The effects of monovalent cations Li+, Na+, K+, NH4+, Rb+ and Cs+ on the solid and solution structures of the nucleic acid components. Metal ion binding and sugar conformation.

The interactions of the monovalent ions Li+, Na+, K+, NH4+, Rb+ and Cs+ with adenosine-5'-monophosphoric acid (H2-AMP), guanosine-5'-monophosphoric acid (H2-GMP) and deoxyguanosine-5'-monophosphoric acid (H2-dGMP) were investigated in aqueous solution at physiological pH. The crystalline salts M2-nucleotide.nH2O, where M = Li+, Na+, K+ NH4+, Rb+ and Cs+, nucleotide = AMP, GMP and dGMP anions and n = 2-4 were isolated and characterized by Fourier Transform infrared (FTIR) and 1H-NMR spectroscopy. Spectroscopic evidence showed that these ions are in the form of M(H2O)n+ with no direct metal-nucleotide interaction, in aqueous solution. In the solid state, Li+ ions bind to the base N-7 site and the phosphate group (inner-sphere), while the NH4+ cations are in the vicinity of the N-7 position and the phosphate group, through hydrogen bonding systems. The Na-nucleotides and K-nucleotides are structurally similar. The Na+ ions bind to the phosphate group of the AMP through metal hydration shell (outer-sphere), whereas in the Na2-GMP, the hydrated metal ions bind to the base N-7 or the ribose hydroxyl groups (inner-sphere). The Na2-dGMP contains hydrated metal-carbonyl and metal-phosphate bindings (inner-sphere). The Rb+ and Cs+ ions are directly bonded to the phosphate groups and indirectly to the base moieties (via H2O). The ribose moiety shows C2'-endo/anti conformation for the free AMP acid and its alkali metal ion salts. In the free GMP acid, the ribose ring exhibits C3'-endo/anti conformer, while a C2'-endo/anti sugar pucker was found in the Na2-GMP and K2-GMP salts and a C3'-endo/anti conformation for the Li+, NH4+, Rb+ and Cs+ salts. The deoxyribose has C3'-endo/anti conformation in the free dGMP acid and O4'-endo/anti in the Na2-dGMP, K2-dGMP and a C3'-endo/anti for the Li+, NH4+, Rb+ and Cs+ salts. An equilibrium mixture of the C2'-endo/anti and C3'-endo/anti sugar puckers was found for these metal-nucleotide salts in aqueous solution.     

Infrared and 31P-NMR studies of the interaction of Mg2+ with phosphatidylserines: effect of hydrocarbon chain unsaturation

Infrared and 31P-NMR spectra of aqueous dispersions of 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and ox brain phosphatidylserine in the presence of excess Mg2+ have been recorded. A consistent picture emerges from the application of infrared and 31P-NMR spectroscopy to Mg2+-PS interactions. Mg2+ forms crystalline complexes with saturated phosphatidylserines, such as DMPS, and probably with POPS. In these crystalline PS-Mg2+ complexes the phosphate group loses its water of hydration but the serine carboxylate remains hydrated. Furthermore, there is formation of an additional hydrogen bond to one of the ester carbonyl groups of DMPS, and interchain interactions appear to be enhanced as reflected by a tighter packing of the fatty acyl chains. One main conclusion of this work is that Mg2+ binding to PS bilayers shows a gradation, the binding is in the order DMPS greater than POPS greater than ox brain PS greater than DOPS. The molecular area increases in the order DMPS less than ox brain PS less than POPS less than DOPS and is apparently an important parameter determining the affinity of PS for Mg2+. The general trend is that with increasing molecular area, and hence spacing of the ligands, the binding of Mg2+ decreases. While PS with two saturated fatty acyl chains forms tightly packed, crystalline Mg2+ complexes with an immobilized headgroup, the unsaturated PS molecules such as ox brain PS and DOPS interact only weakly with Mg2+. Their interaction seems to be restricted to electrostatic shielding, since no major changes in molecular conformation, chain packing and headgroup hydration are found. The interaction of POPS with Mg2+ is intermediate between that of saturated PS and that of DOPS. POPS exhibits a higher affinity for Mg2+ than ox brain PS, although their molecular areas (and the surface charge density) are approximately the same. This apparent anomaly is proposed to be due to a discreteness of charge effect. It is proposed that a lipid surface with regularly spaced polar groups has a higher affinity for binding Mg2+.     

