Electrical excitability of artificial enzyme membranes: II. Electrochemical and enzyme properties of immobilized acetylcholinesterase membranes

This paper deals with aspects of the reciprocal interaction between enzyme activity and the microenvironment or the potential difference in artificial proteinaceous membranes bearing cross-linked acetylcholinesterase. The potential difference resulting from asymmetric substrate injection into the system is recorded as a function of time. The influence of the membrane charge density on both enzyme activity and potential difference is studied by varying the external solution pH. The enzyme specific potential is initiated by local change of pH at the membrane level and the dependence on the buffer strength is studied. The recorded potential difference appears to be the result of the reciprocal interaction between enzyme reaction and the diffusion of substrate or products.     

Enzyme immobilization by the coulomb force

A new immobilization technique has been developed. It involves immobilizing enzymes on a porous polytetrafluoroethylene membrane with a nonporous polyurethane coat by the use of an electrostatic force, i.e. the Coulomb force. The immobilized enzyme can be recovered by supplying a reversed electrical potential. The reaction characteristics of the immobilized amyloglucosidase were studied using maltose as a substrate. The Michaelis constant becomes larger than that of the native enzyme, and depends on the electrical potential gradient in the solution.     

Electric field effects on immobilized urease activity.

The behavior of an enzyme/membrane system containing urease is studied when an external electric field is applied. The device using a potential difference across the enzyme/membrane system is first described. Optimal operating conditions with respect to substrate concentration, ionic strength and pH are studied. Possible mechanisms of the change in membrane activity by electric field are discussed.     

The kinetic behavior of an artificial bienzyme membrane.

Artificial membranes bearing immobilized enzymes can be used to study some effects of membrane structure on enzyme kinetic behavior. The bienzyme system described is a mixture of beta-glucosidase and glucose oxidase. Gluconolactone, the product of thesecond enzyme, is an inhibitor of the first one. The resulting feedback effect has been compared using a mixed two-enzyme membrane, two separated one-enzyme membranes, and astirred bienzyme solution. The feedback effect is quicker and more efficient in the two-enzyme membrane than in solution; it is slower and less efficient in the case of the separated one-enzyme membranes. Effects of enzyme proximity in the structure are discussed. Conclusions are drawn concerning the efficiency of feedback mechanisms when enzymes are embedded within a single structure.     

Membrane microreactor in biocatalytic transesterification of triolein for biodiesel production

Transesterification is a principal chemical reaction that occurs in biodiesel production. We developed a novel biocatalytic membrane microreactor (BMM) for continuous transesterification by utilizing an asymmetric membrane as an enzyme-carrier for immobilization. The BMM was developed by pressure driven filtration of lipase from Pseudomonas fluorescens, which is suitable for highly efficient biocatalytic transesterification. Lipase solution was allowed to permeate through an asymmetric membrane with NMWL 300 kDa composed of polyethersulfone. The performances of BMM were studied in biodiesel synthesis via transesterification of triolein with methanol. Transesterification was carried out by passing a solution of triolein and methanol through the asymmetric membrane. The degree of triolein conversion using this microreactor was ca. 80% with a reaction time of 19 min. The BMM system displayed good stability, with no activity decay over a period of 12 day with continuous operation. Results from triolein transesterification clearly demonstrate the potential of an asymmetric membrane as an enzyme carrier material. Enzyme activity (mmol/h·g_lipase) was approximately 3 fold higher than that of native free lipase.     

