Specific macromolecular interactions between tau and the microtubule system

The microtubule-associated protein Tau, a major component of brain microtubules, shares common repeated C-terminal sequences with the high molecular-weight protein MAP-2. It has been shown that tau peptides V¹⁸⁷-G²⁰⁴ and V²¹⁸-G²³⁵ representing two main repeats, induced brain tubulin assembly in a concentration-dependent fashion. The specific roles of these repeats in the interaction of tau with microtubules, and its antigenic nature were investigated using synthetic tau peptides and site-directed monoclonal antibodies. Tau peptides appeared to compete with MAP-2 incorporation into assembled microtubules. The interactions of the tau fragments with -tubulin peptides bearing the tau binding domain on tubulin were analyzed by fluorescence spectroscopy. The specificity of the binding was further demonstrated by the reactivity of tau and the tau peptides with a monoclonal anti-idiotypic antibody produced after immunization with the -11(422–434) tubulin peptide, as assessed by enzyme-linked immunoassay. Western blots confirmed the interaction of tau with the monoclonal antibody. In addition, immunoassays revealed a competition between the MAP-reacting monoclonal antibody and the tubulin peptide -11(422–434) for their interaction with the tau molecule.     

Preparation and characterization of des-C-terminal tubulin

Tubulin, from which the C-terminal peptide had been removed by limited proteolysis was compared to intact native tubulin. Des-C-terminal tubulin (with a nominal molecular weight of 48,000) was prepared by digestion with 1% subtilisin carlsberg at 25°C for 16 min, and the product was purified by ion-exchange chromatography on cellulose DE-52 followed by Sephadex G-50 chromatography. The purified product was composed of the cores of both the - and -subunits of tubulin and was free from other proteins and peptides containing the COOH-terminal moiety as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Sephadex G-50 and ion exchange DE-52 cellulose chromatographies, and ultracentrifugation analysis. The ultraviolet (UV) absorption and fluorescence spectra of des-C-terminal tubulin were the same as those of native tubulin. The sedimentation coefficient of des-C-terminal tubulin (5.9S) was slightly higher than that of native tubulin reflecting a decrease in axial ratio. The change in circular dichroism in the far UV indicated a decrease of -helical contents by 10–15%. These optical properties of des-C-terminal tubulin indicate that the elimination of the COOH-terminal region from tubulin did not change the conformation of the core tubulin molecule significantly, the decrease in -helix being due to the elimination of the C-terminal peptide. des-C-terminal tubulin bound 2 moles/mole of GTP and 1 mole/mole of colchicine, just as intact tubulin, but its binding ability of ruthenium red was reduced.     

Tubulin evolution: Two major types of α-tubulin

Summary Tubulin subunits have been isolated from a variety of protists and marine invertebrates. The sources were: sperm tails of a tunicate (Ciona intestinalis), an abalone (Haliotis rufescens) and a sea anemone (Tealia crassicornis), the gill cilia of a clam (Mercenaria mercenaria), the cilia of a ciliate (Tetrahymena pyriformis) and the cytoplasm of a slime mold (Physarum polycephalum). All the -tubulins, as characterised by their electropherograms after limited proteolytic cleavage withStaphylococcus aureus protease, were fairly similar. In contrast, two markedly different peptide patterns were found for the -tubulins of (a) metazoan axonemes and (b) protistan axonemes, plant axonemes and slime mold cytoplasm.Metazoan axonemal -tubulin peptide patterns could be further divided into two similar but distinct subtypes which did not correlate with the taxonomic divisions of deuterostomia and protostomia, or to different tubulins within an axoneme, or to different tubulins of flagella and cilia. We have postulated that these small differences may be accounted for by a simple glutamicaspartic acid exchange at a particular position in the -tubulin sequence. Identical peptide patterns were observed for sea urchin and sea anemone sperm tail tubulins, proving that the metazoan type of axonemal tubulin arose before the divergence of bilateral and radial symmetric organisms.The close similarity of the slime mold cytoplasmic -tubulin peptide pattern to protistan and plant axonemal -tubulin patterns suggests that the same type of tubulin might be used to form both axonemal and cytoplasmic types of microtubules in protists and plants. The large structural constraints imposed upon this tubulin molecule probably allowed very little change in its primary structure, thus explaining the similarity of tubulins from organisms which diverged at such an early time in eukaryote history. Duplication and modification of the tubulin gene may then have led to the development of specific axonemal and cytoplasmic microtubules during the evolution of the metazoa.     

