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 Microtubule assembly kinetics. Changes with solution conditions

1987 ◽  
Vol 247 (3) ◽  
pp. 505-511 ◽  
Author(s):  
J S Barton ◽  
D L Vandivort ◽  
D H Heacock ◽  
J A Coffman ◽  
K A Trygg
Keyword(s):  
Activation Energy ◽  
Ionic Strength ◽  
Low Temperature ◽  
Number Concentration ◽  
High Ionic Strength ◽  
Microtubule Assembly ◽  
Microtubule Protein ◽  
Assembly Kinetics ◽  
Low Ionic Strength ◽  
Kinetics Of

The assembly kinetics of microtubule protein are altered by ionic strength, temperature and Mg2+, but not by pH. High ionic strength (I0.2), low temperature (T less than 30 degrees C) and elevated Mg2+ (greater than or equal to 1.2 mM) induce a transition from biphasic to monophasic kinetics. Comparison of the activation energy obtained for the fast biphasic step at low ionic strength (I0.069) shows excellent agreement with the values obtained at high ionic strength, low temperature and elevated Mg2+. From this observation it can be implied that the tubulin-containing reactant of the fast biphasic event is also the species that elongates microtubules during monophasic assembly. Second-order rate constants for biphasic assembly are 3.82(+/- 0.72) x 10(7) M-1.s-1 and 5.19(+/- 1.25) x 10(6) M-1.s-1, and for monophasic assembly the rate constant is 2.12(+/- 0.56) x 10(7) M-1.s-1. The microtubule number concentration is constant during elongation of microtubules for biphasic and monophasic assembly.

scholarly journals MICROTUBULE BIOGENESIS AND CELL SHAPE IN OCHROMONAS

1974 ◽  
Vol 61 (2) ◽  
pp. 514-536 ◽  
Author(s):  
David L. Brown ◽  
G. Benjamin Bouck
Keyword(s):  
Protein Synthesis ◽  
Cell Shape ◽  
Microtubule Assembly ◽  
Short Term ◽  
Protein Pool ◽  
Microtubule Protein ◽  
Kinetics Of ◽  
Mitotic Inhibitor ◽  
Kinetics Of Inhibition

The role of microtubules and microtubule nucleating sites in the unicell, Ochromonas has been examined through the use of two mitotic inhibitors, isopropyl N-phenylcarbamate (IPC) and isopropyl N-3-chlorophenyl carbamate (CIPC). Although IPC and CIPC have little or no effect on intact microtubules, the assembly of three separate sets of microtubules in Ochromonas has been found to be differentially affected by IPC and CIPC. The assembly of flagellar microtubules after mechanical deflagellation is partially inhibited; the reassembly of rhizoplast microtubules after pressure depolymerization is totally inhibited (however, macrotubules may form at the sites of microtubule initiation or elsewhere); and, the reassembly of the beak set of microtubules after pressure depolymerization may be unaffected although similar concentrations of IPC and CICP completely inhibit microtubule regeneration on the rhizoplast. These effects on microtubule assembly, either inhibitory or macrotubule inducing, are fully reversible. The kinetics of inhibition and reversal are found to be generally similar for both flagellar and cell shape regeneration. Incorporation data suggest that neither IPC nor CIPC has significant effects on protein synthesis in short term experiments. Conversely, inhibiting protein synthesis with cycloheximide has little effect on microtubule regeneration when IPC or CIPC is removed. Although the exact target for IPC and CIPC action remains uncertain, the available evidence suggests that the microtubule protein pool or the microtubule nucleating sites are specifically and reversibly affected. Comparative experiments using the mitotic inhibitor colchicine indicate some similarities and differences in its mode of action with respect to that of IPC and CIPC on assembly and disassembly of microtubules in these cells.

scholarly journals Microtubules and nucleoside diphosphate kinase. Comparison of kinetics of GTP- and CTP-induced assembly

1985 ◽  
Vol 232 (3) ◽  
pp. 657-662 ◽  
Author(s):  
K Islam ◽  
R G Burns
Keyword(s):  
Substrate Specificity ◽  
Kinase Activity ◽  
Nucleoside Diphosphate Kinase ◽  
Elongation Rate ◽  
Microtubule Assembly ◽  
Microtubule Protein ◽  
Assembly Kinetics ◽  
Kinetics Of

The kinetics of assembly of MAP2-tubulin microtubule protein were examined as a function of the GTP concentration in order to test the hypothesis that CTP-induced assembly results from the generation of GTP by nucleoside diphosphate kinase. These studies show that (a) there is no assembly below a minimum GTP concentration and that this represents a nucleation requirement, (b) the rate of elongation is inconsistent with a single assembly-species, and (c) the elongation rate increases markedly as the GTP concentration is raised, although GTP is not absolutely required for elongation. These assembly kinetics have been compared with those with increasing CTP concentrations, by using microtubule protein containing a very low nucleoside diphosphate kinase activity of known substrate specificity. Neither nucleation nor the observed rates of elongation can be attributed to the formation of GTP, either (a) in terms of the generation of free GTP and subsequent binding to tubulin or (b) by the direct charging of GDP bound to the tubulin exchangeable site. The results show that nucleoside diphosphate kinase is not required for CTP-induced microtubule assembly, and suggest that CTP directly effects microtubule assembly.

