Visualization of lactoperoxidase binding to microtubule and tubulin

The binding of lactoperoxidase to microtubules and tubulin was shown in both electron micrography and polyacrylamide gel electrophoresis by tracing the enzymatic activity of lactoperoxidase. Lactoperoxidase bound to purified microtubules appeared to distribute evenly on the surface without forming special structures. Both alpha and beta-tubulin separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis bound lactoperoxidase, and could be detected by the use of lactoperoxidase reaction. Electrophoretic study revealed that the interaction between lactoperoxidase and tubulin were not strictly specific and a variety of proteins other than alpha- and beta-tubulin, including actin and neurofilament subunits, bound lactoperoxidase.     

Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure

Tubulin undergoes posttranslational modifications proposed to specify microtubule subpopulations for particular functions. Most of these modifications occur on the C-termini of tubulin and may directly affect the binding of microtubule-associated proteins (MAPs) or motors. Acetylation of Lys-40 on α-tubulin is unique in that it is located on the luminal surface of microtubules, away from the interaction sites of most MAPs and motors. We investigate whether acetylation alters the architecture of microtubules or the conformation of tubulin, using cryo–electron microscopy (cryo-EM). No significant changes are observed based on protofilament distributions or microtubule helical lattice parameters. Furthermore, no clear differences in tubulin structure are detected between cryo-EM reconstructions of maximally deacetylated or acetylated microtubules. Our results indicate that the effect of acetylation must be highly localized and affect interaction with proteins that bind directly to the lumen of the microtubule. We also investigate the interaction of the tubulin acetyltransferase, αTAT1, with microtubules and find that αTAT1 is able to interact with the outside of the microtubule, at least partly through the tubulin C-termini. Binding to the outside surface of the microtubule could facilitate access of αTAT1 to its luminal site of action if microtubules undergo lateral opening between protofilaments.     

Zinc-stimulated microtubule assembly and evidence for zinc binding to tubulin

• 1.1. Microtubule reassembly was studied in supernatant fluids from rat brain. Tubulin in extracts from zinc-deficient animals showed an impaired ability to repolymerize compared to extracts from controls; 10 μM zinc stimulated reassembly of tubulin in extracts from zinc-deficient animals.
• 2.2. Low zinc concentrations (250–900 μM zinc in the presence of 1 mM EGTA) stimulated reassembly oftubulin in brain extracts from control rats; similar concentrations of nickel had no effect whilst cobalt was inhibitory. In the absence of EGTA 20–40 μM zinc stimulated reassembly in brain extracts from normal rats.
• 3.3. Zinc-induced changes in reassembly were associated with changes in the free sulphydryl group content of the assembled crude microtubule protein; increased assembly was associated with a higher free sulphydryl group content, decreased assembly with a lower content.
• 4.4. ⁶⁵Zn was found to bind to tubulin. This binding was partly inhibited by N-ethylmaleimide.

     

The natural naphthoquinone plumbagin exhibits antiproliferative activity and disrupts the microtubule network through tubulin binding

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a naphthoquinone isolated from the roots of Plumbaginaceae plants, has potential antiproliferative activity against several tumor types. We have examined the effects of plumbagin on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human non-small lung epithelium carcinoma cells (A549) indicated that the IC 50 value for plumbagin is 14.6 microM. Immunofluorescence studies using an antitubulin FITC conjugated antibody showed a significant perturbation of the interphase microtubule network in a dose dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by plumbagin with an IC 50 value of 38 +/- 0.5 microM. Its binding to tubulin quenches protein tryptophan fluorescence in a time and concentration dependent manner. Binding of plumbagin to tubulin is slow, taking 60 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 235.12 +/- 36 M (-1) s (-1) and 11.63 +/- 11 M (-1) s (-1) at 25 degrees C respectively. The stoichiometry of plumbagin binding to tubulin is 1:1 (mole:mole) with a dissociation constant of 0.936 +/- 0.71 microM at 25 degrees C. Plumbagin competes for the colchicine binding site with a K i of 7.5 microM as determined from a modified Dixon plot. Based on these data we conclude that plumbagin recognizes the colchicine binding site to tubulin. Further study is necessary to locate the pharmacophoric point of attachment of the inhibitor to the colchicine binding site of tubulin.     

