Involvement of photosynthesis in the action of temperature on plasmalemma transport inNitella

Summary At membrane potentials different fromE _K, the temperature effect on membrane potential ofNitella consists of two components. One of them changes its sign atE _K, the other one does not. This leads to the assignment of these components to changes in the K⁺ channel and in the H⁺ pump, respectively. It is shown that the fast time constant (3 to 30 sec) of the temperature effect on the H⁺ pump measured as a change in membrane potential and that of the temperature effect on the K⁺ channel measured as a change in resistance (having about twice the value of that of the pump) are sensitive to light intensity. Both time constants measured inNitella become smaller if light intensity increases from 0 to 15 Wm^–2. This supports the suggestion of Fisahn and Hansen (J. Exp. Bot. 37:440–460, 1986) that temperature acts on plasmalemma transport via photosynthesis via the same mechanism as light does.     

The action of sinusoidally modulated light on the membrane potential of Acetabularia

Cells of the alga Acetabularia crenulata were illuminated withlight of sinusoidally modulated intensity of different frequenciesand changes in membrane potential were measured with microelectrodes.The frequency response, investigated in the range of frequenciesof 8 to 1/40 cycles/min, displayed the following features: 1.The slope of the frequency response is, according to our results,6 db/octave or steeper. Experiments should be extended to higherfrequencies to gain more information concerning the slope ofthe frequency response and, thus, of the number of reactionsin the series. 2. The time course of changes in membrane potentialcaused by light reveals non-linearities in time-scale and amplitude,indicating that not only first-order reactions are involved.3. Time constants are slightly influenced by the intensity oflight. 4. Further evidence is given that the characteristicof the action of light is logarithmic. 5. A phenomenon similarto resonance is observed, which corresponds to the overshootsgenerated by square-wave illumination. Models show that thisis not necessarily explained by feedback systems, but also bycounteracting parallel pathways (Received October 8, 1970; )     

Very low osmotic water permeability and membrane fluidity in isolated toad bladder granules

Summary Osmotic water permeability of the apical membrane of toad urinary epithelium is increased greatly by vasopressin (VP) and is associated with exocytic addition of granules and aggrephores at the apical surface. To determine the physiological role of granule exocytosis, we measured the osmotic water permeability and membrane fluidity of isolated granules, surface membranes and microsomes prepared from toad bladder in the presence and absence of VP.P _f was measured by stopped-flow light scattering and membrane fluidity was examined by diphenylhexatriene (DPH) fluorescence anisotropy. In response to a 75mm inward sucrose gradient, granule size decreased with a single exponential time constant of 2.3±0.1 sec (sem, seven preparations, 23°C), corresponding to aP _f of 5×10^–4 cm/sec; the activation energy (E _a ) forP _f was 17.6±0.8 kcal/mole. Under the same conditions, the volume of surface membrane vesicles decreased biexponentially with time constants of 0.13 and 1.9 sec; the fast component comprised 70% of the signal. Granule, surface membrane and microsome time constants were unaffected by VP. However, in surface membranes, there was a small decrease (6±2%) in the fraction of surface membranes with fast time constant. DPH anisotropies were 0.253 (granules), 0.224 (surface membrane fluidity is remarkably lower than that of surface and microsomal membranes, and (4) rapid water transport occurs in surface membrane vesicles. The unique physical properties of the granule suggests that apical exocytic addition of granule membrane may be responsible for the low water permeability of the unstimulated apical membrane.     

Current-voltage curve of electrogenic Cl− pump predicts voltage-dependent Cl− efflux inAcetabularia

