scholarly journals Kinetic model of the inositol trisphosphate receptor that shows both steady-state and quantal patterns of Ca2+ release from intracellular stores

2003 ◽  
Vol 370 (2) ◽  
pp. 621-629 ◽  
Author(s):  
Alan P. DAWSON ◽  
Edward J.A. LEA ◽  
Robin F. IRVINE
Keyword(s):  
Steady State ◽  
Kinetic Properties ◽  
Experimental Conditions ◽  
Low Concentrations ◽  
Insp3 Receptors ◽  
Hill Coefficients ◽  
Quantal Responses ◽  
Rapid Transients ◽  
Trisphosphate Receptor ◽  
Insp3 Receptor

The release of Ca2+ from intracellular stores via InsP3 receptors shows anomalous kinetics. Successive additions of low concentrations of InsP3 cause successive rapid transients of Ca2+ release. These quantal responses have been ascribed to all-or-none release from stores with differing sensitivities to InsP3 or, alternatively, to a steady-state mechanism where complex kinetic properties of the InsP3 receptor allow partial emptying of all the stores. We present here an adaptive model of the InsP3 receptor that can show either pattern, depending on the imposed experimental conditions. The model proposes two interconvertible conformational states of the receptor: one state binds InsP3 rapidly, but with low affinity, whereas the other state binds slowly, but with high affinity. The model shows repetitive increments of Ca2+ release in the absence of a Ca2+ gradient, but more pronounced incremental behaviour when released Ca2+ builds up at the mouth of the channel. The sensitivity to InsP3 is critically dependent on the density of InsP3 receptors, so that different stores can respond to different concentration ranges of InsP3. Since the model generates very high Hill coefficients (h7), it allows all-or-none release of Ca2+ from stores of differing receptor density, but questions the validity of the use of h values as a guide to the number of InsP3 molecules needed to open the channel. The model presents a mechanism for terminating Ca2+ release in the presence of positive feedback from released Ca2+, thereby providing an explanation of why elementary Ca2+ signals ('blips’ and ‘puffs') do not inevitably turn into regenerative waves.

scholarly journals Regulatory effects of ATP and luciferin on firefly luciferase activity

1995 ◽  
Vol 305 (3) ◽  
pp. 929-933 ◽  
Author(s):  
N Lembert ◽  
L Å Idahl
Keyword(s):  
Steady State ◽  
Active Site ◽  
Luciferase Activity ◽  
Firefly Luciferase ◽  
Kinetic Properties ◽  
Allosteric Site ◽  
Experimental Conditions ◽  
Regulatory Effects ◽  
High Concentrations ◽  
Firefly Luciferase Activity

ATP and luciferin are not only substrates of firefly luciferase, but can, in addition, modulate its activity. High concentrations of luciferin induce a conformational change of the enzyme that temporarily reduces the catalytic rate. Re-activation takes approx. 20 min and is independent of variation in the concentration of enzyme or ATP, but lengthens with increasing luciferin concentration. High concentrations of albumin reduce this luciferin effect. The kinetic properties of firefly luciferase determined from initial rates and at steady state after 1 min of catalysis have been analysed according to Michaelis-Menten kinetics. There is only one active site for each of the substrates. At steady state the Km and Vmax. values for both substrates are reduced in an uncompetitive manner. Hyperbolic Lineweaver-Burk plots indicate an activation by ATP probably by binding to an allosteric site. A model is presented which incorporates luciferin induced de- and re-activation effects. Experimental conditions to avoid the regulatory effects of substrates during ATP monitoring are proposed.

scholarly journals Kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase

1982 ◽  
Vol 203 (3) ◽  
pp. 617-627 ◽  
Author(s):  
G J Hart ◽  
F M Dickinson
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Kinetic Properties ◽  
Stopped Flow ◽  
Flow Measurements ◽  
Sheep Liver ◽  
Low Concentrations ◽  
Mechanism Of Reaction ◽  
High Concentrations ◽  
Variable Substrate

The kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase (preparations that had been shown to be free from contamination with the corresponding mitochondrial enzyme) were investigated with both propionaldehyde and butyraldehyde as substrates. At low aldehyde concentrations, double-reciprocal plots with aldehyde as the variable substrate are linear, and the mechanism appears to be ordered, with NAD+ as the first substrate to bind. Stopped-flow experiments following absorbance and fluorescence changes show bursts of NADH production in the pre-steady state, but the observed course of reaction depends on the pre-mixing conditions. Pre-mixing enzyme with NAD+ activates the enzyme in the pre-steady state and we suggest that the reaction mechanism may involve isomeric enzyme-NAD+ complexes. High concentrations of aldehyde in steady-state experiments produce significant activation (about 3-fold) at high concentrations of NAD+, but inhibition at low concentrations of NAD+. Such behaviour may be explained by postulating the participation of an abortive complex in product release. Stopped-flow measurements at high aldehyde concentrations indicate that the mechanism of reaction under these conditions is complex.

scholarly journals The steady-state visual evoked potential (SSVEP) reflects the activation of cortical object representations: evidence from semantic stimulus repetition

2020 ◽  
Author(s):  
Elise L. Radtke ◽  
Ulla Martens ◽  
Thomas Gruber
Keyword(s):  
Steady State ◽  
Object Representation ◽  
Sensory Information ◽  
Familiar Object ◽  
Object Representations ◽  
Stimulus Repetition ◽  
Experimental Conditions ◽  
Repetition Suppression ◽  
Task Irrelevant ◽  
Visual Evoked

AbstractWe applied high-density EEG to examine steady-state visual evoked potentials (SSVEPs) during a perceptual/semantic stimulus repetition design. SSVEPs are evoked oscillatory cortical responses at the same frequency as visual stimuli flickered at this frequency. In repetition designs, stimuli are presented twice with the repetition being task irrelevant. The cortical processing of the second stimulus is commonly characterized by decreased neuronal activity (repetition suppression). The behavioral consequences of stimulus repetition were examined in a companion reaction time pre-study using the same experimental design as the EEG study. During the first presentation of a stimulus, we confronted participants with drawings of familiar object images or object words, respectively. The second stimulus was either a repetition of the same object image (perceptual repetition; PR) or an image depicting the word presented during the first presentation (semantic repetition; SR)—all flickered at 15 Hz to elicit SSVEPs. The behavioral study revealed priming effects in both experimental conditions (PR and SR). In the EEG, PR was associated with repetition suppression of SSVEP amplitudes at left occipital and repetition enhancement at left temporal electrodes. In contrast, SR was associated with SSVEP suppression at left occipital and central electrodes originating in bilateral postcentral and occipital gyri, right middle frontal and right temporal gyrus. The conclusion of the presented study is twofold. First, SSVEP amplitudes do not only index perceptual aspects of incoming sensory information but also semantic aspects of cortical object representation. Second, our electrophysiological findings can be interpreted as neuronal underpinnings of perceptual and semantic priming.

scholarly journals Properties of pyruvate kinase and phosphoenolpyruvate carboxykinase in relation to the direction and regulation of phosphoenolpyruvate metabolism in muscles of the frog and marine invertebrates

1978 ◽  
Vol 174 (3) ◽  
pp. 979-987 ◽  
Author(s):  
Victor A. Zammit ◽  
Eric A. Newsholme
Keyword(s):  
Pyruvate Kinase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Marine Invertebrates ◽  
Adductor Muscle ◽  
Sea Anemone ◽  
Anaerobic Conditions ◽  
Fructose Bisphosphate ◽  
Low Concentrations ◽  
Optimum Ph ◽  
Hill Coefficients

