Proton conductance through phospholipid bilayers: Water wires or weak acids?

The proton/hydroxide (H⁺/OH^–) permeability of phospholipid bilayer membranes at neutral pH is at least five orders of magnitude higher than the alkali or halide ion permeability, but the mechanism(s) of H⁺/OH^– transport are unknown. This review describes the characteristics of H⁺/OH^– permeability and conductance through several types of planar phospholipid bilayer membranes. At pH7, the H⁺/OH^– conductances (G _H/OH) range from 2–6 nS cm^–2, corresponding to net H⁺/OH^– permeabilities of (0.4–1.7)×10^–5 cm sec^–1. Inhibitors ofG _H/OH include serum albumin, phloretin, glycerol, and low pH. Enhancers ofG _H/OH include chlorodecane, fatty acids, gramicidin, and voltages >80 mV. Water permeability andG _H/OH are not correlated. The characteristics ofG _H/OH in fatty acid (weak acid) containing membranes are qualitatively similar to the controls in at least eight different respects. The characteristics ofG _H/OH in gramicidin (water wire) containing membranes are qualitatively different from the controls in at least four different respects. Thus, the simplest explanation for the data is thatG _H/OH in unmodified bilayers is due primarily to weakly acidic contaminants which act as proton carriers at physiological pH. However, at low pH or in the presence of inhibitors, a residualG _H/OH remains which may be due to water wires, hydrated defects, or other mechanisms.     

The novel non-heme vanadium bromoperoxidase from marine algae: Phosphate inactivation

Summary Vanadium bromoperoxidase is a naturally occurring vanadium-containing enzyme isolated from marine algae. V-BrPO catalyzes the oxidation of halides by hydrogen peroxide which can result in the halogenation of organic substrates. Bromoperoxidase activity is measured by the halogenation of monochlorodimedone (2-chloro-5,5-dimethyl-1,3-dimedone, MCD). In the absence of an organic substrate, V-BrPO catalyzes the halide-assisted disproportionation of hydrogen peroxide yielding dioxygen. The dioxygen formed is in the singlet excited state (¹O₂). V-BrPO is quite stable to thermal denaturation and denaturation by certain organic solvents which makes V-BrPO an excellent candidate for industrial applications. The stability of V-BrPO in the presence of strong oxidants and in the presence of phosphate is reported. Incubation of V-BrPO in phosphate buffer (1–100 mM at pH 6; 2–10 mM at pH 5) inactivates the enzyme. The inactivity can be fully restored by the addition of vanadate if excess phosphate is removed. The inactivation of V-BrPO by phosphate can be prevented by the presence of H₂O₂ (4–40 mM). We are currently investigating the mechanism of V-BrPO inactivation by phosphate. V-BrPO was not inactivated by HOCl (1 mM) nor H₂O₂. In addition V-BrPO was not inactivated under turnover conditions of 1 mM H₂O₂ with 0.1–1 M Cl^– at pH 5 nor 2 mM H₂O₂ with 0.1 M Br^–.     

Proton/hydroxide conductance through lipid bilayer membranes

Summary A simple method of measuring proton/hydroxide conductance (G _H/OH) through planar lipid bilayer membranes is described. First the total conductance (G _m ) is measured electrically. Then the H⁺/OH^– transference number (T _H/OH) is estimated from the diffusion potential (V _m ) produced by a transmembrane pH gradient. The pH gradient is produced by a pair of buffered solutions which have identical concentrations of all ions except H⁺ and OH^–. Thus,V _m is due entirely to H⁺/OH^– diffusion andG _H/OH can be calculated from the relations,V _m =T _H/OH E _H/OH andG _H/OH=T _H/OH G _m , whereE _H/OH is the equilibrium potential for H⁺ and OH^–. In bilayers made from bacterial phosphatidylethanolamine (PE) inn-decane,G _H/OH is nearly independent of pH, ranging from about 10^–9 S cm^–2 at pH 1.6 to 10^–8 S cm^–2 at pH 10.5. BecauseG _H/OH is nearly independent of pH, the calculated permeability coefficients to H⁺ and/or OH^– are extremely pH dependent, which partly explains the wide range of values reported for phospholipid vesicles and biological membranes.G _H/OH appears to be independent of the membrane surface charge, because titrating either the phosphate or the amino group of PE has little effect onG _H/OH.G _H/OH is reduced about 10-fold when the water activity is reduced 33% by replacement with glycerol. Although the mechanism of H⁺/OH^– conductance is not known, the relation betweenG _H/OH and water activity suggests that several water molecules are involved in the H⁺/OH^– transport process.     

