The voltage-dependent step of the chloride transporter of Valonia utricularis encounters a Nernst-Planck and not an Eyring type of potential energy barrier

Voltage-clamp experiments were performed on cells of the giant marine alga Valonia utricularis to study the voltage dependence of the previously postulated chloride transporter (Wang, J., G. Wehner, R. Benz, and U. Zimmermann. 1991. Biophys. J. 59:235-248). Only one exponential current relaxation (apart from the capacitive spike) could be resolved up to a clamp voltage of ~120 mV within the time resolution of our experimental instrumentation (100 μs). This means that the rate constants of the heterogeneous complexation, k_R (association) and k_D (dissociation), were too fast to be resolved. Therefore, the “Läuger” model for carrier-mediated ion transport with equilibrium heterogeneous surface reaction was used to fit the experimental results. The voltage dependence of the initial membrane conductance was used for the evaluation of the voltage dependence of the translocation rate constant of the complexed carriers, k_AS. The initial conductance was found to be independent on the clamp voltage, which means that the translocation rate constant k_AS is a linear function of the applied voltage and that the voltage dependence of the translocation of charged carriers through the plasmalemma could be explained by a square-type Nernst-Planck barrier. The movement of the complexed form of the carrier through the membrane may be explained by a diffusion process rather than by simple first-order kinetic jump across an Eyring-type potential well. The current relaxation after a voltage clamp was studied as a function of the external chloride concentration. The results allowed an estimation of the stability constant, K, of the heterogeneous complexation reaction and a calculation of the translocation rate constants of the free and the complexed carriers, k_s and k_AS, respectively.     

Kinetic study on unzippering of polynucleotides and order-order transition in polypeptides

Solutions are presented for N + 1 sequential and reversible first-order reactions for which the magnitude of the reverse rate constant, k_b, for all steps except the last is identical. Also the magnitude of the forward rate constant, k_f, for all steps except the first and last is identical. The initial and final steps are nucleation reactions; therefore, the initial and final k_f are modified by the factors σ′ and γ respectively. The final k_b is modified by the factor γ σ. The ratio k_b/k_f is defined as s, which has the same meaning as s in the Zimm-Bragg theory. The mathematical model is intended to apply to polymeric molecules of N segments and allows the calculation of the mole fraction of molecules in state i at any time t, C_i(t). A molecule in state i has i unreacted segments and N – i reacted ones. Because the reactions are sequential, all reacted segments are contiguous. Our numerical results show that when σ′ is much less than unity and the forward reaction is favored, the relaxation curve is sigmoidal. If, however, the forward and reverse reactions are equally favored (i.e., s ? 1) the relaxation curve is a straight line. When s and σ′ are near unity, the curve is exponential for a considerably large fraction of the reaction. Further, in the exponential for a considerably large fraction of the reaction. Further, in the exponential phase of the reaction, the relaxation time is proportional to N² for highly cooperative systems (i.e., Nσ ? 1). As found by Pipkin and Gibbs, if N is sufficiently large and s is less than unity (e.g., N ? 50 and s ?0.9) the relaxation curve is largely linear with a slope inversely proportional to N. Applications are given for the unwinding of double-helical poly(A·U) and the order–order transition in poly-L -proline.     

Transport properties of mobile charges in algal membranes: Influence of pH and turgor pressure

