33K protein--an inhibitory factor of tubulin polymerization in porcine brain

A factor (33K protein) that modulates tubulin polymerization in vitro has been purified to homogeneity from porcine brain by ammonium sulfate fractionation and Whatman DE52, Toyo-pearl HW65C and Bio-Gel A 0.5 m column chromatographies. The purified fraction was free of nucleic acids and sugars. The activity of the purified 33K protein is pronase E sensitive but apparently heat- and trypsin-resistant though it undergoes tryptic digestion. The 33K protein inhibits polymerization of brain microtubule proteins in a dose-dependent manner and partially depolymerizes preformed microtubules. It also inhibits polymerization of purified starfish tubulin and microtubule elongation involving fragellar outer doublet microtubules and purified porcine brain tubulin. This suggests that the target of the 33K protein is tubulin rather than microtubule-associated proteins. The 33K protein causes incomplete depolymerization of microtubules and a new steady state is quickly attained which is apparently independent of microtubule mass concentration. Divalent cations such as calcium and magnesium do not modulate the inhibitory activity of the 33K protein.     

In vitro polymerization of flagellar and ciliary outer fiber tubulin into microtubules.

About 10--20% of the total protein in the outer fiber fraction was solubilized by sonication in a solution containing 5 mM MES, 0.5 mM MgSO4, 1.0 mM EGTA, 1.0 mM GTP, and 0 or 50 mM KC1 at pH 6.7. The sonicated extract was shown by analytical centrifugation to consist largely of a 6 S component (tubulin dimer), having a molecular weight of 103,000, as determined by gel filtration, and possessing a colchicine-binding activity of 0.8 mole per tubulin dimer. The tubulin fraction failed to polymerize into microtubules by itself. Addition of a small amount of the ciliary outer fiber fragments or reconstituted short brain microtubules, however, induced polymerization, as demonstrated by viscosity of flow birefringence changes as well as light or electron microscopic observations. The growth of heterogeneous microtubules upon mixing outer fiber tubulin with DEAE-dextran-decorated brain microtubules was observed by electron microscopy. Microtubules were reconstituted from outer fiber tubulin without addition of any nuclei fraction when a concentrated tubulin fraction was warmed at 35degree. A few doublet-like microtubules or pairs of parallel singlet microtubules that were closely aligned longitudinally could be observed among many singlet microtubules. Unlike other fiber microtubules, the reconstituted polymers were depolymerized by exposure to Ca2+ ions, high or low ionic strength, colchicine, low temperature or SH reagents. No microtubules were assembled under these conditions.     

Human brain tubulin purification: Decrease in soluble tubulin with age

The soluble tubulin of human cerebral cortex, as assessed by [³H]colchicine binding of the 100,000g supernatant fraction, decreases drastically with age, 75 percent from age 0 to age 90. There is also a considerably lower concentration of high molecular weight proteins in the soluble fraction of postmortem human cerebral cortex than in that of nonhuman species. Human brain tubulin can be polymerized into microtubules with DEAE-dextran. The DEAE-dextran induced microtubules are stable to cold temperature (4°) and calcium. However, in the presence of 1 M glutamate, the microtubules become cold labile and depolymerize at 4°. Thus we have developed a novel method for purifying polymerization competent tubulin from fresh or frozen human cerebral cortex. Human brain tubulin purified by our novel method is very similar to tubulin from the brains of other mammals in molecular weight, amino acid composition, polymerization-depolymerization parameters, and structural dimensions of the microtubules formed.Some aspects of this work have been published as an abstract in 1981. Fed. Proc. 40:1548.     

The stabilization of microtubules in isolated spindles by tubulin-colchicine complex

We have analyzed the effect of colchicine and tubulin dimer-colchicine complex (T-C) on microtubule assembly in mitotic spindles. Cold- and calcium-labile mitotic spindles were isolated from embryos of the sea urchin Lytechinus variegatus employing EGTA/glycerol stabilization buffers. Polarization microscopy and measurements of spindle birefringent retardation (BR) were used to record the kinetics of microtubule assembly-disassembly in single spindles. When isolated spindles were perfused out of glycerol stabilizing buffer into a standard in vitro microtubule reassembly buffer (0.1 M Pipes, pH 6.8, 1 mM EGTA, 0.5 mM MgCl2, and 0.5 mM GTP) lacking glycerol, spindle BR decreased with a half-time of 120 s. Colchicine at 1 mM in this buffer had no effect on the rate of spindle microtubule disassembly. Inclusion of 20 microM tubulin or microtubule protein, purified from porcine brain, in this buffer resulted in an augmentation of spindle BR. Interestingly, in the presence of 20 microM T-C, spindle BR did not increase, but was reversibly stabilized; subsequent perfusion with reassembly buffer without T-C resulted in depolymerization. This behavior is striking in contrast to the rapid depolymerization of spindle microtubules induced by colchicine and T-C in vivo. These results support the current view that colchicine does not directly promote microtubule depolymerization. Rather, it is T-C complex that alters microtubule assembly, by reversibly binding to microtubules and inhibiting elongation. In vivo, colchicine can induce depolymerization of nonkinetochore spindle microtubules within 20 s. In vitro, colchicine blocks further microtubule assembly, but does not induce rapid disassembly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of glycerol by microtubule protein.

