Resonance transduction of low level periodic signals by an enzyme: an oscillatory activation barrier model.

The overall rate of an enzyme catalyzed reaction is determined by the activation barrier of a rate-limiting step. If the barrier is oscillatory due to the intrinsic properties of a fluctuating enzyme, this enzymatic reaction will be influenced by a low level periodic electric field through the resonance transduction between the applied field and the oscillatory activation barrier. The ATP hydrolysis activity of a highly purified, detergent solubilized Ecto-ATPase from chicken oviduct was used to test the above concept. At 37 degrees C, this activity (1,800 mumols mg-1 min-1) was stimulated up to 47% (to 2,650 mumols mg-1 min-1) by an alternating electric field (AC), with a frequency window at 10 kHz. The maximal stimulation occurred at 5.0 V (peak-to-peak) cm-1. The potential drop across the dimension of the enzyme was approximately 10 microV (micelle diameter 20 nm). The activation barrier, or the Arrhenius activation energy, of the ATP splitting was measured to be 30 kT and the maximal barrier oscillation was calculated to be approximately 2.5 kT according to the oscillatory activation barrier (OAB) model. With the optimal AC field, full impact of the electric stimulation could be effected in much less than a second. The OAB model is many orders of magnitude more sensitive for deciphering low level periodic signals than the electroconformational coupling (ECC) model, although the latter has the ability to actively transduce energy while the former does not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nanopore-facilitated, voltage-driven phosphatidylserine translocation in lipid bilayers--in cells and in silico

Nanosecond, megavolt-per-meter pulses--higher power but lower total energy than the electroporative pulses used to introduce normally excluded material into biological cells--produce large intracellular electric fields without destructively charging the plasma membrane. Nanoelectropulse perturbation of mammalian cells causes translocation of phosphatidylserine (PS) to the outer face of the cell, intracellular calcium release, and in some cell types a subsequent progression to apoptosis. Experimental observations and molecular dynamics (MD) simulations of membranes in pulsed electric fields presented here support the hypothesis that nanoelectropulse-induced PS externalization is driven by the electric potential that appears across the lipid bilayer during a pulse and is facilitated by the poration of the membrane that occurs even during pulses as brief as 3 ns. MD simulations of phospholipid bilayers in supraphysiological electric fields show a tight association between PS externalization and membrane pore formation on a nanosecond time scale that is consistent with experimental evidence for electropermeabilization and anode-directed PS translocation after nanosecond electric pulse exposure, suggesting a molecular mechanism for nanoelectroporation and nanosecond PS externalization: electrophoretic migration of the negatively charged PS head group along the surface of nanometer-diameter electropores initiated by field-driven alignment of water dipoles at the membrane interface.     

Life Cycle of an Electropore: Field-Dependent and Field-Independent Steps in Pore Creation and Annihilation

Electropermeabilization, an electric field-induced modification of the barrier functions of the cell membrane, is widely used in laboratories and increasingly in the clinic; but the mechanisms and physical structures associated with the electromanipulation of membrane permeability have not been definitively characterized. Indirect experimental observations of electrical conductance and small molecule transport as well as molecular dynamics simulations have led to models in which hydrophilic pores form in phospholipid bilayers with increased probability in the presence of an electric field. Presently available methods do not permit the direct, nanoscale examination of electroporated membranes that would confirm the existence of these structures. To facilitate the reconciliation of poration models with the observed properties of electropermeabilized lipid bilayers and cell membranes, we propose a scheme for characterizing the stages of electropore formation and resealing. This electropore life cycle, based on molecular dynamics simulations of phospholipid bilayers, defines a sequence of discrete steps in the electric field-driven restructuring of the membrane that leads to the formation of a head group-lined, aqueous pore and then, after the field is removed, to the dismantling of the pore and reassembly of the intact bilayer. Utilizing this scheme we can systematically analyze the interactions between the electric field and the bilayer components involved in pore initiation, construction and resealing. We find that the pore creation time depends strongly on the electric field gradient across the membrane interface and that the pore annihilation time is at least weakly dependent on the magnitude of the pore-initiating electric field and, in general, much longer than the pore creation time.     

