scholarly journals Models to determine the kinetic mechanisms of ion-coupled transporters

2019 ◽  
Vol 151 (3) ◽  
pp. 369-380 ◽  
Author(s):  
Juke S. Lolkema ◽  
Dirk J. Slotboom
Keyword(s):  
Steady State ◽  
Kinetic Mechanism ◽  
Ion Concentration ◽  
Coupling Mechanism ◽  
Maximal Rate ◽  
Transport Reaction ◽  
Apparent Affinity ◽  
Kinetic Mechanisms ◽  
Experimental Approaches ◽  
Analyze Data

With high-resolution structures available for many ion-coupled (secondary active) transporters, a major challenge for the field is to determine how coupling is accomplished. Knowledge of the kinetic mechanism of the transport reaction, which defines the binding order of substrate and co-ions, together with the sequence with which all relevant states are visited by the transporter, will help to reveal this coupling mechanism. Here, we derived general mathematical models that can be used to analyze data from steady-state transport measurements and show how kinetic mechanisms can be derived. The models describe how the apparent maximal rate of substrate transport depends on the co-ion concentration, and vice versa, in different mechanisms. Similarly, they describe how the apparent affinity for the transported substrate is affected by the co-ion concentration and vice versa. Analyses of maximal rates and affinities permit deduction of the number of co-ions that bind before, together with, and after the substrate. Hill analysis is less informative, but in some mechanisms, it can reveal the total number of co-ions transported with the substrate. However, prior knowledge of the number of co-ions from other experimental approaches is preferred when deriving kinetic mechanisms, because the models are generally overparameterized. The models we present have wide applicability for the study of ion-coupled transporters.

scholarly journals Role of Ca2+ in pyruvate dehydrogenase interconversion in brain mitochondria and synaptosomes

1985 ◽  
Vol 227 (1) ◽  
pp. 129-136 ◽  
Author(s):  
R G Hansford ◽  
F Castro
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Pyruvate Dehydrogenase ◽  
Maximal Activity ◽  
Brain Mitochondria ◽  
Ruthenium Red ◽  
Ion Concentration ◽  
Maximal Effect ◽  
State Content

The steady-state content of active (dephospho) pyruvate dehydrogenase (PDHA) of suspensions of coupled rat brain mitochondria oxidizing succinate was found to be markedly increased with increasing free Ca2+ ion concentration of the medium, with a half-maximal effect at 10(-6.43) M Ca2+. Other ions were present in these studies at concentrations appropriate for the cytosol. Depolarization of the plasma membrane of synaptosomes caused an increase in the steady-state content of PDHA, with veratridine giving a larger increase than depolarization by 33 mM-KCl. Values were 68 +/- 1% (n = 13) and 81 +/- 1% (n = 19) of maximal activity, for control incubations and incubations in the presence of 30 microM-veratridine, respectively. Measurements of cytosolic free Ca2+ concentrations ([Ca2+]cyt.) in these suspensions of synaptosomes, with the use of the fluorescent Ca2+-indicator Quin-2, indicated an increase on depolarization, with the change due to 30 microM-veratridine being larger in extent than that due to 33 mM-KCl. Values were 217 +/- 21 nM (n = 15), 544 +/- 48 nM (n = 15) and 783 +/- 75 nM (n = 14) for control, KCl-depolarized and veratridine-depolarized synaptosomes respectively. Experiments in which synaptosomes were treated with Ruthenium Red, an inhibitor of mitochondrial Ca2+ uptake, gave much lower resting contents of PDHA (42 +/- 2% of maximal), but failed to prevent totally an increase on depolarization. Addition of an excess of EGTA to the synaptosomal suspension just before the addition of veratridine resulted in a partial diminution in the response of PDHA content. Parallel studies with Quin-2 indicated no increase in [Ca2+]cyt. on addition of veratridine, under these conditions. Thus an increase in [Ca2+]cyt. forms only a part of the mechanism whereby pyruvate dehydrogenase interconversion responds to depolarization. A decrease in the ATP/ADP ratio may also be important, as inferred from the results of experiments with ouabain, which inhibits the Na+ + K+-dependent ATPase.