Summary The current-voltage relationship of carrier-mediated, passive and active ion transport systems with one charge-carrying pathway can exactly be described by a simple reaction kinetic model. This model consists of two carrier states (one inside, one outside) and two pairs (forwards and backwards) of rate constants: a voltage-dependent one, describing the transport of charge and a voltage-insensitive one, summarizing all the other (voltage-independent) reactions. For the electrogenic Cl^– pump inAcetabularia these four rate constants have been determined from electrical measurements of the current-voltage relationship of the pump (Gradmann, Hansen & Slayman, 1981;in: Electrogenic Ion Pumps, Academic Press, New York). The unidirectional Cl^– efflux through the pump can also be calculated by the availiable reaction kinetic parameters.³⁶Cl^– efflux experiments on singleAcetabularia cells with simultaneous electrical stimulation (action potentials) and recording, demonstrate the unidirectional Cl^– efflux to depend on the membrane potential. After subtraction of an efflux portion which bypasses the pump, agreement is found between the measured flux-voltage relationship and the theoretical one as obtained from the reaction kinetic model and its parameters from the electrical data.     

Electrical relaxation processes in black lipid membranes in the presence of a cation-selective ionophore

Summary The time course of relaxation of the electric current following steps in the applied potential across lipid bilayer membranes has been measured. The membranes were made cation-selective by the addition of nonactin. To permit the measurement of very short time constants a voltage clamp device was developed in order to reduce the charging period to less than 1 sec, regardless of the magnitude of the series resistances in the external solutions. It was possible by this method to establish the presence of two electric processes, which were found to behave differently with respect to temperature, applied potential and external solution conditions. The rapid process (10 sec) was interpreted in terms of the electric parameters of the polar part of the membrane according to the theory developed by Hägglund and Sandblom (T.I.T.J. Life Sci. 2: 107, 1972). The second process (100 sec) showed a behavior consistent with the model of Stark, Ketterer, Benz and Läuger (Biophys. J. 11:981, 1971), which considers the different rate constants involved in the net transfer of carriermediated ion transport across bilayer membranes.     

Dependence of the electrical breakdown voltage on the charging time inValonia utricularis

Summary Charge-pulse experiments were performed on giant algal cells ofValonia utricularis. For a charging time of 420 sec the breakdown voltage is about 750 mV (18°C), a value that is in close agreement with earlier results obtained with current pulses (Coster & Zimmermann, 1975;J. Membrane Biol. 22:73). If the membrane is charged to the breakdown voltage in a shorter time, the breakdown voltage is found to be a function of the duration of the charge pulses. Whereas towards smaller pulse lengths down to 10 sec only a small, but significant, increase in the breakdown voltage is observed (1.1 V at 10 sec pulse length and 18°C), a strong increase in the breakdown voltage is found for even shorter charging times. For a pulse length of 800 nsec the breakdown voltage has a value of about 2.4 V (18°C) and a plateau seems to be reached for a pulse duration of 500 nsec. The influence of temperature on the breakdown voltage as observed for short charging times is very similar to that reported earlier for current pulses of 500 sec duration. For charge pulses of 1 to 2 sec duration the breakdown voltage decreases from 3.6 V at 3°C to 1.6 V at 25°C by more than a factor of two.Voltage relaxation studies in the low-field range suggest that the time constants of the two membranes arranged in series, tonoplast and plasmalemma, are similar. From this, it is suggested that both membranes show electrical breakdown, whereby the breakdown voltage of a single membrane is probably half the value of the total breakdown voltage. Its dependence on pulse length is therefore considered to be an intrinsic property of one single membrane. The strong dependence of the breakdown voltage on the charging time of the membrane further supports the interpretation of the breakdown phenomenon on the basis of the electro-mechanical model proposed earlier. In this model it is assumed that the electrical and mechanical compressive forces are counter balanced by elastic restoring forces within the membrane. However, towards very short pulses (less than 800 nsec), where a plateau seems to be reached, other processes may be generated by the application of the electric field. We discuss whether one of these processes is the ion movement through the membranes induced by a high electric field (Born energy).     

Dynamics of Interaction of Phosphatidylcholine/Octadecylamine Liposomes with Human Erythrocyte Membranes: Electron Microscopic Study

Abstract

The interactions of octadecylamine and of positively charged liposomes (egg phosphatidylcholine/octadecylamine in molar ratio from 7:5 to 7:0.5) with human erythrocyte membrane have been studied by freeze-fracture and thin-section electron microscopy.