The possibility for improvement of ceramic membrane ultrafiltration of an enzyme solution

Ultrafiltration represents an attractive downstream processing technique for enzymes concentration and their primary purification. However, the process efficiency is often limited by protein fouling and shear-induced enzyme deactivation, resulting in permeate flux decline and loss of enzyme activity. The objective of this work was to investigate the possibility for improvement of ceramic membrane ultrafiltration of endo-pectinase solution. Experimental investigations were performed on a 5 nm ceramic membrane with the Kenics static mixer placed inside the membrane in order to improve the process performance. The use of the static mixer resulted in the flux improvement of about 45% at a volume concentration factor (VCF) of 3 leading to the reduction of operation time of 25% and the energy saving of about 40%. Although the rejection of endo-pectinase was higher than 96%, the extensive loss of the enzyme activity during operation indicated that the modification of the feed solution is essential for improved ultrafiltration performance. Addition of pectin to the original endo-pectinase solution led to a significant reduction of the enzyme deactivation: the enzyme activity yield was 90% at a VCF of 1.6 during operation with the static mixer.     

Kinetic theory of the positive column of a low-pressure discharge in a transverse magnetic field

The influence of a transverse magnetic field on the characteristics of the positive column of a planar low-pressure discharge is studied theoretically. The motion of magnetized electrons is described in the framework of a continuous-medium model, while the ion motion in the ambipolar electric field is described by means of a kinetic equation. Using mathematical transformations, the problem is reduced to a secondorder ordinary differential equation, from which the spatial distribution of the potential is found in an analytic form. The spatial distributions of the plasma density, mean plasma velocity, and electric potential are calculated, the ion velocity distribution function at the plasma boundary is found, and the electron energy as a function of the magnetic field is determined. It is shown that, as the magnetic field rises, the electron energy increases, the distributions of the plasma density and mean plasma velocity become asymmetric, the maximum of the plasma density is displaced in the direction of the Ampère force, and the ion flux in this direction becomes substantially larger than the counter-directed ion flux.     

On the efficiency and reversibility of active ligand transport induced by alternating rectangular electric pulses.

The stationary-state kinetic properties of a simplified two-state electro-conformational coupling model (ECC) in the presence of alternating rectangular electric potential pulses are derived analytically. Analytic expressions for the transport flux, the rate of electric energy dissipation, and the efficiency of the transducing system are obtained as a function of the amplitude and frequency of the oscillation. These formulas clarify some fundamental concept of the ECC model and are directly applicable to the interpretation and design of experiments. Based on these formulas, the reversibility and the degree of coupling of the system can be studied quantitatively. It is found that the oscillation-induced free energy transduction is reversible and tight-coupled only when the amplitude of the oscillating electric field is infinitely large. In general, the coupling is not tight when the amplitude of the electric field is finite. Furthermore, depending on the kinetic parameters of the model, there may exist a "critical" electric field amplitude, below which free energy transduction is not reversible. That is, energy may be transduced from the electric to the chemical, but not from the chemical to the electric.     

Integration of in vivo and in silico metabolic fluxes for improvement of recombinant protein production

The filamentous fungus Aspergillus niger is an efficient host for the recombinant production of the glycosylated enzyme fructofuranosidase, a biocatalyst of commercial interest for the synthesis of pre-biotic sugars. In batch culture on a minimal glucose medium, the recombinant strain A. niger SKAn1015, expressing the fructofuranosidase encoding suc1 gene secreted 45U/mL of the target enzyme, whereas the parent wild type SKANip8 did not exhibit production. The production of the recombinant enzyme induced a significant change of in vivo fluxes in central carbon metabolism, as assessed by (13)C metabolic flux ratio analysis. Most notably, the flux redistribution enabled an elevated supply of NADPH via activation of the cytosolic pentose phosphate pathway (PPP) and mitochondrial malic enzyme, whereas the flux through energy generating TCA cycle was reduced. In addition, the overall possible flux space of fructofuranosidase producing A. niger was investigated in silico by elementary flux mode analysis. This provided theoretical flux distributions for multiple scenarios with differing production capacities. Subsequently, the measured flux changes linked to improved production performance were projected into the in silico flux space. This provided a quantitative evaluation of the achieved optimization and a priority ranked target list for further strain engineering. Interestingly, the metabolism was shifted largely towards the optimum flux pattern by sole expression of the recombinant enzyme, which seems an inherent attractive property of A. niger. Selected fluxes, however, changed contrary to the predicted optimum and thus revealed novel targets-including reactions linked to NADPH metabolism and gluconate formation.     