Experimental amplitude and frequency control of a self-excited microcantilever by linear and nonlinear feedback

2021 ◽  
Author(s):  
Eisuke Higuchi ◽  
Hiroshi Yabuno ◽  
Yasuyuki Yamamoto ◽  
Sohei Matsumoto
Keyword(s):  
Steady State ◽  
Frequency Control ◽  
High Sensitivity ◽  
High Viscosity ◽  
Measurement Methods ◽  
Nonlinear Feedback ◽  
Force Microscopy ◽  
Atomic Force ◽  
Excited Oscillation ◽  
Experimental Approaches

Abstract In recent years, measurement methods that use resonators as microcantilevers have attracted attention because of their high sensitivity, high accuracy, and rapid response time. They have been widely utilized in mass sensing, stiffness sensing, and atomic force microscopy (AFM), among other applications. In all these methods, it is essential to accurately detect shifts in the natural frequency of the resonator caused by an external force from a measured object or sample. Experimental approaches based on self-excited oscillation enable the detection of these shifts even when the resonator is immersed in a high-viscosity environment. In the present study, we experimentally and theoretically investigate the nonlinear characteristics of a microcantilever resonator and their control by nonlinear feedback. We show that the steady-state response amplitude and the corresponding response frequency can be controlled by cubic nonlinear velocity feedback and cubic nonlinear displacement feedback, respectively. Furthermore, the amplitude and frequency of the steady-state self-excited oscillation can be controlled separately. These results will expand application of measurement methods that use self-excited resonators.

scholarly journals Steady-state kinetic mechanism of the NADP+- and NAD+-dependent reactions catalysed by betaine aldehyde dehydrogenase from Pseudomonas aeruginosa

2000 ◽  
Vol 352 (3) ◽  
pp. 675-683 ◽  
Author(s):  
Roberto VELASCO-GARCÍA ◽  
Lilian GONZÁLEZ-SEGURA ◽  
Rosario A. MUÑOZ-CLARES
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Substrate Inhibition ◽  
Kinetic Mechanism ◽  
Betaine Aldehyde Dehydrogenase ◽  
Betaine Aldehyde ◽  
Metabolic Role ◽  
Steady State Kinetic ◽  
State Kinetic

Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible oxidation of betaine aldehyde to glycine betaine with the concomitant reduction of NAD(P)+ to NADP(H). In Pseudomonas aeruginosa this reaction is a compulsory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. The kinetic mechanisms of the NAD+- and NADP+-dependent reactions were examined by steady-state kinetic methods and by dinucleotide binding experiments. The double-reciprocal patterns obtained for initial velocity with NAD(P)+ and for product and dead-end inhibition establish that both mechanisms are steady-state random. However, quantitative analysis of the inhibitions, and comparison with binding data, suggest a preferred route of addition of substrates and release of products in which NAD(P)+ binds first and NAD(P)H leaves last, particularly in the NADP+-dependent reaction. Abortive binding of the dinucleotides, or their analogue ADP, in the betaine aldehyde site was inferred from total substrate inhibition by the dinucleotides, and parabolic inhibition by NADH and ADP. A weak partial uncompetitive substrate inhibition by the aldehyde was observed only in the NADP+-dependent reaction. The kinetics of P. aeruginosa BADH is very similar to that of glucose-6-phosphate dehydrogenase, suggesting that both enzymes fulfil a similar amphibolic metabolic role when the bacteria grow in choline and when they grow in glucose.

scholarly journals Estimating kinetic mechanisms with prior knowledge II: Behavioral constraints and numerical tests

2018 ◽  
Vol 150 (2) ◽  
pp. 339-354 ◽  
Author(s):  
Marco A. Navarro ◽  
Autoosa Salari ◽  
Mirela Milescu ◽  
Lorin S. Milescu
Keyword(s):  
Prior Knowledge ◽  
Estimation Procedure ◽  
Kinetic Mechanism ◽  
Arbitrary Parameter ◽  
Linear Parameter ◽  
Open Probability ◽  
Behavioral Constraints ◽  
Kinetic Mechanisms ◽  
Mathematical Relationships ◽  
Optimization Mechanism

Kinetic mechanisms predict how ion channels and other proteins function at the molecular and cellular levels. Ideally, a kinetic model should explain new data but also be consistent with existing knowledge. In this two-part study, we present a mathematical and computational formalism that can be used to enforce prior knowledge into kinetic models using constraints. Here, we focus on constraints that quantify the behavior of the model under certain conditions, and on constraints that enforce arbitrary parameter relationships. The penalty-based optimization mechanism described here can be used to enforce virtually any model property or behavior, including those that cannot be easily expressed through mathematical relationships. Examples include maximum open probability, use-dependent availability, and nonlinear parameter relationships. We use a simple kinetic mechanism to test multiple sets of constraints that implement linear parameter relationships and arbitrary model properties and behaviors, and we provide numerical examples. This work complements and extends the companion article, where we show how to enforce explicit linear parameter relationships. By incorporating more knowledge into the parameter estimation procedure, it is possible to obtain more realistic and robust models with greater predictive power.