For the first time a very fast adsorption of liposomes to the cell membranes (less than 1 sec) is shown, and their intensive incorporation into plasma membrane (probably within the first 2-5 sec) without visible changes in cell morphology.

The prolonged incubation of the cells with liposomes results in certain morphologic changes: the transition of diskocytes to stomatocytes (30-80 sec) accompanied by the formation of isolated membrane vesicles in cell matrix (80-120 sec); the formation of pentalaminar contacts between plasma membrane of spherocytes and the membrane of isolated matrix vesicles (2.5-3 min); and the incorporation of matrix vesicle membranes into the spherocyte membranes (5 min+).

Possible molecular mechanisms underlying the observed structural changes are discussed briefly.     

The Transient Outward Current of Isolated Bovine Adrenal Zona Fasciculata Cells: Comparison between Standard and Perforated Patch Recording Methods

Voltage-clamp experiments were performed on single bovine adrenal fasciculata cells in short-term primary culture using either standard (broken membrane) or perforated whole-cell patch clamp recording. The membrane current measured with the perforated method was dominated by a very stable transient outward current. By contrast, the transient outward current recorded using the standard method was unstable. The reversal potential of the transient outward current varied linearly with the logarithm of [K⁺] _e with a slope of 47 mV per decade. The onset of activation was sigmoidal and was fitted with a power function where n= 4. Time constants ranged from 1 to 4 msec with a maximum at −25 mV. The steady-state activation curve spanned the voltage range −50 to +80 mV without reaching a clear maximum. During a pulse, the current decayed in a biexponential manner. Time constants τ₁ and τ₂ were voltage-dependent and ranged from 50 to 200 msec respectively for a voltage step at +50 mV. The steady-state inactivation was dependent on the conditioning pulse duration. Using short conditioning pulses (1.2 sec), the curve which spanned the voltage range −40 to −20 mV, was 15 mV more positive than that obtained with longer conditioning pulses (60 sec). Time constants of this ``very slow inactivation' process (τ<sub>vs</sub>) determined for voltage steps at −60 and −50 mV were 15 and 10 sec respectively. A ``facilitation process' of the peak current was observed when the duration or the amplitude of conditioning pulses were increased in the voltage range −100 to −50 mV. Recovery from inactivation followed a biexponential time course which seemed a mixture of both inactivation processes. In some experimental conditions, isolated cells were able to produce overshooting action potentials. These results are discussed in relation with the membrane electrogenesis of this cell type. Received: 14 November 1994/Revised: 24 October 1995     

Dynamics of Excitation and Inhibition in the Light-Adapted Limulus Eye in situ

The dynamics of spike discharge in eccentric cell axons from the in situ lateral eye of Limulus, under small sinusoidal modulation of light to which the eye is adapted, are described over two decades of light intensity and nearly three decades of frequency. Steady-state lateral inhibition coefficients, derived from the very low-frequency response, average 0.04 at three interommatidial spacings. The gain vs. frequency of a singly illuminated ommatidium is described closely from 0.004 to 0.4 cps by the linear transfer function s^0.25; this function also accounts approximately for the measured phase leads, the small signal adaptation following small step inputs, and for Pinter's (1966) earlier low-frequency generator potential data. We suggest that such dynamics could arise from a summation in the generator potential of distributed intensity-dependent relaxation processes along the dendrite and rhabdome. Analysis of the dynamic responses of an eccentric cell with and without simultaneously modulated illumination of particular neighbors indicates an effect equivalent to self-inhibition acting via a first-order low-pass filter with time constant 0.42 sec, and steady-state gain near 4.0. The corresponding filters for lateral inhibition required time constants from 0.35 to 1 sec and effective finite delay of 50–90 msec.     