Proteolytic modifications of the B800-860 complex of the photosynthetic bacterium Rhodocyclus tenuis: Structural and spectral effects

The modification effects on the absorption and cirular dichroic (CD) spectra of the isolated B800-860 antenna complex of Rhodocyclus tenuis by a number of proteolytic enzymes were investigated. The chymotrypsin modifications of the B800-860 complex led to an about 40% decrease of the 860-nm band and a blue-shift to 841 nm. The biphasic CD signal related to the B860 BChl disappeared and a new double CD signal with a zero-crossing point at 842 nm appeared. These absorption and CD spectral changes suggested that a B800-841 complex resulted after chymotrypsin digestion. The polypeptide components of the chymotrypsin-modified B800-860 complex were separated by reverse-phase chromatography, and their amino acid sequences determined by protein sequencing and mass spectrometry. Sequence analyses showed that the C-terminal 25 residues of the B800-860- polypeptide and the C-terminal 8 residues of the B800-860- polypeptide were cleaved by chymotrypsin, and the remaining , polypeptide fragments apparently form the structural basis for the newly-formed B800-841 complex. No significant spectral change was observed from exposing the isolated B800-860 complex to trypsin, carboxypeptidase A and the combination of carboxypeptidase A and carboxypeptidase B. Short-term proteinase K incubation of the B800-860 complex of Rc. tenuis led to a preferential decrease of the 860-nm absorbance band and its related CD signals, as compared to the 800-nm absorbance and CD bands, suggesting that the C-terminal portions of the antenna polypeptides are possibly exposed to the exterior of the B800-860 complex micelles. Whereas, long-term proteinase K digestion resulted in the spectral collapse of the B800-860 complex and the release of free BChls. Our proteolysis experiments support the hypothesis that the C-terminal portions of the antenna polypeptides play a key role in the redshift and strong molar extinction of the Q_y band of the B850 BChls.Abbreviations B800-860 light-harvesting complex with the absorption maxima (Q_y) at 800 nm and 860 nm - B800-860- -, polypeptide of the B800-860 complex - CD circular dichroism - Deriphat-160 disodium Nlauryl--iminodipropionate - FT Fourier transform - LH light-harvesting - near-IR near infra-red - OG n-Octyl--glucoside - PTH phenylthiohydantoin - Rb. Rhodobacter - Rc. Rhodocyclus - Rp. Rhodopseudomonas - Rsp Rhodospirillum - DSM Deutsche Sammlung für Mikroorganismen     

Molecular and enzymatic properties of 1,2-α-d-mannosidase fromAspergillus saitoi

The molecular properties, such as molecular weight, N-and C-terminal amino acids, amino acid composition, and circular dichroism, of 1,2--mannosidase isolated from the culture filtrate ofAspergillus saitoi were determined.The enzyme had aK _m of 0.67 mM andk _cat of 1.27/s with mannobiose at pH 50.0 and 30°C. The anomeric configuration of the reaction products of the enzyme was examined by studying the -anomer. A single Manl2Man linkage in intact Taka-amylase A fromAspergillus oryzae was hydrolyzed, producing free mannose.     

Microtubule-nucleation sites on nuclei of higher plant cells

Summary The nucleation and the elongation of microtubules from isolated nuclei of higher plant cells were investigated. Isolated intact nuclei failed to nucleate microtubules at their surface when they were incubated with purified tubulin from plant or animal sources. However, frozen and thawed nuclei or nuclear particles obtained by gentle nuclei homogenization nucleated microtubules and nucleated microtubules elongated radially from the surface of nuclei or from the nuclear particles. Microtubules radiating from the nuclear particles were very much shorter than those radiating from frozen and thawed nuclei. The washing of the nuclear particles diminished the ability of the particles to nucleate microtubules. The ability of the washed nuclear particles to nucleate microtubules was restored by the addition of the soluble fraction of a nuclear homogenate. The complexes of radiating microtubules could easily be observed under a phasecontrast microscope. Electron microscopy demonstrated that microtubules in the complexes formed bundles. The staining with a monoclonal antibody specific for plant tubulin of the complexes of radiating microtubules, prepared by successive polymerization of animal tubulin and plant tubulin, revealed that microtubules in the complex incorporated tubulin at their proximal ends. This result indicates that the mode of incorporation of tubulin onto frozen and thawed nuclei or onto the nuclear particles is different from that in pericentriolar bodies in animal cells. Mg²⁺ seems to participate in the regulatory mechanism that determines the length of microtubules on the complexes.Abbreviations MTOC microtubule-organizing center - MES 2-(N-morpholino) ethane-sulfonic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - PMSF phenylmethyl sulfonyl fluoride - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol bis (-aminoethylether)-N,N,N,N-tetraacetic acid - GTP guanosine triphosphate - NP-40 Nonidet P-40 - DMSO dimethylsulfoxide - EPC ethyl N-phenylcarbamate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - DAPI 4,6-diamidiho-2-phenylindole     