1. The properties of pyruvate kinase and, if present, phosphoenolpyruvate carboxykinase from the muscles of the sea anemone, scallop, oyster, crab, lobster and frog were investigated. 2. In general, the properties of pyruvate kinase from all muscles were similar, except for those of the enzyme from the oyster (adductor muscle); the pH optima were between 7.1 and 7.4, whereas that for oyster was 8.2; fructose bisphosphate lowered the optimum pH of the oyster enzyme from 8.2 to 7.1, but it had no effect on the enzymes from other muscles. Hill coefficients for the effect of the concentration of phosphoenolpyruvate were close to unity in the absence of added alanine for the enzymes from all muscles except oyster adductor muscle; it was 1.5 for this enzyme. Alanine inhibited the enzyme from all muscles except the frog; this inhibition was relieved by fructose bisphosphate. Low concentrations of alanine were very effective with the enzyme from the oyster (50% inhibition was observed at 0.4mm). Fructose bisphosphate activated the enzyme from all muscles, but extremely low concentrations were effective with the oyster enzyme (0.13μm produced 50% activation). 3. In general, the properties of phosphoenolpyruvate carboxykinase from the sea anemone and oyster muscles are similar: the Km values for phosphoenolpyruvate are low (0.10 and 0.13mm); the enzymes require Mn2+ in addition to Mg2+ for activity; and ITP inhibits the enzymes and the inhibition is relieved by alanine. These latter compounds had no effect on enzymes from other muscles. 4. It is suggested that changes in concentrations of fructose bisphosphate, alanine and ITP produce a coordinated mechanism of control of the activities of pyruvate kinase and phosphoenolpyruvate carboxykinase in the sea anemone and oyster muscles, which ensures that phosphoenolpyruvate is converted into oxaloacetate and then into succinate in these muscles under anaerobic conditions. 5. It is suggested that in the muscles of the crab, lobster and frog, phosphoenolpyruvate carboxykinase catalyses the conversion of oxaloacetate into phosphoenolpyruvate. This may be part of a pathway for the oxidation of some amino acids in these muscles.

scholarly journals The importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2

1993 ◽  
Vol 291 (1) ◽  
pp. 89-94 ◽  
Author(s):  
P White ◽  
F D C Manson ◽  
C E Brunt ◽  
S K Chapman ◽  
G A Reid
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Cytochrome C ◽  
Kinetic Properties ◽  
Stopped Flow ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Cytochrome C Reductase ◽  
Flavocytochrome B2 ◽  
Cytochrome C Reduction

The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) are connected by a typical hinge peptide. The amino acid sequence of this interdomain hinge is dramatically different in flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala. This difference in the hinge is believed to contribute to the difference in kinetic properties between the two enzymes. To probe the importance of the hinge, an interspecies hybrid enzyme has been constructed comprising the bulk of the S. cerevisiae enzyme but containing the H. anomala flavocytochrome b2 hinge. The kinetic properties of this ‘hinge-swap’ enzyme have been investigated by steady-state and stopped-flow methods. The hinge-swap enzyme remains a good lactate dehydrogenase as is evident from steady-state experiments with ferricyanide as acceptor (only 3-fold less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (2.5-fold slower than in wild-type enzyme). The major effect of the hinge-swap mutation is to lower dramatically the enzyme's effectiveness as a cytochrome c reductase; kcat. for cytochrome c reduction falls by more than 100-fold, from 207 +/- 10 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 1.62 +/- 0.41 s-1 in the mutant enzyme. This fall in cytochrome c reductase activity results from poor interdomain electron transfer between the FMN and haem groups. This can be demonstrated by the fact that the kcat. for haem reduction in the hinge-swap enzyme (measured by the stopped-flow method) has a value of 1.61 +/- 0.42 s-1, identical with the value for cytochrome c reduction and some 300-fold lower than the value for the wild-type enzyme. From these and other kinetic parameters, including kinetic isotope effects with [2-2H]lactate, we conclude that the hinge plays a crucial role in allowing efficient electron transfer between the two domains of flavocytochrome b2.

scholarly journals Participation of plasma membrane proteins in the formation of tight junction by cultured epithelial cells

1983 ◽  
Vol 96 (3) ◽  
pp. 693-702 ◽  
Author(s):  
EB Griepp ◽  
WJ Dolan ◽  
ES Robbins ◽  
DD Sabatini
Keyword(s):  
Protein Synthesis ◽  
Steady State ◽  
Tight Junction ◽  
Messenger Rna ◽  
Freeze Fracture ◽  
Epithelial Cell Line ◽  
Experimental Conditions ◽  
Tight Junction Formation ◽  
Junction Formation ◽  
Spinner Culture