Inhibition of carbonic anhydrase: its effect on carbon dioxide elimination by the lungs

1959 ◽  
Vol 14 (1) ◽  
pp. 109-115 ◽  
Author(s):  
John C. Mithoefer
Keyword(s):  
Carbon Dioxide ◽  
Steady State ◽  
Carbonic Anhydrase ◽  
Normal Animal ◽  
Carbon Dioxide Elimination ◽  
Carbonic Anhydrase Inhibition ◽  
Experimental Approaches

The effect of carbonic anhydrase inhibition on carbon dioxide elimination by the lungs has been studied in dogs by the following four experimental approaches: 1) the alveolar pathway (PaOO2 and PaCOCO2) was measured during the hyperventilation which follows carbonic anhydrase inhibition and compared to the normal pathway at the same hyperventilation. 2) The alveolar gas tensions were measured following carbonic anhydrase inhibition when the ventilation was held constant. 3) The changes in PaCOCO2 were measured when the ventilation was suddenly, artificially changed to a new level in the presence of carbonic anhydrase inhibition and in normal animals. 4) The CO2 output was measured directly during the hyperventilation which results from carbonic anhydrase inhibition, during the same hyperventilation in the normal animal and following carbonic anhydrase inhibition when the ventilation is held constant. These experiments demonstrate an immediate fall in CO2 output relative to the ventilation when carbonic anhydrase is inhibited, resulting in CO2 retention until a new steady state has been reached. An hypothesis is presented to explain the effect of carbonic anhydrase inhibition on CO2 transport. Submitted on March 28, 1958

scholarly journals Benthic phosphorus and iron budgets for three NW African slope sediments: a balance approach

2010 ◽  
Vol 7 (2) ◽  
pp. 469-480 ◽  
Author(s):  
K. Küster-Heins ◽  
G. J. de Lange ◽  
M. Zabel
Keyword(s):  
Steady State ◽  
Pore Water ◽  
Analytical Data ◽  
General Distribution ◽  
Data Set ◽  
Transport Reaction ◽  
Particle Mixing ◽  
Open Questions ◽  
Close Relationship ◽  
Key Issues

Abstract. Despite intensive research on the different domains of the marine phosphorus (P) cycle during the last decades, frequently discussed open questions still exist especially on controlling factors for the benthic behaviour of P and its general distribution in sediment-pore water systems. Steady state or the internal balance of all relevant physical and (bio)geochemical processes are amongst the key issues. In this study we present and discuss an extended data set from surface sediments recovered from three locations on the NW African continental slope. Pore water data and results from sequential sediment extractions give clear evidence to the well-known close relationship between the benthic cycles of P and iron. Accordingly, most of the dissolved phosphate must have been released by microbially catalyzed reductive dissolution of iron (oxhydr)oxides. However, rates of release and association of P and iron, respectively, are not directly represented in profiles of element specific sediment compositions. Results from steady-state based transport-reaction modelling suggest that particle mixing due to active bioturbation, or rather a physical net downward transport of P associated to iron (oxyhydr)oxides, is an essential process for the balance of the inspected benthic cycles. This study emphasizes the importance of balancing analytical data for a comprehensive understanding of all processes involved in biogeochemical cycles.

scholarly journals Import of Non-Coding RNAs into Human Mitochondria: A Critical Review and Emerging Approaches

Cells ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 286 ◽  
Author(s):  
Damien Jeandard ◽  
Anna Smirnova ◽  
Ivan Tarassov ◽  
Eric Barrey ◽  
Alexandre Smirnov ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Nuclear Genome ◽  
Genetic System ◽  
Mitochondrial Import ◽  
In Situ Techniques ◽  
Genome Wide ◽  
Experimental Approaches ◽  
Non Coding Rnas ◽  
Analyze Data

Mitochondria harbor their own genetic system, yet critically depend on the import of a number of nuclear-encoded macromolecules to ensure their expression. In all eukaryotes, selected non-coding RNAs produced from the nuclear genome are partially redirected into the mitochondria, where they participate in gene expression. Therefore, the mitochondrial RNome represents an intricate mixture of the intrinsic transcriptome and the extrinsic RNA importome. In this review, we summarize and critically analyze data on the nuclear-encoded transcripts detected in human mitochondria and outline the proposed molecular mechanisms of their mitochondrial import. Special attention is given to the various experimental approaches used to study the mitochondrial RNome, including some recently developed genome-wide and in situ techniques.

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