Direct photoaffinity labeling of tubulin with colchicine.

Wolff, J.; Knipling, Leslie; Cahnmann, H. J.; Palumbo, G. J.

doi:10.1073/pnas.88.7.2820

Cited by 64 publications

(48 citation statements)

References 29 publications

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“…In late stages of irradiation, or with "damaged" tubulin, label also appeared in a-tubulin (9). These results suggested the possibility that the colchicine-binding domain on 3-tubulin might be near a-tubulin.…”

Section: Example Low-mentioning

confidence: 53%

“…Whether these bands are precursor peptides or provide other binding surfaces remains to be determined. Moreover, the region of a-tubulin that is near enough to the colchicine-binding site on ,B-tubulin to become labeled under mild denaturing conditions (6,9,10) remains to be identified. Nonetheless, our findings identify two regions of 3-tubulin that are important structural elements of the colchicine-binding site.…”

Section: Resultsmentioning

confidence: 99%

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Localization of the colchicine-binding site of tubulin.

Uppuluri

Knipling

Sackett

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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142

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We have previously shown that rat brain tubulin, a heterodimer consisting of an a and fi monomer, can be covalently labeled with [3H]colchicine by near UV irradiation. Most of the label appears in (&tubulin. We show here that (3-tubulin can be separated and purified from SDS preparative gels and analyzed by proteolysis. Chymotrypsin yielded a labeled -4-kDa band that contained two peptides. Tryptic digestion also yielded an =4-kDa band containing two peptides. Sequence analysis revealed a peptide ofresidues 1-36 and 213-242 for chymotrypsin and a peptide of residues 1-46 and 214-241 for trypsin. To identify which peptide carried the label, limited hydrolysis of -tubulin was done with trypsin; this procedure yielded a labeled 16-kDa N-terminal peptide and a 35-kDa C-terminal peptide, as identified by antibodies. Isolation of these peptides and extensive digestion with trypsin yielded two labeled peptides corresponding to residues 1-46 from the 16-kDa N-terminal fragment and residues 214-241 from the 35-kDa C-terminal fragment. These results show that at least two regions in P-tubulin are specifically involved in colchicine binding and that the span of the colchicine molecule, s11 A, bridges these two regions in the native /3 monomer.Colchicine is the most studied of all antimitotic agents. It binds to tubulin with a stoichiometry of one and inhibits microtubule assembly substoichiometrically. Colchicine binding to tubulin exhibits pseudoirreversible kinetics; it displays a fast step followed by a slow step involving conformational changes of both ligand and tubulin. The latter, in turn, promote fluorescence characteristic of the tropolone moiety of colchicine. Many analogs have been studied, resulting in the proposal that the A and C rings of colchicine both bind to specific loci, whereas the B ring is primarily a regulator of binding kinetics (see refs. 1 and 2 and the references therein).Attempts to localize the binding site(s) in the tubulin dimer have led to conflicting results. Studies with bromacetylcolchicine indicated binding to a-tubulin (3), but these results have been criticized on the basis of the nonspecificity of the alkylation reaction. Proteolytic studies of the colchicinetubulin complex yielded a-tubulin-derived, colchicinebearing peptides of 16-18 kDa; because z90% of the label bound to the protein was lost during processing, conclusions are uncertain (4). Others (5) have also shown colchicine localization to a-tubulin by using a photoaffinity label with a long spacer arm. A shorter spacer arm led to colchicine binding on both a-and ,B-tubulins (6), suggesting that localization was a function ofthe spacer length. On the other hand, Luduena and Roach (7) [3H]colchicine that the label is localized almost exclusively on 3-tubulin. In late stages of irradiation, or with "damaged" tubulin, label also appeared in a-tubulin (9). These results suggested the possibility that the colchicine-binding domain on 3-tubulin might be near a-tubulin. That proposal was strengthened by the finding that co...

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Section: Example Low-mentioning

confidence: 53%

Section: Resultsmentioning

confidence: 99%

Localization of the colchicine-binding site of tubulin.