Summary Charge-pulse experiments were performed on giant algal cells ofValonia utricularis. If the tonoplast and plasmalemma in series are charged to voltages of the order of 10mV, the decay of the initial voltage with time can be described by the sum of two or three exponential relaxations. It is not possible to explain the exponential decay of the voltage by twoRC-circuits in series (e.g. tonoplast and plasmalemma), because this would lead to unreasonable values for the specific capacities of the two membranes. The exponential relaxations might be attributable to the transport of mobile negative charges present in both membranes, possibly as a part of a transport system. From an analysis of the experimental results in terms of the proposed model, the translocation rate constantk and the total surface densityN _t of the mobile charges in one membrane could be evaluated. On averagek is of the order of 600 sec^–1 andNt is about 5×10^–12 mol cm^–2 (average turgor pressure 1.6 bar). The transport properties of the mobile charges within the tonoplast and plasmalemma were studied as a function of different parameters such as external pH, glutardialdehyde, electrical breakdown and turgor pressure. When the pH is lowered from 8.2 to 4 or 5 the mobile charges disappear completely, presumably as the result of protonation of the anionic groups. This pH effect was found to be completely reversible. Electrical breakdown causes a reversible disappearance of the relaxation with the longer half-time due to the decrease in membrane resistance. The value of the electrical breakdown voltage determined by injection of charge pulses of 300-sec duration into the cell is pH-independent and therefore is consistent with the mobile charge model and with results previously reported (U. Zimmermann & R. Benz.J. Membrane Biol 53:33–43, 1980). Addition of glutardialdehyde leads also to a disappearance of the mobile charges probably due to cross-linkage. Increase of the turgor pressure from 0.05 bar to 2 bar results in an increase ink by a factor of 2 and inNt by about 30%. The increase ink is in reasonable agreement with that expected on the basis of the assumed compressibility of the membranes. The elastic compressive modulus perpendicular to the membrane plane calculated from the pressure dependence of the translocation rate constantk is in very good agreement with that derived from electrical breakdown experiments (14 and 13 bar, respectively). The presence of charges within the membranes as well as the compressibility of the membranes are discussed in terms of a possible turgor-pressure-sensing mechanism.     

Kinetics of the iodine- and bromine-mediated transport of halide ions: demonstration of an interfacial complexation mechanism.

Stationary and kinetic experiments were performed on lipid bilayer membranes to study the mechanism of iodine- and bromine-mediated halide transport in detail. The stationary conductance data suggested that four different 1:1 complexes between I2 and Br2 and the halides I- and Br- were responsible for the observed conductance increase by iodine and bromine (I3-, I2Br-, Br2I-, and Br3-). Charge pulse experiments allowed the further elucidation of the transport mechanism. Only two of three exponential voltage relaxations predicted by the Läuger model could be resolved under all experimental conditions. This means that either the heterogeneous complexation reactions kR (association) and kD (dissociation) were too fast to be resolved or that the neutral carriers were always in equilibrium within the membrane. Experiments at different carrier and halide concentrations suggested that the translocation of the neutral carrier is much faster than the other processes involved in carrier-mediated ion transport. The model was modified accordingly. From the charge pulse data at different halide concentrations, the translocation rate constant of the complexed carriers, kAS, the dissociation constant, kD, and the total surface concentration of charged carriers, NAS, could be evaluated from one single charge pulse experiment. The association rate of the complex, kR, could be obtained in some cases from the plot of the stationary conductance data as a function of the halide concentration in the aqueous phase. The translocation rate constant, kAS, of the different complexes is a function of the image force and of the Born charging energy. It increases 5000-fold from Br3- to I3- because of an enlarged ion radius.     

Influence of membrane structure on the kinetics of carrier-mediated ion transport through lipid bilayers

Charge-pulse relaxation experiments of valinomycin-mediated Rb⁺ transport have been carried out in order to study the influence of membrane structure on carrier kinetics. From the experimental data the rate constants of association (k_R) and dissociation (k_D) of the ion-carrier complex as well as the rate constants of translocation of the complex (k_MS) and of the free carrier (k_S) could be obtained. The composition of the planar bilayer membrane was varied in a wide range. In a first series of experiments, membranes made from glycerolmonooleate dissolved in different n-alkanes (n-decane to n-hexadecane), as well as solvent-free membranes made from the same lipid by the Montal-Mueller technique were studied. The translocation rate constants k_S and k_MS were found to differ by less than a factor of two in the membranes of different solvent content. Much larger changes of the rate constants were observed if the structure of the fatty acid residue was varied. For instance, an increase in the number of double bonds in the C₂₀ fatty acid from one to four resulted in an increase of k_S by a factor of seven and in an increase of k_MS by a factor of twenty-four. The stability constant K = k_R/k_D of the ion-carrier complex as well as the translocation rate constants k_S and k_MS were found to depend strongly on the nature of the polar headgroup of the lipid. The incorporation of cholesterol into glycerolmonooleate membranes reduced k_R, k_MS and k_S up to seven-fold.     