Relation of growth process to spatial patterns of electric potential and enzyme activity in bean roots

The electric spatial pattern and invertase activity distribution in growing roots of azuki bean (Phaseolus chrysanthos) have been studied. The electric potential near the surface along the root showed a banding pattern with a spatial period of about 2 cm. It was found that the enzyme activity has a peak around 3-7 mm from the root tip, in good agreement with the position of the first peak of the electric potential, which is located a little behind the elongation zone. An inhomogeneous distribution of ATP content was also detected along the root. Experiments on the electric isolation of the elongation zone from the mature zone and acidification treatment showed that H+ is transported from the mature-side to elongation-side regions, causing tip elongation through an acid-growth mechanism. Both acidification and electric disturbance on growing roots affected growth significantly. Simultaneous measurements of electric potential and enzyme activity clearly showed a good correlation between these two quantities and growth speed. From an analogy with the Characean banding, the spatio-temporal organization via the cell membrane in electric potential and enzyme activity can be regarded as a dissipative structure arising far from equilibrium. These experimental results can be interpreted with a new mechanism that the dissipative structure is formed spontaneously along the whole root, accompanied by energy metabolism, to make H+ flow into the root tip.     

The study of metabolite-separation using an electromicrofiltration appratus

This study was performed to investigate the recovery of dissolved metabolic constituents produced during fermentation, using an electromicrofiltration apparatus. Filtration experiments were performed with crude and with permeate from electromicrofiltration and microfiltration. The results of each method were compared with a focus on measuring the enhancement of the filtrate flux with the application of an electric field. Hydraulic electrofiltration was found to significantly improve filtration and serves as an interesting alternative to cross-flow filtration for the separation of advantagenous constituents, such as amino acids and biopolymers from the fermentation broth.     

A Material Perspective of Rechargeable Metallic Lithium Anodes

Rechargeable lithium‐based batteries are long considered as the most promising candidates for application in various electronic devices, electric vehicles, and even electrical grids owing to their ultrahigh energy densities. However, to date, metallic lithium‐based batteries are still far from practical applications due to the low Coulombic efficiency and fast capacity decay of lithium anodes. The poor electrochemical performances of metallic lithium anodes are inherently related to random growth of lithium dendrites and infinite volume charge of lithium anodes. In this review, the failure mechanisms of metallic lithium anodes are summarized and ascribed to the unstable and inhomogeneous solid electrolyte interphase, uneven distributions of electric field, and lithium‐ion flux during the lithium plating processes. Correspondingly, efficient strategies for mitigating these problems, including surficial engineering, electric field, and lithium‐ion flux regulation are discussed from the perspective of anode materials. Finally, an outlook is proposed for the design and fabrication of next‐generation rechargeable metallic lithium anodes that aims to address the intrinsic problems of metallic lithium anodes.     

Energetics of an rf SQUID Coupled to Two Thermal Reservoirs

We study energetics of a Josephson tunnel junction connecting a superconducting loop pierced by an external magnetic flux (an rf SQUID) and coupled to two independent thermal reservoirs of different temperature. In the framework of the theory of quantum dissipative systems, we analyze energy currents in stationary states. The stationary energy flow can be periodically modulated by the external magnetic flux exemplifying the rf SQUID as a quantum heat interferometer. We also consider the transient regime and identify three distinct regimes: monotonic decay, damped oscillations and pulse-type behavior of energy currents. The first two regimes can be controlled by the external magnetic flux while the last regime is robust against its variation.     

