Immobilized cycling enzyme active transport systems. pH feedback control.

Active transport can be induced by applying a pH gradient across a membrane containing a homogeneous mixture of two cycling enzymes. When the two reactions are inversely 'pH active', one producing protons and the other consuming them, a pH feedback control of the functional structure occurs and the active transport function of the membrane can be either stabilized or inhibited according to whether the endogenic pH modifications tend to enhance or reduce the exergonic pH gradient. When it is stabilized, the system looks like a thin active layer surrounded by two diffusive layers, leading to a fairly good model for biological transport systems. Under particular conditions, signals can be emitted.

Theoretical study of mechano-chemical couplings in a compartmental enzyme system. I. Analytical treatment.

This paper deals with theoretical aspects of the volume changes of a system in which diffusion, convection and reaction processes are coupled. This study involves a material able to swell in the presence of a chemical effector produced by an enzyme reaction. Three limiting factors of volume change rate were considered: fluid flow, diffusion or reaction limitations. Dimensionless diffusion-reaction and diffusion-convection parameters were introduced to allow quantitative predictions in limit cases. The steady states appear to be independent of convection processes; however, the transient states depend on diffusion, convection and reaction processes.

pH feedback control of enzyme membranes.

pH feedback on immobilized enzymes is theoretically examined with respect to substrate and pH levels, strength of acids produced by the reaction, buffering and asymmetry of the system. All the productions of proton by the different reactions are taken into account by using a 'symbolic species' H. The system of differential diffusion-reaction equations is then integrated using numerical methods. The local 'effective enzyme activity' modulated by an acidity factor enables us to predict and quantify evolutions of the systems: NonMichaelian behavior of an immobilized Michaelis-Menten-type enzyme is shown, even when pH back-actions are excluded; the analysis of intramembrane pH profiles shows that the shift of the optimal pH is a complex function of the substrate and pH levels, the intrinsic pH dependence of the enzyme, and the membrane characteristics. This study may easily be transposed to other types of effector such as divalent cations and used in examining self-regulations of multienzyme systems where pH-active reactions are involved.

Monitoring of artificial enzyme membrane systems by electric currents. I. Analytical treatment with direct current and buffered conductivity.

Continuous electric fields (E) modify the transport flows and the intramembrane concentration profiles of protons or of ionic substrates or cofactors (inhibitors). These "mediators' induce variations in enzyme activity, quantifiable by a generalized Damköhler group II psi distinguishing electrotransport reactions from diffusion reactions. For three typical reaction schemas, using only one mediator, the steady-state equations have been established. Depending on boundary conditions, the direction of electric current (for asymmetrical systems) and the value of psi, activations, inhibitions or activations followed by inactivations have been found. With buffered conductivity (supporting electrolyte), the limiting concentration profiles (E leads to infinity) are uniformly equal to the boundary values; i.e., diffusion constraints are suppressed and the regime is controlled by the reaction. The calculations give the relative activity variations for partially suppressed transport controls.

Two-enzyme active transport in vitro with pH induced asymmetrical functional structures. III. Discussions based on numerical treatments of the time-dependent evolutions.

Three complementary models have been considered in which pH gradients (step function, linear pH or linear H+) impose asymmetry on a two-enzyme mixture. If the "combined pH dependences" of enzymes is pro-asymmetrical, the pH gradient induces an asymmetrical distribution of potential activities ("latent" asymmetry of functional structure). When substrate is added, "developed" asymmetry of effective activities appears which results in "substrate space wave" and pumping when the catalysed reaction couple is "inversible". It is shown that only one steady state exists for a given boundary condition and is attained when the "combined effective activity" of enzymes is nil; the stationary flux with symmetrical boundaries or the stationary load with moving boundaries is proportional to "effective global activities" of enzymes. "Equivalent square models" could be proposed that would be able to describe "functional" or "permanent" structure pumps as well. These models belong to the thermodynamic branch and the asymmetrical "space wave" substrate concentration profiles obtained must be distinguished from dissipative structures. It appears that such primary active transport pumps are chemical equivalents of heat pumps.

[Temporal oscillations in a structured enzymatic medium. Vectorial transport with space-time oscillations Analytic study]. / Oscillations temporelles en milieu enzymatique structuré. Transport vectoriel à oscillation spatio-temporelle. Etude analytique.

