scholarly journals Partial reactions of the Na,K-ATPase: determination of rate constants.

1994 ◽  
Vol 104 (2) ◽  
pp. 197-240 ◽  
Author(s):  
S Heyse ◽  
I Wuddel ◽  
H J Apell ◽  
W Stürmer
Keyword(s):  
Electric Current ◽  
Rate Constant ◽  
Conformational Changes ◽  
Rate Constants ◽  
Conformational Transition ◽  
Divalent Cations ◽  
Quantitative Description ◽  
Reaction Model ◽  
Na Ions

Experiments were designed to characterize several partial reactions of the Na,K-ATPase and to demonstrate that a model can be defined that reproduces most of the transport features of the pump with a single set of kientic parameters. We used the fluorescence label 5-iodoacetamidofluorescein, which is thought to be sensitive to conformational changes, and the styryl dye RH 421, which can be applied to detect ion-binding and -release reactions. In addition transient electric currents were measured, which are associated mainly with the E1-->E2 conformational transition. Numerical simulations were performed on the basis of a reaction model, that has been developed from the Post-Albers cycle. Analysis of the experimental data allows the determination of several rate constants of the pump cycle. Our conclusions may be summarized as follows: (a) binding of one Na+ ion at the cytoplasmic face is electrogenic. This Na+ ion is specifically bound to a neutral binding site with an affinity of 8 mM in the presence of 10 mM Mg2+. In the absence of divalent cations, the intrinsic binding affinity was found to be 0.7 mM. (b) The analysis of fluorescence experiments with the cardiotonic steroid strophanthidin indicates that the 5-iodoacetamidofluorescein label monitors the conformational transition (Na3)E1-P-->P-E2(Na2), which is accompanied by the release of one Na+ ion. 5-IAF does not respond to the release of the subsequent two Na+ ions, which can be monitored by the RH 421 dye. These experiments indicate further that the conformational transition E1P-->P-E2 is the rate limiting process of the Na+ translocation. The corresponding rate constant was determined to be 22 s-1 at 20 degrees C. From competition experiments with cardiotonic steroids, we estimated that the remaining 2 Na+ ions are released subsequently with a rate constant of at least 5,000 s-1 from their negatively charged binding sites. (c) Comparing the fluorescence experiments with electric current transients, which were performed at various Na concentrations in the absence and presence of strophanthidin, we found that the transition (Na3).E1-P-->P-E2.(Na2) is the major charge translocating step in the reaction sequence Na3.E1-->(Na3).E1-P-->P-E2.(Na2)-->P-E2. The subsequent release of 2 Na+ ions contributed less than 25% to the total electric current transient. (d) The well known antagonism between cardiotonic steroids and K+ binding can be explained by a kinetic model. A quantitative description has been obtained under the assumption that these inhibitors bind only to the states P-E2(Na2) and P-E2(K2).(ABSTRACT TRUNCATED AT 400 WORDS)

Determination of rate constants of redox reactions of second order from current-less potential-time curves by a simplified graphical-numerical method

1983 ◽  
Vol 48 (5) ◽  
pp. 1358-1367 ◽  
Author(s):  
Antonín Tockstein ◽  
František Skopal
Keyword(s):  
Numerical Method ◽  
Explicit Form ◽  
Rate Constant ◽  
Optimization Algorithm ◽  
Rate Constants ◽  
Redox Reactions ◽  
Second Order ◽  
Wide Region ◽  
Recurrent Formula

A method for constructing curves is proposed that are linear in a wide region and from whose slopes it is possible to determine the rate constant, if a parameter, θ, is calculated numerically from a rapidly converging recurrent formula or from its explicit form. The values of rate constants and parameter θ thus simply found are compared with those found by an optimization algorithm on a computer; the deviations do not exceed ±10%.

scholarly journals Effect of Vascular Activity in the Determination of Rate Constants for the Uptake of 18F-Labeled 2-Fluoro-2-Deoxy-D-Glucose: Error Analysis and Normal Values in Older Subjects

1986 ◽  
Vol 6 (6) ◽  
pp. 724-738 ◽  
Author(s):  
A. C. Evans ◽  
M. Diksic ◽  
Y. L. Yamamoto ◽  
A. Kato ◽  
A. Dagher ◽  
...  
Keyword(s):  
Rate Constant ◽  
Gray Matter ◽  
Rate Constants ◽  
Cerebral Blood Volume ◽  
Physiological State ◽  
Control Group ◽  
Vascular Activity ◽  
Regional Cerebral Blood Volume ◽  
Wide Range