Carboxyl terminal sequences of β-tubulin involved in the interaction of HMW-MAPs. Studies using site-specific antibodies

After the finding of the involvement of the C-terminal moieties of tubulin subunits in the interaction of MAPs, different studies have focused on the substructure of the binding domains for the different MAPs. Current biochemical evidence point to the role of a low-homology sequence between and -subunits within the conserved region of the C-terminal domain of tubulin, in the binding of AMP-2 and tau. Another line of studies indicates that a site for interaction of the high molecular weight MAPs is located in the variable region defined by the glutamic-rich C-terminus of -tubulin. Here, we report the usefulness of idiotypic site-directed antibodies, produced by immunization with peptides from different -tubulin isoforms, to study both MAP-1 and MAP-2 binding sites on tubulin. On the basis of these results with site-specific antibodies along with previous structural information (Crosset al. 1991, Biochemistry 30: 4362–4366), we propose the role of consensus sequences, from the invariant -tubulin C-terminal domain in the binding of MAP-2 and from the variable domain in the interactions of MAP-1 and MAP-2.Abbreviations HMW-MAPs High Molecular Weight Microtubule Associated Proteins - PC-tubulin Tubulin Purified by the phosphocellulose chromatographic procedure     

Solid-phase peptide synthesis by fragment condensation: Coupling in swelling volume

The condensation of short peptides to resin-bound fragments was examined with respect to high coupling yields with only a small molar excess of a peptide in the reaction solution. The best results were achieved by the addition of reactants (C-unprotected peptide, DIC, and HOBt) dissolved in a so-called swelling volume of an appropriate solvent to a dry resin with an attached N-deprotected peptide chain. Each coupling step was followed by the end-capping of unreacted resin-bound peptide with 2,4-dinitrofluorobenzene. The substituted dinitroaniline chromophore formed in this reaction made the detection and separation of deletion peptides easy. Both conventional and swelling volume methods were compared on parallel syntheses of the HIV-1 protease C-terminal 78–99 fragment. The yields of the isolated heneicosapeptide were 21 and 81% in favor of the swelling volume procedure.     

Temperature sensitivity of vinblastine-induced tubulin polymerization in the presence of microtubule-associated proteins

The antitumor drug vinblastine has been a useful probe for examining the interaction of tubulin with the microtubule-associated proteins (MAPs), specifically with and MAP 2. Although and MAP 2 can stimulate microtubule assemblyin vitro, their specific interactions with tubulin are known to differ. For example, in the presence of vinblastine, both and MAP 2 cause tubulin to form spirals, but causes formation of clustered spirals of high turbidity, while MAP 2 causes formation of loose spirals of low turbidity [Ludueñaet al., J. Biol. Chem. 259, 12890–12898 (1984)]. Although cold temperatures can inhibit microtubule assembly, cold has no effect on vinblastine-induced tubulin spiral formation. Consequently, we used the vinblastine-tubulin system to examine the interactions of and MAP 2 with tubulin at low temperatures. We found that -tubulin-vinblastine complexes form about as well at 0°C as at 37°C. In contrast, MAP 2-tubulin-vinblastine complexes form much less well at 0°C than at 37°C. We find, however, that MAP 2, at 0°C, will strongly inhibit, and even reverse, formation of the -tubulin-vinblastine complex. This suggests that the temperature-sensitive factor is the MAP 2-stimulated tubulin-tubulin interaction rather than the MAP 2-tubulin interactionper se; this raises the possibility that the tubulin-tubulin interactions stimulated by differ in their temperature sensitivity from those stimulated by MAP 2.     

