Immobilization-dependent fluorescence of colchicine

Colchicine fluoresces when bound to tubulin but not in water, dioxane, or benzene. The basis of the fluorescence has now been investigated. Colchicine fluoresces in higher alcohols and shows a blue shift as a function of chain length. Glycerol produces a higher fluorescence efficiency and a further blue shift. Plots of 1/fluorescence versus T/η yield straight lines for both alcohols and glycerol/water mixtures. Fluorescence in glycerol/dimethyl sulfoxide mixtures, in which the dielectric constant remains unchanged, varies as a function of solvent viscosity. Even highly nonpolar solvents such as dioxane require a threshold viscosity for fluorescence to occur. When solvent polarity was decreased at constant viscosity, there was also an enhancement of colchicine fluorescence, but this effect appeared to be smaller than that obtained with increasing viscosity. Immobilization by covalent attachment of desacetylcolchicine to thyroglobulin, serum albumin, or lysozyme also promotes fluorescence from the drug. By contrast, the highly rigid analogue of colchicine, imerubine, fluoresces in water and is unaffected by viscosity changes. We concluded that a major contribution to colchicine fluorescence stems from immobilization of colchicine in the site and that this response to immobilization depends, in part, on the partially flexible nature of the drug. Since certain other flexible molecules such as auramine O, reduced flavines, and diarylalkanes also require increased viscosity or binding to macromolecules to fluoresce at room temperature, we propose that immobilization-enhanced fluorescence may be more common than heretofore believed

Maytansine binding to the vinblastine sites of tubulin

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Membrane-bound tubulin in brain and thyroid tissue

Brain and thyroid tissue contain membrane-bound colchicine-binding activity that is not due to contamination by loosely bound cytoplasmic tubulin. This activity can be solubilized to the extent of 80 to 90% by treatment with 0.2% Nonidet P-40 with retention of colchicine binding. Extracts so obtained contain a prominent protein band in disc gel electrophoresis that co-migrates with tubulin. Membranes, and the solubilized protein therefrom, exhibit ligand binding properties like tubulin; for colchicine the KA is ~1 x 106 M-1 in brain and ~0.6 x 106 M-1 in thyroid; for vinblastine the KA is ~8 x 106 M-1 for both tissues; and for podophyllotoxin the Ki is ~2 x 10-6 M for both tissues. Displacement by analogues of colchicine is of the same order as for soluble tubulin. Although membrane-bound colchicine-binding activity shows greater thermal stability and a higher optimum binding temperature (54° versus 37°) than soluble tubulin, this appears to be the result of the membrane environment since the solubilized binding activity behaves like the soluble tubulin. Antibody against soluble brain tubulin reacts with membranes and solubulized colchicine-binding activity from both brain and thyroid gland. We conclude that brain and thyroid membrane preparations contain firmly bound tubulin or a very similar protein

Podophyllotoxin as a probe for the colchicine binding site of tubulin

The binding of [3H]podophyllotoxin to tubulin, measured by a DEAE-cellulose filter paper method, occurs with an affinity constant of 1.8 x 106 M-1 (37° at pH 6.7). Like colchicine, ~0.8 mol of podophyllotixin are bound per mol of tubulin dimer, and the reaction is entropy-driven (43 cal deg-1 mol-1). At 37° the association rate constant for podophyllotoxin binding is 3.8 x 106 M-1 h-1, ~10 times higher than for colchicine; this is reflected in the activation energies for binding which are 14.7 kcal/mol for podophyllotoxin and 20.3 kcal/mol for colchicine. The dissociation rate constant for the tubulin-podophyllotoxin complex is 1.9 h-1, and the affinity constant calculated from the ratio of the rates is close to that obtained by equilibrium measurements. Podophyllotxin and colchicine are mutually competitive inhibitors. This can be ascribed to the fact that both compounds have a trimethoxyphenyl ring and analogues of either compound with bulky substituents in their trimethoxyphenyl moiety are unable to inhibit the the binding of either of the two ligands. Tropolone, which inhibits colchicine binding competitively, has no effect on the podophyllotoxin/tubulin reaction. Conversely, podophyllotoxin does not influence tropolone binding. Moreover, the tropolone binding site of tubulin does not show the temperature and pH lability of the colchicine and podophyllotoxin domains, hence this lability can be ascribed to the trimethoxyphenyl binding region of tubulin. Since podophyllotoxin analogues with a modified B ring do not bind, it is concluded that both podophyllotoxin and colchicine each have at least two points of attachment to tubulin and that they share one of them, the binding region of the trimethoxyphenyl moiety

Scaling Analysis of the Site-Diluted Ising Model in Two Dimensions

A combination of recent numerical and theoretical advances are applied to analyze the scaling behaviour of the site-diluted Ising model in two dimensions, paying special attention to the implications for multiplicative logarithmic corrections. The analysis focuses primarily on the odd sector of the model (i.e., that associated with magnetic exponents), and in particular on its Lee-Yang zeros, which are determined to high accuracy. Scaling relations are used to connect to the even (thermal) sector, and a first analysis of the density of zeros yields information on the specific heat and its corrections. The analysis is fully supportive of the strong scaling hypothesis and of the scaling relations for logarithmic corrections.Comment: 15 pages, 3 figures. Published versio

Risso's dolphins alter daily resting pattern in response to whale watching at the Azores

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Tubulin subunit carboxyl termini determine polymerization efficiency

Cleavage of tubulin by subtilisin removes a small (Mr < 2000) fragment from the C-terminal end of both α and β subunits. The resulting protein is much reduced in negative charge. The cleaved, less acidic protein retains its competence to polymerize in a GTP-dependent and cold-, GDP-, and podophyllotoxin-sensitive manner and assembles into sheets or bundles of twisted filaments. The critical concentration for polymerization of the cleaved protein is about 50-fold lower than that for intact tubulin. It is proposed that the C termini of the subunits normally impede polymerization