The rationalization of high enzyme concentration in metabolic pathways such as glycolysis

The cellular concentration of enzymes of some major metabolic pathways, such as glycolysis, can approach millimolarity. This concentration of enzyme can catalyze in vitro rates which are 100-fold higher than maximum pathway flux. In an attempt to understand the need for such high enzyme concentration, an artificial metabolic pathway of five enzymes (apropos the central enzymes of glycolysis) has been modeled. Numerical methods were then used to determine the effect of enzyme concentration on: (1) the change in total free metabolite concentration as the pathway changes from low flux to high flux, (2) the time lag (transient time) in the rate of final product formation upon the transition from low flux to high flux. Both the changes in metabolite pool size and the transient time decreased with increased enzyme concentrations. When all enzymatic reactions were assigned Keq of unity, a concentration for each enzyme of 25 microM is sufficient to provide a transient time of 1 sec. When Keq different from unity are introduced, more enzyme is required to provide comparably short transient times. Under the latter condition, a pathway of sufficiently low transient time would require all the enzyme available in mammalian muscle. It is shown that there is little scope for further increases in either enzyme concentration or of catalytic efficiency of independent enzymes. Therefore, an alternative method of increasing efficiency is considered in which enzyme-bound metabolites can serve directly as substrates for subsequent enzymes in a metabolic pathway.     

Lactate biosensor and energy supply for the enzyme molecule

A biosensor of lactate has been constructed, made, and tested. The lactate biosensor uses the lactate dehydrogenase molecules from muscle. The lactate biosensor works according to the simplest scheme. An immobilized lactate dehydrogenase molecule binds a L-lactate molecule in the absence of the coenzyme NAD+. Then the L-lactate molecule is oxidized by the electric field of a metal electrode of the biosensor to generate an electron. The transfer of this electron between the immobilized lactate dehydrogenase molecule and the metal electrode of the biosensor is carried out without a redox mediator molecule. A new mechanism for the energy supply of the enzyme molecule is proposed to explain this effect. The new mechanism is based on the electric dipole-dipole interactions occurring in the enzyme molecule and surrounding water and on the thermal energy of this water.     

An enzymological approach to mitochondrial energy transduction

An approach to the problem of mitochondrial energy transduction is outlined. The approach is based on the fundamental assumption that there is an intimate relation between the mechanisms of enzyme catalysis and energy transduction. The implications of this assumption for the coupling of two chemical reactions and the coupling of a chemical reaction to an ion flux are discussed.     

Mathematical modelling of metabolic pathways affected by an enzyme deficiency. A mathematical model of glycolysis in normal and pyruvate-kinase-deficient red blood cells

A mathematical model of glycolysis in human erythrocytes is proposed to study the influence of a pyruvate kinase deficiency on the energy metabolism. The model takes into account the main regulatory properties of the non-equilibrium enzymes and the magnesium-complex formation by the adenine nucleotides and by 2,3-bisphosphoglycerate. In the normal case (no enzyme defect) the calculated flux rates and metabolite concentrations are in a good agreement with experimental data. It is shown that a severe pyruvate kinase deficiency manifested in a tenfold diminished activity of that enzyme leads to a remarkable decrease of the glycolytic flux and the ATP concentration of about 50% of the normal values. On the other hand a lowering of the pyruvate kinase activity to half of the normal value, characteristic for the heterozygotes, gives no significant alterations of the metabolite concentrations and the flux rates compared with the normal case which is in accordance with the lack of clinical symptoms for a metabolic disease of these probands. For three patients with known alterations of their pyruvate kinase mutants the calculated metabolite concentrations and the control characteristics permit estimation of the degree of disorder of the glycolytic pathway. The resulting classification corresponds well to other independent experimental and clinical findings. In particular, the calculation demonstrates that there is no simple correlation between the lowered enzyme activity and the reduced flux rate through the affected pathway.     

