scholarly journals The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Johanna Roostalu ◽  
Claire Thomas ◽  
Nicholas Ian Cade ◽  
Simone Kunzelmann ◽  
Ian A Taylor ◽  
...  
Keyword(s):  
Critical Importance ◽  
Binding Region ◽  
Gtp Hydrolysis ◽  
Diverse Range ◽  
Slowing Down ◽  
High Affinity ◽  
Microtubule Stability ◽  
Conformation Changes ◽  
Kinetics Of ◽  
Microtubule Function

Microtubules are cytoskeletal polymers whose function depends on their property to switch between states of growth and shrinkage. Growing microtubules are thought to be stabilized by a GTP cap at their ends. The nature of this cap, however, is still poorly understood. End Binding proteins (EBs) recruit a diverse range of regulators of microtubule function to growing microtubule ends. Whether the EB binding region is identical to the GTP cap is unclear. Using mutated human tubulin with blocked GTP hydrolysis, we demonstrate that EBs bind with high affinity to the GTP conformation of microtubules. Slowing-down GTP hydrolysis leads to extended GTP caps. We find that cap length determines microtubule stability and that the microtubule conformation changes gradually in the cap as GTP is hydrolyzed. These results demonstrate the critical importance of the kinetics of GTP hydrolysis for microtubule stability and establish that the GTP cap coincides with the EB-binding region.

scholarly journals The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability

2019 ◽  
Author(s):  
Johanna Roostalu ◽  
Claire Thomas ◽  
Nicholas I. Cade ◽  
Simone Kunzelmann ◽  
Ian A. Taylor ◽  
...  
Keyword(s):  
Single Molecule ◽  
Critical Importance ◽  
Gtp Hydrolysis ◽  
Diverse Range ◽  
Slowing Down ◽  
High Affinity ◽  
Microtubule Stability ◽  
Kinetics Of ◽  
Microtubule Function

ABSTRACTMicrotubules are bistable cytoskeletal polymers whose function depends on their property to switch between states of growth and shrinkage 1. Growing microtubules are thought to be stabilized by a GTP cap at their ends 2-5. The nature of this cap, however, is still poorly understood. How GTP hydrolysis determines the properties of the GTP cap and hence microtubule stability is unclear. End Binding proteins (EBs) recruit a diverse range of regulators of microtubule function to growing microtubule ends 6. Whether these regulatory platforms at growing microtubule ends are identical to the GTP cap is not known. Using mutated human tubulin with blocked GTP hydrolysis, we demonstrate in microscopy-based in vitro reconstitutions that EB proteins bind with high affinity to the GTP conformation of microtubules. Slowing-down GTP hydrolysis leads to extended GTP caps and consequently hyper-stable microtubules. Single molecule experiments reveal that the microtubule conformation gradually changes in the cap as GTP is hydrolyzed. These results demonstrate the critical importance of the kinetics of GTP hydrolysis for microtubule stability; and establish that the GTP cap coincides with the EB-binding regulatory hub that modulates microtubule cytoskeleton function in cells.

Systems analysis of digoxin kinetics and inotropic response in the rat heart: effects of calcium and KB-R7943

2004 ◽  
Vol 287 (4) ◽  
pp. H1857-H1867 ◽  
Author(s):  
Michael Weiss ◽  
Myoungki Baek ◽  
Wonku Kang
Keyword(s):  
Rat Heart ◽  
Site Response ◽  
High Capacity ◽  
Left Ventricular ◽  
Limiting Factor ◽  
Inotropic Response ◽  
Influx Rate ◽  
High Affinity ◽  
Reverse Mode ◽  
Kinetics Of

