Transient state kinetics of the reactions of isobutyraldehyde with compounds I and II of horseradish peroxidase

Elementary reactions have been studied quantitatively in the complex overall process catalyzed by horseradish peroxidase whereby isobutyraldehyde and molecular oxygen react to form triplet state acetone and formic acid. The rate constant for the reaction of the enol form of isobutyraldehyde with compound I of peroxidase is (8 +/- 1) X 10(6) M-1 s-1 and with compound II (1.3 +/- 0.3) X 10(6) M-1 s-1. Neither the enolate anion nor the keto form is reactive. The reactivity of enols with peroxidase parallels that of unionized phenols and a common mechanism is proposed. The overall catalyzed reaction of isobutyraldehyde and oxygen consists of an initial burst followed by a steady state phase. The burst is caused by the following sequence: 1) an initial high yield of compound I is formed from reaction of native enzyme with the autoxidation product of isobutyraldehyde, a peracid and 2) compound I rapidly depletes the equilibrium pool of enol which is present. After this burst a steady state phase is observed in which the rate-limiting step is the conversion of the keto to the enol form of the aldehyde catalyzed by phosphate buffer. The rate constant for the keto form reacting with phosphate is (8.7 +/- 0.6) X 10(-5) M-1 s-1. All constants were measured in dilute aqueous ethanol at 35 degrees C, pH 7.4, and ionic strength 0.67 M. Both the initial burst of light and the steady state emission from triplet acetone can be observed with the naked eye. Since the magnitude of the burst is a measure of the equilibrium amount of enol, the keto-enol equilibrium constant is readily calculated and hence also the rate constant for conversion of enol to keto. The keto-enol equilibrium constant is unaffected by phosphate which therefore acts as a true catalyst.     

Pattern formation in an immobilized bienzyme system. A morphogenetic model.

Experimental and theoretical studies of a reaction-diffusion model of two immobilized enzymes participating in the cellular acid-base metabolism, namely glutaminase and urease, are presented. The system shows an unstable steady state at pH 6.0, where any perturbation will drive the system towards a more alkaline or more acidic pH, owing to the autocatalytic behaviour with respect to pH exhibited by both enzymes. When diffusion is coupled to reaction by means of immobilization, different patterns of the internal pH profile appear across the membrane. If the bienzymic membrane is subjected to a perturbation at its boundaries, of the same amplitude but in opposite directions, the internal pH evolves through an asymmetric pattern to attain a nearly symmetric distribution of pH. The pH value at the final steady state is more acidic or more alkaline than the initial state according to the initial and boundary conditions. The final nearly symmetric state is attained more rapidly when less enzyme is immobilized (1.8 x 10(-4) M.s-1 as against 3.3 x 10(-4) M.s-1 of total enzyme activity in the membrane volume). The experimental results agree rather well qualitatively with numerical predictions of the model equations.     

Dynamic behavior of the adaptive control system, hypothalamus--anterior pituitary--adrenal cortex. Animal experimental study of the adequate system dynamics of the direct and indirect parameters of the adaptive control system]

The dynamic response of the adaptive closed-loop control system hypothalamus-anterior pituitary-adrenal cortex after application of short-time disturb pulses were examined experimentally in the pig. Blood sampling was performed biologically non-reactive by chronically implanted vascular catheters in the upper vena cava. A circadian biphase periodical dynamics characterizes the distrubance response of the adaptive control system, respectively, its direct and indirect parameters. After distrubance variables induced initial excess the plasma corticosteriods undershoot the basic level over a long period. Conversely, the dynamics of the eosinophils is determined through an initial eosinopenic phase and a subsequent typical overshoot. Periodical dynamic is absent in the steady state. The controller of the adaptive system does not show any spontaneous activity in the steady state without stress. The absence of an endogenic periodicity of the dependent parameters likewise gives evidence of an active oscillator. On the other hand, the controlling unit of the adaptive system constitutes a control response of circadian periodicty after activation by disturbance variables in pulse shape. The high damping of the control system terminates the transient state within 24 hours, and the corticosteriods and the eosinophils reach the steady state level. This control quality of the adaptive control system gains a high importance in the origination of the spontaneous circadian periodicity by corticosteriods and eosinophils and for the biological compensation of exogenous distrubance variables.     

