Concerning the location of the GTP hydrolysis site on microtubules

1985 ◽  
Vol 63 (6) ◽  
pp. 422-429 ◽  
Author(s):  
Michael Caplow ◽  
John Shanks ◽  
Bruna Pegoraro Brylawski
Keyword(s):  
Steady State ◽  
Reaction Scheme ◽  
Product Inhibition ◽  
Microtubule Assembly ◽  
Assembly Process ◽  
Progressive Decrease ◽  
Reaction Conditions ◽  
Gtp Hydrolysis ◽  
Tubulin Subunit ◽  
A Site

The kinetics for GTP hydrolysis associated with microtubule assembly with microtubular protein has been analyzed under reaction conditions where tubulin–GDP does not readily assemble into microtubules. The GTPase rate is only slightly faster during the time when net microtubule assembly occurs, as compared with steady state. The slightly slower steady-state GTPase rate apparently results from GDP product inhibition, since the progressive decrease in the rate can be quantitatively accounted for using the previously determined GTP dissociation constant and the Ki value for GDP. Since the GTPase rate is not a function of the rate for net microtubule assembly, it is concluded that GTP hydrolysis is not required for tubulin subunit incorporation into microtubules. The constancy of the rate indicates that the GTPase reaction occurs at a site, the concentration of which does not change during the assembly process. This result is consistent with a reaction scheme in which GTP hydrolysis occurs primarily at microtubule ends. We propose that hydrolysis occurs at microtubule ends, at the interface between a long core of tubulin–GDP subunits and a short cap of tubulin–GTP subunits.

scholarly journals Dissociation of the Tubulin-sequestering and Microtubule Catastrophe-promoting Activities of Oncoprotein 18/Stathmin

1999 ◽  
Vol 10 (1) ◽  
pp. 105-118 ◽  
Author(s):  
Bonnie Howell ◽  
Niklas Larsson ◽  
Martin Gullberg ◽  
Lynne Cassimeris
Keyword(s):  
Tight Binding ◽  
Terminal Region ◽  
Microtubule Assembly ◽  
Gtp Hydrolysis ◽  
Truncated Protein ◽  
C Terminus ◽  
Dependent Change ◽  
Tubulin Subunit ◽  
Oncoprotein 18

Oncoprotein 18/stathmin (Op18) has been identified recently as a protein that destabilizes microtubules, but the mechanism of destabilization is currently controversial. Based on in vitro microtubule assembly assays, evidence has been presented supporting conflicting destabilization models of either tubulin sequestration or promotion of microtubule catastrophes. We found that Op18 can destabilize microtubules by both of these mechanisms and that these activities can be dissociated by changing pH. At pH 6.8, Op18 slowed microtubule elongation and increased catastrophes at both plus and minus ends, consistent with a tubulin-sequestering activity. In contrast, at pH 7.5, Op18 promoted microtubule catastrophes, particularly at plus ends, with little effect on elongation rates at either microtubule end. Dissociation of tubulin-sequestering and catastrophe-promoting activities of Op18 was further demonstrated by analysis of truncated Op18 derivatives. Lack of a C-terminal region of Op18 (aa 100–147) resulted in a truncated protein that lost sequestering activity at pH 6.8 but retained catastrophe-promoting activity. In contrast, lack of an N-terminal region of Op18 (aa 5–25) resulted in a truncated protein that still sequestered tubulin at pH 6.8 but was unable to promote catastrophes at pH 7.5. At pH 6.8, both the full length and the N-terminal–truncated Op18 bound tubulin, whereas truncation at the C-terminus resulted in a pronounced decrease in tubulin binding. Based on these results, and a previous study documenting a pH-dependent change in binding affinity between Op18 and tubulin, it is likely that tubulin sequestering observed at lower pH resulted from the relatively tight interaction between Op18 and tubulin and that this tight binding requires the C-terminus of Op18; however, under conditions in which Op18 binds weakly to tubulin (pH 7.5), Op18 stimulated catastrophes without altering tubulin subunit association or dissociation rates, and Op18 did not depolymerize microtubules capped with guanylyl (α, β)-methylene diphosphonate–tubulin subunits. We hypothesize that weak binding between Op18 and tubulin results in free Op18, which is available to interact with microtubule ends and thereby promote catastrophes by a mechanism that likely involves GTP hydrolysis.

scholarly journals Kinetochore microtubules shorten by loss of subunits at the kinetochores of prometaphase chromosomes

1991 ◽  
Vol 98 (2) ◽  
pp. 151-158 ◽  
Author(s):  
L. Cassimeris ◽  
E.D. Salmon
Keyword(s):  
Metaphase Plate ◽  
Force Generation ◽  
Microtubule Assembly ◽  
Immunofluorescent Staining ◽  
Chromosome Movement ◽  
Mitotic Spindles ◽  
Subunit Dissociation ◽  
Spindle Poles ◽  
Tubulin Subunit ◽  
A Site

