Binding of dolastatin 10 to tubulin at a distinct site for peptide antimitotic agents near the exchangeable nucleotide and vinca alkaloid sites

Dolastatin 10, a potent antimitotic peptide from a marine animal, strongly inhibits microtubule assembly, tubulin-dependent GTP hydrolysis, and the binding of vinca alkaloids to tubulin. In studies of the binding of [3H]vincristine to the protein, with vinblastine as a control for competitive inhibition (Ki, 6.6 microM), we found that the macrolide antimitotic agents maytansine and rhizoxin were also competitive inhibitors (Ki values, 3.1 and 12 microM). Dolastatin 10 and an unrelated peptide antimitotic, phomopsin A, were more potent but noncompetitive inhibitors (Ki values, 1.4 and 2.8 microM). Since maytansine and, to a much lesser extent, vinblastine interfere with nucleotide exchange on tubulin, all drugs were examined for effects on nucleotide interactions at the exchangeable GTP site. Rhizoxin had effects intermediate between those of vinblastine and maytansine. Both peptides inhibited binding of radiolabeled GTP to tubulin even more strongly than did maytansine, but no drug displaced nucleotide from tubulin. The drugs were evaluated for stabilizing effects on the colchicine binding activity of tubulin. The peptides prevented loss of this activity, and vinblastine provided partial protection, while rhizoxin and maytansine did not stabilize tubulin. A tripeptide segment of dolastatin 10 also effectively inhibits tubulin polymerization and GTP hydrolysis. The tripeptide did not significantly inhibit either vincristine binding or nucleotide exchange, nor did it stabilize colchicine binding. These findings are rationalized in terms of a model with two distinct drug binding sites in close physical proximity to each other and to the exchangeable GTP site on beta-tubulin.     

The interaction of phomopsin A with bovine brain tubulin

Phomopsin A is an anti-mitotic compound from the fungus Phomopsis leptostroniformis which is a potent inhibitor of microtubule assembly in vitro; like maytansine, it is known to compete with vinblastine for binding to tubulin (E. Lacey, J. A. Edgar, and C. C. J. Culvenor (1987) Biochem. Pharmacol. 36, 2133-2138). A major difference between the effects of maytansine and vinblastine is that vinblastine is a potent inhibitor of tubulin decay, whereas maytansine has little or no effect on decay. Since phomopsin A is structurally distinct from either maytansine or vinblastine, tubulin decay may be measured by either the time-dependent loss of the ability to bind to [3H]colchicine or the time-dependent increase in the binding of bis(8-anilinonaphthalene 1-sulfonate) (BisANS) to tubulin. By either method, phomopsin A was found to be a much stronger inhibitor of tubulin decay than is vinblastine or any other drug yet tested, and in fact, when decay is measured by the increase of BisANS binding, phomopsin A appears to stop the process entirely. This may prove to be useful in the determination of the higher-order structure of the tubulin molecule.     

Contrasting effects of maytansine and vinblastine on the alkylation of tubulin sulfhydryls

The ansa macrolide maytansine is a competitive inhibitor of vinblastine for binding to tubulin. Both drugs are potent inhibitors of microtubule assembly in vitro but maytansine, unlike vinblastine, is unable to induce tubulin aggregation or to stabilize colchicine binding. In this study, the effects of maytansine and vinblastine on the accessibility of tubulin's sulfhydryl groups were compared. It was found that 10 μm vinblastine inhibited the reaction of bovine brain tubulin with [¹⁴C]iodoacetamide by 45%. In contrast, maytansine, even up to 100 μm, had no effect on the reaction. However, when the two drugs were tested in combination, maytansine was a potent inhibitor of vinblastine's effect, consistent with the two drugs competing for the same or overlapping sites, but suggesting that the nature of the binding was different. In contrast, maytansine did not affect the suppression of alkylation induced by colchicine and podophylotoxin, consistent with these drugs binding to different sites. Maytansine and vinblastine were each able to increase the formation of $\text{β}{}^1$ by the bifunctional reagent, N,N′-ethylenebis-(iodoacetamide); $\text{β}{}^1$ is the designation for an electrophoretically faster migrating form of β-tubulin which apparently contains an intrachain crosslink. Thus, in at least the portion of the tubulin molecule involved in $\text{β}{}^1$ formation, the two drugs have similar effects. Since maytansine does not appear to suppress any competing alkylation reactions, it is possible that the enhancement of $\text{β}{}^1$ formation represents a genuine conformational effect. Since the sulfhydryl groups of tubulin may be involved in regulating microtubule assembly, it is likely that maytansine and vinblastine differ in the manner in which they inhibit microtubule assembly.     