Effect of membrane voltage on the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae

A novel system for generating large interior positive membrane potentials in proteoliposomes was used to examine the effects of membrane voltage on reconstituted plasma membrane H(+)-ATPase from Saccharomyces cerevisiae. The membrane potential-generating system was dependent upon the lipophilic electron carrier tetracyanoquinodimethane, located within the bilayer, to mediate electron flow from vesicle entrapped ascorbate to external K3Fe(CN)6. Membrane potential formation was followed by the potential-dependent probe oxonol V and was found to rapidly reach a steady-state which lasted at least 90 s. A membrane potential of approximately 254 mV was determined under optimal conditions and ATP hydrolysis by wild-type H(+)-ATPase was inhibited from 34 to 46% under these conditions. In contrast, membrane potential had little effect on pma1-105 mutant enzyme suggesting that it is defective in electrogenic proton translocation. Applied membrane voltage was also found to alter the sensitivity of wild-type enzyme to vanadate at concentrations less than 50 microM. These data suggest a coupling between the charge-transfer and ATP hydrolysis domains and establish a solid basis for future probing of the electrogenic properties of the yeast H(+)-ATPase.     

Glucose sensing based on the intrinsic fluorescence of sol-gel immobilized yeast hexokinase

In this study, we investigated measurements of the intrinsic fluorescence of yeast hexokinase as an assay for glucose and immobilization of the enzyme in a silica sol-gel matrix as a potential in vivo glucose sensor for use in patients with diabetes. The intrinsic fluorescence of hexokinase in solution (excitation=295 nm, emission=330 nm) decreased by 23% at a saturating glucose concentration of 1 mM (Kd=0.3 mM), but serum abolished the glucose-related fluorescence response. When entrapped in tetramethylorthosilicate-derived sol gel, hexokinase retained activity, with a 25% maximal glucose-related decrease in intrinsic fluorescence, and the saturation point was increased to 50 mM glucose (Kd=12.5 mM). The glucose response range was increased further (to 120 mM, Kd=57 mM) by a covering membrane of poly(2-hydroxyethyl) methacrylate. Unlike free enzyme, the fluorescence responses to glucose with sol-gel immobilized hexokinase, with or without covering membrane, were similar for buffer and serum. We conclude that fluorescence monitoring of sol-gel entrapped yeast hexokinase is a suitable system for development as an in vivo glucose biosensor.     

Properties of microfiltration membranes: the effects of adsorption and shear on the recovery of an enzyme

An experimental study of the interaction of the enzyme yeast alcohol dehydrogenase (YADH) with microfiltration membranes has been carried out. Most measurements were made with capillary pore inorganic membranes (Anopore) with some comparative measurements being made with polymeric membranes of low protein affinity (Durapore). It has been shown that the prolonged exposure of the enzyme to the inorganic membrane under low-shear conditions (slow recycle) resulted in a loss of enzyme activity. Under filtration conditions, the membrane permeation rate decreased continuously with time. This decrease could be quantified using the standard blocking filtration law, which describes a decrease in pore volume due to deposition of enzyme on the walls of the pore. No significant loss in activity of permeating enzyme occurred under solution conditions where the enzyme was stable. However, a significant loss of such activity occurred under solution conditions where the enzyme was slightly unstable. The experiments indicate that the likely mechanism for activity loss is a membrane/enzyme interaction resulting from a shear induced deformation of the enzyme structure. Two conclusions of practical importance are drawn from the work. (c) 1992 John Wiley & Sons, Inc.     

Analytical solution of the Poisson-Boltzmann equation for membrane potential

Analytical solution of the one-dimensional Poisson-Boltzmann equation for membrane potential is obtained in an equilibrium state of the Nemst-Planck Poisson system. Approximations, e.g. constant field or Debye-Hückel approximation, need not be used. Two types of solution, arctangenthyperbolic and arccotangenthyperbolic, exist for every value of membrane potential.A new approximate solution is obtained where V and V_m are electric potential inside the membrane and membrane potential respectively; R, T and F have their usual thermodynamic meanings, κ is Debye constant, τ the membrane thickness. This approximate solution fits the numerical solution by Runge-Kutta method (maximum error being less than 5 × 10^?4) around the potential being 50 mV. The fitness is considerably more accurate than that of Debye-Huckel approximation (error being c. 15 % around the potential 50 mV.).     