Evidence for the presence of mobile charges in the cell membrane of Valonia utricularis.

Charge-pulse relaxation studies were performed on cells of the giant marine alga Valonia utricularis with microelectrodes inserted into the vacuole. If the cell was charged by short pulses of 200 ns duration, the decay of the initial membrane voltage could be described by two relaxation processes at normal pH (8.2). The fast exponential relaxation had a time constant of approximately 100 microseconds whereas the the time constant of the slow relaxation ranged between 2 and 15 ms. The ratio of the two amplitudes varied between 10 and 20 and was found to be independent of the initial voltage, up to 400 mV. In contrast to the time constants, the amplitude ratio was a function of the duration of the charge pulse. As the pulse length was increased to 10 ms, the fast relaxation disappeared. A change in pH of the natural sea water from 8.2 to 4 resulted in the disappearance of both exponential processes and the appearance of one single exponential with a 1-ms time constant over the whole pulse-length range. The analysis of the data in terms of a two-membrane model leads to unusual values and a pH-dependence of the specific capacitances (0.6 and 6 microF cm-2) of the two membranes, which can be treated as two serial circuits of a capacitor and a resistor in parallel. The charge-pulse and the current-clamp data are consistent with the assumption that the cell membrane of V. utricularis contains mobile charges with a total surface concentration of approximately 4 pmol cm-2. These charges cross the membrane barrier with a translocation rate constant around 500 s-1 and become neutralized at low pH. From our experimental results it cannot be completely excluded that the tonoplast has also a high specific resistance. But in this case it has to be assumed that the tonoplast and plasmalemma have very similar electrical properties and contain both mobile charges, so that the two membranes appear as a single membrane. Experiments on artificial lipid bilayer membranes in the presence of the lipophilic ion dipicrylamine, support our mobile charge concept for the cell membrane of V. utricularis.     

Mathematical Model of Thermal Destruction of Bacillus stearothermophilus Spores

The experimental survival curves of Bacillus stearothermophilus spores in aqueous suspension, for six constant temperatures ranging from 105 to 130°C, displayed an initial shoulder before a linear decline. To interpret these observations, we supposed that, before the heat treatment, the designated spore suspension contained a countable and mortal N₀ population of activated spores and an M₀ population of dormant spores which remained masked during spore counting and had to be activated before being destroyed by heat. We also hypothesized that the mechanisms of both activation and destruction are, at constant temperature, ruled by first-order kinetics, with velocity constants k_A and k_D, respectively. Mathematical analysis showed that this model could represent not only our experimental survival curves, but also all other shapes (linear and biphasic) of survival curves found in the literature; also, there is an inherent symmetry in the model formulation between the activation and destruction reactions, and we showed that the dormancy rate (τ = M₀/N₀) is the only parameter which permits a distinction between the two reactions. By applying the model to our experimental data and considering that the dormancy rate is not dependent on the treatment temperature, we showed that, for the studied suspension, the limiting reaction was the activation reaction.     