An analytical diffusion-reaction model of chemical time-oscillations is obtained with two Michaelian enzymes and boundaries selectively open to H+ or OH- ions. One reaction produces, and the other one consumes H+ ions. During a half oscillation the high reaction rate is progressively equalled by the rate of diffusion, the substrate is consumed and diffusion through the boundaries brings the pH back toward its original value. Asymmetrical selectivities on the boundaries lead to a new "time oscillatory vectorial transport model".

[Non-enzymatic active transport in vitro. Conversion of chemical to osmotic energy]. / Transport actif non-enzymatique in vitro. Conversion d'énergie chimique en énergie osmotique.

The active transport of ammonia gas was obtained with an asymmetric composite membrane-model using H+ as carrier and the formation of water as chemical energy source. The experimental molecular pump externally limited by two silicone films permeable to gasses but not to liquids or ions is separated into two liquid layers by a central cation-exchange membrane. The first acid layer produces the aspiration of NH3, due to NH4+ formation, through the inlet silicone film; NH4+ is transferred to the second alkaline layer through the ion exchange membrane, by interdiffusion with K+; there NH4+ reacts with OH-, water is formed and NH3 is expelled throught the oulet silicone film. This new pump enlarges the applicability of in vitro active transport to non-enzymatic systems and energy sources different from ATP.

Electrochemical detection and counting of Escherichia coli in the presence of a reducible coenzyme, lipoic acid.

Reduction of lipoic acid by bacteria coupled to oxygen consumption during glucose absorption can be followed potentiometrically with a pair of gold and reference electrodes in a minimal culture medium. The variations in potential as a function of time have the shape of a wave. A theoretical expression was derived relating the size of the original inoculum to the time preceding the appearance of the wave. The validity of that relation was experimentally verified with Escherichia coli, and the time needed for a drop of 100 mV was determined. Detection of small inocula, e.g., down to a range of 10 viable E. coli per liter, is possible in about 11 h by yeast extract stimulation. The method, technically simple and adequately sensitive, suggests the possibility of automated detectors of bacterial contaminations.

[An electrochemical evidence that some hormones influence the transmembrane transport of lipoic acid by Escherichio coli K 12 (author's transl)]. / Mise en évidence électrochimique de l'influence d'hormones sur le transport transmembranaire de l'acide lipoïque chez Escherichia coli K 12.

The influence of diverse substances on the transmembrane transport of lipoic acid can be followed by a recently developed method based on electrochemical measurements of the oxydo-reduction of lipolic acid. This technique was applied to study some hormonal effects on E. coli. The results show that the active transport of lipoic acid (at low lipoic acid concentration) through the E. coli K 12 membrane is increased by hydrocortisone and insulin, but remains unaffected by adrenalin. At high lipoic acid concentration, its passive diffusion through the E. coli membrane is enhanced by adrenalin, but decreased by insulin and hydrocortisone. These results are similar to those reported for rat liver mitochondria and erythrocites.

[Microbial metabolic activity and transmembrane transport phenomena by potentiometric analysis of lipoic acid oxidation-reduction, in a minimal culture medium]. / Mesure de l'activité métabolique microbienne et des phénoménes de transports transmembranaires par analyse potentiométrique de l'oxydoréduction de l'acide lipoïque, dans un milieu de culture minimal.

A method of measuring and studying metabolic bacterial activity is proposed, by following the kinetic evolution of the ratio of the oxidized and reduced forms of an electron transporter as a consequence of decreasing oxidizing power--due to oxygen consumption in the culture,--and increasing. Reduction power of bacterial activity. Namely, with minimum composition using salts and glucose the oxido-reduction of lipoic acid is well indicated by a gold electrode without any major bio-or electrochemical interference. A kinetic diffusion reaction theory takes into account the passive or active transmembrane transport of lipoic acid in good agreement with the experimentally observed shapes of the electrical signal. The various types of antibiotic activities are well reflected by the modifications of the signal.

[Theoretical analysis of potential variations of "Escherichia coli" K12 cultures in presence of an electron carrier (author's transl)]. / Analyse théorique des variations de potentiel dans les cultures de Escherichia coli K12 en présence d'un transporteur d'électrons.

