Modulation of tubulin-nucleotide interactions by metal ions: comparison of beryllium with magnesium and initial studies with other cations.

With microtubule-associated proteins (MAPs) BeSO4 and MgSO4 stimulated tubulin polymerization as compared to a reaction mixture without exogenously added metal ion, while beryllium fluoride had no effect (E. Hamel et al., 1991, Arch. Biochem. Biophys. 286, 57-69). Effects of both cations were most dramatic at GTP concentrations in the same molar range as the tubulin concentration. We have now compared effects of beryllium and magnesium on tubulin-nucleotide interactions in both unpolymerized tubulin and in polymer. Polymer formed with magnesium had properties similar to those of polymer formed without exogenous cation, except for a 20% lower stoichiometry of exogenous GTP incorporated into the latter. In both polymers the incorporated GTP was hydrolyzed to GDP. Stoichiometry of GTP incorporation into polymers formed with beryllium or magnesium was identical, but much of the GTP in the beryllium polymer was not hydrolyzed. The beryllium polymer was more stable than the magnesium polymer. Beryllium also differed from magnesium in only weakly enhancing the binding of GTP in the exchangeable site of unpolymerized tubulin, while neither cation affected GDP exchange at the site. If both cations were present in a reaction mixture, polymer stability was little changed from that of the beryllium polymer, but most of the GTP incorporated into polymer was hydrolyzed. Six additional metal salts (AlCl3, CdCl2, CoCl2, MnCl2, SnCl2, and ZnCl2) also stimulated MAP-dependent tubulin polymerization, but enhanced polymer stability did not correlate with polymer GTP content. We postulate that enhanced polymer stability is a consequence of cation binding directly to tubulin and/or polymer while deficient GTP hydrolysis in the presence of beryllium, as well as aluminum and tin, is a consequence of tight binding of cation to GTP in the exchangeable site.     

Differential effects of magnesium on tubulin-nucleotide interactions

Magnesium-depleted 2-(N-morpholino)ethanesulfonate (Mes), glutamate, tubulin and microtubule-associated proteins were prepared and used to study the effects of exogenously added MgCl2 on tubulin-nucleotide interactions in 0.1 M Mes with microtubule-associated proteins and in 1.0 M glutamate. Endogenous levels of Mg2+ in the systems studied were approximately stoichiometric with the tubulin concentrations and largely derived from the tubulin. We examined the effects of added Mg2+ on tubulin polymerization, GDP inhibition of polymerization, binding of GDP and GTP to tubulin, and GTP hydrolysis. Exogenously added Mg2+ had markedly different effects on these reactions. The order of their sensitivity for a requirement for added Mg2+ was as follows: GTP binding greater than GTP hydrolysis greater than polymerization greater than GDP binding. Inhibition of polymerization by GDP varied inversely with the Mg2+ concentration and was greatest in the absence of the cation. These results indicate that GDP and GDP-Mg2+ interact with similar affinity at the exchangeable site, while GTP-Mg2+ has a higher affinity for tubulin than does free GTP. Nevertheless, under appropriate conditions, free GTP can interact sufficiently well with tubulin to permit both nucleation and elongation reactions.     

Stabilization of microtubules by inorganic phosphate and its structural analogues, the fluoride complexes of aluminum and beryllium

In order to elucidate how the elementary reactions of GTP cleavage and subsequent inorganic phosphate (Pi) release, which accompany microtubule assembly, regulate microtubule dynamics, the effect of Pi and of its structural analogues AlF4- and BeF3- on the stability of GDP-microtubules has been investigated. Inorganic phosphate binds to microtubules with a low affinity (KD = 25 mM) and slows down the rate of GDP-subunit dissociation by about 2 orders of magnitude. AlF4- and BeF3- exhibit phosphate-like effects with 1000-fold higher affinity. Evidence has been obtained for direct binding of BeF3- to microtubules with a stoichiometry of 1 mol of BeF3- per mole of GDP-subunit and an equilibrium dissociation constant of 12-15 microM. AlF4- and Pi compete for this site. Phosphate analogues abolish oscillatory polymerization kinetics and slow down microtubule turnover at steady state. In view of these results, we propose that Pi and its structural analogues bind to the site of the gamma-phosphate of GTP in the E site and reconstitute a GDP-Pi-microtubule, from which tubulin subunits dissociate very slowly. We therefore understand that, following GTP cleavage on microtubules, Pi release in the medium is accompanied by a structural change resulting in a large destabilization of the polymer. A cap of slowly dissociating GDP-Pi-subunits prevents depolymerization of the microtubule GDP-core at steady state. The similarity with the actin system [Carlier, M.-F., & Pantaloni, D. (1988) J. Biol. Chem. 263, 817-825] is underlined.     