When microtubules are purified by polymerization and depolymerization in a buffer containing glycerol, some glycerol becomes bound to the microtubule protein and is not removable by gel filtration or by prolonged dialysis. Both 6s tubulin and larger aggregates containing tubulin and accessory proteins bind glycerol. The 6s fraction has associated with it about 5 moles of glycerol per mole of tubulin dimer; 3 moles are exchangeable upon polymerization-depolymerization and 2 moles are not. The aggregate fraction has associated with it about 22 moles of glycerol per mole of tubulin dimer; approximately 11 moles are exchangeable and 11 moles are not.     

Tubulin and microtubule are potential targets for brain hexokinase binding.

The metabolite-modulated association of a fraction of hexokinase to mitochondria in brain is well documented, however, the involvement of other non-mitochondrial components in the binding of the hexokinase is controversial. Now we present evidence that the hexokinase binds both tubulin and microtubules in brain in vitro systems. The interaction of tubulin with purified bovine brain hexokinase was characterized by displacement enzyme-linked immunosorbent assay using specific anti-brain hexokinase serum (IC(50)=4.0+/-1.4 microM). This value virtually was not affected by specific ligands such as ATP or glucose 6-phosphate. Microtubule-bound hexokinase obtained in reconstituted systems using microtubule and purified hexokinase or brain extract was visualized by transmission and immunoelectron microscopy on the surface of tubules. The association of purified bovine brain hexokinase with either tubulin or microtubules caused about 30% increase in the activity of the enzyme. This activation was also observed in brain, but not in muscle cell-free extract. The possible physiological relevance of the multiple heteroassociation of brain hexokinase is discussed.     

Antimitotic antifungal compound benomyl inhibits brain microtubule polymerization and dynamics and cancer cell proliferation at mitosis, by binding to a novel site in tubulin

The antifungal agent benomyl [methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate] is used throughout the world against a wide range of agricultural fungal diseases. In this paper, we investigated the interaction of benomyl with mammalian brain tubulin and microtubules. Using the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid, benomyl was found to bind to brain tubulin with a dissociation constant of 11.9 +/- 1.2 microM. Further, benomyl bound to at a novel site, distinct from the well-characterized colchicine and vinblastine binding sites. Benomyl altered the far-UV circular dichroism spectrum of tubulin and reduced the accessibility of its cysteine residues to modification by 5,5'-dithiobis-2-nitrobenzoic acid, indicating that benomyl binding to tubulin induces a conformational change in the tubulin. Benomyl inhibited the polymerization of brain tubulin into microtubules, with 50% inhibition occurring at a concentration of 70-75 microM. Furthermore, it strongly suppressed the dynamic instability behavior of individual brain microtubules in vitro as determined by video microscopy. It reduced the growing and shortening rates of the microtubules but did not alter the catastrophe or rescue frequencies. The unexpected potency of benomyl against mammalian microtubule polymerization and dynamics prompted us to investigate the effects of benomyl on HeLa cell proliferation and mitosis. Benomyl inhibited proliferation of the cells with an IC(50) of 5 microM, and it blocked mitotic spindle function by perturbing microtubule and chromosome organization. The greater than expected actions of benomyl on mammalian microtubules and mitosis together with its relatively low toxicity suggest that it might be useful as an adjuvant in cancer chemotherapy.     

A novel 24-kDa microtubule-associated protein purified from sea urchin eggs.

Chromatographic fractionation of a crude extract of sea urchin eggs on a hydrophobic column enabled us to find a new 24-kDa microtubule-associated protein (SU-MAP24) that bound tightly to the column and was eluted under alkaline conditions. Biochemical studies using the purified protein showed its direct binding to microtubules reconstituted from tubulin purified from starfish sperm outer fibers. SU-MAP24 promoted tubulin polymerization in a dose-dependent manner. Immunoblotting analysis showed that SU-MAP24 is present in a microtubule protein fraction obtained from a crude extract using taxol, and immunostaining of paraffin-sectioned metaphase eggs showed its localization in the mitotic apparatus. These results show that SU-MAP24 is a newly identified microtubule-associated protein.     