Active ion uptake and maintenance of cation-anion balance: A critical examination of their role in regulating rhizosphere pH

The processes responsible for maintenance of cation-anion balance in plants and their relation to active ion accumulation and changes in rhizosphere pH are outlined and discussed. The major processes involved are: (1) accumulation and degradation of organic acids which occur in the plant mainly as organic acid anions (and their transfer within the plant) and (2) extrusion of H⁺ or OH^– into the rhizosphere. The relative importance of the two processes is determined by the size of the excess anion or cation uptake. Indeed, plants typically absorb unequal quantities of nutritive cations (NH₄ ⁺+Ca²⁺+ Mg²⁺+K⁺+Na⁺) and anions (NO₃ ^–+Cl^–+SO₄ ^2–+H₂PO₄ ^–) and charge balance is maintained by excretion of an amount of H⁺ or OH^– which is stoichiometrically equal to the respective excess cation or anion uptake. The mechanisms and processes by which H⁺ and in particular OH^– ions are excreted in response to unequal cation-anion uptake are, however, poorly understood.The contemporary view is that primary active extrusion of H⁺, catalyzed by a membrane-located ATPase, is the major driving force for secondary transport of cations and anions across the plasma membrane. However, the fact that net OH^– extrusion often occurs (since excess anion absorption commonly takes place) implies there is a yet-to-be characterized OH^– ion efflux mechanism at the plasma membrane that is associated with anion uptake. There is, therefore, a need for future studies of the uptake mechanisms and stoichiometry of anion uptake; particularly that of NO₃ ^– which is often the predominant anion absorbed. Another related phenonenon which requires detailed study in terms of cation-anion balance is localized rhizosphere acidification which can occur in response to deficiencies of Fe and P.     

Mitochondrial transmembrane potential is diminished in phorbol myristate acetate-stimulated peritoneal resident macrophages isolated from wild-type mice,but not in those from gp91-phox-deficient mice

Macrophages produce superoxide (O₂^–) during phagocytosis or upon stimulation with a variety of agents including phorbol myristate acetate (PMA) through the activation of NADPH oxidase, and the formed O₂^– is converted to other reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂). The aim of the present study was to elucidate the effect of the intracellularly produced ROS on mitochondrial transmembrane potential (MTP) in mouse (C57BL/6) peritoneal resident macrophages stimulated with PMA. Using a fluorescent dye, succinimidyl ester of dichlorodihydrofluorescein (H₂DCFDA), O₂^– was visualized in intracellular compartments in a certain subpopulation of macrophages isolated from wild-type mice. Cells deficient in gp91-phox, one of the membrane components of NADPH oxidase, were negative for the fluorescence. When cells were loaded with both H₂DCFDA and MitoCapture, a fluorescent dye for mitochondria, mitochondrial fluorescence was diminished in O₂^–-producing cells, but not in O₂^–-deficient cells. Flow cytometry also revealed the decrease of mitochondrial fluorescence in wild-type cells, but not in gp91-phox-deficient cells. The loss of mitochondrial fluorescence was prevented by microinjection of catalase into cells. The present findings demonstrate that MTP is diminished by ROS, including the H₂O₂ dismutated from O₂^–, produced intracellularly by activation of the NADPH oxidase in mouse peritoneal resident macrophages stimulated with PMA.     