Uppuluri

Knipling

Sackett

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

142

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“…By direct photoaffinity labeling with vinblastine (17) and with two photoactive derivatives of vinblastine (18,19), there was greater labeling of ␣-tubulin than ␤-tubulin, ranging from 57 to 75% of the incorporated radiolabel being in the ␣-subunit. A photoaffinity analog of maytansine was incorporated in about a 4:5 ratio into ␣-and ␤-tubulin, respectively (20).…”

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confidence: 99%

Direct Photoaffinity Labeling by Dolastatin 10 of the Amino-terminal Peptide of β-Tubulin Containing Cysteine 12

Bai

Covell

Taylor

et al. 2004

Journal of Biological Chemistry

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The subunit protein of microtubules, the ␣␤-tubulin heterodimer, has a number of ligand-binding sites. These include the exchangeable and nonexchangeable GTP-binding sites and at least three reasonably well characterized sites that bind antimitotic drugs. The electron crystallographic model of the tubulin sheet polymer formed in the presence of zinc and paclitaxel and composed of antiparallel protofilaments has provided relatively detailed information about the locations of the nucleotide-binding sites and the paclitaxel site on the ␣␤-dimer (1).

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“…In spite of the intensive study of colchicine-tubulin interaction, there is no consensus about the location of the colchicine binding site on tubulin. Some studies have placed the colchicine binding site on the a subunit [2], some on the p subunit [3] and some at the interface [4].Luduena and co-workers [5] have used cross linkers to crosslink two sulfhydryl groups which are at the colchicine binding site. They have shown that these two sulfhydryl groups are protected from chemical modification by colchicine and its analogs and identified them as Cys239 and Cys354 of the p subunit [6, 71.…”

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confidence: 99%

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confidence: 99%

A study of colchicine tubulin complex by donor quenching of fluorescence energy transfer

Bhattacharyya

Roy

1993

European Journal of Biochemistry

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The utility of collisional quenching of energy donors in fluorescence energy transfer is described. In multi-donor single acceptor systems, which contain different classes of donors (as distinguished by their accessibility towards a collisional quencher), donor quenching may be used to assess the fraction of energy transfer from each class of donor. The tubulin-colchicine complex was used as a donor-acceptor system to show that two inaccessible tryptophans are at or near the colchicine binding site.Tubulin (a heterodimer ap>, the major component of microtubules, binds the plant alkaloid colchicine, specifically and quasi-irreversibly at a single site and inhibits tubulin polymerization [l]. In spite of the intensive study of colchicine-tubulin interaction, there is no consensus about the location of the colchicine binding site on tubulin. Some studies have placed the colchicine binding site on the a subunit [2], some on the p subunit [3] and some at the interface [4].Luduena and co-workers [5] have used cross linkers to crosslink two sulfhydryl groups which are at the colchicine binding site. They have shown that these two sulfhydryl groups are protected from chemical modification by colchicine and its analogs and identified them as Cys239 and Cys354 of the p subunit [6, 71.One approach for localizing a binding site on a protein is to measure distances from fixed points in proteins by fluorescence energy transfer. Colchicine, which is non-fluorescent in solution, fluoresces upon binding to tubulin [8]. Its excitation spectrum overlaps well with the emission spectrum of tryptophan, thus forming a good donor-acceptor pair. The presence of eight tryptophans in tubulin, however, makes it difficult to have even a qualitative assessment of the involvement of various tryptophan residues in energy transfer to bound colchicine. Donor quenching of fluorescence energy transfer has previously been used to estimate distance distribution between a donor-acceptor pair [9]. In this experiment, a collisional quencher is used which quenches donor fluorescence causing a reduction in R , and hence energy transfer efficiency. A study with model peptide and protein with single indole and dansyl moieties has firmly established the feasibility of this approach [9]. In a multi-tryptophan protein, where classes of tryptophan residues differ in accessibility towards a collisional quencher, donor quenching may be used to assess the degree of energy transfer from different classes. We have explored this concept in the colchicine-tubulin complex using acrylamide as the collisional quencher.

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Direct photoaffinity labeling of tubulin with colchicine.

Cited by 64 publications

References 29 publications

Localization of the colchicine-binding site of tubulin.

Localization of the colchicine-binding site of tubulin.

Direct Photoaffinity Labeling by Dolastatin 10 of the Amino-terminal Peptide of β-Tubulin Containing Cysteine 12

A study of colchicine tubulin complex by donor quenching of fluorescence energy transfer

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