Halichondrin B and homohalichondrin B, marine natural products binding in the vinca domain of tubulin. Discovery of tubulin-based mechanism of action by analysis of differential cytotoxicity data

Data generated in the new National Cancer Institute drug evaluation program, which is based on inhibition of cell growth in 60 human tumor cell lines, were used to compare new compounds with agents of known mechanism of action in terms of their differential cytotoxicity. Two marine natural products, halichondrin B and homohalichondrin B, appeared repeatedly when the data base was probed with known antimitotic agents. We confirmed that both compounds were highly cytotoxic (IC50 values for L1210 murine leukemia cells of 0.3 and 1 nM, respectively), with accumulation of cells arrested in mitosis at toxic concentrations, that both inhibited the polymerization of purified tubulin, and that both inhibited microtubule assembly dependent on microtubule-associated proteins. Limited amounts of homohalichondrin B, the less active agent, were available, so only halichondrin B was studied in detail. Halichondrin B did not interfere with colchicine binding to tubulin, but it was a noncompetitive inhibitor of the binding of vinblastine to tubulin (apparent Ki, 5.0 microM). Halichondrin B was therefore compared with other agents which interfere with the binding of vinca alkaloids to tubulin (vinblastine, maytansine, dolastatin 10, phomopsin A, rhizoxin) in terms of its effects on tubulin polymerization, inhibition of GTP hydrolysis, inhibition of nucleotide exchange, and stabilization of tubulin, as well as the quantitative assessment of its effects on vinca alkaloid binding and inhibition of cell growth. Since halichondrin B was originally isolated from the same organism as the phosphatase inhibitor okadaic acid, and since it is about 50-fold more effective than okadaic acid as an inhibitor of L1210 cell growth, perturbations of cellular microtubules observed following treatment with okadaic acid should be interpreted cautiously.     

Distinct and overlapping binding sites for IKP104 and vinblastine on tubulin

IKP104 is one of a group of tubulin-binding drugs whose interaction with tubulin suggests that it may bind to the protein at or close to the region where vinblastine binds. By itself IKP104 is a potent enhancer of tubulin decay as evidenced by the fact that it induces the exposure of the sulfhydryl groups and hydrophobic areas on tubulin. In this respect, IKP104 differs from vinblastine and other drugs such as phomopsin A, dolastatin 10, rhizoxin, and maytansine which are competitive or noncompetitive inhibitors of vinblastine binding. In contrast, however, in the presence of colchicine, IKP104 behaves differently and strongly stabilizes tubulin, to an extent much greater than does colchicine alone. IKP104 appears to have two classes of binding site on tubulin, differing in affinity; the acceleration of decay appears to be mediated by the low-affinity site (Chaudhuriet al., 1998,J. Protein Chem., in press). We investigated the relationship of the binding of IKP104 and vinblastine. We found that the high-affinity site or sites of IKP104 overlap with or interact with the vinblastine-binding sites, but that the low-affinity site is distinctly different.     

Distinct and overlapping binding sites for IKP104 and vinblastine on tubulin

IKP104 is one of a group of tubulin-binding drugs whose interaction with tubulin suggests that it may bind to the protein at or close to the region where vinblastine binds. By itself IKP104 is a potent enhancer of tubulin decay as evidenced by the fact that it induces the exposure of the sulfhydryl groups and hydrophobic areas on tubulin. In this respect, IKP104 differs from vinblastine and other drugs such as phomopsin A, dolastatin 10, rhizoxin, and maytansine which are competitive or noncompetitive inhibitors of vinblastine binding. In contrast, however, in the presence of colchicine, IKP104 behaves differently and strongly stabilizes tubulin, to an extent much greater than does colchicine alone. IKP104 appears to have two classes of binding site on tubulin, differing in affinity; the acceleration of decay appears to be mediated by the low-affinity site (Chaudhuriet al., 1998,J. Protein Chem., in press). We investigated the relationship of the binding of IKP104 and vinblastine. We found that the high-affinity site or sites of IKP104 overlap with or interact with the vinblastine-binding sites, but that the low-affinity site is distinctly different.     