To gain more insight into the mechanistic processes controlling the kinetics of inotropic response of digoxin in the perfused whole heart, an integrated kinetic model was developed incorporating digoxin uptake, receptor binding (Na+-K+-ATPase inhibition), and cellular events linking receptor occupation and response. The model was applied to data obtained in the single-pass Langendorff-perfused rat heart for external [Ca2+] of 0.5 and 1.5 mM under control conditions and in the presence of the reverse-mode Na+/Ca2+ exchange inhibitor KB-R7943 (0.1 μM) in perfusate. Outflow concentration and left ventricular developed pressure data measured for three consecutive doses (15, 30, and 45 μg) in each heart were analyzed simultaneously. While disposition kinetics of digoxin was determined by interaction with a heterogeneous receptor population consisting of a high-affinity/low-capacity and a low-affinity/high- capacity binding site, response generation was >80% mediated by binding to the high-affinity receptor. Digoxin sensitivity increased at lower external [Ca2+] due to higher stimulus amplification. Coadministration of KB-R7943 significantly reduced the positive inotropic effect of digoxin at higher doses (30 and 45 μg) and led to a saturated and delayed receptor occupancy-response relationship in the cellular effectuation model. The results provide further evidence for the functional heterogeneity of the Na+-K+-ATPase and suggest that in the presence of KB-R7943 a reduction of the Ca2+ influx rate via the reverse mode Na+/Ca2+ exchanger might become the limiting factor in digoxin response generation.

The increase in intracellular pH associated with Xenopus egg activation is a Ca(2+)-dependent wave

1992 ◽  
Vol 101 (1) ◽  
pp. 55-67 ◽  
Author(s):  
N. Grandin ◽  
M. Charbonneau
Keyword(s):  
Cell Cycle ◽  
Intracellular Ph ◽  
Egg Activation ◽  
Free Calcium ◽  
Slowing Down ◽  
M Phase ◽  
Xenopus Egg ◽  
General Slowing ◽  
The Impact ◽  
Kinetics Of

In Xenopus eggs, the transient increase in intracellular free calcium ([Ca2+]i), or Ca2+ transient, which occurs 1–3 min after egg activation, is likely to be partly responsible for the release of the cell cycle blockade. In the present study, we have used microinjection of BAPTA or EGTA, two potent chelators of Ca2+, to buffer [Ca2+]i at various steps during Xenopus egg activation and evaluate the impact on some of the associated events. Microinjection of either one of the Ca2+ chelators into unactivated eggs prevented egg activation without, however, lowering [Ca2+]i, suggesting that only physiological [Ca2+]i changes, but not [Ca2+]i levels, were affected by the Ca2+ buffer. When BAPTA was microinjected around the time of occurrence of the Ca2+ transient, the egg activation-associated increase in intracellular pH (pHi) was clearly delayed. That delay was not due to a general slowing down of the cell cycle, since under the same conditions of microinjection of BAPTA the kinetics of MPF (a universal M-phase promoting factor) inactivation were unaffected. These results represent the first indication that the Ca2+ transient participates in determining the time of initiation of the pHi increase during Xenopus egg activation. The present results also demonstrate that the egg activation-associated pHi changes (a slight, transient decrease in pHi followed by a permanent increase in pHi) proceed as a wave propagating from the site of triggering of egg activation. Experiments of local microinjection of BAPTA support the view that the pH wave is a consequence of the Ca2+ wave, which it follows closely.

Probing high-affinity aptamer binding region and development of aptasensor platform for the detection of cylindrospermopsin

2020 ◽  
Vol 412 (19) ◽  
pp. 4691-4701 ◽  
Author(s):  
Raja Chinnappan ◽  
Razan AlZabn ◽  
Amjad K. Fataftah ◽  
Ali Alhoshani ◽  
Mohammed Zourob
Keyword(s):  
Binding Region ◽  
High Affinity

Kinetics of serotonin uptake in isolated rabbit lungs

1989 ◽  
Vol 66 (5) ◽  
pp. 2328-2337 ◽  
Author(s):  
F. A. Peeters ◽  
T. A. Bronikowski ◽  
C. A. Dawson ◽  
J. H. Linehan ◽  
H. Bult ◽  
...  
Keyword(s):  
Endothelial Function ◽  
Bolus Injection ◽  
Rabbit Lung ◽  
Future Studies ◽  
High Affinity ◽  
Lung Physiology ◽  
Extraction Pattern ◽  
Uptake Data ◽  
5 Ht Uptake ◽  
Kinetics Of