Reexamination of the role of nonhydrolyzable guanosine 5'-triphosphate analogues in tubulin polymerization: reaction conditions are a critical factor for effective interactions at the exchangeable nucleotide site

Recently it was proposed [O'Brien, E. T., & Erickson, H. P. (1989) Biochemistry 28, 1413-1422] that tubulin polymerization supported by guanosine 5'-(beta,gamma-imidotriphosphate) [p(NH)ppG], guanosine 5'-(beta,gamma-methylenetriphosphate) [p(CH2)ppG], and ATP might be due to residual GTP in reaction mixtures and that these nucleotides would probably support only one cycle of assembly. Since we had observed polymerization with these three compounds, we decided to study these reactions in greater detail in two systems. The first contained purified tubulin and a high concentration of glycerol, the second tubulin and microtubule-associated proteins (MAPs). In both systems, reactions supported by nucleotides other than GTP were most vigorous at lower pH values. In the glycerol system, repeated cycles of polymerization were observed with ATP and p(CH2)ppG, but not with p(NH)ppG. With p(NH)ppG, a single cycle of polymerization was observed, and this was caused by contaminating GTP. In the MAPs system, repeated cycles of polymerization were observed with both nonhydrolyzable GTP analogues, even without contaminating GTP, but ATP was not active at all in this system. Binding to tubulin of p(NH)ppG, p(CH2)ppG, and, to a lesser extent, ATP was demonstrated indirectly, since high concentrations of the three nucleotides displaced radiolabeled GDP originally bound in the exchangeable site, with p(NH)ppG the most active of the three compounds in this displacement assay. The failure of GTP-free p(NH)ppG to support tubulin polymerization in our glycerol system even though it displaced GDP from the exchangeable site was further investigated by examining the effects of p(NH)ppG on polymerization and polymer-bound nucleotide with low concentrations of GTP. The two nucleotides appeared to act synergistically in supporting polymerization, so that a reaction occurred with a subthreshold GTP concentration if p(NH)ppG was also in the reaction mixture. Analysis of radiolabeled exchangeable-site nucleotide in polymers formed in reaction mixtures containing both GTP and p(NH)ppG demonstrated that p(NH)ppG which entered polymer did so primarily at the expense of GDP originally bound in the exchangeable site rather than at the expense of GTP. It appears that in the glycerol reaction condition, tubulin-p(NH)ppG cannot initiate tubulin polymerization but that it can participate in polymer elongation. ATP and p(CH2)ppG also entered the exchangeable site during polymerization without GTP in glycerol, as demonstrated by displacement of radiolabeled GDP from polymer when these alternate nucleotides were used.(ABSTRACT TRUNCATED AT 400 WORDS)     

Guanosine 5'-O-(3-thiotriphosphate), a potent nucleotide inhibitor of microtubule assembly

Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and the two diastereoisomers of guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) were prepared enzymatically, and their interactions with tubulin and microtubule-associated proteins (MAPs) in 0.1 M 2-(N-morpholino)ethanesulfonate, 0.5 mM MgCl2 were examined. GTP gamma S did not support microtubule assembly but instead inhibited the reaction. This analog was 1.5-2 times more potent than GDP in inhibiting both tubulin polymerization and GTP hydrolysis under conditions in which these reactions were dependent on MAPs. In contrast to the analog's inhibitory effects on polymerization and hydrolysis, however, radiolabeled GTP gamma S was only feebly bound by purified tubulin at 0 degrees C relative to the binding of GDP and GTP. There was a marked increase in the amount of GTP gamma S bound when the reaction temperature was raised to 37 degrees C or when MAPs were included in the reaction mixture. Only when both MAPs were present and the higher reaction temperature was used did the binding of GTP gamma S exceed that of GDP. Since substitution of sulfur for oxygen in a molecule should decrease its hydrophilic properties, these findings suggest that the exchangeable nucleotide binding site of tubulin becomes more hydrophobic at higher temperatures and in the presence of MAPs. The two isomers of GTP beta S were able to support MAP-dependent polymerization, although a 50-100-fold higher concentration of the analogs as compared to GTP was required. Neither isomer of GTP beta S had a significant inhibitory effect on GTP hydrolysis dependent on tubulin + MAPs.     