Infrared and 31P-NMR studies of the effect of Li+ and Ca2+ on phosphatidylserines

Infrared and 31P-NMR spectra of solid samples of 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) have been recorded. Comparison of the spectra of the Na+ salts of these phospholipids with those of complexes formed with Li+ and Ca2+ ions allows the characterization of conformational changes induced by complexation with Li+ and Ca2+. Ca2+ forms tight, crystalline complexes with these phosphatidylserines (PS), irrespective of the degree of unsaturation in the hydrocarbon chains. In these PS-Ca2+ complexes the torsion angles of the two P-O ester bonds exhibit the antiplanar-antiplanar conformation which is significantly different from the standard gauche-gauche conformation commonly found in phosphodiesters. In contrast, complexation with Li+ does not induce this conformational change in the phosphodiester group. It is shown that the degree of unsaturation in the hydrocarbon chains, and related to it, the cross-sectional area of the phospholipid or the surface charge density, determine the affinity of the phosphatidylserine for the metal ion. In general, the affinity of phosphatidylserines for both Li+ and Ca2+ decreases with increasing unsaturation in the hydrocarbon chains or decreasing surface charge density; it is in the order DMPS greater than POPS greater than DOPS.     

Infrared studies of fully hydrated unsaturated phosphatidylserine bilayers. Effect of Li+ and Ca2+

Infrared spectroscopy has been used to characterize the thermal-phase behavior of fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) as well as their interaction with Li+ and Ca2+. The order-disorder transition of POPS-NH4+ is at 17 degrees C; in the presence of Li+ a POPS-Li+ complex is formed, and the transition temperature of this complex is 40 degrees C. DOPS-NH4+ has an order-disorder transition at -11 degrees C, and unlike POPS the addition of Li+ has no effect on the thermal behavior of DOPS-NH4+. This indicates that the binding of Li+ to DOPS is negligible or very weak. Li+ binds to the phosphate and carboxylate groups of POPS, and as a result these groups lose their water of hydration. Li+ binding induces a conformational change, probably in the glycerol backbone of POPS; however, the conformation of the two P-O ester bonds remains gauche-gauche as in POPS-NH4+. Both POPS and DOPS form crystalline complexes with Ca2+. As a result of Ca2+ binding to the phosphate, this group loses its water of hydration and there is a conformational change in the P-O ester bonds from gauche-gauche to antiplanar-antiplanar. In contrast to the POPS-Li+ complex, the carboxylate group remains hydrated in the Ca2+ complexes. Furthermore, in these PS-Ca2+ complexes a new hydrogen bond is formed between one of the ester C=O groups and probably water. Such a situation is not found in the NH4+ and Li+ salts of phosphatidylserine.     

Interactions of metal ions with phosphatidylserine bilayer membranes: effect of hydrocarbon chain unsaturation

A combination of surface monolayer, scanning calorimetry, 31P NMR, and spin-label ESR techniques has been used to monitor the interactions of monovalent (NH4+, Na+, and Li+) and divalent (Ca2+) cations with phosphatidylserines (PS) differing in their levels of chain unsaturation. Comparisons are made between the disaturated dimyristoyl-, dipalmitoyl-, and dihexadecyl-PS (DMPS, DPPS, and DHPS), saturated cis-monounsaturated palmitoyloleoyl-PS (POPS) (and bovine brain PS), di-trans-monounsaturated dielaidoyl-PS (DEPS), and di-cis-monounsaturated dioleoyl-PS (DOPS). Na+ and NH4+ cations interact weakly with all PS monolayers and bilayers without significant changes in molecular conformation, chain packing, or headgroup dynamics and without dependence on chain composition. In contrast, considering these structural and dynamic parameters, Li+ shows a gradation in its interaction with PS (DMPS greater than POPS approximately bovine brain PS greater than DOPS), suggesting that Li+-PS interactions depend on the interfacial properties of the PS molecules (e.g., surface area). Finally, Ca2+ interacts strongly with all PS monolayers and bilayers, without obvious chain selectivity. Thus, ion binding to PS depends not only on the properties of the cation (Na+ vs Li+ vs Ca2+) but also on the molecular details of the PS membrane surface.     