Kinetic analysis of carrier-mediated ion transport by the charge-pulse technique

Summary The charge-pulse technique has been used previously for the study of quasistationary processes in membranes which required only a moderate time resolution. It is shown here that a time resolution of about 400 nsec may be achieved with this technique and that it may be applied to the kinetic analysis of carrier-mediated ion transport. By this method we have studied the transport of alkali ions through optically black monoolein membranes in the presence of the ion carrier valinomycin. All three relaxation processes that are predicted by theory have been resolved. From the relaxation times and the relaxation amplitudes the rate constants for the association (k _R ) and the dissociation (k _D ) of the ioncarrier complex, as well as the translocation rate constants of the complex (k _MS ) and the free carrier (k _S ) could be obtained. For 1m Rb⁺ at 25° C the values arek _R =3×10⁵ m ^–1 sec^–1,k _D =2×10⁵ sec^–1,k _MS =3×10⁵ sec^–1,k _S =4×10⁴ sec^–1. The activation energies of the single rate constants which have been estimated from experiments at two different temperatures range between 50 and 90 kJ/mol.     

A mathematical description of short-term effects of β-lactam antibiotics on bacterial growth in vitro

Bacterial growth at moderately high concentrations of β-lactam antibiotics and within the first few hours of exposure cannot be described by a simple exponential equation. The quadratic function InN _t=InN ₀+k₀t?1/2at², in whichk ₀ is the initial growth rate constant anda a rate inhibition constant, is a better approximation. Whena is used as a single parameter, the effect of a particular antibiotic can be described as a concentration-effect relation. This approach also permits comparison of antibiotics, e.g., nafcillin and cloxacillin. The relation betweenk ₀ anda in the interaction between nafcillin and chloramphenicol shows that chloramphenicol antagonizes the effect of nafcillin.     

The State of Water in Muscle Tissue as Determined by Proton Nuclear Magnetic Resonance

The nuclear magnetic resonance (NMR) of water protons in live and glycerinated muscle, suspensions of glycerinated myofibrils, and solutions of several muscle proteins has been studied. T₁ and T₂, measured on partially hydrated proteins by pulsed spin-echo techniques, decreased as the ratio of water to protein decreased, showing that the water which is tightly bound by the protein has short relaxation times. In live muscle fibers the pulse techniques showed that, after either a 180 or a 90° pulse, the relaxation of the magnetization is described by a single exponential. This is direct evidence that a fast exchange of protons occurs among the phases of the intracellular water. The data can be fitted with a model in which the bulk of the muscle water is in a phase which has properties similar to those of a dilute salt solution, while less than 4-5% of the total water is bound to the protein surface and has short relaxation times. Measurements of T₁ and T₂ in protein solutions showed that no change in the proton relaxation times occurred when heavy meromyosin was bound to actin, when myofibrils were contracted with adenosine triphosphate (ATP), or when globular actin was polymerized.     

Solvent and solute effects on “inside-outside” conformations and rotation about the peptide bond in a miniature protein model

The physicochemical parameters affecting protein unfolding in relation to peptide bond rotations are briefly reviewed. As a suitable model for the study of solvent and solute effects on amide rotation and inside-outside conformations, the 2,2′-biphenyl analog of N-benzoyl-l-phenylalanine methyl ester (I) was synthesized and resolved enzymatically with α-chymotrypsin. The optically pure substance exists as conformer I_a (R,S configuration) with an axial methoxycarbonyl in the crystalline state. Rotation about the biphenyl axis leads to the equatorial conformer I_b (S,S configuration) in various solvents. In polar solvents, rotation about the amide is rate limiting. Accurate measurements of this rotation were accomplished by following the rate of change in the maximum amplitude of the biphenyl Cotton band at 256 nm. The high sensitivity of the method allowed rate and equilibrium measurements at 10^?3M in the absence of intermolecular association. Small differences of the order of 100 cal/mole in ΔG^≠ or ΔG_eq could thus be detected accurately. It was found that k_obs or k₁ (forward step) for equilibration was linearly related (correlation coefficient of 0.96 for k_obs) with E_T, the solvent polarity index on Reichardt and Dimroth's scale. Rotation was slowest in water and fastest in carbon tetrachloride, δΔG^≠, being 2.4 kcal/mole. Chaotropic anions, cations, and guanidinium chloride accelerated the rate in water. However, the inside-outside (axial-equatorial; I_aI_b) ratio at equilibrium did not correlate in any simple manner with the solvent E_T values. Rather, correlation within groups of solvents appeared to exist. It was suggested that solvent association with the amide differs quantitatively in the inside and outside conformations. The position of the equilibrium in water was affected by chaotropic ions but not by urea or quanidinium chloride. Some possible mechanisms are briefly outlined.     