Comparative molecular field analysis of colchicine inhibition and tubulin polymerization for combretastatins binding to the colchicine binding site on beta-tubulin

A molecular modeling study using Comparative Molecular Field Analysis (CoMFA) was undertaken to develop a predictive model for combretastatin binding to the colchicine binding site of tubulin. Furthermore, we examined the potential contribution of lipophilicity (log P) and molecular dipole moment and were unable to correlate these properties to the observed biological data. In this study we first confirmed that tubulin polymerization inhibition (IC50) correlated (R2 = 0.92) with [3H]colchicine displacement. Although these data correlated quite well, we developed two independent models for each set of data to quantify structural features that may contribute to each biological property independently. To develop our predictive model we first examined a series of molecular alignments for the training set and ultimately found that overlaying the respective trimethoxyphenyl rings (A ring) of the analogues generated the best correlated model. The CoMFA yielded a cross-validated R2 = 0.41 (optimum number of components equal to 5) for the tubulin polymerization model and an R2 = 0.38 (optimum number of components equal to 5) for [3H]colchicine inhibition. Final non-cross-validation generated models for tubulin polymerization (R2 of 0.93) and colchicine inhibition (R2 of 0.91). These models were validated by predicting both biological properties for compounds not used in the training set. These models accurately predicted the IC50 for tubulin polymerization with an R2 of 0.88 (n = 6) and those of [3H]colchicine displacement with an R2 of 0.80 (n = 7). This study represents the first predictive model for the colchicine binding site over a wide range of combretastatin analogues.     

The synthesis and tubulin binding activity of thiophene-based analogues of combretastatin A-4

A number of analogues of combretastatin A-4 (1), containing a thiophene ring interposed between the two phenyl groups, have been prepared. The synthesis of these compounds employed a combination of palladium-mediated coupling and iodocyclization techniques. The thiophene compounds 11, 14, 18, and 19 also represent non-benzofused analogues of some recently described tubulin binding benzo[b]thiophenes 3-5. The most active thiophene compounds identified in this study were 11, 14, and 18. Overall they are less active than 1 but exhibit comparable activity to the most active of the benzo[b]thiophenes 3-5. A structure-activity relationship of these compounds is considered.     

Interaction between chromium GTP and tubulin. Stereochemistry of GTP binding, GTP hydrolysis, and microtubule stabilization

Chromium GTP (CrGTP) has been used to probe the stereochemistry of metal-GTP binding to exchangeable site of tubulin and to examine the fate and role of nucleotide-bound metal ion in GTP hydrolysis associated with microtubule assembly. The absolute stereoconfiguration of the two pairs of diastereomers of beta,gamma-bidentate CrGTP has been determined by comparison of their visible circular dichroism spectra with those of the beta,gamma-CrATP isomers whose configurations have been established (Lin, I., and Dunaway-Mariano, D. (1988) J. Am. Chem. Soc. 110, 950-956). Tubulin binds metal-GTP preferentially in the delta pseudoaxial configuration. CrGTP-tubulin shows a high propensity to undergo tubulin-tubulin interactions with associated hydrolysis of CrGTP. Hydrolysis of CrGTP in microtubule assembly develops in two consecutive steps: cleavage of the gamma-phosphate followed by release of Pi and chromium. In contrast to other NTPases (actin, hexokinase) tubulin appears able to catalyze the dissociation of the stable chromium-phosphate bonds, which implies a highly nucleophilic environment of the binding site of the metal-triphosphate moiety of GTP. Microtubules assembled from CrGTP-tubulin are made of 90% GDP subunits, and their stability is linked to a 10% proportion of CrGDP-Pi subunits, scattered along the microtubule, from which Pi does not dissociate. The possibility is evoked that some tubulin variants do not catalyze release of Pi and metal ion efficiently, and their presence could affect microtubule dynamics.     

Interaction of tubulin with bifunctional colchicine analogues: an equilibrium study

The interaction of tubulin with simple analogues of colchicine that contain both its tropolone and trimethoxyphenyl rings has been characterized, and the results were analyzed in terms of the simple bifunctional ligand model developed for the binding of colchicine [ Andreu , J. M., & Timasheff , S. N. (1982) Biochemistry 21, 534-543] on the basis of interactions of tubulin with single-ring analogues. The compound 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6- cycloheptatrien -1-one has been found to bind reversibly to 0.86 +/- 0.06 site of purified calf brain tubulin with an equilibrium constant of (4.9 +/- 0.3) X 10(5) M-1 (25 degrees C), delta H degrees app = -1.6 +/- 0.7 kcal mol-1, and delta S degrees app = 20.5 +/- 2.5 eu. The binding appears specific for the colchicine site. The closely related compound 2-methoxy-5-[[3-(3,4,5-trimethoxyphenyl)-propionyl]amino] -2,4,6- cycloheptatrien -1-one interacts weakly with tubulin. Binding of the first analogue is accompanied by ligand fluorescence appearance, quenching of protein fluorescence, perturbation of the far-ultraviolet circular dichroism of tubulin, and induction of the tubulin GTPase activity, similarly to colchicine binding. Substoichiometric concentrations of the analogue inhibit microtubule assembly in vitro. Excess analogue concentration under microtubule-promoting conditions induces an abnormal cooperative polymerization of tubulin, similar to that of the tubulin-colchicine complex.     