Structure of the squid axon membrane as derived from charge-pulse relaxation studies in the presence of absorbed lipophilic ions

Summary Charge-pulse relaxation studies were performed on squid giant axons in the presence of membrane absorbed lipophilic anions, dipicrylamine (DPA) and tetraphenylborate (TPhB), and of specific blockers of sodium and potassium active currents. With the instrumentation used in this work a time resolution of 5 to 10 sec was easily obtained without any averaging, although the voltage relaxations were always smaller than 5 mV in amplitude in order to keep the membrane voltage in a range where the used theory cyn be linearized. Two well distinguishable linear relaxations were invariably observed in the presence of the lipophilic anions. With DPA the fast relaxation (time constants between 8 and 70 sec) was attributed to the redistribution of the lipophilic ions within the membrane following the change in membrane potential. The long relaxation process (time constant in the millisecond range) corresponds to the normal voltage relaxation of the passive squid axon membrane slightly modified by the process of redistribution of the extrinsic ions.The results support the same model for the translocation of lipophilic ions within the nerve membrane proposed earlier for artificial lipid bilayers. The fit of the data with a single barrier model yields the translocation rate constant,K, and the total concentration,N _t , of membrane absorbed ions, from which the membrane-solution partition coefficient, , can be derived. Both for DPA and TPhB,K had values close to those measured for solvent-free artificial lipid bilayers. The axon membrane appears as fluid mosaic membrane with a thickness of about 2.5 nm for the lipid bilayer part.In axons treated with DPA the dependence of relaxation data upon the holding membrane potential, , provided information on the asymmetry of the membrane structure. The data were best fitted by assuming that nearly 100% of the membrane potential drops between the two free energy minima where the extrinsic ions are located, indicating that these minima lie very close to the membrane-solution interfaces, in the region of the phospholipid polar heads. The asymmetry voltage,E _o, at which the extrinsic ions are expected to be equally distributed between the two sides of the membrane was found to range between –35 and –65 mV (inside negative), depending on the assumed shape of the free energy barrier describing the ion translocation process. This voltage is of the same sign and of the same order of magnitude as the equilibrium voltages for the open-close transitions of the gates of sodium and potassium channels, suggesting that all these voltages result from the same membrane asymmetry. A similar analogy was found between the asymmetry of the free energy barrier which best fitted DPA relaxation data and the asymmetrical voltage dependence of the gating of ionic channels. Our data were best fitted by assuming that about 70% of the potential drop occurs between the free energy minimum on the intracellular membrane face and the top of the barrier.     

Electrogenic and diffusive components of the membrane ofHydrodictyon africanum

Summary In cells of the freshwater algaHydrodictyon africanum, in solutions where [K⁺]₀=0.1mm and pH₀>7.0, the membrane in the light is hyperpolarized. The membrane potential difference {ie179-1} has values from –180 to –275 mV, more negative than any ion diffusion potential difference, and is predominantly a function of pH₀, and independent of [K⁺]₀. The hyperpolarization of the membrane appears to arise from an electrogenic efflux of H⁺, estimated from voltage-clamp data to be about 8 nmol m^–2 sec^–1 when pH₀=8.5. In the light the membrane conductanceg _m is about 0.084 S m^–2. At light-off, {ie179-2} becomes less negative, with a halftime for change of 15 to 30 sec andg _m decreases by about 0.052 S m^–2. After dark periods of up to 300 sec, {ie179-3} is largely independent of pH₀ for values greater than 6.0 and usually behaves as a combined K⁺ and Na⁺ diffusion potential with permeability ratioP _Na/P _K=0.05 to 0.2. The membrane potassium conductanceg _K has either a low value of 2–6×10^–2 Sm^–2, or a high value of up to 18×10^–2 S m^–2 depending on [K⁺]₀, the transition from low to high values occurring when {ie179-4} moves over a threshold value that is more negative than {ie179-5}, the electrochemical equilibrium potential for K⁺. The time for half-change of the transition is about 30 sec. The results are consistent with a model of the membrane in which the pump electromotive force and conductance are in parallel with diffusive electromotive forces and conductances. When the pump is operating its properties determine membrane properties, and when it is inoperative, or running at a diminished rate, the membrane properties are determined more by the diffusive pathways. Changes in both pump rate andg _K can account for a variety of characteristic changes in membrane PD and conductance occurring in response to ligh-dark changes, changes in light intensity, pasage of externally applied electric current across the membrane and changes in ionic constituents of the external medium.     