Enhancement of tubulin assembly as monitored by a rapid filtration assay

The early kinetics of microtubule formation from lamb brain tubulin isolated by affinity chromatography can be followed by a newly developed filter assay. The rapid collection of microtubules on glass fiber filters permits the calculation of the moles of tubulin polymerized. The filter assay gives both a rate and extent of polymerization that are identical to those obtained by turbidity or sedimentation analysis, respectively. The microtubules trapped by the filter are readily depolymerized by cold (t12= 3 min) and slowly by colchicine (t1/2= 32min). Tubulin purified by affinity chromatography requires a high protein concentration (>4 mg/ml) for polymerization. Although 5m glycerol allows polymerization to occur at tubulin concentrations below 2 mg/ml, the maximum amount of microtubule formation is observed at low tubulin concentration when microtubule-associated proteins are present. These proteins are not retained by the affinity resin; however, they can be eluted from diethylaminoethyl-Sephadex by solutions containing 0.3m KCl. Microtubule-associated proteins enhance both the rate of polymerization and the total amount of tubulin polymerized as assessed by the filter assay, suggesting that they are involved in both initiation and elongation of microtubules.     

Perturbation of microtubule polymerization by quercetin through tubulin binding: a novel mechanism of its antiproliferative activity

The dietary flavonoid quercetin has a broad range of biological activities, including potent antitumor activity against several types of tumors. Recently, it has been shown that quercetin inhibits cancer cells proliferation by depleting cellular microtubules and perturbing cellular microtubule functions. However, the direct interactions of quercetin with tubulin and microtubules have not been examined so far. Here, we found that quercetin inhibited polymerization of microtubules and depolymerized microtubules made from purified tubulin in vitro. The binding of quercetin with tubulin was studied using quercetin fluorescence and intrinsic tryptophan fluorescence of tubulin. Quercetin bound to tubulin at a single site with a dissociation constant of 5-7 microM, and it specifically inhibited colchicine binding to tubulin but did not bind at the vinblastine site. In addition, quercetin perturbed the secondary structure of tubulin, and the binding of quercetin stimulated the intrinsic GTPase activity of soluble tubulin. Further, quercetin stabilized tubulin against decay and protected two cysteine residues of tubulin toward chemical modification by 5,5'-dithiobis-2-nitrobenzoic acid. Our data demonstrated that the binding of quercetin to tubulin induces conformational changes in tubulin and a mechanism through which quercetin could perturb microtubule polymerization dynamics has been proposed. The data suggest that quercetin inhibits cancer cells proliferation at least in part by perturbing microtubule functions through tubulin binding.     

Light-microscopic observations of individual microtubules reconstituted from brain tubulin.

The course of polymerization of individual brain microtubules could be observed with a light microscope employing dark-field illumination. Statistical analysis of the increase in microtubule length during the polymerization was in accordance with the time course of viscosity change of the tubulin solution. After a plateau level in viscosity was attained, there was no significant change in histograms showing length distribution. These observations were confirmed with fixed and stained microtubules, using a phase-contrast microscope. Observations with dark-field illumination revealed that reconstituted microtubules depolymerized and disappeared immediately upon exposure to buffer containing CaCl2 or sulphydryl reagents such as p-chloromercuriphenyl sulphonic acid (PCMPS) and p-chloromercuribenzoic acid (PCMB). They were also cold-labile. The growth of heterogeneous microtubules which were assembled by mixing purified tubulin dimers with ciliary outer fibres could also be followed with these optical systems.     

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.     

Proteins associated with tubulin.

The distribution of proteins on SDS-urea polyacrylamide (7.5%) disc gel electrophoresis is studied from rat brain tubulin purified by three different procedures, including ammonium sulfate precipitation followed by DEAE cellulose chromotography, three cycles of polymerization-depolymerization and colchicinecontaining agarose affinity columns. Three tubulin-associated proteins other than the principal tubulin dimer are identified and characterized with respect to molecular weight, behavior on gel filtration chromatography and method of tubulin purification. One of these proteins (H₁) is released from the tubulin complex when colchicine is irreversibly bound to tubulin. These proteins may participate in processes related to microtubule assembly or function.     