Interaction of tubulin with octyl glucoside and deoxycholate. 2. Protein conformation, binding of colchicine ligands, and microtubule assembly

The structural change induced by binding of mild detergents to cytoplasmic calf brain tubulin and the effects on the functional properties of this protein have been characterized. Massive binding of octyl glucoside or deoxycholate monomers induces circular dichroism changes indicating a partial alpha-helix to disordered structure transition of tubulin. The protein also becomes more accessible to controlled proteolysis by trypsin, thermolysin, or V8 protease. This is consistent with the looser protein structure proposed in previous binding and hydrodynamic studies [Andreu, J. M., & Mu?oz, J. A. (1986) Biochemistry (preceding paper in this issue)]. Micelles of octyl glucoside and deoxycholate bind colchicine and its analogue 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC). This impedes the determination of colchicine binding in the presence of detergents. Both detergents cause a reduction in the number of tubulin equilibrium binding sites for the colchicine site probe MTC. Deoxycholate monomers bind poorly to the tubulin-colchicine complex, but deoxycholate above the critical micelle concentration effectively dissociates the complex. Microtubule assembly in glycerol-containing buffer is inhibited by octyl glucoside, which raises the critical protein concentration. Low concentrations of deoxycholate enhance tubulin polymerization, allowing it to proceed without glycerol. The polymers formed are microtubules, pairwise associated open microtubular sheets, and macrotubules possibly generated by helical folding of the sheets, as indicated by the optical diffraction patterns. Saturation of tubulin with octyl glucoside, followed by full dissociation of the detergent, allowed the recovery of binding to the colchicine site and microtubule assembly, indicating the reversibility of the protein structural change.     

Enhanced microtubule binding and tubulin assembly properties of conformationally constrained paclitaxel derivatives

Microtubule binding and tubulin assembly promotion by a series of conformationally restricted paclitaxel (PTX) derivatives was investigated. In these derivatives, the C-4 acetate of the taxane is tethered to the C-3' phenyl at ortho and meta positions with different length linkers. The apparent affinity of these derivatives for GMPCPP-stabilized microtubules was assessed by a competition assay, and their influence on microtubule polymerization was evaluated by measuring the critical concentration of GDP-tubulin in the presence of the respective molecule. In general, taxane derivatives with higher apparent affinity for microtubules induced tubulin assembly more efficiently. Among the derivatives, molecules with the shortest tether display the strongest affinity for microtubules. These derivatives exhibited enhanced microtubule stabilization properties and efficiently induced GDP-tubulin assembly into microtubules at low temperature of 12 degrees C and in the absence of Mg2+ ions in 0.1 M PIPES. Based on molecular dynamics simulations, we propose that the enhanced ability to assemble microtubules by these taxane derivatives is linked to their ability to effectively shape the conformation of the M-loop of tubulin for cross-protofilament interaction.     

Interaction of rat brain microtubule proteins and 6S tubulin with rabbit skeletal muscle actomysin.

The interaction between actomyosin from rabbit skeletal muscle and microtubule proteins or 6S tubulin from rat brain was investigated with respect to the change in ATPase activity and physicochemical properties. Myosin bound to both microtubule proteins and 6S tubulin at low ionic strength. In the aggregates the molar ratio of microtubule proteins or 6S tubulin to myosin was 0.5–1.5 or 1.5–2.5. The superprecipitation of actomyosin was inhibited by 6S tubulin. The degree of superprecipitation inhibition was dependent on the mixing order of myosin, actin, 6S tubulin, and ATP. When myosin was preincubated first with 6S tubulin, the inhibition was most marked. The actin activation of myosin Mg-ATPase was inhibited by both microtubule proteins and 6S tubulin with stronger effects by the latter. The preincubation of myosin with 6S tubulin prior to the addition of actin induced not only greater inhibition of ATPase but also the binding of a larger quantity of 6S tubulin to myosin than the preincubation of myosin with actin. The similar results were obtained with microtubule proteins.     