Novel inhibition mechanism of Bacillus cereus sphingomyelinase by beryllium fluoride

Phosphate analogs have been known to inhibit competitively various phosphatases and phospholipase C and D. We found for the first time that only beryllium fluoride (BeF(x)) among the phosphate analogs studied inhibits Bacillus cereus sphingomyelinase (SMase) activity. The active inhibitory species proved to be not BeF(3)(-) but BeF(2) by the measurement of SMase activity and of (19)F NMR spectroscopy in the presence of a fixed concentration of BeCl(2) and different concentrations of NaF, although both the species have been reported for other kinds of enzymes. The result of kinetic experiment also indicated that the BeF(x) binds in the vicinity of the essential binding site for the substrate and that the Mg(2+) binding to SMase is essential for the binding of BeF(x) to the enzyme.     

Mechanism of GTP hydrolysis in tubulin polymerization: characterization of the kinetic intermediate microtubule-GDP-Pi using phosphate analogues

Beryllium fluoride (BeF3-) has previously been shown to bind tightly to microtubules as a structural analogue of Pi and to mimic the GDP-Pi transient state in tubulin polymerization [Carlier, M.-F., Didry, D., Melki, R., Chabre, M., & Pantaloni, D. (1988) Biochemistry 27, 3555-3559]. The interaction of BeF3- with tubulin is analyzed here in greater detail. BeF3- binds to and dissociates from microtubule GDP subunits at very slow rates (k+ congruent to 100 M-1 s-1; k- congruent to 6 x 10(-4) s-1), suggesting that a slow conformation change of tubulin, linked to the stabilization of the microtubule structure, follows BeF3- binding. The possibility is evoked that BeF3- acts as a transition-state analogue in the GTPase reaction of tubulin. BeF3- does not bind to dimeric nor to oligomeric GDP-tubulin with high affinity. Substoichiometric binding of BeF3- to microtubules provides extensive stabilization of the structure. An original mechanistic model that accounts for the data is proposed. The kinetic parameters for microtubule elongation in the presence of GTP- and GDP-tubulin with and without BeF3- have been determined. Data support the following views: (i) Microtubules at steady state and in a regime of slow growth in the presence of GTP are stabilized by a cap of GDP-Pi subunits functionally similar to GDP-BeF3 subunits. (ii) In the presence of BeF3-, microtubules elongate from GDP-tubulin within the following sequence of reactions: initial nonproductive binding of GDP-tubulin to microtubule ends is followed by the binding of BeF3- and the associated conformation change allowing sustained elongation.     

Stimulation of Tubulin-Dependent ATPase Activity in Microtubule Proteins from Porcine Brain by Taxol

Taxol, an antimitotic agent that induces microtubule assembly, stimulated tubulin-dependent Mg2+-ATPase activity of microtubule-associated proteins (MAPs). A concentration-dependent increase in the rate of ATP hydrolysis was observed. Taxol acted through its binding to the tubulin molecule on MAP ATPase, and maximal stimulation, which was found at approximately equal concentrations of taxol and tubulin, reached about 140% of the original level in the absence of taxol. Taxol enhanced ATP hydrolysis by a mixture of MAPs and tubulin, and this continued at a steady linear rate even when the polymerization had approached a plateau. In the presence of taxol, a large portion of ATPase activity and protein was recovered in the pellet after centrifugation at 70,000 g for 60 min at 25 degrees C. Both colchicine and podophyllotoxin inhibited taxol-stimulated ATPase activity via the same mechanism by which they inhibited taxol-induced microtubule polymerization. The stimulation by taxol was not found in the presence of Ca2+ alone but required Mg2+. We conclude that tubulin effectively stimulates Mg2+-ATPase activity of MAPs under conditions that induce tubulin polymerization.     

