Interaction of 6-mercapto-GTP with bovine brain tubulin. Equilibrium aspects.

Fishback, James L.; Yarbrough, Lynwood R.

doi:10.1016/s0021-9258(17)43502-2

Cited by 29 publications

(16 citation statements)

References 30 publications

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“…Fluorescence Stopped-Flow Studies of Binding of S6-GTP to Tu(f). Binding of S6-GTP to Tu(-) is accompanied by an approximately 15% quenching of intrinsic tubulin fluorescence (Fishback & Yarbrough, 1984). Using fluorescence stopped-flow technique, we have examined the binding of S6-GTP FIGURE 2: Dependence of 1 /r, on S6-GTP concentration.…”

Section: Resultsmentioning

confidence: 99%

“…Chemicals. S6-GTP and S6-GDP were synthesized as described previously (Fishback & Yarbrough, 1984). All other chemicals were of reagent grade.…”

Section: Methodsmentioning

confidence: 99%

“…we have synthesized the analogues S6-GTP and S6-GDP and used them to characterize the binding process. We have previously described the equilibrium aspects of this interaction (Fishback & Yarbrough, 1984). In this paper we describe the kinetic aspects of binding and exchange of these analogues.…”

mentioning

confidence: 99%

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Kinetics of interaction of 2-amino-6-mercapto-9-.beta.-ribofuranosylpurine 5'-triphosphate with bovine brain tubulin

Yarbrough¹,

Fishback²

1985

Biochemistry

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The binding of the guanine nucleotide analogue 2-amino-6-mercapto-9-beta-ribofuranosylpurine 5'-triphosphate (S6-GTP) to tubulin from which the associated proteins and exchangeably bound nucleotide have been removed produces about a 15% decrease in intrinsic tubulin fluorescence. Using a fluorescence stopped-flow technique, we have examined the kinetics and mechanism of this process. Analysis of the data reveals that the binding is complex, involving at least one conformational change subsequent to nucleotide binding. The bimolecular association rate constant for binding of S6-GTP to tubulin is approximately 6 X 10(5) M-1 s-1, suggesting that the orientation requirements are stringent. The kinetic parameters for dissociation of GDP, S6-GTP, and S6-GDP from the exchangeable nucleotide binding site have also been determined. S6-GDP and GDP were found to have comparable rates of dissociation; S6-GTP dissociated approximately twice as slowly as either GDP or S6-GDP. Glycerol produces a significant decrease in the rates of nucleotide dissociation. The mechanism whereby glycerol produces such an effect is not known; however, it may involve slight changes in the conformation of the tubulin protomer.

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Section: Resultsmentioning

confidence: 99%

“…Chemicals. S6-GTP and S6-GDP were synthesized as described previously (Fishback & Yarbrough, 1984). All other chemicals were of reagent grade.…”

Section: Methodsmentioning

confidence: 99%

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Kinetics of interaction of 2-amino-6-mercapto-9-.beta.-ribofuranosylpurine 5'-triphosphate with bovine brain tubulin

Yarbrough¹,

Fishback²

1985

Biochemistry

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“…The binding constants of mant-GTP to the tubulin dimer and the adduct were measured employing an anisotropy fluorescence assay as described. 51 The anisotropy of a 2 μM solution of mant-GTP in 10 mM NaPi/1.5 mM MgCl 2 pH 7.0 was measured as described 51 in the presence of growing amounts of apo-tubulin or apo-tubulin− zampanolide-adduct (depleted of the E-site nucleotide with active charcoal as described 52 ). The fraction of intact E-site binding sites in apo-tubulin (0.29 ± 0.04) and apo-tubulin−zampanolide-adduct (0.30 ± 0.02) after the active charcoal treatment was determined by measuring the recovery of GTP binding of the treated protein.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