Blockade of a KCa channel with synthetic peptides from noxiustoxin: A K+ channel blocker

Using the outside-out configuration of the patch-clamp method, we studied the effect of several synthetic peptides corresponding to various segments from the N-terminal region of noxiustoxin (NTX) on single Ca²⁺-activated K⁺ (K_Ca) channels of small conductance obtained from cultured bovine aortic endothelial cells. These peptides induced diverse degrees of fast blockade in the endothelial K_Ca channel. The most effective blockers were the peptides NTX_1–39 (IC₅₀=0.5 m) and NTX_1–20 comprising the first 20 amino acids from the native toxin (IC₅₀ 5 m), while less effective was the hexapeptide NTX_1–6, from the first six amino acid residues of NTX (IC₅₀ = 500 m). This was the minimum sequence required to block the channel.By testing overlapping sequences from the entire molecule, specially those corresponding to the N-terminal region of NTX, we have been able to determine their different apparent affinities for the K_Ca channel. Synthetic peptides from the C-terminal region produced no effect on the K_Ca channel at the concentrations tested (up to 1 mm). These results confirm that in the N-terminal region of the NTX is located part of the sequence that may recognize K⁺ channels, as we have suggested previously from in vivo experiments. The blockade induced by native NTX was poorly affected by changes in membrane potential; however, the blockage induced by synthetic peptides lacking the C-terminal region was partially released by depolarization.This study was supported by grant HL-45880 from the National Institutes of Health, and by grant 900946 from the American Heart Association to D.L.K. and Howard Hughes Medical Institute No. 75191-527104, CONACyT-Mexico No. 0018-N9105, and DGAPA-UNAM No. IN 202689 to L.D.P. This work was partially supported by a Grant-in-aid No. 92014250 from the American Heart Association to L.V.     

Antibodies from Trypanosoma cruzi infected mice recognize the second extracellular loop of the β1-adrenergic and M2-muscarinic receptors and regulate calcium channels in isolated cardiomyocytes

Sera from T. cruzi infected mice were tested in an enzyme immunoassay on peptides corresponding to the second extracellular loops of the –, the ₂-adrenergic receptor and the M2 muscarinic receptor. All sera of mice (4/4) in the acute phase recognized the ₁-adrenergic receptor and the M2 muscarinic receptor peptides but not the ₂-adrenergic receptor peptide. The same peptides were recognized during the chronic phase in half of the mice (6/12). The immunoglobulin fractions of the mice were tested for their activity on L-type Ca^–+ channels of isolated guinea-pig cardiomyocytes using the whole-cell patch clamp technique. The immunoglobulin fractions of acute phase mice were able to activate the Ca^–+ channels by stimulation of the -adrenergic receptors, as assessed by inhibition with propranolol. Those of the chronic phase mice reduced the Ca⁺⁺ current by stimulation of the muscarinic receptors, as assessed by inhibition with atropine.These results confirm the existence of functional epitopes on the second extracellular loops of both receptors. They suggest that, as in humans, the parasite is able to elicit functional autoantibodies against these epitopes. They give evidence that these autoantibodies mediate their physiological effects by modulating the cAMP activated Ca^+– channels.     

Acetylated tubulin is found in all microtubule arrays of two species of pine

Summary The distribution of acetylated tubulin in microtubule arrays of conifer cells was investigated by immunofluorescence techniques with 6-11B-1, a monoclonal antibody specific for posttranslationally acetylated -tubulins. In methacrylate sections ofPinus radiata andPinus conforta root tip cells, acetylated tubulin was detected in mitotic spindles, phragmoplasts, and cortical microtubules. Furthermore, staining of isolated, intact cells ofP. radiata andP. contorta indicated that all microtubule structures, including preprophase bands, prophase, metaphase and anaphase spindles, and phragmoplasts, contained some acetylated tubulin, and that the intensity of staining with 6-11B-1 was variable. For example, preprophase bands were lightly labelled, kinetochore fibres of anaphase spindles and phragmoplasts were heavily stained, and metaphase spindles had a granular appearance suggesting discontinuous acetylation of their constituent microtubules. This first report of the presence of acetylated tubulin in conifer cells is in contrast to our results with two species of angiosperms where no acetylated tubulin was detected. The significance of this and the variability of the intensity of staining in conifer arrays is discussed in terms of microtubule dynamics.     