Contribution of the Juxtatransmembrane Intracellular Regions to the Time Course and Permeation of ATP-gated P2X7 Receptor Ion Channels

P2X7 receptors are ATP-gated ion channels that contribute to inflammation and cell death. They have the novel property of showing marked facilitation to repeated applications of agonist, and the intrinsic channel pore dilates to allow the passage of fluorescent dyes. A 60-s application of ATP to hP2X7 receptors expressed in Xenopus oocytes gave rise to a current that had a biphasic time course with initial and secondary slowly developing components. A second application of ATP evoked a response with a more rapid time to peak. This facilitation was reversed to initial levels following a 10-min agonist-free interval. A chimeric approach showed that replacement of the pre-TM1 amino-terminal region with the corresponding P2X2 receptor section (P2X7–2Nβ) gave responses that quickly reached a steady state and did not show facilitation. Subsequent point mutations of variant residues identified Asn-16 and Ser-23 as important contributors to the time course/facilitation. The P2X7 receptor is unique in having an intracellular carboxyl-terminal cysteine-rich region (Ccys). Deletion of this region removed the secondary slowly developing current, and, when expressed in HEK293 cells, ethidium bromide uptake was only ∼5% that of WT levels, indicating reduced large pore formation. Dye uptake was also reduced for the P2X7–2Nβ chimera. Surprisingly, combination of the chimera and the Ccys deletion (P2X7–2NβdelCcys) restored the current rise time and ethidium uptake to WT levels. These findings suggest that there is a coevolved interaction between the juxtatransmembrane amino and carboxyl termini in the regulation of P2X7 receptor gating.     

Bimolecular systems: I. Linear systems of complexes

A vast number of biologically important processes are based upon bimolecular systems. In these systems intermediate complexes are formed. Bimolecular systems in which no complex-complex interactions occur are called linear systems of complexes. A definition and some characteristic properties of these systems are given here. There may exist a contradiction of Onsager's principle of detailed balancing in these systems; however, no principal differences are found between the steady state behavior of an open system and that of a closed system. It is shown that the steady state behavior of a linear system of complexes of arbitrary complexity has some similarities with the steady state behavior of a simple bimolecular system, e.g., Michaelis-Menten enzymatic reaction. Multiplicity of action of the substances participating in biomolecular processes may produce some qualitative differences in the steady state behavior of the system.     

Current-Voltage Relations in the Lobster Giant Axon Membrane Under Voltage Clamp Conditions

The sucrose-gap method introduced by Stämpfli provides a means for the application of a voltage clamp to the lobster giant axon, which responds to a variety of different experimental procedures in ways quite similar to those reported for the squid axon and frog node. This is particularly true for the behavior of the peak initial current. However, the steady state current shows some differences. It has a variable slope conductance less than that of the peak initial current. The magnitude of the steady state slope conductance is related to the length of the repolarization phase of the action potential, which does not have an undershoot in the lobster. The steady state outward current is maintained for as long as 100 msec.; this is in contrast to a decline of about 50 per cent in the squid axon. Lowering the external calcium concentration produces shifts in the current-voltage relations qualitatively similar to those obtained from the squid axon. On the basis of the data available, there is no reason to doubt that the Hodgkin and Huxley analysis for the squid giant axon in sea water can be applied to the lobster giant axon.     

A xylazine infusion regimen to provide analgesia in sheep.

The efficacy of continuous low-dose xylazine infusion following an initial loading dose in providing analgesia in sheep was examined using an algesimetry method based on a leg lifting response to an electrical stimulus. Sheep received a 5 mg intramuscular injection of xylazine followed by continuous infusion of intravenous xylazine (2mg/h) for 90 min. This treatment resulted in significant increases in the level of current required to elicit a leg lifting response (287% of baseline) and steady state analgesia was maintained from 10 min after the start of the infusion until the end of the experimental period. This protocol appears to be a simple and effective regimen for providing steady state analgesia in sheep.     

