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.     

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.     

Interactions of Bovine Brain Tubulin with Pyridostigmine Bromide and N,N′-Diethyl-m-Toluamide

Pyridostigmine bromide (PB), an inhibitor of acetylcholinesterase, has been used as a prophylactic for nerve gas poisoning. N,N-diethyl-m-toluamide (DEET) is the active ingredient in most insect repellents and is thought to interact synergistically with PB. Since PB can inhibit the binding of organophosphates to tubulin and since organophosphates inhibit microtubule assembly, we decided to examine the effects of PB and DEET on microtubule assembly as well as their interactions with tubulin, the subunit protein of microtubules. We found that PB binds to tubulin with an apparent K _d of about 60 M. PB also inhibits microtubule assembly in vitro, although at higher concentrations PB induces formation of tubulin aggregates of high absorbance. Like PB, DEET is a weak inhibitor of microtubule assembly and also induces formation of tubulin aggregates. Many tubulin ligands stabilize the conformation of tubulin as measured by exposure of sulfhydryl groups and hydrophobic areas and stabilization of colchicine binding. PB appears to have very little effect on tubulin conformation, and DEET appears to have no effect. Neither compound interferes with colchicine binding to tubulin. Our results raise the possibility that PB and DEET may exert some of their effects in vivo by interfering with microtubule assembly or function, although high intracellular levels of these compounds would be required.     

Modulation of cytosolic and nuclear Ca2+ and Na+ transport by taurine in heart cells

Brain membranes contain tubulin that can be isolated as a hydrophobic compound by partitioning into Triton X-114. We have previously postulated: (a) that this kind of tubulin is a peripheral membrane protein that arises from microtubules that in vivo interact with membranes and (b) that the hydrophobic behaviour is due to the interaction of tubulin with a membrane component. Here we report the in vitro conversion of hydrophilic into hydrophobic tubulin by incubating microtubule associated proteins (MAPs) free taxol-stabilized microtubules with Triton X-100 solubilized membranes. After incubation, the microtubules were sedimented, depolymerized and subjected to partition into Triton X-114. Part of the tubulin was isolated in the detergent phase and contained, as observed in native membranes, a high proportion of the acetylated isotype. Because of the high proportion of acetylated tubulin the in vitro conversion resembles the in vivo interaction. Electrophoretic analysis of the detergent phase shows, besides tubulin, two major protein bands of 29 and 100 kDa molecular mass. The ability of the solubilized membranes to convert hydrophilic into hydrophobic tubulin is greatly diminished if the solubilized membrane preparation is preincubated in the presence of trypsin or heated at 90°C for 5 min, indicating that the membrane component that confers the hydrophobic behaviour to tubulin is of proteinaceous nature.     

Studies on lipid peroxidation in the rat brain

The aim of this study was to set up a simple procedure for assessing lipid peroxidation (L.P.) and testing the activity of antioxidant compounds. L. P. was determined in rat brain homogenates by measuring the endogenous and stimulated accumulation of malonaldehyde (MDA). MDA was assayed by an HPLC method. Homogenates spontaneously formed appreciable amounts of MDA. The addition of increasing concentrations of FeCl₂ resulted in a linear accumulation of MDA, up to 16.6-fold at 50 M. An organic form of iron (Fe-saccharate) was less active on MDA formation (11.4-fold increase at 100 M). The addition of xanthine-xanthine oxidase resulted in only a 2.4-fold increase in MDA formation. Various antioxidant or chelating compounds effectively inhibited L.P., with IC₅₀ between 0.1 M (phenoxazine) and 4–50 M (-tocopherol). Their potencies depended on the iron concentration and time of preincubation with the homogenates. In conclusion, this is a simple and reliable procedure for studying L.P. and inhibiting agents, provided that the experimental conditions are carefully assessed.     

G beta gamma mediates the interplay between tubulin dimers and microtubules in the modulation of Gq signaling

Agonist stimulation causes tubulin association with the plasma membrane and activation of PLC beta 1 through direct interaction with, and transactivation of, G alpha q. Here we demonstrate that G beta gamma interaction with tubulin down-regulates this signaling pathway. Purified G beta gamma, alone or with phosphatidylinositol 4,5-bisphosphate (PIP2), inhibited carbachol-evoked membrane recruitment of tubulin and G alpha q transactivation by tubulin. Polymerization of microtubules elicited by G beta gamma overrode tubulin translocation to the membrane in response to carbachol stimulation. G beta gamma sequestration of tubulin reduced the inhibition of PLC beta 1 observed at high tubulin concentration. G beta 1 gamma 2 interacted preferentially with tubulin-GDP, whereas G alpha q was transactivated by tubulin-GTP. Prenylation of the gamma 2 polypeptide was required for G beta gamma/tubulin interaction. Both confocal microscopy and coimmunoprecipitation studies revealed the spatiotemporal pattern of G beta gamma/tubulin interaction during carbachol stimulation of neuroblastoma SK-N-SH cells. In resting cells G beta gamma localized predominantly at the cell membrane, whereas tubulin was found in well defined microtubules in the cytosol. Within 2 min of agonist exposure, a subset of tubulin translocated to the plasma membrane and colocalized with G beta. Fifteen min post-carbachol addition, tubulin and G beta colocalized in vesicle-like structures in the cytosol. G beta/tubulin colocalization increased after pretreatment of cells with the microtubule-depolymerizing agent, colchicine, and was inhibited by taxol. Taxol also inhibited carbachol-induced PIP2 hydrolysis. It is suggested that G beta gamma/tubulin interaction mediates internalization of membrane-associated tubulin at the offset of PLC beta 1 signaling. Newly cytosolic G beta gamma/tubulin complexes might promote microtubule polymerization attenuating further tubulin association with the plasma membrane. Thus G protein-coupled receptors might evoke G alpha and G beta gamma to orchestrate regulation of phospholipase signaling by tubulin dimers and control of cell shape by microtubules.     