The single-pass, bolus-injection method was used to study the effect of serotonin (5-HT) concentration on the extraction of 5-HT by isolated perfused rabbit lungs. The extraction pattern suggested that an uptake model, which includes multiple parallel uptake processes, provided a better representation of the data than the simple Michaelis-Menten equation, which has commonly been used to fit the saturable uptake data in previous studies. In particular, the rabbit lung data could be fit with two such parallel pathways. Since the 5-HT uptake could virtually be completely blocked by imipramine, both pathways can be considered to be carrier-mediated processes. The high-affinity pathway was saturable within the range of concentrations studied, with a Km and Vmax of approximately 0.84 microM and 0.21 nmol.s-1.g wet lung wt-1. The Km for the low-affinity pathway was larger than concentrations for which accurate uptake measurements are practical in the perfused organ. Thus, for the low-affinity pathway, only Vmax/Km was identifiable. Vmax/Km values for the high- and low-affinity pathways were approximately 2.87 and 0.35 ml/s, respectively. The results suggest that it will be worthwhile to investigate the behavior of these uptake parameters in response to changes in lung physiology and endothelial function in future studies.

Intrinsic, apparent, and effective affinities of co- and countertransport systems

1986 ◽  
Vol 250 (4) ◽  
pp. C523-C533 ◽  
Author(s):  
W. D. Stein
Keyword(s):  
Dissociation Constant ◽  
Chemical Reaction ◽  
Binding Sites ◽  
Kinetic Schemes ◽  
High Affinity ◽  
Dependent Atpase ◽  
Conformation Changes ◽  
Rapid Equilibrium

Solutions to kinetic schemes for the simple carrier, the countertransporter (antiport, exchanger), and the rapid equilibrium cases of the cotransporter (symport) and co-chemiporter (cation-dependent ATPase) are listed. A distinction is made between the intrinsic, apparent, and effective affinities of the transporters for their substrates. Effective pumping requires that the active transporter binds the pumped substrate, at high affinity, realized at the “whence side” (from which pumping takes place) and, at low affinity, at the “whither side” (to which pumping takes place). It is demonstrated how effective pumping might be achieved by appropriate design of the transporter or chemiporter in terms of the energies of the intrinsic binding sites, the energies of the conformation changes that the pump protein undergoes, the dissociation constant of the chemical reaction that drives the co-chemiport, and the order of binding of the cosubstrates, appropriate at different prevailing levels of the driving substrate.

RV11 (Propionyl Erythromycin Mercaptosuccinate) Pharmacokinetics in Bronchial Secretions

1986 ◽  
Vol 14 (3) ◽  
pp. 137-141 ◽  
Author(s):  
E Concia ◽  
P Marone ◽  
G C Moreo ◽  
C Sardi ◽  
R Braschi
Keyword(s):  
Oral Dose ◽  
Plasma Concentrations ◽  
Bronchial Secretion ◽  
Dose Equivalent ◽  
Bronchial Secretions ◽  
Definitive Evidence ◽  
High Affinity ◽  
Diagnostic Bronchoscopy ◽  
Erythromycin Base ◽  
Kinetics Of

The kinetics of RV11 (propionyl erythromycin mercaptosuccinate) in serum and bronchial secretions was investigated in heterogeneous bronchopneumopathic patients requiring diagnostic bronchoscopy. A single oral dose, equivalent to 500 mg of erythromycin base, was administered to all patients and the bronchial secretion and plasma concentrations were determined after 2, 3 and 4 hr. The bronchial secretion and plasma levels consistently exceeded those reported previously for erythromycin per os, suggesting that RV11 may have an unusually high affinity for bronchial secretions in humans. The results of this study also suggested that RV11 might have different kinetics in bronchial secretions and serum, though further studies are required to provide definitive evidence.

scholarly journals Kinetics of GTP hydrolysis during the assembly of chick brain MAP2-tubulin microtubule protein

1991 ◽  
Vol 277 (1) ◽  
pp. 239-243 ◽  
Author(s):  
R G Burns
Keyword(s):  
Initial Rate ◽  
Molar Concentration ◽  
Microtubule Assembly ◽  
Chick Brain ◽  
Gtp Hydrolysis ◽  
Microtubule Protein ◽  
Rate Of Hydrolysis ◽  
Kinetics Of