Effects of serotonin on neuronal response induced in the sensorimotor cortex by tactile and conditioned acoustic stimuli

Neuronal firing response in the sensorimotor cortex to tactile (non-conditioned) and acoustic (conditioned) stimuli was investigated in trained cats before and after iontophoretic application of serotonin and lysergide. Three functionally distinct groups of neurons were identified from the response produced by presenting tactile and acoustic stimuli. Applying serotonin was found to facilitate preliminary and residual spike response induced by tactile stimulation; it also facilitates and modulates response in many cortical neurons to conditioned stimuli. Facilitation takes the form of reduced latency of response and increased numbers of spikes in response to conditioned stimulus presentation, especially at the initial phase of response to sound and immediately after the onset of conditioned reflex motion. Additional neurons formerly unresponsive to acoustic stimuli joined in the reaction under the effects of serotonin. Changed response patterns often evolve following minor fluctuations in background activity level. It is suggested that facilitation of response following iontophoretic serotonin application in the neocortex is associated with activation of excitatory serotonin receptors (S₂). The lysergide-induced increase in background and evoked activity noted during experimentation can apparently be put down to blockade of inhibitory serotonon (S_1B) receptors.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 3, pp. 337–347, May–June, 1990.     

Emergence of homochirality in far-from-equilibrium systems: mechanisms and role in prebiotic chemistry

Since the model proposed by Frank (Frank FC, Biochem Biophys Acta 1953;11:459-463), several alternative models have been developed to explain how an asymmetric non-racemic steady state can be reached by a chirally symmetric chemical reactive system. This paper explains how a stable non-racemic regime can be obtained as a symmetry breaking occurring in a far-from-equilibrium reactive system initiated with an initial imbalance. Departing from the variations around the original Frank's model that are commonly described in the literature, i.e. open-flow systems of direct autocatalytic reactions, we discuss recent developments emphasizing both an active recycling of components and an autocatalytic network of simple reactions. We will present our APED model as the most natural realization of such thermodynamic openness and non-equilibrium, of recycling and of network autocatalysis, each of these in prebiotic conditions. The different experimental and theoretical models in the literature will be classified according to mechanism. The place and role of such self-structured networks responsible for the presence of homochirality in the primitive Earth will be detailed.     

Enzyme reactions at the surface of living cells. II. Destabilization in the membranes and conduction of signals

The dynamics of a bound-enzyme reaction is studied when the diffusion of both the substrate and the product is coupled to their electric repulsion and to enzyme reaction. Contrary to what is occurring when substrate diffusion is uncoupled with electric repulsion and enzyme reaction, no hysteresis loop of the partition coefficient exists. The electric partition coefficient monotonically declines as substrate or product concentration is increased in the reservoir. The random perturbation of a steady state may generate a localized destabilization of substrate and product concentration. This destabilization must propagate in the membrane and may be viewed as the conduction of a signal. These conduction phenomena are entirely due to electric effects. In the absence of these effects, the system is homeostatic, that is it returns back to its initial steady state after a perturbation. Obviously under these conditions conduction of signals cannot occur. Increasing the ionic strength of the external milieu tends to stabilize the system and to suppress conduction effects in the membrane.     

Fast odor learning improves reliability of odor responses in the locust antennal lobe

Recordings in the locust antennal lobe (AL) reveal activity-dependent, stimulus-specific changes in projection neuron (PN) and local neuron response patterns over repeated odor trials. During the first few trials, PN response intensity decreases, while spike time precision increases, and coherent oscillations, absent at first, quickly emerge. We examined this "fast odor learning" with a realistic computational model of the AL. Activity-dependent facilitation of AL inhibitory synapses was sufficient to simulate physiological recordings of fast learning. In addition, in experiments with noisy inputs, a network including synaptic facilitation of both inhibition and excitation responded with reliable spatiotemporal patterns from trial to trial despite the noise. A network lacking fast plasticity, however, responded with patterns that varied across trials, reflecting the input variability. Thus, our study suggests that fast olfactory learning results from stimulus-specific, activity-dependent synaptic facilitation and may improve the signal-to-noise ratio for repeatedly encountered odor stimuli.     