Purification of bovine adrenocortical and brain tubulin. A comparative study

Microtubules from the cow adrenal cortex and brain were purified by three cycles of the temperature-dependent polymerization-depolymerization procedure. Whereas tubulin comprised approximately 8--10% of soluble brain protein, it comprised only 0.5-1.0% of the soluble adrenocortical protein. The partially purified tubulin from both sources gave similar results in the following studies: (1) [3H]colchicine binding examined by Scatchard analysis revealed an apparent Ka of 1 . 10(6) M-1 and a colchicine/tubulin molar binding ratio of 0.4-0.6; (2) tyrosylation studies using a specific tubulin-tyrosine ligase (which adds a tyrosine residue to the C-terminal glutamate or glutamine of the alpha-chain) in conjunction with carboxypeptidase A (which recovers the tyrosine) and (3) amino acid analysis. Examination of protein bands, in addition to the tubulin doublet of 55 000 molecular weight, on sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a difference between the two tubulin preparations. The adrenocortical preparation had protein bands corresponding to apparent molecular weight of 36 000, 60 000, and 68 000. In contrast the brain preparation had only proteins of molecular weights greater than 200 000 (these bands were absent in all adrenal preparations). It would thus appear that if proteins which copurify with tubulin through repeated cycles of polymerization-depolymerization play a role in either microtubule formation or function there is a distinct difference between neural and non-neural tissue.     

Putative involvement of a 49 kDa protein in microtubule assembly in vitro

In higher plant cells, thus far only a few molecules have been inferred to be involved in microtubule organizing centers (MTOCs). Examination of a 49 kDa tobacco protein, homologous to a 51 kDa protein involved in sea urchin MTOCs, showed that it also accumulated at the putative MTOC sites in tobacco BY-2 cells. In this report, we show that the 49 kDa protein is likely to play a significant role in microtubule organization in vitro. We have established a system prepared from BY-2 cells, capable of organizing microtubules in vitro. The fraction, which was partially purified from homogenized miniprotoplasts (evacuolated protoplasts) by salt extraction and subsequent ion exchange chromatography, contained many particles of diameters about 1 micron after desalting by dialysis. When this fraction was incubated with purified porcine brain tubulin, microtubules were elongated radially from the particles and organized into structures similar to the asters observed in animal cells, and therefore also termed "asters" here. Since we could hardly detect BY-2 tubulin molecules in this fraction, the microtubules in "asters" seemed to be solely composed of the added porcine tubulin. Tubulin molecules were newly polymerized at the ends of the microtubules distal to the particles, and the elongation rate of microtubules was more similar to the reported rate of the plus-ends than that of the minus-ends in vitro. By fluorescence microscopy, the 49 kDa protein was shown to be located at the particles. Thus, its location at the centers of the "asters" suggests that the protein plays a role in microtubule organization in vitro.     

Interaction of oncodazole (R 17934), a new antitumoral drug, with rat brain tubulin.

Oncodazole (R 17934), methyl [5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl] carbamate (I), a new synthetic drug with anti-tumoral activity, inhibits the polymerization of rat brain tubulin $\text{in vitro}$. It has no depolymerizing effect on preformed microtubules $\text{in vitro}$. Binding studies by means of molecular sieving and equilibrium dialysis indicates that the drug binds to purified rat brain tubulin in a mole to mole ratio. Finally the drug competitively inhibits colchicine binding to purified rat brain tubulin. From these results the conclusion may be drawn that oncodazole is a true microtubule inhibitor.     

Microtubule-associated proteins-dependent colchicine stability of acetylated cold-labile brain microtubules from the Atlantic cod, Gadus morhua

Assembly of brain microtubule proteins isolated from the Atlantic cod, Gadus morhua, was found to be much less sensitive to colchicine than assembly of bovine brain microtubules, which was completely inhibited by low colchicine concentrations (10 microM). The degree of disassembly by colchicine was also less for cod microtubules. The lack of colchicine effect was not caused by a lower affinity of colchicine to cod tubulin, as colchicine bound to cod tubulin with a dissociation constant, Kd, and a binding ratio close to that of bovine tubulin. Cod brain tubulin was highly acetylated and mainly detyrosinated, as opposed to bovine tubulin. When cod tubulin, purified by means of phosphocellulose chromatography, was assembled by addition of DMSO in the absence of microtubule-associated proteins (MAPs), the microtubules became sensitive to low concentrations of colchicine. They were, however, slightly more stable to disassembly, indicating that posttranslational modifications induce a somewhat increased stability to colchicine. The stability was mainly MAPs dependent, as it increased markedly in the presence of MAPs. The stability was not caused by an extremely large amount of cod MAPs, since there were slightly less MAPs in cod than in bovine microtubules. When "hybrid" microtubules were assembled from cod tubulin and bovine MAPs, these microtubules became less sensitive to colchicine. This was not a general effect of MAPs, since bovine MAPs did not induce a colchicine stability of microtubules assembled from bovine tubulin. We can therefore conclude that MAPs can induce colchicine stability of colchicine labile acetylated tubulin.     