Potassium transport in Neurospora. Evidence for a multisite carrier at high pH

At low extracellular pH (4–6), net uptake of potassium by Neurospora is a simple exponential process which obeys Michaelis kinetics as a function of [K]_o. At high pH, however, potassium uptake becomes considerably more complex, and can be resolved into two distinct exponential components. The fast component (time constant = 1.2 min) is matched quantitatively by a rapid loss of sodium; it is attributed to ion exchange within the cell wall, since it is comparatively insensitive to low temperature and metabolic inhibitors. By contrast, the slower component (time constant = 10.9 min) is inhibited markedly at 0°C and by CN and deoxycorticosterone, and is thought to represent carrier-mediated transport of potassium across the cell membrane. This transport process exhibits sigmoid kinetics as a function of [K]_o; the data can be fitted satisfactorily by two different two-site models (one involving a carrier site and a modifier site, the other an allosteric model). Either of these models could also accommodate the simple Michaelis kinetics at low pH.     

Quantitative Transcription Factor Binding Kinetics at the Single-Molecule Level

We investigated the binding interaction between the bacteriophage λ-repressor CI and its target DNA using total internal reflection fluorescence microscopy. Large stepwise changes in the intensity of the red fluorescent protein fused to CI were observed as it associated with and dissociated from individually labeled single-molecule DNA targets. The stochastic association and dissociation were characterized by Poisson statistics. Dark and bright intervals were measured for thousands of individual events. The exponential distribution of the intervals allowed direct determination of the association and dissociation rate constants (k_a and k_d, respectively). We resolved in detail how k_a and k_d varied as a function of three control parameters: the DNA length L, the CI dimer concentration, and the binding affinity. Our results show that although interactions with nonoperator DNA sequences are observable, CI binding to the operator site is not dependent on the length of flanking nonoperator DNA.     

Electron paramagnetic resonance study of the oxidation of N-methyl-substituted aromatic amines catalyzed by hemeproteins

The oxidation of N-mono- and dimethyl-substituted toluidines and aniline by H₂O₂, catalyzed by horseradish peroxidase or metmyoglobin, produces organic free radicals, detectable by electron paramagnetic resonance spectroscopy at room temperature. The radical cation of N,N-dimethyl-p-toluidine was conclusively identified, but the other resolved EPR signals were assigned to radical cations of radical dimerization products, e.g., N,N,N′,N′-tetramethylbenzidine formed from N,N-dimethylaniline. The N-demethylase activities of metmyoglobin were found to be uniformly smaller than those of horseradish peroxidase, consistent with the much faster reaction of the latter hemeprotein with H₂O₂. Detection of the monomeric radical cation of N,N-demethyl-p-toluidine correlated with the largest rate of N-demethylation among this class of compounds. These findings emphasize the importance of radical stability (provided, for example, by the para methyl substituent) on subsequent competing reactions of the radical cation of the N-methyl substrate, i.e., one-electron oxidation leading to formaldehyde release or radical dimerization, which becomes more probable for the less stable radical intermediates. Attempts were made to correlate these results with data obtained for the $\text{O}{}_2\text{NADPH}\text{-supported}$ oxidation of these same substrates by liver microsomal cytochrome P-450. However, pronounced differences in physical state and kinetic properties of this heterogeneous, membrane-associated microsomal hemeprotein and the soluble “model” hemeprotein systems precluded firm conclusions concerning a radical mechanism of N-demethylation monooxygenase activities of microsomal fractions.     