Steady-state voltage-dependent gating and conduction kinetics of single K+ channels in the membrane of cytoplasmic drops ofChara australis

Summary Cytoplasmic drops, covered by a membrane derived from the tonoplast, were obtained from the internodal cells ofChara australis. Patch-clamp measurements were made on this membrane using the droplet-attached configuration with the membrane patch voltage clamped at values from –250 to 50 mV. Single-channel records, filtered at 5 kHz, were analyzed to elucidate the kinetics of the ion gating reaction of the K⁺-selective channel. The current-voltage characteristics for single channels exhibit saturation and are shown to be consistent with Läuger's theory of diffusion-limited ion flow through pores (P. Läuger,Biochim. Biophys. Acta 455:493–509, 1976). The time-averaged behavior of the K⁺ conductance has a maximum at –100 to –150 mV which is produced by the combination of two distinct mechanisms: (1) The channel spending more time in long-lived closed states at positive voltages and (2) a large decrease in the mean open lifetime at more negative voltages. The channel activity shows bursting behavior with opening and closing rates that are voltage-dependent. The mean open time is the kinetic parameter most sensitive to membrane potential, showing a maximum between –100 to –150 mV. The distribution of open times is dominated by one exponential component (time constant 0.3 to 10 msec). In some cases an additional rapidly decaying exponential component was detectable (time constant=0.1 msec). The closed distributions contained were observed to obtain up to four exponential components with time constants over the range 0.1 to 200 msec. However, the voltage dependence of the closed-time distributions suggests an eight-state model for this channel.     

Rapid effect of light on the K+ channel in the plasmalemma ofNitella

Summary Chlorophyll fluorescence, plasmalemma potential and resistance were measured simultaneously and subjected to a kinetic analysis. It was found that the light-induced changes of all three signals have two time constants in common. The faster one (₄=ca. 20 sec) was assigned to the action of light-induced proton uptake across the thylakoid membrane on the plasmalemma H⁺ pump. The slower one (_5a=40 sec) is related to the light action of an unknown photosynthetic process on the potassium channel. The action on the K⁺ channel was revealed from the reversal potential of the related effect on membrane potential. The comparison of the data with findings of other authors led to the hypothesis that the unknown photosynthetic mechanism is the depletion of NADP⁺, which stimulates the uptake of Ca²⁺ from the cytosol, which is required for the NAD-kinase. The resulting change in cytosolic Ca²⁺ modulates the number of open K⁺ channels.     

A multivariate population density model of the dLGN/PGN relay

Using a population density approach we study the dynamics of two interacting collections of integrate-and-fire-or-burst (IFB) neurons representing thalamocortical (TC) cells from the dorsal lateral geniculate nucleus (dLGN) and thalamic reticular (RE) cells from the perigeniculate nucleus (PGN). Each population of neurons is described by a multivariate probability density function that satisfies a conservation equation with appropriately defined probability fluxes and boundary conditions. The state variables of each neuron are the membrane potential and the inactivation gating variable of the low-threshold Ca²⁺ current I_T. The synaptic coupling of the populations and external excitatory drive are modeled by instantaneous jumps in the membrane potential of postsynaptic neurons. The population density model is validated by comparing its response to time-varying retinal input to Monte Carlo simulations of the corresponding IFB network composed of 100 to 1000 cells per population. In the absence of retinal input, the population density model exhibits rhythmic bursting similar to the 7 to 14 Hz oscillations associated with slow wave sleep that require feedback inhibition from RE to TC cells. When the TC and RE cell potassium leakage conductances are adjusted to represent cholingergic neuromodulation and arousal of the network, rhythmic bursting of the probability density model may either persists or be eliminated depending on the number of excitatory (TC to RE) or inhibitory (RE to TC) connections made by each presynaptic cell. When the probability density model is stimulated with constant retinal input (10–100 spikes/sec), a wide range of responses are observed depending on cellular parameters and network connectivity. These include asynchronous burst and tonic spikes, sleep spindle-like rhythmic bursting, and oscillations in population firing rate that are distinguishable from sleep spindles due to their amplitude, frequency, or the presence of tonic spikes. In this context of dLGN/PGN network modeling, we find the population density approach using 2,500 mesh points and resolving membrane voltage to 0.7 mV is over 30 times more efficient than 1000-cell Monte Carlo simulations. Action Editor: David Golomb     