Purification of tau,a microtubule-associated protein that induces assembly of microtubules from purified tubulin

Tau, a microtubule-associated protein which copurifies with tubulin through successive cycles of polymerization and depolymerization, has been isolated from tubulin by phosphocellulose chromatography and purified to near homogeneity. The purified protein is seen to migrate during electrophoresis on acrylamide gels as four closely spaced bands of apparent molecular weights between 55,000 and 62,000. Specific activity for induction of microtubule formation from purified tubulin has been assayed by quantitative electron microscopy and is seen to be enhanced three- to fourfold in the purified tau when compared with the unfractionated microtubule-associated proteins. Nearly 90% of available tubulin at 1 mg/ml is found to be polymerizable into microtubules with elevated levels of tau. Moreover, the critical concentration for polymerization of the reconstituted tau + tubulin system is seen to be a function of tau concentration and may be lowered to as little as 30 μg of tubulin per ml. Under depolymerizing conditions, 50% of the tubulin at only 1 mg/ml may be driven into ring structures. A separate purification procedure for isolation of tau directly from cell extracts has been developed and data from this purification suggest that tau is present in the extract in roughly the same proportion to tubulin as is found in microtubules purified by cycles of assembly and disassembly. Tau is sufficient for both nucleation and elongation of microtubules from purified tubulin and hence the reconstituted tau + tubulin system defines a complete microtubule assembly system under standard buffer conditions. In an accompanying paper (Cleveland et al., 1977) the physical and chemical properties of tau are discussed and a model by which tau may function in microtubule assembly is presented.     

Isolation and characterization of a unique 15 kilodalton trypanosome subpellicular microtubule-associated protein.

A protein of 15 kDa (p15) was isolated from Trypanosoma brucei subpellicular microtubules by tubulin affinity chromatography. The protein bound tubulin specifically both in its native form and after SDS-PAGE in tubulin overlay experiments. p15 promoted both the in vitro polymerization of purified calf brain tubulin and the bundling of preformed mammalian microtubules. Immunolabeling identified p15 at multiple sites along microtubule polymers comprising calf brain tubulin and p15 as well as on the subpellicular microtubules of cryosectioned trypanosomes. Antibodies directed against p15 did not cross react with mammalian microtubules. It is suggested that p15 is a trypanosome-specific microtubule-associated protein (MAP) that contributes to the unique organization of the subpellicular microtubules.     

Mechanism of inhibition of microtubule polymerization by colchicine: inhibitory potencies of unliganded colchicine and tubulin-colchicine complexes.

The tubulin-colchicine binding reaction appears to involve a number of intermediate steps beginning with rapid formation of a transient preequilibrium complex that is followed by one or more slow steps in which conformational changes in tubulin and colchicine lead to formation of a poorly reversible final-state complex. In the present study, we investigated the relative ability of unliganded colchicine and preformed final-stage tubulin-colchicine complex to incorporate at microtubule ends and to inhibit addition of tubulin at the net assembly ends of bovine brain microtubules in vitro. Addition of 0.1 microM final-stage tubulin-colchicine complex to suspensions of microtubules at polymer-mass steady-state resulted in rapid incorporation of one to two molecules of tubulin-colchicine complex per microtubule net assembly end concomitant with approximately 50-60% inhibition of tubulin addition. Incorporation of colchicine-tubulin complex continued slowly with time, without significant additional change in the rate of tubulin addition. In contrast, addition of unliganded colchicine to microtubule suspensions resulted in incorporation of small numbers of colchicine molecules at microtubule ends and inhibition of tubulin addition only after periods of time that varied from several minutes to approximately 20 min depending upon the concentration of colchicine. Inhibition of tubulin addition beginning with unliganded colchicine increased slowly with time, concomitant with increases in the concentration of final-state tubulin-colchicine complex and the amount of colchicine bound per microtubule end. The results indicate that inhibition of tubulin incorporation at microtubule ends is caused by colchicine-liganded tubulin in the form of a final-state complex.(ABSTRACT TRUNCATED AT 250 WORDS)