Small air ions: Their effect on blood levels of serotonin in terms of modern physical theory

Under stringently controlled environmental conditions in which only the small air ion content of the ambient air was varied, positive ions raised blood levels of 5-hydroxytryptamine in the mouse and negative ions depressed them. Examination of the experimental evidence in terms of the modern physical theory of air ion formation and composition led to the hypothesis that the ions H⁺(H₂O) and(H₃O)⁺ (H₂O)n were the physiologically active constituents of positively ionized air,while O₂ ^–(H₂O)n and OH^–(H₂O)n were the active agents in negatively ionized air. Further tests were conducted with positive molecular ions of CO₂, O₂ and N₂, and with negative molecular ions of CO₂ and O₂ emitted separately into pure air containing water vapor. The effedts on the blood level of 5-HT in mice supported our initial hypothesis and were in accord with physical theory.

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Zusammenfassung Unter streng kontrollierten Umgebungsbedingungen,bei denen lediglich die Zahl der kleinen Luftionen variiert wurde, bewirkten positive Ionen eine Erhöhung des Blutspiegel von 5-Hydroxytryptamin bei der Maus und negative Ionen eine Senkung. Die Prüfung des experimentellen Ergebnisses nach der modernen physikalischen Theorie der Bildung und Zusammensetzung der Luftionen führte zu der Hypothese, dass die Ionen H⁺(H₂O) und (H₃O)⁺(H₂O)n die physiologisch aktiven Anteile der positiv ionisierten Luft waren, während O₂ ^–(H₂O)n und OH^–(H₂O)n die aktiven Anteile der negativ ionisierten Luft darstellten. Weitere Versuche wurden mit positiven molekularen Ionen von CO₂, O₂ und N₂ sowie mit negativen molekularen Ionen von CO₂ und O₂ durchgeführt, die getrennt in reine Luft abgegeben wurden,die Wasserdampf enthielt. Die Wirkungen auf den Blutspiegel von 5-HT bei der Maus unterstützten die Ausgangshypothese und standen in Übereinstimmung mit der physikalischen Theorie.

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Resume En expérimentant dans des conditions de milieu ambiant strictement controlées et dans lesquelles seul le nombre des petits ions variait, on a pu démontrer que les ions positifs provoquaient une augmentation de la 5-hydrocytryptamine chez la souris. Les ions négatifs la faisaient au contraire diminuer.L'examen des résultats expérimentaux selon la théorie physique moderne de la formation et de la composition des ions de l'air conduit à l'hypothèse que les ions H⁺(H₂O) et (H₃O)+(H₂O)n représentaient la part physiologiquement active de l'air ionisé positivement alors que les ions O₂ ^– (H₂O)n et OH^–(H₂O)n formaient la partie active de l'air ionisé négativement. D'autres essais furent effectués avec des ions moléculaires positifs de CO₂, O₂ et N₂ ainsi qu'avec des ions moléculaires négatifs ce CO₂ et O₂.Ceux-ci furent projetés séparément dans de l'air pur contenant de la vapeur d'eau. Leur influence sur le taux de 5-HT dans le sant des souris appuie notre hypothèse dedépart et concorde avec la théorie physique.

     

On the mechanism of hydrogen peroxide-, superoxide-, and ultraviolet light-induced DNA cleavages of inactive bleomycin-iron (III) complex

The ³²P-labeled DNA cleavage experiments showed that the biological activity of the bleomycin(BLM)-Fe(III)OH^? complex is evidently induced by addition of H₂O₂ and KO₂, or by irradiation of UV light. Hydrogen peroxide contributes to the conversion from the inactive BLM-Fe(III)OH^? complex to the active BLM-Fe(III)O₂H^? complex, and UV light to the reduction of the BLM-Fe(III)OH^? complex to the BLM-Fe(II) complex. The proposed hypothetical mechanism for cyclic function of BLM-iron complex is similar to that of certain heme-oxygenases and heme-oxidases.     