Effects of localised,low-voltage pulsed electric fields on the development and inhibition of Pseudomonas aeruginosa biofilms

Abstract

This work describes the use of low-voltage (0.5 – 5 V) pulsed electric fields to prevent Pseudomonas aeruginosa biofilm development. Interdigitated electrodes (IDEs) with 29-μm spacing between 22-μm-wide electrodes, were used as a platform where the effect of localised, high-strength electric fields could be tested. Alternating current, square-wave pulses were applied to the IDEs in 1 sec intervals. A two-level, three-variable factorial design experiment was used to detect the effects of applied voltage, frequency, and pulse duty ratio (i.e. percentage of pulsing time over one cycle) on the inhibition of biofilm formation. The observations indicated that a pulse configuration of 1% duty ratio, 5 V, and 200 Hz frequency reduced the area of the electrodes covered by biofilm by 50%. In general, the application of low-duty ratio pulses had a positive effect on preventing biofouling. Comparatively, frequency and applied voltage were observed to have less influence on biofouling.     

Ion transport and displacement currents with membrane-bound carriers

Summary The standard carrier model for ion transport by a one-to-one mechanism is developed to predict the time-dependent currents for systems that are symmetrical at zero applied potential. The complete solution for ions and carriers bearing any charge is derived by assuming that the concentration of ions in the membrane is low and either that the applied potential is small or that the applied potential affects equally all of the association and dissociation reactions between the ions and the carriers. The response to an abruptly applied potential is then given by the sum of a constant and two declining exponential terms. The time constants of these relaxations are described by the equations derived for neutral carriers by Stark, Ketterer, Benz and Läuger in 1971 (Biophys. J. 11:981). The sum of the amplitudes of the exponentials for small applied potentials obeys a relation like that first derived by Markin and Liberman in 1973 (Biofizika 18:453). For small applied potentials expressions are also provided for the voltage transients in charge-pulse experiments and for the membrane admittance.     

Pump and displacement currents of reconstituted ATP synthase on black lipid membranes

Summary Purified ATP synthase (F ₀ F ₁) fromRhodospirillum rubrum was reconstituted into asolectin liposomes which were than adsorbed to a planar lipid bilayer. After the addition of an inactive photolabile ATP derivative (caged ATP), ATP was released after illumination with UV light, which led to a transient current in the system. The transient photocurrent indicates that the vesicles and the planar membrane are capacitatively coupled. Stationary pump currents were obtained after addition of protonophores. These currents are specifically inhibited by oligomycin and stimulated threefold by inorganic phosphate (P _i ). In analogy oligomycin-sensitive pump currents in the reverse direction coupled to net ATP synthesis were induced by a light-induced concentration jump of ADP out of caged ADP, demonstrating the reversibility of the pump. For this, a preformed proton motive force and P _i were necessary.In a second series of experiments, proteoliposomes containing both ATP synthase and bacteriorhodopsin were adsorbed to a planar bilayer. The system was excited by a laser flash. The resulting photocurrents were measured with a time resolution of 2 sec. In the presence of ADP, the signal was modulated by the electrical activity of ATP synthase. ADP-induced charge displacements in ATP synthase, with time constants of 11 and 160 sec were obtained. The kinetics of the charge movements were slowed down byF ₀ specific inhibitors (DCCD or oligomycin) and were totally absent if ADP binding toF ₁ is prevented by the catalytic site-blocking agent NBD-Cl. The charge displacement of ATP synthase is coupled only to the membrane potential induced by the electrical activity of bacteriorhodopsin. The charge movements are interpreted as conformational transitions during early steps of the reaction cycle of ATP synthase.     