Dolastatin 15 binds in the vinca domain of tubulin as demonstrated by Hummel-Dreyer chromatography.

The antimitotic depsipeptide dolastatin 15 was radiolabeled with tritium in its amino-terminal dolavaline residue. Dolastatin 15, although potently cytotoxic, is a relatively weak inhibitor of tubulin assembly and does not inhibit the binding of any other ligand to tubulin. The only methodology found to demonstrate an interaction between the depsipeptide and tubulin was Hummel-Dreyer equilibrium chromatography on Sephadex G-50 superfine. The average apparent Kd value obtained in these studies was about 30 microM, with no difference observed when column size or tubulin concentration was varied. This relatively high dissociation constant is consistent with the apparent weak interaction of dolastatin 15 with tubulin demonstrated indirectly in the assembly assay. We attempted to gain insight into the binding site for dolastatin 15 on tubulin by studying inhibitory effects of other drugs when the gel filtration column was equilibrated with both [3H]dolastatin 15 and a second, nonradiolabeled drug. No inhibition was detected with either the colchicine site agent combretastatin A-4 or with an analog of the antimitotic marine peptide diazonamide A (both the analog and diazonamide A are potent inhibitors of tubulin assembly). Weak inhibition was observed with cemadotin, a structural analog of dolastatin 15, and with the depsipeptide cryptophycin 1. Moderate inhibition occurred with vinblastine and vincristine, and strong inhibition with maytansine, halichondrin B, and the peptides dolastatin 10 and phomopsin A. These observations suggest that the binding site(s) for peptide and depsipeptide antimitotic drugs may consist of a series of overlapping domains rather than a well-defined locus on the surface of beta-tubulin.     

Maytansine inhibits nucleotide binding at the exchangeable site of tubulin

The antineoplastic drug maytansine inhibits the binding of exogenously added radiolabeled GDP and GTP to tubulin (50% inhibition at 9-10 microM drug at 0 degrees). Vinblastine was 1/10-th as inhibitory. Neither maytansine nor vinblastine displaced GDP from tubulin, and both drugs virtually eliminated dissociation of radiolabeled GDP from the exchangeable site. Maytansine also inhibits binding of nucleotides to a vacant exchangeable site. Maytansine thus prevents nucleotide exit and entry at the exchangeable site because of a direct physical obstruction or a conformational change in the tubulin molecule.     

