchanged. PGE₂ was without effect on the heart of the rabbit but was as potent as PGI₂ in decreasing the CPP in the guinea pig heart. 6-oxo-PGF_1α (up to 3 × 10⁻⁶ M) did not affect any of the parameters measured.