scholarly journals Phosphorylation of the microtubule-associated protein MAP2 by GTP

1984 ◽  
Vol 224 (2) ◽  
pp. 623-627 ◽  
Author(s):  
R G Burns ◽  
K Islam
Keyword(s):  
Steady State ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinetic Parameters ◽  
Microtubule Assembly ◽  
Chick Brain ◽  
Chemically Modified ◽  
Microtubule Protein ◽  
Independent Protein

The chick brain microtubule-associated protein MAP2 can be phosphorylated in vitro to the extent of 12 mol/mol with GTP at the same sites as can be labelled by the cyclic AMP-independent protein kinase utilizing [gamma-32P]ATP as the phosphoryl donor. Consequently, the microtubule protein is chemically modified by the conditions usually employed for studies of microtubule assembly, so that the derived kinetic parameters may not relate to steady-state conditions.

scholarly journals Assembly of chick brain MAP2-tubulin microtubule protein. Analysis of tubulin subunit flux rates by immunofluorescence microscopy

1991 ◽  
Vol 277 (1) ◽  
pp. 245-253 ◽  
Author(s):  
M F Symmons ◽  
R G Burns
Keyword(s):  
Close Agreement ◽  
Immunofluorescence Microscopy ◽  
Dynamic Instability ◽  
Theoretical Models ◽  
Chick Brain ◽  
Gtp Hydrolysis ◽  
Tubulin Subunit ◽  
Microtubule Protein ◽  
Cap Model ◽  
Microscopy Method

A filter-based immunofluorescence-microscopy method for obtaining microtubule lengths has been developed and evaluated. Kinetic constants and mean lengths obtained show close agreement with those obtained by complementary methods applied to chick brain MAP2-tubulin microtubule protein in NaCl-supplemented buffer. The filter-based method has been used to estimate tubulin subunit flux (Jon) resulting from isothermal dilution of microtubule populations to various free tubulin concentrations, (c). This experimental Jon(c) plot is significantly different from that predicted by a variety of theoretical models, but is consistent with a ‘lateral cap’ model of dynamic instability [Bayley, Schilstra & Martin (1990) J. Cell. Sci. 95, 33-48] adapted to accommodate the observed vectorial GTP hydrolysis.

Kinetics of hydrolysis of peroxodisulphate ions in acidic medium to peroxomonosulphuric acid

1981 ◽  
Vol 46 (5) ◽  
pp. 1229-1236 ◽  
Author(s):  
Jan Balej ◽  
Milada Thumová
Keyword(s):  
Ionic Strength ◽  
Rate Constants ◽  
Acidic Medium ◽  
Measured Data ◽  
Molar Ratios ◽  
Starting Solution ◽  
Kinetics Of Hydrolysis ◽  
Rate Of Hydrolysis ◽  
Kinetics Of ◽  
Hydrolysis Of

The rate of hydrolysis of S2O82- ions in acidic medium to peroxomonosulphuric acid was measured at 20 and 30 °C. The composition of the starting solution corresponded to the anolyte flowing out from an electrolyser for production of this acid or its ammonium salt at various degrees of conversion and starting molar ratios of sulphuric acid to ammonium sulphate. The measured data served to calculate the rate constants at both temperatures on the basis of the earlier proposed mechanism of the hydrolysis, and their dependence on the ionic strength was studied.

scholarly journals Thermodynamic and detailed kinetic study of the formation of orthophthalaldehyde-agmatine complex by fluorescence intensities

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Khémesse Kital ◽  
Moumouny Traoré ◽  
Diégane Sarr ◽  
Moussa Mbaye ◽  
Mame Diabou Gaye Seye ◽  
...  
Keyword(s):  
Complex Formation ◽  
Thermodynamic Parameters ◽  
Initial Rate ◽  
Reaction Medium ◽  
Standard Entropy ◽  
Step Size ◽  
Formation Reaction ◽  
Different Temperatures ◽  
Kinetics Of ◽  
Complex Formation Process

Abstract The aim of this work is to determine the thermodynamic parameters and the kinetics of complex formation between orthophthalaldehyde (OPA) and agmatine (AGM) in an alkaline medium (pH 13). Firstly, the association constant (Ka) between orthophthalaldehyde and agmatine was determined at different temperatures (between 298 K and 338 K) with a step size of 10 K. Secondly, the thermodynamic parameters such as standard enthalpy (ΔH°), standard entropy (ΔS°),and Gibbs energy (∆G) were calculated, where a positive value of ΔH° (+45.50 kJ/mol) was found, which shows that the reaction is endothermic. In addition, the low value of ΔS°(+0.24 kJ/mol) indicates a slight increase in the disorder in the reaction medium. Furthermore, the negative values of ΔG between −35.62 kJ/mol and −26.02 kJ/mol show that the complex formation process is spontaneous. Finally, the parameters of the kinetics of the reaction between OPA and AGM were determined as follows: when the initial concentration of AGM (5 × 10−6 M) is equal to that of the OPA, the results show that the reaction follows an overall 1.5 order kinetics with an initial rate of 5.1 × 10−7Mmin−1 and a half-life of 8.12 min. The partial order found in relation to the AGM is 0.8. This work shows that the excess of OPA accelerates the formation reaction of the complex.