GTP analogues interact with the tubulin exchangeable site during assembly and upon binding

The question of whether nonhydrolyzable nucleotide analogues and other nucleoside triphosphates support tubulin assembly was addressed. Tubulin which contained residual GTP at the exchangeable site polymerized in the absence of added GTP in the presence of DMSO or glycerol. After maximum absorbance was reached, disassembly occurred at a slow rate. When 0.5 mM GMPPCP, GMPPNP, or ATP was included in the assembly reaction, disassembly did not occur, and about 0.1 mol of these nucleotides per mole of tubulin was incorporated into the protein. When 5 mM nucleotide was used or alkaline phosphatase was included in the case of the nonhydrolyzable analogues, a greater amount of assembly occurred and about 0.7-0.8 mol of analogue was incorporated. The products of the assembly reaction were cold-labile microtubules and protofilament ribbons. After cold-depolymerization of the microtubules and ribbons, a second cycle of assembly produced some microtubules, but cold-stable amorphous polymers were the major product. In addition, when GTP at the exchangeable site was first removed by a cycle of assembly, followed by depolymerization, assembly in the presence of GMPPCP, GMPPNP, or ATP produced a mixture of microtubules and cold-stable polymers, both of which contained bound analogue. Incorporation of GMPPCP, GMPPNP, or ATP into polymerized tubulin always occurred at the expense of GDP at the exchangeable site, the content of which decreased correspondingly. Incubation of tubulin with 5 mM GMPPCP, GMPPNP, or ATP under nonassembly conditions also displaced GDP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural immunocytochemical distribution of tubulin in cultured cells treated with microtubule inhibitors.

Microtubules in tissue cultured cells are stained immunocytochemically with the PAP-method using a purified antitubulin antibody. Treatment of the cells with microtubule inhibitors (colchicine, nocodazole, vinblastine) results in the disappearance of microtubules. The diffuse cytoplasmic staining is strongly increased in the cells by colchicine and nocodazole. Vinblastine produces paracrystalline aggregates that are strongly stained and macrotubules that are unstained. The diffuse staining is much less in vinblastine-treated cells. The bundles of intermediate filaments that are induced by all microtubule inhibitors do not bind the antitubulin antibody.     

A new family of quinoline and quinoxaline analogues of combretastatins

The 3-hydroxy-4-methoxyphenyl ring of combretastatin A-4 can be replaced by a 2-naphthyl moiety without significant loss of cytotoxicity and inhibition of tubulin polymerization potency. In this paper we show that the 6- or 7-quinolyl systems can in turn replace both cyclic moieties, keeping in the first case most of the potency as cytotoxic agent and in the second case as inhibitor of tubulin polymerization, related to the activities displayed by model compounds.     

Gamma-tubulin colocalizes with microtubule arrays and tubulin paracrystals in dividing vegetative cells of higher plants

Summary The distribution of -tubulin throughout cell division is studied in several taxa of higher plants. -Tubulin is present along the whole length of microtubules (Mts) in every cell stage-specific Mt array such as the preprophase band, the preprophase-prophase perinuclear Mts, the kinetochore Mt bundles, the phragmoplast, and the telophase-interphase transition Mt arrays. -Tubulin follows with precision the Mt pattern, being absent from any other, Mt-free, cell site. In cells treated with anti-Mt drugs, -tubulin is present only on degrading or on reappearing Mt arrays, while it is totally absent from cells devoid of Mts. -Tubulin is also present in tubulin paracrystals, which are formed in colchicine-treated cells. These observations support the view that in higher plants -tubulin may not be a microtubule-organizing-center-specific protein, but it may play a certain structural and/or functional role being related to - and -tubulin.Abbreviations Mt microtubule - MTOC microtubule-organizing center - PPB preprophase band     

Interaction of nerve growth factor with tubulin. Studies on binding and induced polymerization