Synthesis and structural chemistry of novel heteropolymolybdates and -tungstates

The chemistry of polynuclear oxometalate anions is dominated by molybdenum and tungsten in their highest oxidation state. During the past twenty years this class of compounds has attracted much attention because of their variable applications, e.g. as reagents in analytical procedures, as industrial catalysts and as potential anticancer drugs.In order to obtain model systems for the investigation of the catalytic activity of heteropolyanions we have synthesized and structurally characterized some organo derivatives of polyoxoanions. We secondly focus on Sb(III) and Bi(III) heteropolytungstates to examine the important influence of the unshared electron pair on the resulting structures and properties. Some of these compounds may be regarded as supramolecular aggregates showing inclusion phenomena.In 9 two [SbW₉O₃₃]^9– anions are linked by a set of six sodium ions forming a nearly planar hexagon. The sodium ions are enveloped by an oxygen cage formed by terminal oxygen atoms of the polyanions and by water molecules. Furthermore, the four anions [Sb₂W₂₂O₇₄(OH)₂]^12–, [Sb₂W₂₀Fe₂O₇₀(H₂O)₆]^8–, [Sb₂W₂₀Co₂O₇₀(H₂O)₆]^10– and [Bi₂W₂₀Fe₂O₆₈(OH)₂(H₂O)₆]^6– (in10, 11, 12, 13) may be regarded as transition metal complexes of novel [Sb₂W₂₀O₇₀]^14– or [Bi₂W₂₀O₇₀]^14– anions which are serving as ligands. The octahedral coordination sphere of each transition metal is formed by three oxygen atoms of the anion and completed by three water molecules. The Sb(III) heteropolyanion, [Na₂Sb₈W₃₆O₁₃₂(H₂O)₄]^22– in (14) includes two sodium and four antimony ions besides four water molecules. Each anion consists of four -B-SbW₉-Keggin fragments linked together by four SbO₄-groups, incorporating two sodium and four water molecules effecting an additional connection of the subunits.     

pH equilibration in human erythrocyte suspensions

Summary A stopped-flow rapid reaction apparatus was used to study the rate of pH equilibration in human red cell suspensions. Flux of OH^– or H⁺ was determined over a wide range of extracellular pH (4–11) in CO₂-free erythrocyte suspensions. In these experiments, an erythrocyte suspension at pH 7.3 is rapidly mixed with an equal volume of NaCl solution at 3.0>pH>11.5. The pH of the extracellular fluid of the mixture changes rapidly as OH^– or H⁺ moves across the red cell membrane. Flux and velocity constants can be calculated from the initiald pH/dt using the known initial intra- and extracellular pH. It was found that the further the extracellular pH is from 7.3 (in either direction from 4–11), the greater the absolute value of total OH^– and/or H⁺ flux. Pretreatment with SITS (4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid), a potent anion exchange inhibitor, greatly reduces flux over the entire pH range, while exposure to valinomycin, a potassium ionophore, has no measurable effect. These data suggest that (i) both H⁺ and OH^– may be moving across the red cell membrane at all pH; (ii) the species dominating pH equilibration is probably dependent on the extracellular pH, which determines the magnitude of the driving gradient for each ion; and (iii) the rapid exchange pathway of the erythrocyte membrane may be utilized for both H⁺ and OH^– transport during CO₂-free pH equilibration.     

Die Erzeugung von Chemilumineszenzen wÄ\riger TlI-Salzlösungen durch Röntgenstrahlen