Influence of Mg2+ and inorganic phosphates on the assembly of tubulin depleted of its exchangeable guanine nucleotide.

After the removal of the exchangeable guanine nucleotides by chromatography on phenyl-Sepharose [Hanssens, I., Baert, J., and Van Cauwelaert, F. (1990) Biochemistry 29, 5160-5165] tubulin polymerizations with GTP, GDP, tripolyphosphate, pyrophosphate or orthophosphate as possible stimulants are compared. It is demonstrated that, besides GTP and pyrophosphate, also tripolyphosphate stimulates the assembly into microtubules at high concentrations (4.65 mM) of Mg2+. The influence of Mg2+ is more pronounced in combination with pyrophosphate and tripolyphosphate than with GTP. The microtubules assembled in combination with Mg2+ and tripolyphosphate or pyrophosphate are short, suggesting that especially the nucleation step of microtubule assembly is favoured.     

Tubulin-zinc interactions: binding and polymerization studies

The binding of Zn2+ to tubulin and the ability of this cation to promote the polymorphic assembly of the protein were examined. Equilibrium binding showed the existence of more than 60 potential Zn2+ binding sites on the dimer, including a number of high-affinity sites. The number of high-affinity sites, estimated by using a standard amount of phosphocellulose to remove more weakly bound Zn2+, reached a maximum of 6-7.5 with increasing levels of Zn2+ in the incubation solution. The number also increased with time of incubation at a single Zn2+ concentration. It is suggested that tubulin is slowly denatured in the presence of Zn2+, exposing more binding sites. Cu+ and Cd2+ were effective inhibitors of Zn2+ binding; Mg2+, Mn2+, and Co2+ were much less effective, and Ca2+ was without effect. Zn2+ does not replace the tightly bound Mg2+. GTP reduces the amount of Zn2+ binding under equilibrium conditions and the amount bound to high-affinity sites. Zinc-induced protofilament sheets are produced at a Zn2+/tubulin ratio of 5 in the presence of 0.5 mM GTP, conditions where about two to three Zn2+ ions would be bound to the dimer. At higher GTP concentrations, less assembly occurred, and the products were narrower sheets and microtubules. Zn2+-tubulin, isolated from phosphocellulose, will not assemble unless Mg2+ and dimethyl sulfoxide (Me2SO) or more Zn2+ is added. Broad protofilament sheets, formed from Zn2+-tubulin in the presence of Mg2+ and Me2SO, contain slightly more than one Zn2+ per dimer. It is concluded that Zn2+ stimulates tubulin assembly by binding directly to the protein, not via a ZnGTP complex.     

The binding of Ruthenium red to tubulin

The inhibition of microtubule assembly by Ruthenium red (Deinum, J., Wallin, M., Kanje, M. and Lagercrantz, C. (1981) Biochim. Biophys. Acta 675, 209-213) could be counteracted by either taxol or dimethyl sulfoxide. Ruthenium red remained bound to the assembled microtubules. Microtubules assembled in the presence of Ruthenium red and taxol showed the typical taxol-dependent stability. The dimethyl sulfoxide-induced microtubules showed normal assembly characteristics, e.g., were GTP dependent, could be disassembled by cold, colchicine and Ca2+ and had no alterations in ultrastructure. The absolute disassembly induced by Ca2+ in the presence of dimethyl sulfoxide and Ruthenium red was dependent on the microtubule protein concentration, but independent in the absence of Ruthenium red. Ruthenium red was strongly bound to purified tubulin also in the presence of 8% (v/v) dimethyl sulfoxide. The dimethyl sulfoxide-induced assembly of purified tubulin in the presence of Ruthenium red was slightly stimulated, although the critical protein concentration was the same. It was found by resonance Raman spectroscopy with a flow technique that Ruthenium red did not bind to a specific calcium binding site on tubulin, although binding to a GTP binding site cannot be excluded. The wavenumbers of the lines in the region 375-500 cm-1 differ from those found for Ruthenium red bound to typical calcium-binding proteins such as calmodulin. Although Ruthenium red binds to serum albumin as well, the spectrum with albumin resembled that of the free dye.     