Interaction of tubulin with a new fluorescent analogue of vinblastine

Vinblastine is an antimitotic agent that has been used extensively in cancer chemotherapy. The biological effects of the drug are believed to be the result of its interaction with tubulin, the major component of cellular microtubules. Fluorescence spectroscopy is a powerful and versatile technique for studying drug-tubulin interactions, but it rarely has been applied to studies involving vinca alkaloids. We have prepared a new fluorescent derivative of vinblastine designed to retain high affinity for tubulin while possessing a fluorophore that absorbs and emits visible light. A coumarin derivative of vinblastine, 17-deacetyl-O-(3-carbonylamino-7-diethylaminocoumarin) vinblastine (F-VLB), was prepared by reaction of 17-deacetylvinblastine with 7-diethylaminocoumarin-3-carbonyl azide. F-VLB was a potent inhibitor of in vitro microtubule assembly (IC(50) = 0.5 microM). F-VLB binding to tubulin was inhibited by vinblastine. Tubulin binding induced an increase in the F-VLB emission intensity and shifted the emission maximum to higher energy (from 500 to 480 nm). The Stokes shift of tubulin-bound F-VLB was about the same as the Stokes shift of the molecule in ethanol, indicating that the tubulin-bound fluorophore is probably on the exterior of the vinblastine binding site. Unlike vinblastine, F-VLB failed to induce self-assembly of tubulin that could be detected by light scattering or electron microscopy, although some self-association could be detected by analytical ultracentrifugation. Equilibrium binding parameters were quantitatively determined by monitoring the change in fluorescence anisotropy of F-VLB upon tubulin binding. The apparent equilibrium constant for F-VLB binding to tubulin [K(a)(app) = (7.7 +/- 0.5) x 10(4) M(-1) at 25 degrees C] was identical to the equilibrium constant for vinblastine binding to 2 microM tubulin (K(1)) measured under similar buffer and temperature conditions using ultracentrifugation [Vulevic, B., Lobert, S., and Correia, J. J. (1997) Biochemistry 36, 12828-12835]. Binding allocolchicine to tubulin did not significantly affect F-VLB's affinity for the protein [K(a)(app) = (9.1 +/- 0.4) x 10(4) M(-1) at 25 degrees C]. Analysis of the steady-state emission spectra yielded a distance between the colchicine and vinca binding sites on tubulin of approximately 40 A. F-VLB bound to paclitaxel- and glutaraldehyde-stabilized microtubules, with approximately equal affinity. We conclude that F-VLB can be used to obtain information about the vinblastine binding site on tubulin under equilibrium conditions.     

Binding selectivity of rhizoxin, phomopsin A, vinblastine, and ansamitocin P-3 to fungal tubulins: differential interactions of these antimitotic agents with brain and fungal tubulins.

The binding of four potent antimitotic agents, rhizoxin (RZX), phomopsin A (PMS-A), ansamitocin P-3 (ASMP-3), and vinblastine (VLB), to tubulins from RZX-sensitive and -resistant strains of Aspergillus nidulans, Schizosaccharomyces pombe, and Saccharomyces cerevisiae was investigated. Mycelial extracts to which RZX could bind contained beta-tubulin with Asn as the 100th amino acid residue (Asn-100) in all cases, and those without affinity for RZX contained beta-tubulins with either Ile-100 or Val-100. Though PMS-A shares the same binding site as RZX and ASMP-3 on porcine brain tubulin (Asn-100), only ASMP-3 bound Asn-100 fungal tubulins in a competitive manner with respect to RZX. PMS-A and VLB, which strongly bind to porcine brain tubulin, did not bind to any of the fungal mycelial extracts examined. The results indicate differential interactions of these antimitotic agents with brain and fungal tubulins.     

Induced transposition of Ds by a stable Ac in crosses of transgenic tobacco plants

Summary Rhizoxin and ansamitocin P-3 (a maytansinoid compound), potent inhibitors of mammalian brain tubulin assembly, inhibit growth of a variety of fungi including Aspergillus nidulans. Mutants of A. nidulans, benA10 which is a benomyl resistant -tubulin gene mutant and tubAl which is a benomyl supersensitive a-tubulin gene mutant, were both sensitive to rhizoxin and ansamitocin P-3 to the same extent as wild-type strains. We isolated 18 rhizoxin resistant mutants of A. nidulans. All of these mutants were cross-resistant to ansamitocin P-3, but not to benzimidazole antimitotic drugs. These mutants mapped to two loci, rhiA and rhiB, and all of those with high resistance mapped to rhiA. The fact that the protein extracts of rhiA mutants lost rhizoxin binding affinity and that rhiA was closely linked to benA, the major -tubulin gene in A. nidulans, indicated that rhiA must be a structural gene for -tubulin and that rhiA mutants are a new class of -tubulin gene mutants. All of this suggested that, in A. nidulans, these antimitotic drugs bind to -tubulin, and that rhizoxin and ansamitocin P-3 share the same binding site but the site does not overlap with the benzimidazole binding site. Protein extracts from a rhiB mutant retained rhizoxin binding affinity, therefore this rhizoxin resistance mechanism should not be a tubulin mediated process.     