Zusammenfassung Die Lumineszenz wÄ\riger Tl⁺-Lösungen unter Einwirkung von 8,5-keV_eff-Röntgenstrahlen wird untersucht, und zwar 1. in neutraler Lösung, 2. in saurer Lösung, 3. in alkalischer Lösung, 4. bei Zusatz von NaCl.Bei Änderung des pH-Wertes nimmt die Lichtausbeute sowohl auf der sauren Scite (beipH=3,4) als auch auf der alkalischen Scite (beipH=12) ein Maximum an; sie erreicht dort den 2- bis 3fachen Wert der Ausbeute in neutraler Lösung. Andererseits ist bei Zusatz von 1 M NaCl in neutraler Lösung die Ausbeute fünfmal höher als bei Abwesenheit von NaCl; die Maxima im sauren und alkalischen Bereich verschwinden jedoch bei 1 M NaCl vollkommen, und man erhÄlt bei gro\en und bei kleinen pH-Werten lediglich eine Löschung der Lumineszenz.Die Lumineszenz kommt durch Bildung von Tl^+* bei der Anlagerung von e_aq an Tl⁺⁺ zustande. Tl⁺⁺ entsteht jedoch nicht unmittelbar gemÄ\ Tl⁺ + OH Tl⁺⁺ + OH^–, sondern zunÄchst bildet sich Tl⁺OH, und dieses dissoziiert erst nach einer Verzögerung von > 10^–7 sec in Tl⁺⁺ und OH^–. Die Sensibilisierung durch H⁺ wird durch die Reaktion Tl⁺OH + H⁺ Tl⁺⁺ + H₂O und die durch OH^– durch Tl⁺OH + OH^– Tl⁺⁺O^– + H₂O erklÄrt. Die Reaktion der Komplexe Tl⁺Cl^– und Tl⁺Cl₂ ^– mit OH erfolgt offenbar ohne zeitliche Verzögerung, d. h. Tl⁺⁺Cl^–– bzw. Tl⁺⁺Cl₂ ^–– wird dabei innerhalb einer Zeit 10^–7 sec gebildet.

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Chemiluminescences of aqueous Tl^I solutions produced by irradiation with 8.5-keV-X-rays

Summary The luminescence of aqueous solutions of Tl⁺ during irradiation with 8.5-keV-X-rays has been investigated. In particular we have studied the Tl⁺-luminescence in 1. neutral solutions, 2. acid solutions, 3. alkaline solutions, 4. solutions containing various concentrations of NaCl.Varying thepH the luminescence yield passes a maximum on the acid side as well as on the alkaline side (atpH=3.4 respectivelypH=12). Compared with neutral solutions the luminescence yield is increased by a factor 2 to 3 at these pH-values.By adding 1 M NaCl to neutral Tl⁺-solutions the luminescence yield is enhanced by a factor five, however in dependence ofpH no further increase has been observed, but only quenching of the luminescence at low and highpH.The luminescence origins from the formation of Tl^+* by the reaction of e_aq with Tl⁺⁺ formed by oxidation of Tl⁺ by OH. However, Tl⁺⁺ is not formed directly by Tl⁺+OH Tl⁺⁺ + OH^– but via dissociation of the intermediate Tl+OH after a delay > 10^–7 sec.We explain the increase of the luminescence yield in acid solutions by the reaction Tl⁺OH + H⁺ Tl⁺⁺ + H₂O and in alkaline solutions by the reaction Tl⁺OH + OH^– Tl⁺⁺O^– + H₂O. In alkaline solutions the luminescence spectrum shifts to longer wavelengths; we conclude, that this spectrum is attributed to Tl^+*O^–. Evidently the reaction of Tl+Cl^– and Tl+Cl₂ ^–– with OH leads to formation of Tl⁺⁺Cl^– and Tl⁺⁺Cl₂ ^–– without any efficient delay.

     

Passive H+/OH− permeability in epithelial brush border membranes

Passive H⁺/OH^– permeability across epithelial cell membranes is rapid and leads to partial dissipation of H⁺/OH^– gradients produced by H⁺ pumps and ion gradient-coupled H⁺/OH^– transporters. A heterogeneous set of H⁺/OH^– transport mechanisms exist in biological membranes: lipid solubility/diffusion, protein-mediated transport by specific proteins or by slippage through ion-coupled H⁺/OH^– transporters, and transport at the protein/lipid interface or through protein-dependent defects in the lipid structure. A variety of methods are available to study protein transport mechanisms accurately in cells and biomembrane vesicles including pH electrode recordings, pH-sensitive fluorescent and magnetic resonance probes, and potentiometric probes. In brush border vesicles from the renal proximal tubule, the characteristics of passive H⁺/OH^– permeability are quite similar to those reported for passive H⁺/OH^– permeability through pure lipid bilayers; slippage of protons through the brush border Na⁺/H⁺ antiporter or through brush border water channels is minimal. In contrast, passive H⁺/OH^– permeability in brush border vesicles from human placenta is mediated in part by a stilbene-sensitive membrane protein. To demonstrate the physiological significance of passive renal brush border H⁺/OH^– transport, proximal tubule acidification and cell pH regulation mechanisms are modeled mathematically for states of normal and altered H⁺/OH^– permeabilities.     