Fourier transform infrared study of fully hydrated dimyristoylphosphatidylglycerol. Effects of Na+ on the sn-1' and sn-3' headgroup stereoisomers

Molecular packing and the thermotropic phase behavior of fully hydrated ammonium salts of 1,2-dimyristoyl-sn-glycero-3-phosphatidyl-sn-1'-glycerol (1'-DMPG) and the corresponding 3' stereoisomer (3'-DMPG) as well as the effects of 300 mM NaCl on these lipids were studied by Fourier transform infrared (FTIR) spectroscopy. The ammonium salts of both stereoisomer show similar thermotropic phase behavior and have an order-disorder phase transition at approximately 21 degrees C. While complexing with Na+, however, an incubation of liposomes at +6 degrees C for 3 days results in significant structural differences between liposomes of 1'-DMPG and 3'-DMPG. In the presence of 300 mM NaCl the infrared spectra for 3'-DMPG reveal the appearance of a more solidified lipid nominated here as the highly crystalline phase with a transition into the liquid-crystalline state at a significantly higher temperature (approximately at 33 degrees C) than that for 1'-DMPG (approximately at 23 degrees C). Crystal field splitting resulting from interchain vibrational coupling is observed in the CH2 scissoring mode of the 3'-DMPG(Na+) complex in the highly crystalline phase (T less than 33 degrees C); i.e., the acyl chains are packed in a rigid orthorhombic- or monoclinic-like crystal lattice. At temperatures above the transition at 33 degrees C the acyl chains of 3'-DMPG(Na+) give rise to infrared spectra indicative of hexagonal packing. The latter type of hydrocarbon chain packing is also found for the ammonium salts of 1'-DMPG and 3'-DMPG without Na+ as well as for 1'-DMPG with Na+. In addition, the binding of Na+ to 3'-DMPG causes narrowing of the bands associated with the interfacial and polar headgroup regions of 3'-DMPG and thus reveals reduced motional freedom. This demonstrates that Na+ binds tightly to 3'-DMPG, leading to the immobilization of the entire phospholipid polar headgroup. Such effects by Na+ are not observed for 1'-DMPG.     

Hydrogen bonds between protein side chains and phosphates and their role in biological calcification

Poly(L-histidine)-phosphate (H2PO4-, HPO4(2-)) and poly(L-glutamate)-phosphate systems (residue/phosphate, 1:1) in the presence of Ca2+ are studied by infrared spectroscopy. In the poly(L-histidine)-phosphate systems N...HOP in equilibrium NH+...O-P hydrogen bonds are formed where most phosphate protons are found at the histidine ring. With an increase in the degree of hydration the proportion of the proton limiting structure NH+...O-P increases. In the poly(L-glutamate)-dihydrogen phosphate system most phosphate protons are found at the carboxylate groups. Different behavior is observed for poly(L-glutamate)-hydrogen phosphate mixtures, where the residence time of the phosphate proton at the hydrogen acceptor carboxylate group is very short. This residence time increases, however, with increasing humidity. All these results support the triphasic theory of biological calcification involving a tripartite protein-calcium-phosphate complex where these hydrogen bonds can be present. The behavior of these hydrogen bonds can also explain the formation of a nidus of calcium phosphate salts in calcium oxalate-containing urinary calculi.     

Detection of secondary structure in glycosaminoglycans via the H n.m.r. signal of the acetamido NH group.

Two simple methods for dissolving salts of acid glycosaminoglycans with inorganic cations (e.g. Li+ and Na+) in dry dimethyl sulphoxide are described. Complete n.m.r. spectra of, e.g., Na+ and Li+ salts of chondroitin sulphate and keratan sulphate were obtained on these solutions. In [2H6]dimethyl sulphoxide the NH resonance of 2-acetamido-2-deoxy hexosides is in the range 7.2-8.0 delta, but is downfield (8.3-9.3 delta) when the NH is H-bonded to -CO2-. Heparan sulphate shows two NH resonances, of which one (at 8.3 delta) is probably indicative of H-bonding. Space-filling models show that a very close approach of NH to -CO2- across the alpha-glucosaminidic bond is possible, and a solution configuration for heparan sulphate is proposed. The n.m.r. results are entirely compatible with interpretations of periodate-oxidation kinetics, based on H-bonded secondary structures present in hyaluronate and chondroitin sulphates, but not in dermatan (or keratan) sulphate.     