Analysis of ionic channels by a flash spectrophotometric technique applicable to thylakoid membranes: CF0, the proton channel of the chloroplast ATP synthase,and, for comparison,gramicidin

Summary We previously introduced a flash spectrophotometric method to analyze proton conduction by CF₀ in vesicles derived from thylakoid membranes (H. Lill, S. Engelbrecht, G. Schönknecht & W. Junge, 1986,Eur. J. Biochem. 160:627–634). The unit conductance of CF₀, as revealed by this technique, was orders of magnitude higher than that theoretically expected for a hydrogen-bonded chain. We scrutinized the validity of this method. Small vesicles were derived from thylakoids by EDTA treatment. The intrinsic electric generators in the membrane were stimulated by short flashes of light and the relaxation of the voltage via ionic channels was measured through electrochromic absorption changes of intrinsic pigments. The voltage decay was stimulated by a statistical model. As the vesicle-size distribution had only a minor influence, the simulation required only two fit parameters, the first proportional to the unit conductance of an active channelG, and the second denoting the average number of active channels per vesiclen. This technique was applied to CF₀, the proton channel of the chloroplast ATP synthase, and to gramicidin, serving as a standard. For both channels we found the above two fit parameters physically meaningful. They could be independently varied in predictable wasy, i.e.n by addition of known inhibitors of F₀-type proton channels andG via the temperature. for gramicidin, the unit conductance (2.7 pS) was within the range described in the literature. This established the competence of this method for studies on the mechanism of proton conduction by CF₀, whose conductance so far has not been accessible to other, more conventional approaches. The time-averaged unit conductance of CF₀ was about 1 pS, equivalent to the turnover of 6×10⁵ H⁺/(CF₀·sec) at 100 mV driving force.     

Evaluation of droplet-based microfluidic platforms as a convenient tool for lipases and esterases assays

Abstract

The accurate estimation of kinetic parameters is of fundamental importance for biochemical studies for research and industry. In this paper, we demonstrate the application of a modular microfluidic system for execution of enzyme assays that allow determining the kinetic parameters of the enzymatic reactions such as V_max – the maximum rate of reaction and K_M – the Michaelis constant. For experiments, the fluorogenic carbonate as a probe for a rapid determination of the kinetic parameters of hydrolases, such as lipases and esterases, was used. The microfluidic system together with the method described yields the kinetic constants calculated from the concentration of enzymatic product changes via a Michaelis–Menten model using the Lambert function W(x). This modular microfluidic system was validated on three selected enzymes (hydrolases).     

Direct ab initio dynamics studies of the hydrogen abstraction reactions of hydrogen atom with n-propyl radical and isopropyl radical

The kinetics of the hydrogen abstraction reactions of hydrogen atom with n-propyl radical and isopropyl radical were studied using the direct ab initio dynamics approach. BHandHLYP/cc-pVDZ method was employed to optimize the geometries of stationary points as well as the points on the minimum energy path (MEP). The energies of all the points for the two reactions were further refined at the QCISD(T)/cc-pVTZ level of theory. No barrier was found at the QCISD(T)/cc-pVTZ//BHandHLYP/cc-pVDZ level of theory for both reactions. The forward and reverse rate constants were evaluated with both canonical variational transition state theory (CVT) and microcanonical variational transition state theory ( VT) in the temperature range of 300–2,500 K. The fitted three-parameter Arrhenius expression of the calculated CVT rate constants at the QCISD(T)/cc-pVTZ//BHandHLYP/cc-pVDZ level of theory are k^CVT (n – C₃H₇)=1.68×10^–14 T^0.84 e^(319.5/T) cm³ molecule^–1 s^–1 and k^CVT (iso-C₃H₇)=4.99×10^–14 T^0.90 e^(159.5/T) cm³ molecule^–1 s^–1 for reactions of n-C₃H₇ + H and iso-C₃H₇ + H, respectively, which are in good agreement with available literature data. The variational effects were analysed.Figure Comparison of the calculated forward rate constants at the QCISD(T)/cc-pVTZ//BHandHLYP/cc-pVDZ level of theory and the available experimental and theoretical data of the reaction vs 1,000/T for the two reactions.