Measurements of the transepithelial electrical resistance correlated with freeze-fracture observations have been used to study the process of tight junction formation under various experimental conditions in monolayers of the canine kidney epithelial cell line MDCK. Cells derived from previously confluent cultures and plated immediately after trypsin- EDTA dissociation develop a resistance that reaches its maximum value of several hundred ohms-cm(2) after approximately 24 h and falls to a steady-state value of 80-150 ohms- cm(2) by 48 h. The rise in resistance and the development of tight junctions can be completely and reversibly prevented by the addition of 10 μg/ml cycloheximide at the time of plating, but not when this inhibitor is added more than 10 h after planting. Thus tight junction formation consists of separable synthetic and assembly phases. These two phases can also be dissociated and the requirement for protein synthesis after plating eliminated if, following trypsinization, the cells are maintained in spinner culture for 24 h before plating. The requirement for protein synthesis is restored, however, if cells maintained in spinner culture are treated with trypsin before plating. Actinomycin D prevents development of resistance only in monolayers formed from cells derived from sparse rather than confluent cultures, but new mRNA synthesis is not required if cells obtained from sparse cultures are maintained for 24 h in spinner culture before plating. Once a steady-state resistance has been reached, its maintenance does not require either mRNA or protein synthesis; in fact, inhibition of protein synthesis causes a rise in the resistance over a 30-h period. Following treatments that disrupt the junctions in steady- state monolayers recovery of resistance also does not require protein synthesis. These observations suggest that proteins are involved in tight junction formation. Such proteins, which do not turn over rapidly under steady-state conditions, are destroyed by trypsinization and can be resynthesized in the absence of stable cell-cell or cell-substratum contact. Messenger RNA coding for proteins involved in tight junction formation is stable except when cells are sparsely plated, and can also be synthesized without intercellular contacts or cell-substratum attachment.

scholarly journals Temperature uniformity in the CERN CLOUD chamber

2017 ◽  
Vol 10 (12) ◽  
pp. 5075-5088 ◽  
Author(s):  
António Dias ◽  
Sebastian Ehrhart ◽  
Alexander Vogel ◽  
Christina Williamson ◽  
João Almeida ◽  
...  
Keyword(s):  
Steady State ◽  
Air Temperature ◽  
Elevated Temperatures ◽  
Trace Gases ◽  
Vertical Direction ◽  
Cloud Chamber ◽  
Temperature Uniformity ◽  
Atmospheric Conditions ◽  
Cloud Droplets ◽  
Experimental Conditions

Abstract. The CLOUD (Cosmics Leaving OUtdoor Droplets) experiment at CERN (European Council for Nuclear Research) investigates the nucleation and growth of aerosol particles under atmospheric conditions and their activation into cloud droplets. A key feature of the CLOUD experiment is precise control of the experimental parameters. Temperature uniformity and stability in the chamber are important since many of the processes under study are sensitive to temperature and also to contaminants that can be released from the stainless steel walls by upward temperature fluctuations. The air enclosed within the 26 m3 CLOUD chamber is equipped with several arrays (strings) of high precision, fast-response thermometers to measure its temperature. Here we present a study of the air temperature uniformity inside the CLOUD chamber under various experimental conditions. Measurements were performed under calibration conditions and run conditions, which are distinguished by the flow rate of fresh air and trace gases entering the chamber at 20 and up to 210 L min−1, respectively. During steady-state calibration runs between −70 and +20 °C, the air temperature uniformity is better than ±0.06 °C in the radial direction and ±0.1 °C in the vertical direction. Larger non-uniformities are present during experimental runs, depending on the temperature control of the make-up air and trace gases (since some trace gases require elevated temperatures until injection into the chamber). The temperature stability is ±0.04 °C over periods of several hours during either calibration or steady-state run conditions. During rapid adiabatic expansions to activate cloud droplets and ice particles, the chamber walls are up to 10 °C warmer than the enclosed air. This results in temperature differences of ±1.5 °C in the vertical direction and ±1 °C in the horizontal direction, while the air returns to its equilibrium temperature with a time constant of about 200 s.

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