Crystallization of phosphatidylserine bilayers induced by lithium

Utilizing differential scanning calorimetry and x-ray diffraction, 1,2-dimyristoyl-L-glycero-3-phospho-L-serine (DMPS) was shown to form hydrated bilayer membrane structures exhibiting a gel leads to liquid crystalline transition at 39 degrees C (delta H = 7.2 kcal/mol). Addition of up to molar concentrations of the alkali halides NaCl, KCl, Rl Cl, and CsCl produced relatively minor changes in DMPS bilayer structure or stability. For example, in the presence of 0.5 M NaCl, the transition temperature (Tc = 42 degrees C) and transition enthalpy (delta H = 7.0 kcal/mol) show only minor changes. In marked contrast, addition of LiCl results in "'crystallization" of the DMPS bilayer membrane structure. At 0.5 M LiCl, the crystalline DMPS exhibits a bilayer gel leads to liquid crystal transition at 89 degrees C accompanied by a high enthalpy change, delta H = 16.0 kcal/mol. Thus, Li+ induces an isothermal crystallization of DMPS bilayers, the hydrocarbon chains adopting a more ordered packing mode than the "hexagonal" arrangement of the gel state. In view of the widespread use of lithium in the treatment of manic-depressive illness, we also raise the possibility that interaction of Li+ with anionic membrane phospholipids could play a role in its pharmacological action.     

Infrared characterization of conformational differences in the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine

Fourier transform infrared spectroscopy was used to characterize the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine (1,3-DPPC), a positional isomer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-DPPC). The molecule exists in three distinct phases over the temperature interval 0-70 degrees C. In the low-temperature (LC) phase, the spectra are indicative of acyl chains packed in an orthorhombic subcell, while the carbonyl groups and phosphate ester at the head group show evidence of only partial hydration. The transition from the low-temperature (LC) phase to the intermediate-temperature (L beta) phase at 25 degrees C corresponds to a temperature-induced head-group hydration in which the hydration of the phosphate and carbonyl ester groups results in the reorganization of the hydrocarbon chain-packing subcell from orthorhombic to hexagonal. The transition from the intermediate (L beta) to the high-temperature (L alpha) phase at 37 degrees C is a gel-to-liquid-crystalline phase transition analogous to the 41.5 degrees C transition of 1,2-DPPC. The spectra of the acyl-chain carbonyl groups show evidence of significant differences in molecular conformation at the carbonyl esters in the LC phase. In the L beta and L alpha phases, the carbonyl band contour becomes much more symmetric. However, two components are clearly present in the spectra indicating that the sn-1 and sn-3 carbonyls experience slightly different environments. The observed differences are likely due to a preferred conformation of the phosphocholine group relative to the glycerol backbone. Indications from the infrared spectra of differences in the structure of the C = O groups provide a possible explanation for the selection of the sn-1 chain of 1,3-DPPC by phospholipase A2 on the basis of a preferred head group conformation.     

Inhibition by K+ of Na+-Dependent D-Aspartate Uptake into Brain Membrane Saccules

Na+-dependent uptake of dicarboxylic amino acids in membrane saccules, due to exchange diffusion and independent of ion gradients, was highly sensitive to inhibition by K+. The IC50 was 1-2 mM under a variety of conditions (i.e., whole tissue or synaptic membranes, frozen/thawed or fresh, D-[3H]aspartate (10-1000 nM) or L-[3H]glutamate (100 nM), phosphate or Tris buffer, NaCl or Na acetate, presence or absence of Ca2+ and Mg2+). The degree of inhibition by K+ was also not affected on removal of ion gradients by ionophores, or by extensive washing with H2O and reloading of membrane saccules with glutamate and incubation medium in the presence or absence of K+ (3 mM, i.e., IC70). Rb+, NH4+, and, to a lesser degree Cs+, but not Li+, could substitute for K+. [K+] showed a competitive relationship to [Na+]2. Incubation with K+ before or after uptake suggested that the ion acts in part by allowing net efflux, thus reducing the internal pool of amino acid against which D-[3H]aspartate exchanges, and in part by inhibiting the interaction of Na+ and D-[3H]aspartate with the transporter. The current model of the Na+-dependent high-affinity acidic amino acid transport carrier allows the observations to be explained and reconciled with previous seemingly conflicting reports on stimulation of acidic amino acid uptake by low concentrations of K+. The findings correct the interpretation of recent reports on a K+-induced inhibition of Na+-dependent "binding" of glutamate and aspartate, and partly elucidate the mechanism of action.     