High field 1H NMR Studies of Sol-Gel Kinetics

1986 ◽  
Vol 73 ◽  
Author(s):  
Bruce D. Kay ◽  
Roger A. Assink
Keyword(s):  
Equilibrium Distribution ◽  
Sol Gel ◽  
Nmr Studies ◽  
H Nmr ◽  
Rate Limiting Step ◽  
Rate Of Hydrolysis ◽  
Acid Catalyzed ◽  
Product Species ◽  
Rate Limiting ◽  
Kinetics Of

ABSTRACTHigh resolution 1H NMR spectroscopy at high magnetic fields is employed to study the reaction kinetics of the Si(OCH3)4:CH3OH:H2O sol-gel system. Both the overall extent of reaction as a function of time and the equilibrium distribution of species are measured. In acid catalyzed solution, condensation is the rate limiting step while in base catalyzed solution, hydrolysis becomes rate limiting. A kinetic model in which the rate of hydrolysis is assumed to be independent of the adjacent functional groups is presented. This model correctly predicts the distribution of product species during the initial stages of the sol-gel reaction.

The mechanism of the oxidation of thiocyanate ion by peroxomonosulphate in aqueous solution. II. Kinetics of the reaction

1966 ◽  
Vol 19 (8) ◽  
pp. 1365 ◽  
Author(s):  
RH Smith ◽  
IR Wilson
Keyword(s):  
Aqueous Solution ◽  
Initial Rate ◽  
Stopped Flow ◽  
Thiocyanate Ion ◽  
First Order ◽  
Conductance Method ◽  
Rate Of Reaction ◽  
Kinetics Of

Initial rates of reaction for the above oxidation have been measured by a stopped-flow conductance method. Between pH 2 and 3.6, the initial rate of reaction, R, is given by the expression R{[HSO5-]+[SCN-]} = {kb+kc[H+]}[HSO5-]0[SCN-]20+ka[H+]-1[HSO5]20[SCN-]0 As pH increases, there is a transition to a pH-independent rate, first order in each thiocyanate and peroxomonosulphate concentrations.

scholarly journals The assembly of microtubule protein in vitro. The kinetic role in microtubule elongation of oligomeric fragments containing microtubule-associated proteins

1985 ◽  
Vol 227 (2) ◽  
pp. 439-455 ◽  
Author(s):  
P M Bayley ◽  
F M M Butler ◽  
D C Clark ◽  
E J Manser ◽  
S R Martin
Keyword(s):  
Self Assembly ◽  
Protein Concentration ◽  
Wavelength Dependence ◽  
Electrophoretic Analysis ◽  
Slow Phase ◽  
Condensation Polymerization ◽  
Fast Phase ◽  
Microtubule Protein ◽  
Kinetics Of

The kinetics of assembly were studied for bovine and pig microtubule protein in vitro over a range of conditions of pH, temperature, nucleotide and protein concentration. The kinetics are in general biphasic with two major processes of similar amplitude but separated in rate by one order of magnitude. Rates and amplitudes are complex functions of solution conditions. The rates of the fast phase and the slow phase attain limiting values as a function of increasing protein concentration, and are more stringently limited at pH 6.5 than pH 6.95. Such behaviour indicates that mechanisms other than the condensation polymerization of tubulin dimer become rate-limiting at higher protein concentration. The constancy of the wavelength-dependence of light-scattering and ultrastructural criteria indicate that microtubules of normal morphology are formed in both phases of the assembly process. Electrophoretic analysis of assembling microtubule protein shows that MAP- (microtubule-associated-protein-)rich microtubules are formed during the fast phase. The rate of dissociation of oligomeric species on dilution of microtubule protein closely parallels the fast-phase rate in magnitude and temperature-dependence. We propose that the rate of this process constitutes an upper limit to the rate of the fast phase of assembly. The kinetics of redistribution of MAPs from MAP-rich microtubules may be a factor limiting the slow-phase rate. A working model is derived for the self-assembly of microtubule protein incorporating the dissociation and redistribution mechanisms that impose upper limits to the rates of assembly attainable by bimolecular addition reactions. Key roles are assigned to MAP-containing fragments in both phases of microtubule elongation. Variations in kinetic behaviour with solution conditions are inferred to derive from the nature and properties of fragments formed from oligomeric species after the rapid temperature jump. The model accounts for the limiting rate behaviour and indicates experimental criteria to be applied in evaluating the relative contributions of alternative pathways.

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