The function and activity of certain membrane enzymes when localized on – and off – the membrane

A group of enzymes known to be involved in group translocation-type transport mechanisms for the uptake of a variety of nucleotide precursors are enzymatically active both in their natural membrane milieu and in aqueous solution. The activity in aqueous solution markedly differ, however, from the enzymatic activity when the enzyme is membrane localized. The adenine phosphoribosyltransferase (PRT) of E. coli (Hochstadt-Ozer and Stadtman, 1971 a) is capable of carrying out an exchange reaction between the base moieties of adenine and AMP without requiring P-ribose-PP as an intermediate; the enzyme in aqueous solution requires P-ribose-PP, indicating a different reaction mechanism in the two environments. Like the adenine PRT of E. coli, the hypo-xanthine PRT of Salmonella typhimurium (Jackman and Hochstadt, 1976) also carried out an exchange reaction on the membrane only and also is more sensitive to a number of inhibitors in aqueous solution relative to the sensitivity when embedded in the membrane. In addition, however, the hypoxanthine PRT, while restricted to hypoxanthine as a substrate in the membrane, also accepts guanine as substrate in its soluble form. The membrane capacities reflect the in situ capacities of the enzyme and the gain of guanine specificity was determined in a guanine PRT deletion strain (Jackman and Hochstadt, 1976). Finally, in mammalian cell lines purine nucleoside phosphorylase, which translocates the ribose moiety of inosine across the plasma membrane of mouse fibroblasts undergoes a 30-fold increase in substrate turnover number upon liberation from the membrane. These data raise two important caveats with respect to study of membrane enzymes and transport. Firstly, an enzyme once solubilized and found to differ kinetically from substrate transport in situ cannot be excluded from participating in translocations in the membrane on the basis of its activity in aqueous solution. Secondly, an enzyme which “appears” largely soluble upon cell rupture cannot be assumed to be a cycloplasmic enzyme because the majority of the solubilized activity may represent only a small fraction of the enzyme molecules highly activated concomitant to their solubilization. In this latter case the ability to activate enzyme still residing on the membrane (e.g., with detergents) would be necessary in order to estimate total membrane associated activity after cell rupture.     

Theoretical and experimental analysis of a soluble enzyme membrane reactor.

Recently enzyme immobilization techniques have been proposed that are mainly founded on the formation of an enzyme-gel layer onto the active surface of an ultrafiltration membrane within an unstirred ultrafiltration cell. If the membrane molecular-weight cutoff is less than the enzyme molecular weight and hence such as to completely prevent enzyme permeation (once the enzyme solution has been charged into the test cell and pressure applied to the system), a time progressive increase in enzyme concentration takes place at the upstream membrane surface that can eventually lead to gelation and hence to enzyme immobilization. However, depending on the total enzyme amount fed, the maximum enzyme concentration achieved in the unsteady state could be less than the gelation level. In this situation, no immobilization occurs and the enzyme still remains in the soluble form although it is practically confined within a limited region immediately upstream the membrane and at fairly high concentrations. In this paper, the experimental conditions that allow gelling to occur are discussed together with a theoretical analysis of the soluble enzyme membrane reactor which is obtained when no gelling takes place. Such a system could be usefully employed in performing kinetic analyses at high enzyme concentration levels that are still in the soluble form.     

Electrochemical method for continuous determination of enzymatic acetylocholine hydrolysis

An electrochemical method was elaborated for the continuous determination of enzymatic hydrolysis of acetylcholine. In the electrochemical system applied the aqueous solution of the enzyme is separated from the aqueous solutions of substrates by a semipermeable membrane. In this way cholinesterase is used many times for reactions. Changes in the concentration of hydrogen ions were determined with molybdenum electrodes, one of which was used as an indicator and immersed in enzyme solution and the other served for comparison and was immersed in the solution of acetylcholine flowing to the measuring system.     