The site of tubulin subunit dissociation was determined during poleward chromosome movement in prometaphase newt lung cell mitotic spindles using fluorescence photobleaching techniques and nocodazole-induced spindle shortening. Synchronous shortening of all kinetochore microtubules was produced by incubating cells in 17 microM nocodazole to block microtubule assembly. Under these conditions the spindle poles moved towards the metaphase plate at a rate of 3.6 +/− 0.4 microns min-1 (n = 3). On the basis of anti-tubulin immunofluorescent staining of cells fixed after incubation in nocodazole, we found that nonkinetochore microtubules rapidly disappeared and only kinetochore fibers were present after 60–90 s in nocodazole. To localize the site of tubulin subunit dissociation, a narrow bar pattern was photobleached across one half-spindle in prometaphase-metaphase cells previously microinjected with 5-(4,6-dichlorotriazin-2-yl) amino fluorescein (DTAF)-labeled tubulin. Immediately after photobleaching, cells were perfused with 17 microM nocodazole to produce shortening of kinetochore microtubules. Shortening was accompanied by a decrease in the distance between the bleach bar and the kinetochores. In contrast, there was little or no decrease in the distance between the bleach bar and the pole. Compared to their initial lengths, the average kinetochore to pole distance shortened by 18%, the bleach bar to kinetochore distance shortened by 28% and the average bleached bar to pole distance shortened by 1.6%. The data provide evidence that tubulin subunits dissociate from kinetochore microtubules at a site near the kinetochore during poleward chromosome movement. These results are consistent with models of poleward force generation for chromosome movement in which prometaphase-metaphase poleward force is generated in association with the kinetochore.

scholarly journals Site-Selective C-C Modification of Proteins at Neutral pH Using Organocatalyst-Mediated Cross Aldol Ligations

2018 ◽  
Author(s):  
Martin A. Fascione ◽  
Richard J. Spears ◽  
Robin L. Brabham ◽  
Darshita Budhadev ◽  
Tessa Keenan ◽  
...  
Keyword(s):  
Small Molecules ◽  
Stable Carbon ◽  
Biological Mechanisms ◽  
Large Excess ◽  
Reaction Conditions ◽  
Chemical Methods ◽  
Neutral Ph ◽  
Chemical Modification Of Proteins ◽  
A Site ◽  
Site Selective

The bioconjugation of proteins with small molecules has proved an invaluable strategy for probing and perturbing dynamic biological mechanisms. The general use of chemical methods for the functionalisation of proteins remains limited however by the frequent requirement for complicated reaction partners to be present in large excess, and harsh reaction conditions which are incompatible with many protein scaffolds. Herein we describe a site-selective organocatalyst-mediated protein aldol ligation (OPAL) that affords stable carbon-carbon linked bioconjugates at neutral pH under biocompatible conditions. OPAL enables rapid chemical modification of proteins within an hour using simple aldehyde probes in minimal excess, and is utilised here in the selective affinity tagging of proteins in cell lysate. Furthermore we demonstrate that the b-hydroxy aldehyde product of the OPAL can be functionalised a second time at neutral pH in a subsequent organocatalyst-mediated oxime ligation. This tandem strategy is showcased in the ‘chemical mimicry’ of a previously inaccessible natural dual post-translationally modified protein integral to the pathogenesis of the neglected tropical disease Leishmaniasis. <br>

scholarly journals Complejación de metales por sustancias húmicas acuáticas como proceso natural, tomando como caso de estudio el Lago de Xochimilco

2022 ◽  
Vol 61 (1) ◽  
pp. 55-65
Author(s):  
María de Jesús González-Guadarrama ◽  
Silvia Elena Castillo-Blum ◽  
María Aurora Armienta
Keyword(s):  
Trace Elements ◽  
Natural Habitat ◽  
Reaction Conditions ◽  
Aquatic Humic Substances ◽  
Direct Competition ◽  
Ph Conditions ◽  
Ecological Importance ◽  
Ph Buffer ◽  
Complexation Reactions ◽  
A Site

  Abstract 22 This work discusses the importance of organic matter, specifically Aquatic Humic Substances (SHA) 23 within the speciation and distribution of metals within an aquatic system, in this case Xochimilco 24 Lake, a site with great ecological importance. This lake is the natural habitat of the endemic species 25 “axolotl” (ajolote). In this research, complexation reactions between SHA and metals (Cu, Mn, Pb 26 and Zn) were carried out under different reaction conditions, the source of AHS was water samples 27 taken in Xochimilco Lake in presence and absence of pH buffer dissolution and varying the 28 concentration of metals. The results show that there is a direct competition between the major 29 elements and trace elements to react with the AHS. Under the pH conditions of Xochimilco Lake 30 complexes formation is possible. 31

scholarly journals Temperature tunability in Sr1−xCaxFeO3−δ for reversible oxygen storage: a computational and experimental study

2020 ◽  
Vol 8 (5) ◽  
pp. 2602-2612 ◽  
Author(s):  
Eric J. Popczun ◽  
De Nyago Tafen ◽  
Sittichai Natesakhawat ◽  
Chris M. Marin ◽  
Thuy-Duong Nguyen-Phan ◽  
...  
Keyword(s):  
Experimental Study ◽  
Oxygen Storage ◽  
Oxygen Carriers ◽  
Reaction Conditions ◽  
Specific Reaction ◽  
A Site

Sr1−xCaxFeO3−δ oxygen carriers can be designed for specific reaction conditions through selective Ca2+ inclusion at the A-site.

The effect of GTP hydrolysis and transpeptidation on the arrangement of aminoacyl-tRNA at the A-site of Escherichia coli 70 S ribosomes

FEBS Letters ◽  
1985 ◽  
Vol 181 (2) ◽  
pp. 367-372 ◽  
Author(s):  
S.N. Vladimirov ◽  
D.M. Graifer ◽  
G.G. Karpova ◽  
Yu.P. Semenkov ◽  
V.I. Makhno ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gtp Hydrolysis ◽  
A Site

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.

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