Chemical Bases of Nonspecific Immunity

Our knowledge on the nature and quantity of reactive O₂forms generated in phagocytes, particularly in neutrophil leucocytes, and their role in nonspecific immunity is reviewed. In thermodynamical terms, oxygen is a very reactive molecule and, hence, can react with most chemical elements and many organic molecules. In kinetic terms, O₂is rather inert. Its reactivity can be increased either by reduction or excitation. After accepting four electrons, O₂is finally reduced to H₂O. Partial reduction resulting in highly reactive intermediates, namely, superoxide anion (O₂ ^·–), hydrogen peroxide (H₂O₂), and hydroxyl radical (^·OH), is possible. Singlet oxygen (¹O₂) is the product of O₂excitation. Phagocytes acting like agents of nonspecific immunity generate such reactive forms of O₂.     

Abiotic synthesis of organic molecules from minerals containing traces of dissolved H2O,CO2 and N2 part I: Basic principles

Oxides and silicates which crystallize in the presence of H₂O, CO₂ and N₂, especially at high pressures, are expected to dissolve traces of these fluid components. In doing so atomic and electronic rearrangements occur between the host lattice and the dissolved molecules which lead to different solute species. These may be simply hydroxyl OH^–, carbonate CO₃ ^2– of similar ions, but other species can also form which are chemically reduced such as molecular H₂, CO₂ ^2– and CO^– or oxidized such as peroxy ions, O₂ ^2–. When the minerals containing such solute species are decompressed and cooled, they eventually become supersaturated with respect to their dissolved impurities. If the relative mobilities of the different solute species are different, diffusion and subsurface segregation lead to spatial separation: the surface and subsurface zone become enriched in the reduced species while the oxidized species remain in the bulk. Upon heating or during chemical dissolution (weathering) the reduced species contained in the surface react leading to the formation of a wide variety of H–C–O–N molecules: not only H₂O, CO₂ and possibly N₂ but also organic molecules. No external reduced atmosphere is needed, according to this hypothesis.     

Inactivation of mushroom tyrosinase by hydrogen peroxide

Hydrogen peroxide (H₂O₂) inactivates mushroom tyrosinase in a biphasic manner, with the rate being faster in the first phase than in the second one. The inactivation of the enzyme is dependent on H₂O₂ concentration (in the range of 0.05–5.0 mM), but independent of the pH (in the range of 4.5–8.0). The rate of inactivation of mushroom tyrosinase by H₂O₂ is faster under anaerobic conditions (nitrogen) than under aerobic ones (air). Substrate analogues such as L-mimosine, L-phenylalanine, p-fluorophenylalanine and sodium benzoate protect the enzyme against inactivation by H₂O₂. Copper chelators such as tropolone and sodium azide also protect the enzyme. Under identical conditions, apotyrosinase is not inactivated by H₂O₂, unlike holotyrosinase. The inactivation of mushroom tyrosinase is not accelerated by an OH^?dot generating system (Fe²⁺-EDTA-H₂O₂) nor is it protected by OH^dot scavengers such as mannitol, urate, sodium formate and histidine. Exhaustive dialysis or incubation with catalase does not restore the activity of H₂O₂-inactivated enzyme. The data suggest that Cu²⁺ at the active site of mushroom tyrosinase is essential for the inactivation by H₂O₂. The inactivation does not occur via the OH^dot radical in the bulk phase but probably via an enzyme-bound OH^dot.     