The kinetics of GTP hydrolysis during microtubule assembly have been examined using chick brain MAP2-tubulin microtubule protein in a NaCl-supplemented buffer. The elongating microtubules terminate in a ‘GTP cap’, since the kinetics of GTP hydrolysis are slower than those of subunit addition. GTP hydrolysis is (a) stoichiometric, (b) occurs as a vectorial wave as the initial rate of hydrolysis is proportional to the molar concentration of microtubule ends and not to the initial rate of subunit addition, and (c) either does not occur, or occurs only at a much lower rate, in the terminal subunits.

scholarly journals Kinetics of binding, endocytosis, and recycling of EGF receptor mutants

1992 ◽  
Vol 117 (1) ◽  
pp. 203-212 ◽  
Author(s):  
S Felder ◽  
J LaVin ◽  
A Ullrich ◽  
J Schlessinger
Keyword(s):  
Point Mutation ◽  
Phorbol Ester ◽  
Egf Receptor ◽  
Receptor Internalization ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Low Concentrations ◽  
Internalization Rate ◽  
Kinetics Of

This report describes analysis of factors which regulate the binding of EGF to EGF receptor, receptor internalization, and receptor recycling. Three different methods were used to inhibit high-affinity EGF binding as measured at equilibrium: treatment of cells with an active phorbol ester (PMA), binding of a mAb directed against the EGF receptor (mAb108), and truncation of most of the cytoplasmic domain of the receptor. These treatments reduced the rate at which low concentrations of EGF bound to cells, but did not affect the rate of EGF dissociation. We conclude that high-affinity EGF binding on living cells results from a difference in the apparent on rate of EGF binding. We then used these conditions and cell lines to test for the rate of EGF internalization at different concentrations of EGF. We demonstrate that internalization of the EGF receptor is stimulated roughly 50-fold at saturating concentrations of EGF, but is stimulated an additional two- to threefold at low concentrations (less than 1 nM). Four treatments reduce the rate of internalization of low concentrations of EGF to the rate seen at saturating EGF concentrations. Phorbol ester treatment and mAb108 binding to "wild type" receptor reduce this rate (and reduce high-affinity binding). Point mutation at Lys721 (kinase negative EGF receptor) and point mutation at Thr654 (removing a major site of protein kinase C phosphorylation) reduce the internalization rate, without affecting high-affinity binding. We suggest that while EGF stimulates endocytosis for all receptors, high-affinity receptors bind and are internalized more quickly than low-affinity receptors. Tyrosine kinase activity and the Thr654 region appear necessary for this response.

scholarly journals Hexokinase Regulates Kinetics of Glucose Transport and Expression of Genes Encoding Hexose Transporters inSaccharomyces cerevisiae

2000 ◽  
Vol 182 (23) ◽  
pp. 6815-6818 ◽  
Author(s):  
Thomas Petit ◽  
Jasper A. Diderich ◽  
Arthur L. Kruckeberg ◽  
Carlos Gancedo ◽  
Karel Van Dam
Keyword(s):  
Glucose Transport ◽  
Mrna Levels ◽  
High Affinity ◽  
Expression Of Genes ◽  
Hexose Transporters ◽  
Genes Encoding ◽  
High Concentrations ◽  
Kinetics Of ◽  
Very High ◽  
Null Strain

ABSTRACT Glucose transport kinetics and mRNA levels of different glucose transporters were determined in Saccharomyces cerevisiaestrains expressing different sugar kinases. During exponential growth on glucose, a hxk2 null strain exhibited high-affinity hexose transport associated with an elevated transcription of the genesHXT2 and HXT7, encoding high-affinity transporters, and a diminished expression of the HXT1 andHXT3 genes, encoding low-affinity transporters. Deletion ofHXT7 revealed that the high-affinity component is mostly due to HXT7; however, a previously unidentified very-high-affinity component (Km = 0.19 mM) appeared to be due to other factors. Expression of genes encoding hexokinases from Schizosaccharomyces pombe orYarrowia lipolytica in a hxk1 hxk2 glk1 strain prevented derepression of the high-affinity transport system at high concentrations of glucose.

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