High-pressure infrared study of phosphatidylserine bilayers and their interactions with the local anesthetic tetracaine

High-pressure Fourier-transform infrared (FT-IR) spectroscopy was used to study the barotropic behavior of phosphatidylserine bilayers and their interactions with the local anesthetic tetracaine. The model membrane systems studied were multilamellar aqueous dispersions of 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) in the absence and the presence of tetracaine at pH 5.5 and 9.5. The infrared spectra were measured at 28 degrees C in a diamond anvil cell as a function of pressure up to 25 kbar. The results show that the barotropic behavior of the negatively charged phosphatidylserine bilayers is very similar to that observed for zwitterionic phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, with corresponding acyl chains. The results also indicate that the local anesthetic partitions into phosphatidylserine bilayers in an environment close to the membrane-water interface and interacts electrostatically with the lipid head group. Application of high hydrostatic pressure on the lipid-anesthetic systems results in the pressure-induced expulsion of the anesthetic from a membrane to an aqueous environment. The pressures required for expulsion of anesthetic from bilayers are much higher for the unsaturated lipid (DOPS) than for the saturated lipid (DMPS) (approximately 6 kbar vs approximately 2 kbar, respectively). Whereas incorporation of the anesthetic into DOPS bilayers does not affect significantly the structural and dynamic properties of the disordered acyl chains in the liquid-crystalline phase, it orders the DMPS acyl chains in the gel phase.     

Thermotropic phase behavior of model membranes composed of phosphatidylcholines containing iso-branched fatty acids. 2. Infrared and 31P NMR spectroscopic studies

The polymorphic phase behavior of aqueous dispersions of a number of representative phosphatidylcholines with methyl iso-branched fatty acyl chains was investigated by Fourier transform infrared (FT-IR) and phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy. For the longer chain phosphatidylcholines, where two transitions are resolved on the temperature scale, the higher temperature event can unequivocally be assigned to the melting of the acyl chains (i.e., a gel/liquid-crystalline phase transition), whereas the lower temperature event is shown to involve a change in the packing mode of the methylene and carbonyl groups of the hydrocarbon chains in the gel state (i.e., a gel/gel transition). The infrared spectroscopic data suggest that the methyl iso-branched phosphatidylcholines assume a partially dehydrated, highly ordered state at low temperatures, resembling the Lc phase recently described for the long-chain n-saturated phosphatidylcholines. At higher temperatures, some branched-chain phosphatidylcholines appear to assume a fully hydrated, loosely packed gel phase similar to but not identical with the P beta, phase of their linear saturated analogues. Thus, the iso-branched phosphatidylcholine gel/gel transition corresponds, at least approximately, to a summation of the structural changes accompanying both the subtransition and the pretransition characteristic of the longer chain n-saturated phosphatidylcholines. The infrared spectroscopic data also show that, in the low-temperature gel state, there are significant differences between the odd- and even-numbered isoacylphosphatidylcholines with respect to their hydrocarbon chain packing modes as well as to their head group and interfacial hydration states.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular acidification-induced alkali metal cation/H+ exchange in human neutrophils

Pretreatment of isolated human neutrophils (resting pHi congruent to 7.25 at pHo 7.40) with 30 mM NH4Cl for 30 min leads to an intracellular acidification (pHi congruen to 6.60) when the NH4Cl prepulse is removed. Thereafter, in 140 mM Na+ medium, pHi recovers exponentially with time (initial rate, approximately 0.12 pH/min) to reach the normal resting pHi by approximately 20 min, a process that is accomplished mainly, if not exclusively, though an exchange of internal H+ for external Na+. This Na+/H+ countertransport is stimulated by external Na+ (Km congruent to 21 mM) and by external Li+ (Km congruent to 14 mM), though the maximal transport rate for Na+ is about twice that for Li+. Both Na+ and Li+ compete as substrates for the same translocation sites on the exchange carrier. Other alkali metal cations, such as K+, Rb+, or Cs+, do not promote pHi recovery, owing to an apparent lack of affinity for the carrier. The exchange system is unaffected by ouabain or furosemide, but can be competitively inhibited by the diuretic amiloride (Ki congruent to 8 microM). The influx of Na+ or Li+ is accompanied by an equivalent counter-reflux of H+, indicating a 1:1 stoichiometry for the exchange reaction, a finding consistent with the lack of voltage sensitivity (i.e., electroneutrality) of pHi recovery. These studies indicate that the predominant mechanism in human neutrophils for pHi regulation after intracellular acidification is an amiloride-sensitive alkali metal cation/H+ exchange that shares a number of important features with similar recovery processes in a variety of other mammalian cell types.     