Effects of imidazole and zinc on the interaction of some amino acid compounds of copper(II) with hydrogen peroxide

A novel effect of the inhibition of the decomposition of amino acids to carbonates on addition of imidazole (HIm) to a reacting system containing equimolar amounts of copper and zinc metal powders, an amino acid [glycine (Hgly), aspartic acid (H₂Asp) or glycylglycine (H₂gg)] (1:1:2) and excess hydrogen peroxide (H₂O₂) resulting in formation of a mixed metal mixed ligand peroxo complex compound was observed, because in the absence of imidazole the corresponding reaction system yields only a mixed metal peroxo carbonate. For the resulting complex compounds, the homogeneity, i.e. [Cu(Zn)(O₂ ^2–)(Gly)₂(HIm)(H₂O)], [Cu(Zn)(O₂ ^2–)(Asp)(HIm)(H₂O)₂] or [Cu(Zn)₂(O₂ ^2–)₂(gg)(HIm)(H₂O)₄], molecular formula, presence of peroxo group and coordination environment were established by combined physicochemical evidence from elemental and thermogravimetric analysis in air and argon atmospheres, electron spin resonance and electronic and IR spectral data. It is noteworthy to mention that the corresponding carboxylic acids of the above-mentioned amino acids, i.e. acetic and succinic acids, either do not decompose to carbonates in the absence of imidazole or form novel homogeneous peroxo mixed metal mixed ligand complex compounds as described above in the presence of imidazole. This suggests an important and significant mutual influence (in vitro) of biologically active chromophores like peroxo ions, imidazole and amino groups in the above-mentioned chemical reactions containing bioactive metals such as copper and zinc.     

Inactivation of Escherichia coli by superoxide radicals and their dismutation products.

Escherichia coli cells are inactivated by the products of the reaction between dialuric acid and oxygen, of which the primary product is Superoxide. The rate of inactivation is decreased by Superoxide dismutase, by catalase, and by EDTA, whereas it is increased by addition of cupric ions or hydrogen peroxide. It is concluded that a toxic product is formed in a reaction involving Superoxide, hydrogen peroxide, and metal ions, which might be the Haber-Weiss reaction, O₂^? + H₂O₂ → OH + OH^? + O₂. In radiation chemical experiments it is shown that this reaction does not occur in the absence of metal ions.     

Hydrogen peroxide-mediated cell-wall stiffening in vitro in maize coleoptiles

It has recently been proposed that H₂O₂-dependent peroxidative formation of phenolic cross-links between cell-wall polymers serves as a mechanism for fixing the viscoelastically extended wall structure and thus confers irreversibility to wall extension during cell growth (M. Hohl et al. 1995, Physiol. Plant. 94: 491–498). In the present paper the isolated cell wall (operationally, frozen/thawed maize coleoptile segments) was used as an experimental system to investigate H₂O₂-dependent cell-wall stiffening in vitro. Hydrogen peroxide inhibited elongation growth (in vivo) and decreased cell-wall extensibility (in vitro) in the concentration range of 10–10000 mol·1^–1. In rheological measurements with a constant-load extensiometer the stiffening effect of H₂O₂ could be observed with both relaxed and stressed cell walls. In-vitro cell-wall stiffening was a time-dependent reaction that lasted about 60 min in the presence of saturating concentrations of H₂O₂. The presence of peroxidase in the growth-limiting outer epidermal wall of the coleoptile was shown by histochemical assays. Peroxidase inhibitors (azide, ascorbate) suppressed the wall-stiffening reaction by H₂O₂ in vitro. Hydrogen peroxide induced the accumulation of a fluorescent, insoluble material in the cell walls of living coleoptile segments. These results demonstrate that primary cell walls of a growing plant organ contain all ingredients for the mechanical fortification of the wall structure by H₂O₂-inducible phenolic cross-linking.Supported by Deutsche Forschungsgemeinschaft. I thank Ms. Bärbel Huvermann for expert technical assistance.     