Oxidation of N-Nitrosoalkylamines by Human Cytochrome P450 2A6: SEQUENTIAL OXIDATION TO ALDEHYDES AND CARBOXYLIC ACIDS AND ANALYSIS OF REACTION STEPS*

Cytochrome P450 (P450) 2A6 activates nitrosamines, including N,N-dimethylnitrosamine (DMN) and N,N-diethylnitrosamine (DEN), to alkyl diazohydroxides (which are DNA-alkylating agents) and also aldehydes (HCHO from DMN and CH₃CHO from DEN). The N-dealkylation of DMN had a high intrinsic kinetic deuterium isotope effect (^Dk_app ∼ 10), which was highly expressed in a variety of competitive and non-competitive experiments. The ^Dk_app for DEN was ∼3 and not expressed in non-competitive experiments. DMN and DEN were also oxidized to HCO₂H and CH₃CO₂H, respectively. In neither case was a lag observed, which was unexpected considering the k_cat and K_m parameters measured for oxidation of DMN and DEN to the aldehydes and for oxidation of the aldehydes to the carboxylic acids. Spectral analysis did not indicate strong affinity of the aldehydes for P450 2A6, but pulse-chase experiments showed only limited exchange with added (unlabeled) aldehydes in the oxidations of DMN and DEN to carboxylic acids. Substoichiometric kinetic bursts were observed in the pre-steady-state oxidations of DMN and DEN to aldehydes. A minimal kinetic model was developed that was consistent with all of the observed phenomena and involves a conformational change of P450 2A6 following substrate binding, equilibrium of the P450-substrate complex with a non-productive form, and oxidation of the aldehydes to carboxylic acids in a process that avoids relaxation of the conformation following the first oxidation (i.e. of DMN or DEN to an aldehyde).     

Harmonic system analysis of the algae Valonia utricularis: contribution of an electrogenic transport system to gain and phase-shift of the transfer function

Cell membrane properties of the giant marine alga Valonia utricularis were measured in the frequency domain between 1 Hz and 10 MHz by harmonic system analysis. Harmonic analysis was performed by imposing a sinusoidal electrical voltage on the cell interior via an internal microelectrode. Gain and phase-shift of the resulting sinusoidal membrane voltage were measured over the whole frequency range with an internal voltage microelectrode. Bode plots of gain and phase-shift allowed the determination of the electrical parameters of the equivalent electronic circuits of the cell membrane of V. utricularis, which showed dynamic and passive properties dependent on the pH of the external aqueous solution. The dynamic components of the membrane impedance were caused by an electrogenic transport system for chloride described previously (Wang, J., G. Wehner, R. Benz, and U. Zimmermann. 1991. Biophys. J. 59:235-248). The kinetic and equilibrium parameters of the transport system could be evaluated from the fit of Bode plots of gain and phase-shift. The frequency domain technique revealed complete agreement of transport parameters with previously published results. The data demonstrate that an electrogenic transport system can be driven by an oscillating electric field.     

Electrical noise from lipid bilayer membranes in the presence of hydrophobic ions

Summary In the presence of the hydrophobic ion dipicrylamine, lipid bilayer membranes exhibit a characteristic type of noise spectrum which is different from other forms of noise described so far. The spectral density of current noise measured at zero voltage increases in proportion to the square of frequency at low frequencies and becomes constant at high frequencies. The observed form of the noise spectrum can be interpreted on the basis of a transport model for hydrophobic ions in which it is assumed that the ions are adsorbed in potential-energy minima at either membrane surface and are able to cross the central energy barrier by thermal activation. Accordingly, current-noise results from random fluctuations in the number of ions jumping over the barrier from right to left and from left to right. On the basis of this model the rate constantk _i for the translocation of the hydrophobic ion across the barrier, as well as the mean surface concentrationN _t of adsorbed ions may be caluculated from the observed spectral intensity of current noise. The values ofk _i obtained in this way closely agree with the results of previous relaxation experiments. A similar, although less quantitative, agreement is also found for the surface concentrationN _t .     