Effect of the medium composition on the resting membrane potential in the earthworm longitudinal somatic muscle fibers

Norepinephrine or increased extracellular K+ hyperpolarize the membrane of the earthworm somatic muscle fibre, whereas removal of Cl- from external solution or a hypotonic solution depolarize the membrane. The dependence of the membrane resting potential on the extracellular K+ is quite characteristic against the background of ouabain action. A preliminary membrane depolarisation by ouabain eliminates the above effects on the membrane resting potential. The data obtained suggest that the ouabain-sensitive active ion pump directly contributes to the membrane resting potential value. This hypothesis is discussed with respect to existence of active Cl- transport combined with Na+, K(+)-pump which presumably takes part in the intracellular osmotic pressure regulation in the earthworm somatic muscle.     

Effect of enzyme organization on the stability of Yates-Pardee pathways

Yates-Pardee-type metabolic pathways in a heterogenous cell milieu are modeled as a system of coupled non-linear partial differential equations. A numerical solution to this systmm is described and some properties of such a physiological system are studied. Confinement with and without a membrane is considered and it is shown how confinement results in an increase in the stability of the metabolite concentrations. These results suggest that the enzyme organization may contribute to the stability of the cellular metabolism.     

A quantitative electrochemical theory of the electrolyte permeability of mosaic membranes composed of selectively anion-permeable and selectively cation-permeable parts,and its experimental verification. II. A quantitative test of the theory in model systems which do not involve the use of auxiliary electrodes

The theory of the electrolyte permeability of mosaic membranes composed of ideally anion-selective and ideally cation-selective parts in juxtaposition is tested in a model which consists of an all-electrolytic cyclic arrangement of four component parts: dilute solution/anion-selective membrane/concentrated solution/cation-selective membrane/dilute solution. In this system cations move from the concentrated to the dilute solution across the cation-selective membrane and an equivalent number of anions move through the anion-selective membrane. This movement of ions corresponds to a flow of current in the system. According to the theory, the number of equivalents of electrolyte which penetrate in any given time across the membranes must be identical with the number of faradays of electricity which flow during the same period. The system is essentially a combination of two menbrane-concentration cells arranged in series in a short-circuited state without the presence of electrodes. Experimentally the magnitude of the current was determined by measuring with probe electrodes the potential across an element of the circuit whose resistance was known and constant. The number of faradays of electricity (determined from time-current data) flowing in the system during a measured time was compared with the analytically determined number of equivalents of electrolyte which moved across the membranes during the same period. In a variety of experimental systems the two values show a 1:1 ratio with a mean deviation of ± 1.8 per cent.     

Mass transfer of CO2 across membranes: Facilitation in the presence of bicarbonate ion and the enzyme carbonic anhydrase

A theoretical and experimental analysis of facilitated transport of CO₂ across membranes containing NaHCO₃ and the enzyme carbonic anhydrase (carbonate hydro-lyase EC 4.2.1.1) is presented. The necessary diffusion reaction equations are derived and the system constraints defined. For the CO₂HCO₃^? system, mathematical simplifications based on the magnitude of various reaction and concentration terms are made to make the equations tractable to solution. The resultant equations are solved by a number of analytical and numerical techniques, each having a limited, though useful, range of validity.The experimental arrangement consists of a liquid membrane (created by soaking a porous filter paper in the test solution), a diffusion chamber, and gas metering and analysis equipment. Conditions were selected to cover the range from diffusion- to reaction-dominated behavior.The flux of CO₂ across a membrane containing 1 M NaHCO₃ was measured at various partial pressures of CO₂ (2–28 in Hg) and with membrane thicknesses of 0.02, 0.06 and 0.10 cm. The extent of facilitation (defined as the ratio of reaction-related flux to the expected Fick's Law flux in the absence of reaction) ranged from near zero to nearly 5 in these experiments. The agreement between model calculations and experimental observation was found to be excellent over the entire range of near-diffusion to near-equilibrium behavior.In the presence of enzyme carbonic anhydrase (0.10 mg/ml, activity approx. 80%) and 1 M NaHCO₃, the CO₂ flux across a 0.02 cm membrane was 3–10-fold higher than the corresponding flux in the absence of enzyme. From experiments at various enzyme concentrations and membrane thicknesses, it appeared that the apparent CO₂ reaction rate was directly proportional to the enzyme concentration. The model calculations for the enzyme-catalyzed reactions agreed with the experimental observations to within $\text{±10\%}$.     