Fluorescent and photoaffinity labeling derivatives of rhizoxin

A fluorescent probe (D-RZX) and a photoreactive fluorescent probe (AD-RZX) for studying the rhizoxin binding site on tubulin were prepared by the derivatization of rhizoxin (RZX). D-RZX consists of a rhizoxin moiety and a dansyl moiety. AD-RZX has a 5-azidonaphthalene-1-sulfonyl moiety instead of the dansyl moiety of D-RZX. Both D-RZX and AD-RZX bound tubulin in a mutually competitive manner with rhizoxin, indicating their binding to the rhizoxin site on tubulin. AD-RZX bound the rhizoxin site covalently after UV-irradiation, thus showing its usefulness as a photo-affinity probe for labeling of the rhizoxin site.     

Dietary antioxidant curcumin inhibits microtubule assembly through tubulin binding

Curcumin, a component of turmeric, has potent antitumor activity against several tumor types. However, its molecular target and mechanism of antiproliferative activity are not clear. Here, we identified curcumin as a novel antimicrotubule agent. We have examined the effects of curcumin on cellular microtubules and on reconstituted microtubules in vitro. Curcumin inhibited HeLa and MCF-7 cell proliferation in a concentration-dependent manner with IC(50) of 13.8 +/- 0.7 microm and 12 +/- 0.6 microm, respectively. At higher inhibitory concentrations (> 10 microm), curcumin induced significant depolymerization of interphase microtubules and mitotic spindle microtubules of HeLa and MCF-7 cells. However, at low inhibitory concentrations there were minimal effects on cellular microtubules. It disrupted microtubule assembly in vitro, reduced GTPase activity, and induced tubulin aggregation. Curcumin bound to tubulin at a single site with a dissociation constant of 2.4 +/- 0.4 microm and the binding of curcumin to tubulin induced conformational changes in tubulin. Colchicine and podophyllotoxin partly inhibited the binding of curcumin to tubulin, while vinblastine had no effect on the curcumin-tubulin interactions. The data together suggested that curcumin may inhibit cancer cells proliferation by perturbing microtubule assembly dynamics and may be used to develop efficacious curcumin analogues for cancer chemotherapy.     

The effect of TN-16 on the alkylation of tubulin

The synthetic anti-tumor drug 3-(1-anilinoethylidene)-5-benzylpyrrolidine-2,4-dione (TN-16) is known to block microtubule assembly and colchicine binding to tubulin, although its structure does not resemble those of either colchicine, podophyllotoxin, or nocodazole (Arai, FEBS Lett. 155:273-276 (1983]. We have found that TN-16 affects the intra-chain cross-linking of beta-tubulin by N,N'-ethylene-bis(iodoacetamide) in a manner identical to that of colchicine, podophyllotoxin, and nocodazole, but different from that of vinblastine or maytansine. TN-16 also inhibits alkylation of tubulin by iodo[14C]acetamide, as do colchicine and its congeners. TN-16 appears to bind to tubulin at the colchicine binding site and one of its phenyl groups is likely to bind at the site on tubulin where colchicine's A ring binds.     

Photoaffinity labeling of tubulin subunits with a photoactive analogue of vinblastine

A photoactive, radioactive analogue of vinblastine, N-(p-azido[3,5-3H]benzoyl)-N'-(beta-amino-ethyl)vindesine ([ 3H]NABV), was used to localize the Vinca alkaloid binding site(s) on calf brain tubulin after establishing that its in vitro interactions with tubulin were comparable to those of vinblastine. Microtubule assembly was inhibited by 50% with 2 microM NABV or vinblastine. At higher drug concentrations, NABV and vinblastine both induced tubulin aggregation, and both drugs inhibited tubulin-dependent GTP hydrolysis. Vinblastine and NABV inhibited each other's binding to tubulin, but the binding of neither drug was inhibited by colchicine. Two classes of binding sites for NABV and vinblastine were found on calf brain tubulin. High-affinity sites had apparent KD values of 4.2 and 0.54 microM for NABV and vinblastine, respectively, whereas the low-affinity binding sites showed apparent KD values of 26 and 14 microM for NABV and vinblastine, respectively. Mixtures of tubulin and [3H]NABV were irradiated at 302 nm and analyzed for incorporation of radioactivity into protein. Photolabeling of both the alpha- and beta-subunits of tubulin with increasing concentrations of [3H]NABV exhibited a biphasic pattern characteristic of specific and nonspecific reactions. Nonspecific labeling was determined in the presence of excess vinblastine. Saturable specific covalent incorporation into both subunits of tubulin was observed, with an alpha:beta ratio of 3:2 and maximum saturable incorporation of 0.086 and 0.056 mol of [3H]NABV/mol of alpha-tubulin and beta-tubulin, respectively. Such photolabeling of the tubulin subunits will permit precise localization of Vinca alkaloid binding sites, including identification of the amino acid residues involved, an essential requirement for understanding the interactions of these drugs with tubulin.     