Oxygen consumption and reductive hydrogenation of lipoic acid, linked to the exponential growth of Escherichia coli in a "minimal" medium containing a non-limiting amount of glucose, produce reproducible and intelligible variations of the electric potential between a gold electrode and a reference electrode. A simple quasi-stationary mathematical model leads to analytical expressions of the electric potential in function of the experimental conditions and of time. The effects of active or passive transport of lipoic acid are distinguishable. An experimental method results in detecting and following bacterial activity.

Experimental procedure for a hydrogen peroxide assay based on the peroxidase-oxidase reaction.

A low-cost assay method that is able to measure H2O2 concentrations as low as the nano-molar range is described. The assay solution contains NADH, horseradish peroxidase, and superoxide dismutase at PH 7.5. After the addition of the sample, the decrease in NADH concentration measured by spectrophotometry is proportional to the H2O2 concentration. Because of superoxide dismutation, a high amplification factor defined as moles NADH oxidised per mole H2O2 added is obtained, which allows the sensitivity limit of the method to be greatly improved. We have established the conditions under which the amplification factor can be stabilised at a high level: the best compromise is to increase both the horseradish peroxidase and superoxide dismutase concentrations. Finally, we have also shown that coupled to specific oxidases, our assay method is suitable for measuring very low concentrations of biochemicals that can be oxidized by oxygen with H2O2 production.

[Regulation and electric excitation of enzyme membranes in vitro]. / Régulation et excitation électriques de membranes d'enzymes in vitro.

Enzyme membranes can be activated or inhibited by applying continuous or alternating electrical fields. The field can modify the transport or reaction term of the transport-reaction by action on the displacement of charged species including those giving pH effects or inducing volume flows. A first experimental example is given: the progressive supression of the inhibition of hexokinase by the product when increasing alternating fields are applied. In the same way the apparent optimal pH approaches that of the soluble enzyme. In addition to its theoretical and practical implications electrical regulation can lead to the monitoring of enzyme reaction-driven mechanochemical fibers.

Bacteriological analysis of water by potentiometric measurement of lipoic acid reduction: preliminary assays for selective detection of indicator organisms.

The practical task of adapting an original potentiometric technique to the bacteriological analysis of water is discussed. Various laboratory strains of organisms belonging to the usual aquatic flora were inoculated one by one in a minimal lactose broth supplied with lipoic (thioctic) acid. The time evolution of the redox potential of the cultures was followed during incubation by combined gold versus reference electrodes. When the incubation temperature was regulated at 36 degrees C, most organisms were able to grow and to reduce the coenzyme, generating changes in the redox potential of the culture. However, very few organisms developed significant reductive activity when the temperature was increased to 41 degrees C and when the broth was provided with sodium deoxycholate. Among the fecal coliform organisms, only Escherichia coli and Klebsiella pneumoniae exhibited early but reproducible potential-time responses. Positive potentiometric responses were also recorded with Acinetobacter calcoaceticus. E. coli showed rapid potentiometric signals as compared with K. pneumoniae. The time required for 100-mV shift of potential to be detected was related to the logarithm of the initial concentration of E. coli or K. pneumoniae in the culture broth. Experiments on natural surface water samples showed the the potentiometric method, associated with the selective incubation conditions, mainly detected E. coli among the bacterial flora of the tested environmental water. The calibration curve relating the time required for a 100-mV shift of potential to be detected to the number of fecal coliforms, as determined by control fecal coliform-selective plate counts, was consistent with the composite standard curve of detection times obtained with six different laboratory strains of E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)

[Demonstration of hormonal actions on Escherichia coli K 12 by potentiometry in the presence of lipoic acid; correlation with measurements of microbial growth and glucose consumption]. / Mise en évidence d'actions hormonales sur Escherichia coli K 12 par potentiométrie en présence d'acide lipoïque; corrélation avec des mesures de croissance microbienne et de consommation de glucose.

The action of hormones on Escherichia coli K 12 was followed by potential-time measurements in the presence of lipoic acid (LA), photometric determinations of growth and glucose titrations. Bovine growth hormone (5 to 60 IU/1) increased O2 consumption. The (10 IU/1) insulin-facilitated transport of LA was stimulated by glucose. Adrenaline (0.5 to 15 mg/l) increased more glucose and O2 consumption than growth.