The interaction of the nerve growth factor with the neurotubule protein has been studied with the aim of elucidating the nature of the large complexes that they form when incubated together and the factors that control this event. The results show that the binding of nerve growth factor to tubulin is followed by the formation of large structures that, in certain experimental conditions, accelerate the rate of tubulin polymerization to form microtubules or catalyze their assembly in conditions where this process does not occur spontaneously. The formation of large nerve growth factor-tubulin complexes starts to occur only at a molar ratio of 1.0–1.5 NaCl or GTP strongly inhibit this process without a detectable effect on NGF binding. Two hypotheses are postulated to explain these finding. Firstly, that tubulin has two sites with different affinity for nerve growth factor and the polymerization occurs only when the second NGF molecule has interacted with the microtubule protein. Alternatively, free tubulin in solution is the limiting factor of the polymerization by hindering a site of tubulin-factor complexes present in solutio at a 1 : 1 molar ratio. In both cases, GTP, Na⁺ or H⁺ will affect the formation of large unsoluble, tubulin-NGF complexes, by changing their conformation or by decreasing electrostatic interactions.     

Interaction of benzimidazole anthelmintics with Haemonchus contortus tubulin: binding affinity and anthelmintic efficacy

The ability of various benzimidazoles (BZs) to bind tubulin under different conditions was assessed by determining their IC50 values (the concentration of unlabeled drug required to inhibit 50% of the labeled drug binding), Ka (the apparent equilibrium association constant) and Bmax (the maximum binding at infinite [BZ] = [drug-receptor]). The ability of unlabeled benzimidazoles--fenbendazole, mebendazole (MBZ), oxibendazole (OBZ), albendazole (ABZ), rycobendazole (albendazole sulfoxide, ABZSO), albendazole sulfone, oxfendazole (OFZ), and thiabendazole--to bind tubulin was determined from their ability to inhibit the binding of [3H]MBZ or [3H]OBZ to tubulin in supernatants derived from unembryonated eggs or adult worms of Haemonchus contortus. The binding constants (IC50, Ka, and Bmax) correlated with the known anthelmintic potency (recommended therapeutic doses) of the BZ compounds except for OFZ and ABZSO whose Ka values were lower than could be expected from anthelmintic potency. The binding of [3H]ABZ or [3H]OFZ to tubulin in supernatants derived from BZ-susceptible and BZ-resistant H. contortus was compared. [3H]ABZ demonstrated saturable high-affinity binding but [3H]OFZ bound with low affinity. The high-affinity binding of [3H]ABZ was reduced for the R strain. Tubulin bound BZ drugs at 4 degrees C with lower apparent Ka than at 37 degrees C.     

The binding of isocolchicine to tubulin. Mechanisms of ligand association with tubulin

Isocolchicine is a structurally related isomer of colchicine altered in the methoxytropone C ring. In spite of virtual structural homology of colchicine and isocolchicine, isocolchicine is commonly believed to be inactive in binding to tubulin and inhibiting microtubule assembly. We have found that isocolchicine does indeed bind to the colchicine site on tubulin, as demonstrated by its ability to competitively inhibit [3H]colchicine binding to tubulin with a KI approximately 400 microM. Isocolchicine inhibits tubulin assembly into microtubules with an I50 of about 1 mM, but the affinity of isocolchicine for the colchicine receptor site, 5.5 +/- 0.9 x 10(3) M-1 at 23 degrees C, is much less (approximately 500-fold) than that of colchicine. Unlike colchicine, isocolchicine binds rapidly, and the absorption and fluorescence properties of the complex are only modestly altered compared to free ligand. It is proposed that the binding of isocolchicine to tubulin may be rationalized either in terms of conformational states of colchicinoids when liganded to tubulin or by the structural requirements for C-10 substituents for high affinity binding to the colchicine receptor.     

Interaction of substance P with tubulin

Binding of the peptide neurotransmitter substance P to brain tubulin in vitro inhibits self-assembly of the protein into microtubules and disrupts preassembled microtubules. This cooperative inhibition of the maximum extent of self-assembly by substance P is explicable in terms of preferential binding to the protomer state as compared to the polymer state of tubulin. The inhibition is relieved by the microtubule-associated protein MAP2, which evidently acts in a mixed competitive-noncompetitive fashion. Substance P interacts directly with the isolated C-terminal 4-kDa peptide fragment of tubulin, which appears to contain the specific binding area for MAP2, but is without effect on the self-assembly of the larger (48-kDa) part of the tubulin molecule called S-tubulin. The results are consistent with the C-terminal fragment having a binding site for the cationic substance P as well as for MAP2. However, factors other than electrostatic interaction must be operative, since the sulfoxide of substance P, a derivative with oxidized methionine but similar electrostatic characteristics, is inactive in inhibiting the extent of microtubule assembly.