A Change in Tubulin Synthesis in the Process of Tracheary Element Differentiation and Cell Division of Isolated Zinnia Mesophyll Cells

Changes in tubulin synthesis in the process of cytodifferentiationinto tracheary elements and cell division were investigatedusing a culture of single cells isolated from the mesophyllof Zinnia elegans. The tubulin content was measured by a sensitiveimmunoblotting method using a mouse monoclonal antibody to -or ß-tubulin as a probe and mung bean tubulin as astandard. Freshly isolated mesophyll cells had only small amountsof tubulin, but the content increased rapidly between 24 and48 h of culture before morphological differentiation and celldivision. The content rose more than sixfold during 48 h cultureand then decreased slightly. This pattern of increase closelyresembled that of the increase in cortical microtubules (MTs)estimated by electron microscopic analysis. The - and ß-tubulincontents in the cultured cells were almost the same and changedin coordination during culture. The activity of tubulin synthesis was determined by densitometricscanning of spots corresponding to tubulin subunits on an autoradiogramof a two-dimensional polyacrylamide gel of [³⁵S]-methionine-labeledproteins. Tubulin synthesis began as early as between 4 and8 h of culture and its rate increased similarly to the increasein the tubulin content, with the former always preceding thelatter, indicating that the increase in content resulted fromnew tubulin synthesis. (Received December 16, 1986; Accepted February 25, 1987)     

Tubulin Stability and Decay: Mediation by Two Distinct Classes of IKP104-Binding Sites

IKP104, a novel antimitotic drug, has two classes of binding sites on bovine brain tubulin with different affinities. IKP104, by itself, enhances the decay of tubulin, but in the presence of colchicine or podophyllotoxin, it stabilizes tubulin instead of opening up the hydrophobic areas [Luduena et al. (1995), Biochemistry 34, 15751–15759], Here, we have dissected these two apparently contradictory effects of IKP104 by cleaving the C-terminal ends of both and subunits of tubulin with subtilisin. We have found that the selective removal of the C-terminal ends from both the and subunits of tubulin lowers the sulfhydryl titer by approximately 1.5 mol/mol of dimer. Interestingly, IKP104 does not increase either the sulfhydryl liter or the exposure of hydrophobic areas of this subtilisin-treated tubulin (_ss). Moreover, IKP104 lowers the sulfhydryl titer of _ss tubulin approximately by 1 mol/mol and appears to inhibit completely the time-dependent decay of _ss tubulin. The cleavage at the C-terminal ends of both and modulates the effect of IKP104 on the subunit, but not on the subunit. Fluorometric binding data analysis suggests that IKP104 binds to the _ss tubulin only at the high-affinity site; the low-affinity site(s) disappear almost completely. The sulfhydryl titer data for and and the fluoromelric data therefore suggest that the interaction of IKP104 at the high-affinity site on tubulin is not regulated by the C-terminal domains of and and the effect of the high-affinity site is restricted largely to the subunit, while the low-affinity-site binding is modulated by the C-terminal domain of . It also appears that the stabilization and the acceleration of the decay of tubulin are mediated by distinct interactions of IKP104 with its high- and low-affinity sites on tubulin, respectively.     

Effect of trypsin on D1/D2-cytochrom b 559 Photosystem 2 reaction center complex and reaction center from Rhodopseudomonas viridis

Proteolytic enzyme (trypsin) was used to structurally alter the RCs isolated from plant and bacterium as a way of probing the relation between structure (chromophore-apoprotein interactions) and function (photochemical activity). It was found that neither spectral characteristics (absorption spectrum, the 4th derivative of absorption spectrum) nor photochemical activity (pheophytine photoreduction, P680 photooxidation, etc.) were changed dramatically in D₁/D₂/cytochrom b ₅₅₉ PS 2 reaction center complex digested with trypsin. The PS 2 RC treated with trypsin migrates by one green band during electrophoresis with dodecylmaltoside. The peptides with a molecular mass higher than 3–4 kDa were not separated from PS 2 RC. These data indicate that digestion of D1 and D2 proteins does not disturb yet the conformation of peptides or their interactions in so-called core of RC and the native state of pigments. In contrast to that, the RC from Rhodopseudomonas viridis treated with enzyme has changed absorption spectrum and lost photochemical activity. The stability of the bacterial RC increased after exchange of LDAO by dodecylmaltoside.Abbreviations Chl chlorophyll a - Cyt cytochrome - DPC diphenylcarbazide - Dodecylmaltoside dodecyl--D-maltoside - LDAO lauryldimethylamino oxide - Pheo pheophytine - PS 2 Photosystem 2 - RC reaction center - SiMo silicomolybdate - SD sodium dodecyl sulfate     

