The binding of vincristine, vinblastine and colchicine to tubulin

Owellen, Richard J.; Owens, Albert H.; Donigian, Douglas W.

doi:10.1016/0006-291x(72)90546-3

Cited by 258 publications

(100 citation statements)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3C). This is in agreement with published values, which vary between 0.7 and 1.0 (12,13). This variation is probably due to the known tendency of tubulin to lose colchicine-binding activity during storage.…”

Section: Methodssupporting

confidence: 93%

“…The value obtained by the isotopic method was 1.8 MM-1. Both constants are in good agreement with previously published values (12,14,15).…”

Section: Methodssupporting

confidence: 92%

“…3 with refs. 12, 14, and 15), the stoichiometry (12,13), the pH'optimum (22,25), and the effect of analogues (22,23). Because of the relatively low quantum efficiency of fluorescence (0.03), it is probable that the radioactive method is more sensitive, although it, in turn, is limited by the flow rate at high protein concentrations.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Promotion of Fluorescence upon Binding of Colchicine to Tubulin

Bhattacharyya

Wolff

1974

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

223

166

View full text Add to dashboard Cite

Colchicine, which does not fluoresce in aqueous media and organic solvents, exhibits marked fluorescence on combination with brain tubulin, with a corrected excitation maximum at 362 nm, an emission maximum at 435 nm, and a quantum yield of about 0.03.From fluorescence measurements it was found that rat brain tubulin binds 0.83 moles of colchicine per dimer (molecular weight 110,000) with an association constant of 3.2 ,M-1 at pH 7.0 A large body of evidence supports the view that microtubules from various sources are largely, if not completely, composed of tubulin, a dimeric protein of molecular weight 110,000-120,000 composed of two similar, but not identical, monomers (1). One of the most critical characteristics of tubulin is its ability to bind colchicine, an agent that leads to disaggregation of the polymerized form of this protein. The binding reaction of colchicine to tubulin has generally been studied by the use of labeled colchicine and subsequent separation of bound from free ligand by Sephadex gel filtration or ion exchange separation on DEAE-cellulose impregnated filter disc (2). Arai and Okuyama (3) recently pointed out that colchicine becomes fluorescent when bound to tubulin. We report here that fluorescence measurements offer a convenient method for measuring the colchicine tubulin interaction, which is based on the fact that colchicine fluoresces only in the bound form and not in the free state so that separation is not required. Thus, kinetic and thermodynamic parameters are easily obtained under equilibrium conditions. METHODS AND MATERIALSTubulin was prepared from rat brains in 10 mM Na phosphate (pH 7.0), 10 mM MgCl2, and by the procedure of Weisenberg et al. (2) except that DEAE-cellulose was used in place of DEAE-Sephadex. The purified tubulin solution was rapidly frozen in small aliquots and stored at -20°. The protein used gave a single band in sodium dodecyl sulfate-polyacrylamide gels (4), and its amino-acid analysis agreed well with that of Weisenberg et al. (2). The concentration of protein was determined by the method of Lowry et al. (5) spectrofluorometer, which gives corrected spectra in quanta band width. The quantum yields of colchicine-tubulin complexes were calculated by comparing the absorbances, at the exciting wavelength and the area of the emission spectra, of the colchicine-tubulin complex with quinine sulfate in 1.0 M H2SO4 whose quantum yield was taken as 0.546 at 250 (8).For convenience, routine analyses of tubulin-colchicine fluorescence were done with the Perkin-Elmer fluorometer. Calculation of Binding Data. Binding constants were obtained from fluorescence data by standard Scatchard analysis: r = (F,/FO) (CO/PO) where r = moles of colchicine boutd per mole of tubulin, Fc is the fluorescence of a given solution of colchicine-tubulin complex, and Fo is the fluorescence of an equal concentration of colchicine in excess tubulin, such that all the colchicine is bound, CO = the total colchicine concentration, and PO = the total protein concentration. From the ...

show abstract

Section: Methodssupporting

confidence: 93%

“…The value obtained by the isotopic method was 1.8 MM-1. Both constants are in good agreement with previously published values (12,14,15).…”

Section: Methodssupporting

confidence: 92%

See 1 more Smart Citation

Promotion of Fluorescence upon Binding of Colchicine to Tubulin

Bhattacharyya

Wolff

1974

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

223

166

View full text Add to dashboard Cite

show abstract

“…The trend represented by the plot of Figure 4 indicates that higher scoring complexes are generally among those with more favorable free energies of binding, while lower scoring complexes are generally those with unfavorable binding. The correlation equation: (1) has an r 2 = 0.5818 and a standard error of ± 0.52 kcal mol −1 . A better correlation is observed after omitting the outlier JG-05-3A from the correlation.…”

Section: Structure-activity-binding Relationshipsmentioning

confidence: 99%

“…Microtubules are essential elements of the cytoskeleton and extremely important in mitosis and cell division. Colchicine, the first drug known to bind to the tubulin protein [1,2], inhibits microtubule formation and causes loss of cellular microtubules. In contrast, paclitaxel and its analogues actually promote microtubule polymer formation [3][4][5], albeit by acting at a different site on tubulin than colchicine.…”

Section: Introductionmentioning

confidence: 99%

Docking and hydropathic scoring of polysubstituted pyrrole compounds with antitubulin activity

Tripathi

Fornabaio

Kellogg

et al. 2008

Bioorganic & Medicinal Chemistry

View full text Add to dashboard Cite

Compounds that bind at the colchicine site of tubulin have drawn considerable attention with studies indicating that these agents suppress microtubule dynamics and inhibit tubulin polymerization. Data for eighteen polysubstituted pyrrole compounds are reported, including antiproliferative activity against human MDA-MB-435 cells and calculated free energies of binding following docking the compounds into models of αβ-tubulin. These docking calculations coupled with HINT interaction analyses are able to represent the complex structures and the binding modes of inhibitors such that calculated and measured free energies of binding correlate with an r 2 of 0.76. Structural analysis of the binding pocket identifies important intermolecular contacts that mediate binding. As seen experimentally, the complex with JG-03-14 (3,5-dibromo-4-(3,4-dimethoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester) is the most stable. These results illuminate the binding process and should be valuable in the design of new pyrrole-based colchicine site inhibitors as these compounds have very accessible syntheses.

show abstract