Regional cerebral blood volume (CBV) can be calculated using data obtained during the kinetic analysis of 18F-labeled 2-fluoro-2-deoxy-d-glucose (FDG) uptake measured by positron emission tomography (PET). As a result the influence of vascular activity upon the determination of FDG rate constants can be minimized. The method is investigated by simulation experiments and by analysis of PET studies on seven older, healthy human volunteers aged 52–70 years. The accuracy of measured FDG rate constants k1, k2, and k3, obtained either by omitting the early portion of the uptake curve or by explicit inclusion of CBV as a fit parameter, is compared. The root mean square error in measured rate constant for the latter method is equivalent to that obtained by omitting the first 2.5–3 min of tissue data and neglecting the CBV term. Hence, added information about the physiological state of the tissue is obtained without compromising the accuracy of the (FDG) rate constant measurement. In hyperemic tissue the explicit determination of the vascular fraction results in more accurate estimates of the FDG rate constants. The ratio of CBV determined by this method to CBV obtained using C15O in six subjects with CBV in the normal range was 0.92 ± 0.32. A comparison of the CBV image obtained by this method with that obtained using C15O in an arteriovenous malformation case demonstrates the accuracy of the approach over a wide range of CBV values. The mean value for CBV fraction in gray matter obtained by this method in the older control group was 0.040 ± 0.014. Average gray matter rate constants obtained were k1 = 0.084 ± 0.012, k2 = 0.150 ± 0.071, and k3 = 0.099 ± 0.045 min−1.

scholarly journals Kinetic Study of CaCO3 Precipitation Accelerated by High Voltage Impulse

2021 ◽  
Vol 43 (2) ◽  
pp. 125-134
Author(s):  
Damha Kim ◽  
In-Soung Chang
Keyword(s):  
Rate Constant ◽  
High Voltage ◽  
Rate Constants ◽  
Reaction Order ◽  
Reaction Rate Constant ◽  
Formation Reaction ◽  
Different Temperatures ◽  
Synthetic Solution ◽  
Scale Control

Objectives : Determination of reaction order (n) and rate constants (k) of the CaCO<sub>3</sub> scale formation reaction that was accelerated by the HVI (high voltage impulse) induction.Methods : HVI was inducted to the synthetic solution containing 2.5 mM of Ca<sup>2+</sup> ion at different temperatures of 25, 40, 60℃. The concentration of Ca<sup>2+</sup> ion has been monitored as voltages of the HVI increased from 0 to 5, 10, 15 kV. Reaction order and the rate constants of the CaCO<sub>3</sub> formation reaction were determined with the experimental dataset of Ca<sup>2+</sup> concentration vs. time plots.Results and Discussion : The CaCO<sub>3</sub> formation was determined to follow two-molecules 2<sup>nd</sup> order reaction. The reaction rate constant, k increased as temperature and the applied voltages of HVI increased. The rate constant, k at 25℃ and 15 kV of HVI was 8.2×10<sup>-3</sup> L/(mmol･hr), which was 2.7 times greater than the k of the control at 25℃, 3.0×10<sup>-3</sup> L/(mmol･hr).Conclusions : The reaction of CaCO<sub>3</sub> formation was accelerated by HVI as the applied voltages of HVI increased, indicating that the HVI could be used as an alternative desalting technology for scale control.

scholarly journals Glutamic acid 327 in the sheep α 1 isoform of Na+,K+-ATPase is a pivotal residue for cation-induced conformational changes

1995 ◽  
Vol 309 (1) ◽  
pp. 187-194 ◽  
Author(s):  
C L Johnson ◽  
T A Kuntzweiler ◽  
J B Lingrel ◽  
C G Johnson ◽  
E T Wallick
Keyword(s):  
Glutamic Acid ◽  
Conformational Changes ◽  
Conformational Transition ◽  
Cation Binding ◽  
Monovalent Cations ◽  
Wild Type ◽  
Binding Studies ◽  
Ouabain Binding ◽  
Na Ions ◽  
Inhibition Curve