Different stability of posttranslationally modified brain microtubules isolated from cold-temperate fish

Microtubule proteins were isolated by a temperature-dependent assembly-disassembly method from brain tissue of for cold-temperate fish; one fresh water fish (Oncorhynchus mykiss), and three marine fish (Labrus berggylta, Zoarces viviparus andGadus morhua). The -tubulins from all four fish species were acetylated. The -tubulins from the marine fish were composed of a mixture of tyrosinated and detyrosinated tubulin, while the fresh water fish tubulin only reacted with an antibody against detyrosinated tubulin. The isolated microtubules had a similar MAP composition. A 400 kD protein and a MAP2-like protein were found, but MAP1 was missing. All microtubules disassembled upon cooling to 0°C. In spite of these common characteristics, the assembly of microtubules fromLabrus berggylta was inhibited by colchicine and calcium, in contrast to the assembly of microtubules fromOncorhynchus mykiss andZoarces viviparus. For the latter, colchicine was not completely inhibitory even at a concentration as high as 1 mM, and calcium induced the formation of both loosely and densely coiled ribbons. The effects of calcium and colchicine on microtubules fromOncorhynchus mykiss andZoarces viviparus were modulated by either fish or cow MAPs, indicating that the effects are due to intrinsic properties of the fish tubulins and not the MAPs. In view of these findings, our results suggest that there is not correlation between colchicine sensitivity, inability of calcium to inhibit microtubule assembly, and acetylation and detyrosination.     

The relationship of hydrophobic tubulin with membranes in neural tissue

Brain membrane preparations contain tubulin that can be extracted with Triton X-114. After the extract is allowed to partition, 8% of the total brain tubulin is isolated as a hydrophobic compound in the detergent-rich phase. Cytosolic tubulin does not show this hydrophobic behaviour since it is recovered in the aqueous phase. Membrane tubulin can be released by 0.1 M Na₂CO₃ treatment at pH11.5 in such a way that the hydrophobic tubulin is converted into the hydrophilic form. These results suggest that tubulin exists associated with some membrane component that confers the hydrophobic behaviour to tubulin. If the tissue is homogenized in microtubule-stabilizing buffer containing Triton X-100, the hydrophobic tubulin is isolated from the microtubule fraction. This result indicates that the hydrophobic tubulin isolated from membrane preparations belongs to microtubules thatin vivo are associated to membranes. Therefore, hydrophobic tubulin (tubulin-membrane component complex) can be obtained from membranes or from microtubules depending on the conditions of brain homogenization.Abbreviations TBS Tris-buffered saline - Mes 2-(N-morpholine) ethane sulfonic acid     

Cloning,Purification, and Polymerization of Capsicum annuum Recombinant α and β Tubulin

α and β tubulin genes were cloned from the Capsicum annuum leaves using rapid amplification of cDNA ends (RACE)-PCR. Nucleotide sequence analysis revealed that 1,353 bp Capsicum annuum α?β-tubulin (CAnm α?β-TUB) encodes a protein of 450 amino acids (aa) each. The recombinant α?β tubulin was overexpressed mainly as an inclusion body in Escherichia coli BL21 (DE3), upon induction with 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and its content was as high as 50% of the total protein content. Effective fusion protein purification and refolding are described. The average yields of α and β tubulin were 2.0 and 1.3 mg/l of culture respectively. The apparent molecular weight of each tubulin was estimated to be 55 kDa by SDS-polyacrylamide gel electrophoresis (PAGE). The tubulin monomers were found to be assembly competent using a standard dimerization assay, and also retained antigenicity with anti-His/T7 antibodies. The purified tubulins were polymerized to microtubule-like structures in the presence of 2 mM guanosine 5′-triphosphate (GTP).