Metabolic flux control analysis of branch points: an improved approach to obtain flux control coefficients from large perturbation data

An overview of published approaches for the metabolic flux control analysis of branch points revealed that often not all fundamental constraints on the flux control coefficients have been taken into account. This has led to contradictory statements in literature on the minimum number of large perturbation experiments required to estimate the complete set of flux control coefficients C(J) for a metabolic branch point. An improved calculation procedure, based on approximate Lin-log reaction kinetics, is proposed, providing explicit analytical solutions of steady state fluxes and metabolite concentrations as a function of large changes in enzyme levels. The obtained solutions allow direct calculation of elasticity ratios from experimental data and subsequently all C(J)-values from the unique relation between elasticity ratio's and flux control coefficients. This procedure ensures that the obtained C(J)-values satisfy all fundamental constraints. From these it follows that for a three enzyme branch point only one characterised or two uncharacterised large flux perturbations are sufficient to obtain all C(J)- values. The improved calculation procedure is illustrated with four experimental cases.     

Resonance electroconformational coupling: A proposed mechanism for energy and signal transductions by membrane proteins

Recent experiments show that membrane ATPases are capable of absorbing free energy from an applied oscillating electric field and converting it to chemical bond energy of ATP or chemical potential energy of concentration gradients. Presumably these enzymes would also respond to endogenous transmembrane electric fields of similar intensity and waveform. A mechanism is proposed in which energy coupling is achieved via Coulombic interaction of an electric field and the conformational equilibria of an ATPase. Analysis indicates that only an oscillating or fluctuating electric field can be used by an enzyme to drive a chemical reaction away from equilibrium.In vivo, the stationary transmembrane potential of a cell must be modulated to become locally oscillatory if it is to derive energy and signal transduction processes.     

Identification and biochemical characterization of two novel UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronic acid 2-epimerases from respiratory pathogens

For a long period lactate was considered as a dead-end product of glycolysis in many cells and its accumulation correlated with acidosis and cellular and tissue damage. At present, the role of lactate in several physiological processes has been investigated based on its properties as an energy source, a signalling molecule and as essential for tissue repair. It is noteworthy that lactate accumulation alters glycolytic flux independently from medium acidification, thereby this compound can regulate glucose metabolism within cells. PFK (6-phosphofructo-1-kinase) is the key regulatory glycolytic enzyme which is regulated by diverse molecules and signals. PFK activity is directly correlated with cellular glucose consumption. The present study shows the property of lactate to down-regulate PFK activity in a specific manner which is not dependent on acidification of the medium. Lactate reduces the affinity of the enzyme for its substrates, ATP and fructose 6-phosphate, as well as reducing the affinity for ATP at its allosteric inhibitory site at the enzyme. Moreover, we demonstrated that lactate inhibits PFK favouring the dissociation of enzyme active tetramers into less active dimers. This effect can be prevented by tetramer-stabilizing conditions such as the presence of fructose 2,6-bisphosphate, the binding of PFK to f-actin and phosphorylation of the enzyme by protein kinase A. In conclusion, our results support evidence that lactate regulates the glycolytic flux through modulating PFK due to its effects on the enzyme quaternary structure.     

Factors affecting the activity of citrate synthase of Acetobacter xylinum and its possible regulatory role.

The citrate synthase activity of Acetobacter xylinum cells grown on glucose was the same as of cells grown on intermediates of the tricarboxylic acid cycle. The activity of citrate synthase in extracts is compatible with the overall rate of acetate oxidation in vivo. The enzyme was purified 47-fold from sonic extracts and its molecular weight was determined to be 280000 by gel filtration. It has an optimum activity at pH 8.4. Reaction rates with the purified enzyme were hyperbolic functions of both acetyl-CoA and oxaloacetate. The Km for acetyl-CoA is 18 mum and that for oxaloacetate 8.7 mum. The enzyme is inhibited by ATP according to classical kinetic patterns. This inhibition is competitive with respect to acetyl-CoA (Ki = 0.9 mM) and non-competitive with respect to oxaloacetate. It is not affected by changes in pH and ionic strength and is not relieved by an excess of Mg2+ ions. Unlike other Gram-negative bacteria, the A. xylinum enzyme is not inhibited by NADH, but is inhibited by high concentrations of NADPH. The activity of the enzyme varies with energy charge in a manner consistent with its role in energy metabolism. It is suggested that the flux through the tricarboxylic acid cycle in A. xylinum is regulated by modulation of citrate synthase activity in response to the energy state of the cells.