Structural polymorphism of hydrated monoacylated maltose glycolipids

The physico-chemical properties of three fully hydrated monoacyl maltoside glycolipids were investigated with Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and small-angle X-ray scattering (SAXS). The different synthesized maltoside glycoconjugates vary in the length and saturation of the fatty acid moiety, whereas the constant head group region contains a beta-linked maltose with a OC(2)-NH spacer. The compounds with saturated acyl chains showed a complex pattern of temperature-dependent behaviour, regarding the adopted three-dimensional aggregate structure of the molecules and the main phase transition from the gel to liquid crystalline phase of the acyl chains. A substitution of the saturated acyl chain with an unsaturated acyl chain led to a complete change of the structural preferences, from a high ordered stacking of the bilayers to an unilamellar arrangement of completely disordered and fluid membranes. The presence of the NH group in the spacer, compared to the compounds lacking the NH group allows the formation of a hydrogen bonding network, which influences the observed phase properties. The results of these studies of the hydrated monoacylated maltose glycolipids are discussed in relation to the thermotropic phase properties of the pure compounds in the absence of water.     

Interaction of external H+ with the Na+-H+ exchanger in renal microvillus membrane vesicles

We examined the effects of external H+ on the kinetics of Na+-H+ exchange in microvillus membrane vesicles isolated from the rabbit renal cortex. The initial rate of Na+ influx into vesicles with internal pH 6.0 was optimal at external pH 8.5 and was progressively inhibited as external pH was reduced to 6.0. A plot of 1/V versus [H+]o was linear and yielded apparent KH = 35 nM (apparent pK 7.5). In vesicles with internal pH 6.0 studied at external pH 7.5 or 6.6, apparent KNa was 13 or 54 mM, Ki for inhibition of Na+ influx by external Li+ was 1.2 or 5.2 mM, Ki for inhibition by external NH4+ was 11 or 50 mM, and Ki for inhibition by external amiloride was 7 or 25 microM, respectively. These findings were consistent with competition between each cation and H+ at a site with apparent pK 7.3-7.5. Lastly, stimulation of 22Na efflux by external Na+ (i.e. Na+-Na+ exchange) was inhibited as external pH was reduced from 7.5 to 6.0, also consistent with competition between external H+ and external Na+. Thus, in contrast with internal H+, which interacts at both transport and activator sites, external H+ interacts with the renal microvillus membrane Na+-H+ exchanger at a single site, namely the external transport site, where H+, Na+, Li+, NH4+, and amiloride all compete for binding.     

Inhibition of Na+-H+ exchange by N,N'-dicyclohexylcarbodiimide in isolated rat renal brush border membrane vesicles

The inactivation of rat renal brush border membrane Na+-H+ exchange by the covalent carboxylate reagent N,N'-dicyclohexylcarbodiimide (DCCD) was studied by measuring 1 mM Na+ influx in the presence of a pH gradient (pHi = 5.5; pHo = 7.5) and H+ influx in the presence of a Na+ or Li+ gradient ([Na+]i = 150 mM; [Na+]o = 1.5 mM). In the presence of DCCD, the rate of Na+ uptake decreased exponentially with time and transport inhibition was irreversible. At all DCCD concentrations the loss of activity was described by a single exponential, consistent with one critical DCCD-reactive residue within the Na+-H+ exchanger. Among several carbodiimides the most hydrophobic carbodiimide, DCCD, was also the most effective inhibitor of Na+-H+ exchange. With 40 nmol of DCCD/mg of protein, at 20 degrees C for 30 min, 75% of the amiloride-sensitive 1 mM Na+ uptake was inhibited. Neither the equilibrium Na+ content nor the amiloride-insensitive Na+ uptake was significantly altered by the treatment. The Na+-dependent H+ flux, measured by the change in acridine orange absorbance, was also decreased 80% by the same DCCD treatment. If 150 mM NaCl, 150 mM LiCl, or 1 mM amiloride was present during incubation of the brush border membranes with 40 nmol of DCCD/mg of protein, then Li+-dependent H+ flux was protected 50, 100, or 100%, respectively, compared to membranes treated with DCCD in the absence of Na+-H+ exchanger substrates. The combination of DCCD and an exogenous nucleophile, e.g. ethylenediamine and glycine methyl ester, increased Na+-dependent H+ flux in the presence of 80 nmol of DCCD/mg of protein, compared to the transport after DCCD treatment alone. These findings suggest that the Na+-H+ exchanger contains a single carboxylate residue in a hydrophobic region of the protein, and the carboxylate and/or a nearby endogenous nucleophilic group is critical for exchange activity.     