Reversible high temperature exchange of carbonate and hydroxyl ions in tooth enamel and synthetic hydroxyapatite

Parallel experiments on human tooth enamel with sp.g. >2.95 and synthetic hydroxyapatite have been carried out to study the substitution of CO ₃ ^2– for OH^–, produced at 1000°C in dry CO₂, with the complementary use of neutron diffraction, x-ray diffraction, infrared spectroscopy and thermochemical techniques. It was verified that the substitution (i) is CO ₃ ^2– 2(OH^– and is completely reversible on specimen exposure to H₂O vapor at 1000°C, (ii) takes place with the carbon atom on or near the hexad axis, (ii) places one CO ₃ ^2– group per unit cell in an ordered fashion and so changes the space group fromP6 ₃/m to one without a screw axis, (iii) was consistent, by its incompleteness, with the occurrence of substitution of O^2– for 2(OH^–) in 25 to 40% of the unit cells, (iv) produced similar marked changes in the neutron powder diffraction patterns of both tooth enamel and hydroxyapatite, and (v) took place at a site where only a minor portion of the CO ₃ ^2– in normal untreated human tooth enamel occurs. From comparative Rietveld analysis results from x-ray and neutron powder diffraction patterns it is suggested that the carbon atom of this A-site CO ₃ ^2– is near 0,0,0.12 and the CO ₃ ^2– plane makes an angle of 18° with thec direction.On being heated at 400°C in H₂O vapor, tooth enamel retained much of its CO ₃ ^2– but its a lattice parameter changed from 9.445(3) Å to 9.420(1) Å, essentially that of hydroxyapatite. After once being heated at high temperatures, tooth enamel and hydroxyaptite showed similar responses to various treatments, including carbonation. After heating, more -Ca₃(PO₄)₂ was found in the tooth enamel specimen. Comparative weight change, IR, and other data for tooth enamel and hydroxyapatite heated in He, then in CO₂, and then in H₂O vapor showed a 20% or more deficiency of structural OH^– in the untreated tooth enamel.     

Induction of astaxanthin formation by reactive oxygen species in mixotrophic culture of Chlorococcum sp.

The induction of astaxanthin formation by reactive oxygen species in mixotrophic culture of Chlorococcum sp. was investigated. H₂O₂ (0.1 mM) enhanced the total astaxanthin formation from 5.8 to 6.5 mg g^–1 cell dry wt. Fe²⁺ (0.5 mM) added to the medium with H₂O₂ (0.1 mM) further promoted astaxanthin formation to 7.1 mg g^–1 cell dry wt. Similarly, Fe²⁺ (0.5 mM) together with methyl viologen (0.01 mM) promoted astaxanthin formation to 6.3 mg g^–1 cell dry wt. In contrast, an addition of KI (1 mM), a specific scavenger for hydroxyl radicals (OH), together with H₂O₂ (0.1 mM) and Fe²⁺ (0.5 mM), to the medium decreased astaxanthin formation to 1.8 mg g^–1 cell dry wt. KI (1 mM) also inhibited the enhancement of carotenogenesis by superoxide anion radicals (O₂ ^–), with a decrease of astaxanthin formation to 1.7 mg g^–1 cell dry wt. This suggested that O₂ ^– might be transformed to OH before promoting carotenogenesis in Chlorococcum sp.     

Electrically silent anion transport through bilayer lipid membrane induced by tributylin and triethyllead

Summary The method of the measurement of the nonelectrogenic fluxes of hydrogen (or hydroxyl) ions (J _H) based on the local proton gradients formation in the unstirred layers near a bilayer lipid membrane (BLM) is applied for recording the nonelectrogenic anion/OH^– exchange on BLM induced by tributyltin (TBT) and a novel carrier (Hager, A., Moser, I., & Berthold, W. 1987.Z. Naturforsch.,42C1116–1120), triethyllead (TEL). This method has been used previously for measuring the cation fluxes through BLM. TBT and TEL are shown to be equally efficient in the induction of Cl^–/OH^– exchange.J _H induced by TBT is constant at 4J _H decreases at pH<4 and pH>7. Both ionophores have a transport sequence: I^–> Br^–>Cl^–>F^–. The quatitative measurements reveal that TEL better discriminates these four anions than TBT. It is concluded that this method may prove helpful in a search and study of anion/OH^–-exchangers isolated from natural membranes.