Note on the validity of constant field assumption in relation to the exact solution of Nernst--Planck equations

By means of the exact solution of the Nernst-Planck equations, it can be demonstrated that the constant electric field assumption is incompatible with the stationary flows of ions in the homogeneously charged membrane. The exact solution of the Nernst-Planck equation system is readily given by the power series expansion method. The constant field assumption is valid for membrane in which the fixed charge can move freely and neutralize the charge of permeating ions.The parabolical electric potential is shown to be incompatible with the homogeneously charged membranes including the neutral membranes.     

Evidence for the Functional Association of Enzyme I and HPr of the Phosphoenolpyruvate-Sugar Phosphotransferase System With the Membrane in Sealed Vesicles of Escherichia coli

Several independent assay procedures were used to estimate the activities of the enzyme constituents of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) in osmotically shocked bacterial membrane vesicles. The soluble enzymes of the system were found to be in association with the membrane by several criteria. Phosphoenolpyruvate-dependent sugar phosphorylation was catalyzed by this membrane-bound enzyme system far more efficiently than by a mixture of the individual enzymes at corresponding concentrations. By contrast, the rates of the phosphoryl exchange reactions catalyzed by enzyme I and the enzyme II complexes were essentially the same for the associated and dissociated forms of the system. Functional association of the PTS-enzyme complex was stabilized by Mg⁺⁺ and phosphoenolpyruvate and could be destroyed by detergent treatment, sonication, or by passage of the vesicle preparation through a French pressure cell. These results lead to the possibility that in the intact bacterial cell the soluble enzymes of the phosphotransferase system exist, in part, as peripheral membrane constituents associated with the integral membrane enzyme II complexes.     

Brownian dynamics simulation of the lateral distribution of charged membrane components

Brownian dynamics simulations were performed to study the contribution of electric interactions between charged membrane components to their lateral distribution in a two-dimensional viscous liquid (bilayer lipid membrane). The electrostatic interaction potential was derived from an analytical solution of the linearized Poisson-Boltzmann equation for point charges in an electrolyte solution — membrane — electrolyte solution system. Equilibrium as well as dynamic quantities were investigated. The lateral organization of membrane particles, modelled by mobile cylinders in a homogeneous membrane separating two electrolyte solutions was described by spatial distribution functions, diffusion coefficients and cluster statistics. Disorder, local order and crystal-like arrangements were observed as a function of the particle charge, the closest possible distances between the charges and the particle density. The simulations revealed that the system is very sensitive to the position of the charges with respect to the electrolyte solution — membrane interface. Electrostatic interactions of charges placed directly on the membrane surface were almost negligible, whereas deeper charges demonstrated pronounced interaction. Biologically relevant parameters corresponded at most to local and transient ordering. It was found that lateral electric forces can give rise to a preferred formation of clusters with an even number of constituents provided that the closest possible charge-charge distances are small. It is concluded that lateral electrostatic interactions can account for local particle aggregations, but their impact on the global arrangement and movement of membrane components is limited. Correspondence to: D. Walther     

Analysis of the effect of medium and membrane conductance on the amplitude and kinetics of membrane potentials induced by externally applied electric fields.

The kinetics and amplitudes of membrane potential induced by externally applied electric field pulses are determined for a spherical lipid bilayer using a voltage-sensitive dye. Several experimental parameters were systematically varied. These included the incorporation of gramicidin into the membrane to alter its conductivity and the variation of the external electrolyte conductivity via changes in salt concentration. The ability of the solution to Laplace's equation for a spherical dielectric shell to quantitatively describe the membrane potential induced on a lipid bilayer could thus be critically evaluated. Both the amplitude and the kinetics of the induced potential were consistent with the predictions of this simple model, even at the extremes of membrane conductance or electrolyte concentration. The success of the experimental approach for this system encourages its application to more complex problems such as electroporation and the influences of external electric fields in growth and development.