Glutamic acid-dihydrogen phosphate hydrogen-bonded networks: their proton polarizability as a function of cations present. Infrared investigations.

Glutamic acid [(L-glu)n] + dihydrogen phosphate systems are studied by infrared (IR) spectroscopy dried and hydrated at 75% relative humidity, as a function of both the phosphate-glutamic acid residue (Pi/glu) ratio and the type of cations present. It is shown that the glutamic acid groups form hydrogen-bonded chains with the phosphates. In these chains the positive charge fluctuates, and they show very large proton polarizability which increases in the series Li+,Na+,K+ systems. These chains are cross-linked via phosphate-phosphate hydrogen bonds, in which the proton is almost localized at one Pi. The comparison of the (L-glu)n + dihydrogen phosphate systems with the results obtained earlier in the case of (L-glu)n + hydrogen phosphate systems shows that the behavior of (L-glu)n + Pi systems strongly depends on the pH. Only with decreasing pH the conducting chains are formed. Finally, a hypothesis is discussed with regard to the charge conduction in the F0 subunit of the H+-ATPase in mitochondria.     

Fourier transform infrared spectroscopy of aqueous dispersions of phosphatidylserine-cholesterol mixtures

The effect of cholesterol on vibrational spectra in the non polar and in the polar region of dimyristoyl phosphatidylserine (DMPS) and of phosphatidylserine from bovine spinal cord (PS) has been investigated. The small shifts in the methylene CH stretching frequencies after taking into account the contribution of the cholesterol spectrum were interpreted as a combined effect of cholesterol on the conformation of the chains and of the lesser contributions of the cholesterol methyl groups. Cholesterol also influences the ratio of the trans (1465 cm^–1) to the lower wavelength (1457 cm^–1) CH₂ bending bands. No significant direct effect of cholesterol on the vibration of the polar residues was discerned. The small shift of the carboxylate band observed below the phase transition is probably due to the change in the intermolecular zwitterions when the average distance between the neighboring polar groups increases due to incorporation of cholesterol molecules.Abbreviations PS phosphatidylserine natural - DMPS dimyristoyl phosphatidylserine - DPPC dipalmitoyl phosphatidylcholine - FTIR Fourier transform infrared spectroscopy - DSC differential scanning calorimetry - PE phosphatidylethanolamine Offprint requests to: D. Bach     

The effect of metal cations on the phase behavior and hydration characteristics of phospholipid membranes

To characterize the specificity of ion binding to phospholipids in terms of headgroup structure, hydration and lyotropic phase behavior we studied 1-palmitoyl-2-oleoyl-phosphatidylcholine as a function of relative humidity (RH) at 25 degrees C in the presence and absence of Li+, Na+, K+, Be2+, Mg2+, Ca2+, Sr2+, Ba2+, Zn2+ and Cu2+ ions by means of infrared (IR) spectroscopy. All divalent cations and Li+ shift the gel-to-liquid crystalline phase transition towards bigger RH values indicating stabilization of the gel state. The observed shift correlates in a linearly fashion with the electrostatic solvation free energy for most of the ions in water that in turn, is inversely related to the ionic radius. This interesting result was interpreted in terms of the excess chemical potential of mixing of hydrated ions and lipids. Calcium, zinc and partially lithium, cause a positive deviation from the linear relationship. IR spectral analysis shows that the carbonyl groups become more accessible to the water in the presence of Mg2+, Ca2+, Sr2+ and Ba2+ probably because of their involvement into the hydration shell of the ions. In contrast, Be2+, Zn2+ and Cu2+ dehydrate the carbonyl groups at small and medium RH. The ability of the lipid to take up water is distinctly reduced in the presence of Zn2+ and, partially, of Cu2+ meaning that the headgroups have become less hydrophilic. The binding mode of Be2+ to lipid headgroups involves hydrolyzed water. Polarized IR spectra show that complex formation of the phosphate groups with divalent ions gives rise to conformational changes and immobilization of the headgroups. The results are discussed in terms of the lyotropic Hofmeister series and of fusogenic activity of the ionic species.