The mechanism of action of vinblastine. Binding of [acetyl-3H]vinblastine to embryonic chick brain tubulin and tubulin from sea urchin sperm tail outer doublet microtubules.

Tritium-labeled viblastine, specific activity 107 Ci/mol, was prepared by acetylation of desacetylvinblastine with [3H]acetic anhydride, and has been employed in a study of vinblastine binding to tubulin. There are two high affinity vinblastine-binding sites per mole of embryonic chick brain tubulin (KA = 3-5 X 10(5) l./mol). Binding to these sites was rapid, and relatively independent of temperature between 37 and 0degreeC. Vincristin sulfate and desacetylvinblastine sulfate, two other active vinca alkaloid derivatives, competitively inhibited the binding of vinblastine. The inhibition constant for vincristine was 1.7 X 10(-5) M; and for desacetylvinblastine, 2 X 10(-5) M. The vinblastine binding activity of tubulin decayed upon aging, but this property was not studied in detail. Vinblastine did not depolymerize stable sea urchin sperm tail outer doublet microtubules, nor did it bind to these microtubules. However, tubulin solubilized from the B subfiber of the outer doublet microtubules possessed the two high affinity binding sites (KA = 1-3 X 105 l./mol). These data suggest that vinblastine destroys microtubules in cells primarily by inhibition of microtubule polymerization, and does not directly destroy preformed microtubules.     

A radioligand binding assay for antitubulin activity in tumor cells

The benzamide RH-5854 is shown to be highly potent toward tumor cells and to arrest nuclear division by a highly specific covalent binding to the beta-subunit of tubulin in the colchicine binding region. Binding of 3H-RH-5854 to beta-tubulin in HCT-116 colon cancer cells is saturable and has been exploited in the development of a cell-based competitive binding assay, which allows antitubulin effects to be detected in whole cells. 3H-RH-5854 binding is strongly inhibited by preincubating the cells with compounds that bind to the colchicine site and with paclitaxel. Binding of 3H-RH-5854 is enhanced by preincubating the cells with vinblastine but not by other agents that bind at or near the vinblastine site (ansamitocin P-3 and phomopsin A). Various cytotoxic agents that do not act on tubulin do not affect binding of 3H-RH-5854 in HCT-116 cells, demonstrating specificity of the assay for detection of antitubulin activity. As an alternative to traditional assays that employ isolated brain tubulin, the 3HRH-5854 binding assay enables screening for antitubulin effects directly in tumor cells, providing an assay that accounts for cell-specific criteria that influence sensitivity such as different tubulin isotypes, tubulin mutations, drug metabolism, and efflux mechanisms.     

Interaction of tubulin and cellular microtubules with the new antitumor drug MDL 27048. A powerful and reversible microtubule inhibitor

We have characterized the binding of trans-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2-methyl-2- propen- 1-one (MDL 27048) to purified procine brain tubulin, and the inhibition of microtubule assembly by this compound in vitro and using cultured cells. Binding measurements were performed by difference absorption and fluorescence spectroscopy. MDL 27048 binds to one site/tubulin heterodimer with an apparent equilibrium constant Kb = (2.8 +/- 0.8) X 10(6) M-1 (50 mM 2-(N-morpholino)ethanesulfonic acid, 1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, 0.5 mM MgCl2, 0.1 mM GTP buffer, pH 6.7, at 25 degrees C). Podophyllotoxin displaced the binding of MDL 27048, suggesting an overlap with the colchicine-binding site. Assembly of purified tubulin into microtubules was inhibited by substoichiometric concentrations of MDL 27048, which also induced a slow depolymerization of preassembled microtubules. The cytoplasmic microtubules of PtK2 cells were disrupted in a concentration and time-dependent manner by MDL 27048, as observed by indirect immunofluorescence microscopy. Maximal depolymerization took place with 2 X 10(-6) M MDL 27048 in 3 h. When the inhibitor was washed off from the cells, fast microtubule assembly (approximately 8 min) and complete reorganization of the cytoplasmic microtubule network (15-30 min) were observed. MDL 27048 also induced mitotic arrest in SV40-3T3 cell cultures. Due to all these properties, this anti-tumor drug constitutes a new and potent microtubule inhibitor, characterized by its specificity and reversibility.     