Zampanolide Binding to Tubulin Indicates Cross-Talk of Taxane Site with Colchicine and Nucleotide Sites

et al. 2017

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The marine natural product zampanolide and analogues thereof constitute a new chemotype of taxoid site microtubule-stabilizing agents with a covalent mechanism of action. Zampanolide-ligated tubulin has the switch-activation loop (M-loop) in the assembly prone form and, thus, represents an assembly activated state of the protein. In this study, we have characterized the biochemical properties of the covalently modified, activated tubulin dimer, and we have determined the effect of zampanolide on tubulin association and the binding of tubulin ligands at other binding sites. Tubulin activation by zampanolide does not affect its longitudinal oligomerization but does alter its lateral association properties. The covalent binding of zampanolide to β-tubulin affects both the colchicine site, causing a change of the quantum yield of the bound ligand, and the exchangeable nucleotide binding site, reducing the affinity for the nucleotide. While these global effects do not change the binding affinity of 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC) (a reversible binder of the colchicine site), the binding affinity of a fluorescent analogue of GTP (Mant-GTP) at the nucleotide E-site is reduced from 12 ± 2 × 10 M in the case of unmodified tubulin to 1.4 ± 0.3 × 10 M in the case of the zampanolide tubulin adduct, indicating signal transmission between the taxane site and the colchicine and nucleotide sites of β-tubulin.

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“…These values ranged from 1.5 to 6.7, with an average of 2.7 (a dimensionless constant). It thus appears that the affinity of tubulin for GTP [in 0.1 M Mes (pH 7.0)-0.2 mM MgCl2] is about 3 times greater than its affinity for GDP, a conclusion not greatly different from that reached by several other groups, generally by more indirect methods (Aral et al, 1975;Carlier & Pantaloni, 1978;Zeeberg & Caplow, 1979;Fishback & Yarbrough, 1984).…”

Section: Figure Ib Demonstrates the Progressive Inhibition Of Po-mentioning

confidence: 69%

Interrelationships of tubulin-GDP and tubulin-GTP in microtubule assembly

Lin¹,

Hamel²

1987

Biochemistry

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We previously reported that direct incorporation of GDP (i.e., without an initial hydrolysis of GTP) into microtubules occurs throughout an assembly cycle in a constant proportion. The exact proportion varied with reaction conditions, becoming greater under all conditions in which tubulin-GDP increased relative to tubulin-GTP (low Mg2+ and GTP concentrations, high tubulin concentrations, and in the presence of exogenous GDP). These findings led us to explore further interrelationships of tubulin-GDP and tubulin-GTP in microtubule assembly. We have now determined the minimum amount of tubulin-GTP required for the initiation of microtubule assembly and the relative efficiency with which tubulin-GDP participates in microtubule elongation. When GTP, GDP, and tubulin concentrations were varied at a constant Mg2+ concentration (0.2 mM), initiation of assembly required that 35% of the nucleotide-bearing tubulin be in the form of tubulin-GTP, and incorporation of tubulin-GDP into microtubules during elongation was only 60% as efficient as would be predicted on the basis of its proportional concentration in the reaction mixtures. Very different results were obtained when the Mg2+ concentration was varied. Even though Mg2+ enhances the binding of GTP to tubulin (the equilibrium constant for the exchange of GTP for GDP was 0.2 in the absence of exogenous Mg2+, 3 with 0.2 mM Mg2+, 5 with 0.5 mM Mg2+, and 11 with 2 and 4 mM Mg2+), as Mg2+ was increased the proportion of tubulin-GTP required for the initiation of microtubule assembly rose greatly, and the direct incorporation of tubulin-GDP into microtubules during elongation became progressively more efficient. In the absence of exogenous Mg2+, only 20% tubulin-GTP was required for initiation, and tubulin-GDP was directly incorporated into microtubules half as efficiently as would be predicted on the basis of its concentration in the reaction mixture. At the highest Mg2+ concentration examined (4 mM), 80% tubulin-GTP was required for initiation of assembly, and tubulin-GDP was incorporated into microtubules as efficiently as tubulin-GTP.

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Interaction of 6-mercapto-GTP with bovine brain tubulin. Equilibrium aspects.

Cited by 29 publications

References 30 publications

Kinetics of interaction of 2-amino-6-mercapto-9-.beta.-ribofuranosylpurine 5'-triphosphate with bovine brain tubulin

Kinetics of interaction of 2-amino-6-mercapto-9-.beta.-ribofuranosylpurine 5'-triphosphate with bovine brain tubulin

Zampanolide Binding to Tubulin Indicates Cross-Talk of Taxane Site with Colchicine and Nucleotide Sites

Interrelationships of tubulin-GDP and tubulin-GTP in microtubule assembly

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