Kainate reversibly aggregates brain tubulin in vitro

The effect of kainate, an heterocyclic analogue of glutamate on tubulin polymerization was studied. We demonstrate that kainate induces a dose-dependent aggregation of rat brain tubulin either purified by DEAE-Sephadex A-50 or in the presence of MAPs into a mesh-like structure. Such polymer is cold-, CaCl2- and colchicine-insensitive. Removal of kainate from the incubation medium yields free tubulin competent for polymerizing into normal microtubules in the presence of GTP.     

Assembly properties of tubulin after carboxyl group modification

By chemically modifying carboxyl groups we have investigated the role of the highly acidic COOH-terminal domains of alpha- and beta-tubulin in regulating microtubule assembly. Using a carbodiimide-promoted amidation reaction, as many as 25 carboxyl groups were modified by the addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and an amine nucleophile, [14C] glycine ethyl ester or [3H]methylamine, to assembled microtubules. Modification occurred primarily in the carboxyl-terminal region as demonstrated by limited proteolysis of modified tubulin by trypsin, chymotrypsin, subtilisin, and carboxypeptidase Y. Modified tubulin polymerized into microtubules with a critical concentration that was 15% of that for unmodified tubulin. Assembly of modified tubulin and microtubules formed from modified tubulin were less sensitive to Ca2+ and high ionic strength. Ca2+ binding studies under low ionic strength conditions indicated that modified tubulin does not contain the high affinity Ca2+ binding site. While assembly of unmodified tubulin was stimulated by Mg2+ up to 10 mM, assembly of the modified protein was inhibited by concentrations greater than 1 mM. When 24 residues were modified, polymerization was no longer stimulated by microtubule-associated proteins (MAPs) or polylysine and incorporation of high molecular weight MAPs into the polymers was reduced by about 70% compared to unmodified tubulin. These studies demonstrate that chemical modification of carboxyl groups in tubulin, most of which are localized in the COOH-terminal region, leads to an enhanced ability to polymerize and a decrease in interaction with MAPs and other positively charged species.     

Mg2+ dependence of guanine nucleotide binding to tubulin

The relationship between the concentration of Mg2+ and the binding of GDP and GTP to tubulin dimers was investigated by measuring the displacement of the nucleotide bound at the exchangeable site (E-site) by radiolabeled GDP and GTP. A wide range of concentrations of GTP, GDP, and Mg2+ was explored. In the near absence of Mg2+, the affinity of tubulin for GDP was found to be much greater than its affinity for GTP. In the presence of 1.0 mM Mg2+, however, its affinity for GDP was slightly less than for GTP. The results could be quantitatively described in terms of a small number of reversible equilibria. Equilibrium constants, pertaining to measurements at 0 degrees C, in 0.1 M piperazine-N,N'-bis(2-ethanesulfonic acid), 0.2 mM dithioerythritol, 2 mM EGTA, pH 6.9, were obtained by nonlinear least squares fitting of the data. When the association constant of tubulin for GDP uncomplexed with Mg2+ was taken to be 1.6 X 10(7) M-1, that for uncomplexed GTP was found to be no larger than 1.4 x 10(4) M-1, at least 1100-fold smaller. The association constant of tubulin for the GDP.Mg2+ complex was found to be 2.5-2.7 x 10(7) M-1, while that for the GTP.Mg2+ complex is 6.4-9.0 x 10(7) M-1.     

Characterization of the aluminum and beryllium fluoride species which activate transducin. Analysis of the binding and dissociation kinetics.