Interaction of vinblastine with calf brain tubulin: multiple equilibria

The binding of the anticancer drug vinblastine to calf brain tubulin was measured by a batch gel filtration method in PG buffer (0.01 M NaPi, 10(-4) M GTP, pH 7.0) at three different protein concentrations. The Scatchard binding isotherms obtained were curvilinear. The binding of the first vinblastine molecule to each tubulin alpha-beta dimer (Mr 110,000) was enhanced by an increase in the protein concentration. Additional binding of vinblastine to the protein was independent of the protein concentration. Theoretical ligand binding isotherms were calculated for a ligand-induced macromolecule self-association involving various ligand stoichiometries and association schemes. Fitting of the experimental data to these isotherms showed that the system can be described best by a one-ligand-induced isodesmic indefinite self-association. The pathway giving the best fit consists of a ligand-mediated plus -facilitated self-association mechanism. The self-association-linked bound vinblastine binds specifically at a site with an intrinsic binding constant K1 = 4 X 10(4) M-1. Additional vinblastine molecules can bind less strongly to tubulin in probably nonspecific fashion, and the previous reports of two specific sites on alpha-beta tubulin for binding vinblastine are incorrect. The self-association constant K2 for liganded tubulin is 1.8 X 10(5) M-1. This analysis is fully consistent with the conclusions derived earlier from the linked function analysis of the vinblastine-induced tubulin self-association [Na, G. C., & Timasheff, S. N. (1980) Biochemistry 19, 1347-1354; Na, G. C., & Timasheff, S. N. (1980) Biochemistry 19, 1355-1365].     

Binding of gossypol to purified tubulin and inhibition of its assembly into microtubules

Gossypol is a polyphenolic pigment, which is employed as a male antifertility drug. It inhibits, among other reported effects, the growth of cultured mammalian cells, spermiogenesis, flagellar motility in Trypanosoma and sperm, dynein ATPase and the lactate dehydrogenase X (LDH-X) isozyme. We have characterized the non-covalent binding of gossypol to purified calf brain tubulin in 10 mM phosphate buffer, 0.1 mM GTP pH 7.0 at 25 degrees C. Equilibrium measurements were performed by difference spectroscopy. A peak at 435 nm was produced by the perturbation of gossypol light absorption upon binding to tubulin. The experimental isotherm was fitted by 1.96 +/- 0.06 gossypol binding sites per tubulin molecule, with identical apparent equilibrium binding constants of (7.5 +/- 1.1) X 10(4) M-1. The complex formed could be separated from free gossypol by gel chromatography. Binding of gossypol was independent of the presence of 0.1 mM GTP in the buffer. Gossypol did not affect the binding of ligands to the colchicine site. Gossypol interacted with vinblastine but apparently did not bind to the vinblastine sites of tubulin. Gossypol did not displace anilinonaphthalene sulphonate (ANS) bound to tubulin, but caused a strong (fivefold) quenching of its fluorescence. This indicated that gossypol probably binds in the vicinity of the ANS site of tubulin. Gossypol inhibited in vitro microtubule assembly at the same concentration range employed in the binding studies. An increase in the critical protein concentration required for polymerisation was observed, most simply interpreted by a stoichiometric mechanism. Gossypol did not induce any noticeable distortion of the microtubules observed under the electron microscope. This compound constitutes a new tubulin ligand and an inhibitor of microtubule assembly in vitro.