The cation binding characteristics of the mutant E327A formed in the sheep alpha 1 isoform of the Na+,K(+)-ATPase were examined using [3H]ouabain binding as a function of monovalent cation concentrations. Equilibrium competition binding assays in the presence of Mg2+, inorganic phosphate and various amounts of unlabelled ouabain indicated that both wild-type sheep alpha 1 protein and the E327A mutant expressed in 3T3 cells had similar affinities for ouabain (KD = 1.53 and 1.31 nM respectively). Sodium inhibition of ouabain binding appeared competitive in both enzymes. However, binding of three Na+ ions was required to explain the steep character of the Na+ inhibition curve for the wild-type Na+,K(+)-ATPase (Ki = 12.8 +/- 1.6 mM), whereas the binding of two Na+ ions was detected for the mutant E327A (Ki = 19.2 +/- 2.5 mM). Potassium binding of [3H]ouabain binding displayed a partially competitive nature with Hill coefficients of 2 for both wild-type sheep alpha 1 (Ki = 0.743 +/- 0.044 mM) and E327A (Ki = 0.875 +/- 0.067 mM). At concentrations of K+ above 10 mM, the sheep alpha 1 competition curve levelled off whereas the inhibition curve for E327A displayed a stimulation in ouabain binding. This stimulation in [3H]ouabain binding also occurred with Rb+, Cs+ and Li+, but was never observed with choline or Na+, suggesting that this effect was not due to ionic strength. From these [3H]ouabain-binding studies, it is obvious that the mutant enzyme E327A in the presence of Mg2+, Pi and ouabain, interacts with monovalent cations in a unique fashion. One interpretation of these data is that the glutamic acid residue at position 327 is involved in a conformational transition induced by the binding of monovalent cations to the Na+,K+-ATPase and that this transition is inhibited by the mutation of E327A.

scholarly journals A Two-part Approach to the Determination of Intrinsic Rate Constants of an Alpha-amylase Catalysed Reaction

2020 ◽  
pp. 8-21
Author(s):  
Ikechukwu I. Udema
Keyword(s):  
Rate Constant ◽  
Rate Constants ◽  
Catalytic Efficiency ◽  
Intrinsic Rate ◽  
Product Formation ◽  
Intermolecular Potential ◽  
Diffusion Controlled ◽  
Quantitative Results ◽  
Intrinsic Rate Constant

Background: There is a need for equations with which to calculate the intrinsic rate constants that can further characterise enzyme catalysed reactions despite what seems to be conventional differences in methodology in the literature. Methods: Theoretical, experimental (Bernfeld method), and computational methods. Objectives: 1) To derive an alternative intrinsic rate constant equations consistent with their dimension, 2) derive electrostatic intermolecular potential energy equation, (xe), 3) calculate the intrinsic rate constants for forward (k1) and reverse (k2) reactions, and 4) define the dependence or otherwise of kinetic constants on diffusion and deduce the catalytic efficiency. Results and Discussion: The ultimate quantitative results were ~ 64.69 ±  0.49 exp (+3)/ min (k2) (and kd (s) = ~ 60.66 exp (+3)/ min), ~ 1594.48 ± 11.99 exp (+3) exp (+3) L/mol.min (k1) (and ka (s) = ~1482.47 exp (+3) L/mol.min), ~ 58.00 ± 10.83 exp (+3) /min, the apparent rate constant for reverse reaction (kb), and ~ 75.83 ± 10.83 exp (+3) /min, the rate constant for product formation (k3). The catalytic efficiency was: 3.025 exp (+ 9) L / mol.     Conclusion: The relevant equations were derived. Based on the derived equations the intrinsic rate constants can be calculated. Since k3 is > kb, then k3 is diffusion controlled and it appears that the enzyme has reached kinetic perfection. The evaluation of rate constants either from the perspective of diffusion dependency or independency cannot be valid without Avogadro number.

Thermal effects in flash photolysis with special reference to Br atom recombination

1968 ◽  
Vol 46 (20) ◽  
pp. 3229-3234 ◽  
Author(s):  
George Burns
Keyword(s):  
Rate Constant ◽  
Rate Constants ◽  
Thermal Effects ◽  
Reaction Rate ◽  
Flash Photolysis ◽  
Reaction Rate Constants ◽  
A Value ◽  
Recombination Rate Constant ◽  
Reaction Vessels