Aluminofluoride and beryllofluoride complexes can activate the heterotrimeric G-proteins by binding next to GDP in the nucleotide site of their G alpha subunit and acting as analogs of the gamma-phosphate of a GTP. However, the exact structures of the activatory complexes in solution as well as those of the bound complexes in the nucleotide site are still disputed. We have studied, by monitoring the activation-dependent tryptophan fluorescence of transducin T alpha subunit, the pF (-log[F-]) and pH dependencies of the kinetics of activation and deactivation of T alpha GDP in the presence of NaF and aluminum or beryllium salts. Comparisons were made with the calculated pF and pH dependencies of the distribution of the metallofluoride complexes, in order to identify the activating species. We observed that the contribution of a magnesium-dependent mechanism of activation by fluoride (Antonny, B., Bigay, J., and Chabre, M. (1990) FEBS Lett. 268, 277-280) and effects due to slow equilibration kinetics between various aluminofluoride complexes could give rise to puzzling kinetics that had caused misinterpretations of previous results. Once corrected for these effects, our results suggest that with aluminum AlF3(OH)- is, rather than AlF4-, the main activating species and that the bound form of the complex is tetracoordinated GDP-AlF3. Deactivation kinetics depend on the free fluoride concentration in the medium, suggesting that the simple bimolecular scheme: T alpha GDP-AlF3 in equilibrium with T alpha GDP+AlF3(OH) does not fully describe the interaction. Fluorides in the bound complexes can also exchange with free F- ions in solution. With beryllium, two complexes are activatory: BeF3-.H2O and BeF2(OH)-.H2O. In the nucleotide site these give two tetracoordinated complexes, GDP.BeF3 and GDP.BeF2(OH), as shown by their different dissociation rates.     

Promotion of tubulin assembly by poorly soluble taxol analogs

Promotion or inhibition of tubulin assembly into microtubules is the standard in vitro assay for evaluating potential antimicrotubule agents. Many agents to be tested are poorly soluble in aqueous solution and require a cosolvent such as dimethyl sulfoxide (DMSO). However, DMSO itself can promote tubulin assembly, and its inclusion in assays for compounds that induce tubulin assembly complicates interpretation of the results. Substituting GDP for GTP in the exchangeable nucleotide binding site of tubulin produces a less active form of the protein, tubulin-GDP. Here it is shown that tubulin-GDP can be assembled into normal microtubules in DMSO concentrations up to 15% (v/v), and polymerization assays performed under these conditions can be compared with assays run under more standard conditions. Assays for measuring the effective concentration of a ligand for promotion of tubulin assembly (EC(50)), measuring the concentration for inhibition of tubulin assembly (IC(50)) by a colchicine site ligand, and measuring tubulin critical concentrations in the presence of poorly soluble taxol derivatives are illustrated.     

Band-3 mediated uptake of beryllofluoride complexes by human erythrocytes.

Beryllium forms several multivalent fluoride complexes in aqueous solution; the relative concentration of each is governed by the relative concentrations of the constituent ions and pH. In 9Be NMR spectra the 9Be (spin = 3/2) and 19F (spin = 1/2) spin coupling gave rise to an overlapping resonance triplet, quartet, and quintet of BeF2, BeF3-, and BeF4(2-), respectively. The low frequency shift of the quartet (0.31 ppm) and the quintet (0.62 ppm) from the triplet correlated with an increase in the number of 19F-ions in each complex. 19F NMR spectra of the complexes showed that the spin-coupled quartet of each complex was progressively shifted to higher frequency with an increase in the number of F- ions in the complex. Using 9Be and 19F NMR, the multiple equilibrium mixture of complexes was found to shift substantially to favor the BeF3- and BeF4(2-) with a relative increase of NaF concentration. The association constants for BeF2, BeF3-, and BeF4(2-) at 25 degrees C were determined directly from the peak intensities of the spectra, and by a numerical fitting procedure for multiple spectra, and were 0.51 +/- 0.17 mM-2, 0.26 +/- 0.03 mM-1, and 1.0 x 10(-2) +/- 0.1 x 10(-2) mM-1, respectively. 19F NMR spectra of human erythrocytes to which Be2+ and F- were added showed separate resonances from the intracellular populations of the complexes and these were shifted to higher frequency from their extracellular counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct incorporation of guanosine 5'-diphosphate into microtubules without guanosine 5'-triphosphate hydrolysis