Spike Triggered Hormone Secretion in Vasopressin Cells; a Model Investigation of Mechanism and Heterogeneous Population Function

Vasopressin neurons generate distinctive phasic patterned spike activity in response to elevated extracellular osmotic pressure. These spikes are generated in the cell body and are conducted down the axon to the axonal terminals where they trigger Ca²⁺ entry and subsequent exocytosis of hormone-containing vesicles and secretion of vasopressin. This mechanism is highly non-linear, subject to both frequency facilitation and fatigue, such that the rate of secretion depends on both the rate and patterning of the spike activity. Here we used computational modelling to investigate this relationship and how it shapes the overall response of the neuronal population. We generated a concise single compartment model of the secretion mechanism, fitted to experimentally observed profiles of facilitation and fatigue, and based on representations of the hypothesised underlying mechanisms. These mechanisms include spike broadening, Ca²⁺ channel inactivation, a Ca²⁺ sensitive K⁺ current, and releasable and reserve pools of vesicles. We coupled the secretion model to an existing integrate-and-fire based spiking model in order to study the secretion response to increasing synaptic input, and compared phasic and non-phasic spiking models to assess the functional value of the phasic spiking pattern. The secretory response of individual phasic cells is very non-linear, but the response of a heterogeneous population of phasic cells shows a much more linear response to increasing input, matching the linear response we observe experimentally, though in this respect, phasic cells have no apparent advantage over non-phasic cells. Another challenge for the cells is maintaining this linear response during chronic stimulation, and we show that the activity-dependent fatigue mechanism has a potentially useful function in helping to maintain secretion despite depletion of stores. Without this mechanism, secretion in response to a steady stimulus declines as the stored content declines.     

High extracellular concentrations of potassium and rubidium modulate the synaptic transmission in the frog labyrinth

High extracellular K or Rb levels (20 mM) produce an increase in the resting EPSP and spike frequencies recorded intra cellularly from single fibres of the posterior nerve in the isolated frog labyrinth. The afferent discharge facilitation proved to be inversely related to the fibre's initial resting activity. The K effect is systematically larger than the Rb effect. High sensitive and scarcely sensitive units may be identified with respect to K and Rb action. The present findings suggest that, according to previous models of hair cell functioning, the K and Rb effects are mediated by a raise in intracellular Ca concentration which sustains an increased transmitter release at the cyto-neural junction.     

A tale of two stories: astrocyte regulation of synaptic depression and facilitation

Short-term presynaptic plasticity designates variations of the amplitude of synaptic information transfer whereby the amount of neurotransmitter released upon presynaptic stimulation changes over seconds as a function of the neuronal firing activity. While a consensus has emerged that the resulting decrease (depression) and/or increase (facilitation) of the synapse strength are crucial to neuronal computations, their modes of expression in vivo remain unclear. Recent experimental studies have reported that glial cells, particularly astrocytes in the hippocampus, are able to modulate short-term plasticity but the mechanism of such a modulation is poorly understood. Here, we investigate the characteristics of short-term plasticity modulation by astrocytes using a biophysically realistic computational model. Mean-field analysis of the model, supported by intensive numerical simulations, unravels that astrocytes may mediate counterintuitive effects. Depending on the expressed presynaptic signaling pathways, astrocytes may globally inhibit or potentiate the synapse: the amount of released neurotransmitter in the presence of the astrocyte is transiently smaller or larger than in its absence. But this global effect usually coexists with the opposite local effect on paired pulses: with release-decreasing astrocytes most paired pulses become facilitated, namely the amount of neurotransmitter released upon spike i+1 is larger than that at spike i, while paired-pulse depression becomes prominent under release-increasing astrocytes. Moreover, we show that the frequency of astrocytic intracellular Ca(2+) oscillations controls the effects of the astrocyte on short-term synaptic plasticity. Our model explains several experimental observations yet unsolved, and uncovers astrocytic gliotransmission as a possible transient switch between short-term paired-pulse depression and facilitation. This possibility has deep implications on the processing of neuronal spikes and resulting information transfer at synapses.     