Thermal effects, which accompany flash photolyses, are known to interfere with the determination of reaction rate constants. There are two approximate models currently being used in literature to estimate the magnitude of these effects (1, 8). The first model (1) is the more widely accepted. It is based on the assumption that thermal effects are due to the cooling of reacting gas at the walls of the reaction vessel. The second model (8) is based on the assumption that thermal effects are due to nonuniformity in the concentrations of free radicals produced in flash photolysis; it neglects the heat exchange at the wall of the reaction vessel.It is shown that the second model can be used to calculate the magnitude of thermal effects in reaction vessels of reasonable length. The model was applied to calculate [Formula: see text], the rate constant for the reaction 2Br + Br2 → 2Br2. The value of [Formula: see text], is found to be very sensitive to the choice of model for thermal effects. At room temperature the most reasonable value of [Formula: see text], using the second model, is (4.3 ± 1.3) × 1010 l2 mole−2 s−1. This value agrees very well with independent determinations of [Formula: see text] using a stationary photochemical technique. The first model for treatment of thermal effects (1) was used previously to show that such effects do not influence the measured rates of chemical reactions, and calculations of rate constants using this model have not usually been attempted. In one case (5), however, the first model (1) for thermal effects was employed to calculate a value for [Formula: see text] which was found to be six times larger than our value. Consequently, the second model (8) appears to be a better approximation for quantitative evaluation of thermal effects.Using the raw data (8) and [Formula: see text] = 43 × 109 l2 mole−2 s−1, the value of kAr, the recombination rate constant of Br atoms in excess of argon, was found to be (3.0 ± 0.2) × 109 l2 mole−2 s−1, which agrees well with data available in the literature.

scholarly journals Conformational kinetics reveals affinities of protein conformational states

2015 ◽  
Vol 112 (30) ◽  
pp. 9352-9357 ◽  
Author(s):  
Kyle G. Daniels ◽  
Yang Suo ◽  
Terrence G. Oas
Keyword(s):  
Ligand Binding ◽  
Conformational Change ◽  
Conformational Changes ◽  
Rate Constants ◽  
Conformational Dynamics ◽  
Forward Rate ◽  
Binding Affinities ◽  
Conformational States ◽  
Kinetics Of

Most biological reactions rely on interplay between binding and changes in both macromolecular structure and dynamics. Practical understanding of this interplay requires detection of critical intermediates and determination of their binding and conformational characteristics. However, many of these species are only transiently present and they have often been overlooked in mechanistic studies of reactions that couple binding to conformational change. We monitored the kinetics of ligand-induced conformational changes in a small protein using six different ligands. We analyzed the kinetic data to simultaneously determine both binding affinities for the conformational states and the rate constants of conformational change. The approach we used is sufficiently robust to determine the affinities of three conformational states and detect even modest differences in the protein’s affinities for relatively similar ligands. Ligand binding favors higher-affinity conformational states by increasing forward conformational rate constants and/or decreasing reverse conformational rate constants. The amounts by which forward rate constants increase and reverse rate constants decrease are proportional to the ratio of affinities of the conformational states. We also show that both the affinity ratio and another parameter, which quantifies the changes in conformational rate constants upon ligand binding, are strong determinants of the mechanism (conformational selection and/or induced fit) of molecular recognition. Our results highlight the utility of analyzing the kinetics of conformational changes to determine affinities that cannot be determined from equilibrium experiments. Most importantly, they demonstrate an inextricable link between conformational dynamics and the binding affinities of conformational states.

scholarly journals The use of Spectroquant Merck BOD photometric test to evaluate the stabilityof organic matters in soil

2011 ◽  
Vol 51 (No. 1) ◽  
pp. 46-50
Author(s):  
L. Kolář ◽  
S. Kužel ◽  
A. Hanušová ◽  
J. Gergel ◽  
R. Ledvina ◽  
...  
Keyword(s):  
Rate Constant ◽  
Rate Constants ◽  
High Time ◽  
New Method ◽  
Dilution Method ◽  
Organic Matters ◽  
Biochemical Oxidation ◽  
Volumetric Determination ◽  
Vacuum Method

We proposed a new method to determine the rate constant of biochemical oxidation of soil organic matters that makes it possible to evaluate their stability by Spectroquant Merck BOD photometric tests; if smaller samplings are used and individual determinations are performed, it can replace the method of BOD vacuum measuring by an Oxi Top Control Merck system. The time and labour consumption of this vacuum method is sometimes criticised, but it has demonstrable work advantages for a large series of samples and so it is still recommended. The two methods are identical as for the correctness and coincidence of results and they do not differ by either lower or higher results and therefore we recommend the new method with photometric tests for smaller series of samples. For the high time and labour consumption we must warn against the use of traditional BOD dilution method with volumetric determination of oxygen for the determination of rate constants.

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