Using highly purified calf brain tubulin bearing [8-14C]guanosine 5'-diphosphate (GDP) in the exchangeable nucleotide site and heat-treated microtubule-associated proteins (both components containing negligible amounts of nucleoside diphosphate kinase and nonspecific phosphatase activities), we have found that a significant proportion of exchangeable-site GDP in microtubules can be incorporated directly during guanosine 5'-triphosphate (GTP) dependent polymerization of tubulin, without an initial exchange of GDP for GTP and subsequent GTP hydrolysis during assembly. The precise amount of GDP incorporated directly into microtubules is highly dependent on specific reaction conditions, being favored by high tubulin concentrations, low GTP and Mg2+ concentrations, and exogenous GDP in the reaction mixture. Minimum effects were observed with changes in reaction pH or temperature, changes in concentration of microtubule-associated proteins, alteration of the sulfonate buffer, or the presence of a calcium chelator in the reaction mixture. Under conditions most favorable for direct GDP incorporation, about one-third of the GDP in microtubules is incorporated directly (without GTP hydrolysis) and two-thirds is incorporated hydrolytically (as a consequence of GTP hydrolysis). Direct incorporation of GDP occurs in a constant proportion throughout elongation, and the amount of direct incorporation probably reflects the rapid equilibration of GDP and GTP at the exchangeable site that occurs before the onset of assembly.     

Tubulin exchanges divalent cations at both guanine nucleotide-binding sites

The tubulin heterodimer binds a molecule of GTP at the nonexchangeable nucleotide-binding site (N-site) and either GDP or GTP at the exchangeable nucleotide-binding site (E-site). Mg2+ is known to be tightly linked to the binding of GTP at the E-site (Correia, J. J., Baty, L. T., and Williams, R. C., Jr. (1987) J. Biol. Chem. 262, 17278-17284). Measurements of the exchange of Mn2+ for bound Mg2+ (as monitored by atomic absorption and EPR) demonstrate that tubulin which has GDP at the E-site possesses one high affinity metal-binding site and that tubulin which has GTP at the E-site possesses two such sites. The apparent association constants are 0.7-1.1 x 10(6) M-1 for Mg2+ and approximately 4.1-4.9 x 10(7) M-1 for Mn2+. Divalent cations do bind to GDP at the E-site, but with much lower affinity (2.0-2.3 x 10(3) M-1 for Mg2+ and 3.9-6.6 x 10(3) M-1 for Mn2+). These data suggest that divalent cations are involved in GTP binding to both the N- and E-sites of tubulin. The N-site metal exchanges slowly (kapp = 0.020 min-1), suggesting a mechanism involving protein "breathing" or heterodimer dissociation. The N-site metal exchange rate is independent of the concentration of protein and metal, an observation consistent with the possibility that a dynamic breathing process is the rate-limiting step. The exchange of Mn2+ for Mg2+ has no effect on the secondary structure of tubulin at 4 degrees C or on the ability of tubulin to form microtubules. These results have important consequences for the interpretation of distance measurements within the tubulin dimer using paramagnetic ions. They are also relevant to the detailed mechanism of divalent cation release from microtubules after GTP hydrolysis.     

Purification of tubulin from bovine brain and its interaction with guanine nucleotides.

A rapid and sensitive assay for [3H]GTP binding activity of tubulin has been developed. This assay method is based on the quantitative retention of [3H]GTP. Tubulin complex on a nitrocellulose membrane filter. It was also found that bovine brain tubulin is markedly stablized by glycerol and GTP against denaturation. A large-scale purification of bovine brain tubulin was achieved using the new assay procedure and by the inclusion of glycerol and GTP in a buffer solution used for column chromatograph. The purified tubulin could be stored at -80degrees in the presence of glycerol and GTP for at least a year without any apprecialbe loss of [3H]GTP- and [3H]colchicine binding activities. The interaction of tubulin with guanine nucleotides was also studied using the nitorcellulose membrane filter procedure. It was found that the binding of [3H]GTP to tubulin with an empty exchangeable site proceeded promptly within k sec while the exchange of [3H]GTP- with a GTP-tubulin complex in which the exchangeable site had been occupied with unlabeled GTP occured more slowly. The dissociation constants for GTP and GDP at the exchangeable site of tubulin were determined as 0.5 times 10-6M and 1.9 times 10-6M, respectively. 5'-Guanylylimidodiphosphate could interact, although less strongly, with tubulin at this site, whereas the interaction of other nucleoside triphosphates includint ATP, CTP, UTP, and 5'-guanylyl methylenediphosphonate was very weak, if it occured at all. The presence of Mg2+ and a free sulfhydryl group was found to be essential for binding of [3H]GTP to tubulin. Ca2+ was found to replace Mg2+ in this binding reaction.