Hill coefficient for estimating the magnitude of cooperativity in gating transitions of voltage-dependent ion channels

A frequently used measure for the extent of cooperativity in ligand binding by an allosteric protein is the Hill coefficient, obtained by fitting data of initial reaction velocity (or fractional binding saturation) as a function of substrate concentration to the Hill equation. Here, it is demonstrated that the simple two-state Boltzmann equation that is widely used to fit voltage-activation data of voltage-dependent ion channels is analogous to the Hill equation. A general empiric definition for a Hill coefficient (n(H)) for channel gating transitions that is analogous to the logarithmic potential sensitivity function of Almers is derived. This definition provides a novel framework for interpreting the meaning of the Hill coefficient. In considering three particular and simple gating schemes for a voltage-activated cation channel, the relation of the Hill coefficient to the magnitude and nature of cooperative interactions along the reaction coordinate of channel gating is demonstrated. A possible functional explanation for the low value of the Hill coefficient for gating transitions of the Shaker voltage-activated K(+) channel is suggested. The analogy between the Hill coefficients for ligand binding and for channel gating transitions further points to a unified conceptual framework in analyzing enzymes and channels behavior.     

Numerical integration of a stochastic model for the Volterra-Lotka reaction

Time-integration of the master equation governing the birth-and-death model of the Volterra-Lotka reaction is carried out for three different initial conditions, with the results:

1. Fluctuations destroy the deterministic steady state in a manner quantitatively predicted from a cumulant expansion;
2. The sustained oscillatory behavior predicted by the deterministic model degenerated after 1/4 cycle in the stochastic model;
3. It is possible to select initial distributions such that the asymptotic distribution is a spike at the origin of the plane of reactants.

     

Calculation of diffusion-limited kinetics for the reactions in collision coupling and receptor cross-linking

Both enzyme (e.g., G-protein) activation via a collision coupling model and the formation of cross-linked receptors by a multivalent ligand involve reactions between two molecules diffusing in the plasma membrane. The diffusion of these molecules is thought to play a critical role in these two early signal transduction events. In reduced dimensions, however, diffusion is not an effective mixing mechanism; consequently, zones in which the concentration of particular molecules (e.g., enzymes, receptors) becomes depleted or enriched may form. To examine the formation of these depletion/ accumulation zones and their effect on reaction rates and ultimately the cellular response, Monte Carlo techniques are used to simulate the reaction and diffusion of molecules in the plasma membrane. The effective reaction rate at steady state is determined in terms of the physical properties of the tissue and ligand for both enzyme activation via collision coupling and the generation of cross-linked receptors. The diffusion-limited reaction rate constant is shown to scale with the mean square displacement of a receptor-ligand complex. The rate constants determined in the simulation are compared with other theoretical predictions as well as experimental data.     

Relationship between thermodynamic driving force and one-way fluxes in reversible processes

Chemical reaction systems operating in nonequilibrium open-system states arise in a great number of contexts, including the study of living organisms, in which chemical reactions, in general, are far from equilibrium. Here we introduce a theorem that relates forward and reverse fluxes and free energy for any chemical process operating in a steady state. This relationship, which is a generalization of equilibrium conditions to the case of a chemical process occurring in a nonequilibrium steady state in dilute solution, provides a novel equivalent definition for chemical reaction free energy. In addition, it is shown that previously unrelated theories introduced by Ussing and Hodgkin and Huxley for transport of ions across membranes, Hill for catalytic cycle fluxes, and Crooks for entropy production in microscopically reversible systems, are united in a common framework based on this relationship.