A fast quadrature-based numerical method for the continuous spectrum biphasic poroviscoelastic model of articular cartilage

A new and efficient method for numerical solution of the continuous spectrum biphasic poroviscoelastic (BPVE) model of articular cartilage is presented. Development of the method is based on a composite Gauss–Legendre quadrature approximation of the continuous spectrum relaxation function that leads to an exponential series representation. The separability property of the exponential terms in the series is exploited to develop a numerical scheme that can be reduced to an update rule requiring retention of the strain history at only the previous time step. The cost of the resulting temporal discretization scheme is O(N) for N time steps. Application and calibration of the method is illustrated in the context of a finite difference solution of the one-dimensional confined compression BPVE stress-relaxation problem. Accuracy of the numerical method is demonstrated by comparison to a theoretical Laplace transform solution for a range of viscoelastic relaxation times that are representative of articular cartilage.     

Effects of variation of ion and methylation of carrier on the rate constants of macrotetralide-mediated ion transport in lipid bilayers

Summary The effects of methylation on the rate constants of carrier-mediated ion transport have been studied on monooleindecane bilayers with K⁺, Rb⁺, NH ₄ ⁺ , and TI⁺ ions, using the series of homologue carriers, nonactin, monactin, dinactin, trinactin, and tetranactin, each member of the series differing from the previous one by only one methyl group. Measurements of the amplitude and time constant of the current relaxation after a voltage jump over a large domain of voltage and permeant ion concentration, together with a computer curve-fitting procedure, have allowed us, without the help of steady-state current-voltage data, to deduce and compare the values of the various rate constants for ion transport: formation (k _Ri) and dissociation (k _Di) of the ion-carrier complex at the interface, translocation across the membrane interior of the carrier (k _s) and the complex (k _is). With the additional information from steady-state low-voltage conductance measurements, we have obtained the value of the aqueous phase-membrane and torus-membrane partition coefficient of the carrier ({ie191-1} and {ie191-2}). From nonactin to tetranactin with the NH ₄ ⁺ ion,k _is, and {ie191-3} are found to increase by factors of 5 and 3, respectively,k _Di and {ie191-4} to decrease respectively by factors 8 and 2, whilek _Ri andk _s are practically invariant. Nearly identical results are found for K⁺, Rb⁺, and Tl⁺ ions.k _Ri,k _s andk _is are quite invariant from one ion to the other except for Tl⁺ wherek _Ri is about five times larger. On the other hand,k _Di depends strongly on the ion, indicating that dissociation is the determining step of the ionic selectivity of a given carrier. The systematic variations in the values of the rate constants with increasing methylation are interpreted in terms of modifications of energy barriers induced by the carrier increasing size. Within this framework, we have been able to establish and verify a fundamental relationship between the variations ofk _is andk _Di with methylation.     

Phagocytic killing of microorganisms by radical processes:consequences of the reaction of hydroxyl radicals with chloride yielding chlorine atoms

Chloride anions and hydrogen peroxide serve as substrates for myeloperoxidase (MPO) in order to produce hypochlorous acid (HOCl) as one of the major killing agents of phagocytic leukocytes. Apart from this role of being a substrate for the MPO-reaction the chloride anion has been considered as unreactive and has not been implicated in radical reactions which contribute to the killing process. From the inherent reactivities of the pertinent radicals (as determined by pulse radiolysis experiments), the great abundance of chloride, and the most probable distribution of reactants within the phagosome, we deduce estimates for the average life-time and free diffusion path-length in this milieu and arrive at a model according to which chloride ions enter into radical chains and influence the killing of ingested bacteria to an extraordinarily high extent. We propose that hydroxyl radicals—despite some controversial arguments in the literature—may still be considered as important contributors to cell killing especially since we show that their reactions are made more effective by producing chlorine radicals in a cyclic process. We furthermore present arguments how the phagocyte may protect itself from harmful actions of HOCl and H₂O₂ after the superoxide-generating activity of NADPH oxidase is turned off.