Removal of the projection domain of microtubule-associated protein 2 alters its interaction with tubulin

Microtubule-associated proteins (MAPs) can promote microtubule assemblyin vitro. One of these MAPs (MAP2) consists of a short promoter domain which binds to the microtubule and promotes assembly and a long projection domain which projects out from the microtubule and may interact wth other cytoskeletal elements. We have previously shown that MAP2 and another MAP, tau, differ in their interactions with tubulin in that tau, but not MAP2, promotes extensive aggregation of tubulin into spiral clusters in the presence of vinblastine and that microtubules formed with MAP2 are more resistant than those formed with tau to the antimitotic drug maytansine [Luduena, R. F.,et al. (1984),J. Biol. Chem. 259, 12890–12898; Fellous, A.,et al. (1985),Cancer Res. 45, 5004–5010]. Here we have used chymotryptic digestion to remove the projection domain of MAP2 and examined the interaction of the digested MAP2 (ctMAP2) with tubulin in the presence of vinblastine and maytansine. We have found that ctMAP2 behaves very much like tau, but not like undigested MAP2, in the presence of vinblastine, in that ctMAP2 causes tubulin to polymerize into large clusters of spirals. In contrast, microtubule assembly in the presence of ctMAP2 is much more resistant to maytansine inhibition than is assembly in the presence of tau or undigested MAP2. Our results suggest that the projection domain of MAP2 may play a role in the interaction of tubulin with MAP2 during microtubule assembly.Abbreviations MAPs microtubule-associated proteins - ctMAP2 MAP2 digested with-chymotrypsin - nMAP2 untreated MAP2 - PMSF phenylmethylsulfonyl fluoride - GMPCPP guanosine-5-(,-methylene)triphosphate     

Lignans and neolignans as lead compounds

Many lignans and neolignans have served as lead compounds for the development of new drugs. Perhaps the best known example is podophyllotoxin, an antimitotic compound that binds to tubulin. Etoposide and teniposide are derived from podophyllotoxin, but their antitumoural activity is due to inhibition of topoisomerase II. Combination of both pharmacophores has led to compounds with a dual mechanism of action, such as azatoxin. Dihydrobenzofuran neolignans, based on the natural lead 3,4-di-O-methylcedrusin, have also been investigated as potential antitumoural agents; the dimerisation product of caffeic acid methyl ester was the most active compound. Here too, he cytotoxic activity was due to inhibition of tubulin polymerisation. In addition, the same compounds showed antiangiogenic activity. Podophyllotoxin, as well as other types of lignans, such as dibenzylbutyrolactones related to arctigenin, dibenzocyclooctadiene-type lignans, and dibenzylbutanes, have been explored as leads for antiviral agents (also including HIV). Synthetic 8.O.4-neolignans have been evaluated for their antileishmanial and antifungal properties. Detailed study of the antifungal properties of the phenylpropanoid moieties has resulted in the design of highly active arylpropanoid derivatives. Other examples where lignans have been used as lead compounds include enzyme inhibitors of phosphodiesterase IV and V, and 5-lipoxygenase, and for the development of hypolipidemic and antirheumatic agents.     

Ethacrynic acid inhibition of microtubule assembly in vitro.

Ethacrynic acid (ECA) is a sulfhydryl reactive diuretic drug. Recent studies show that ocular administration of ECA may have potential efficacy for treatment of glaucoma. ECA affects cell shape in cultured cells from the eye outflow pathway and the microtubule system is disrupted. We have studied the effect of ECA on microtubule protein (MTP) (tubulin and microtubule-associated proteins) and purified tubulin assembly. Fifty percent inhibition of MTP (1.8 mg/ml) assembly was found at 70 microM ECA in buffer and 410 microM ECA in 30% glycerol in buffer. If all sulfhydryl groups were attributed to tubulin, then approximately two sulfhydryls were blocked at 50% inhibition. Tubulin (2 mg/ml) assembly showed 50% inhibition at 175 microM ECA and approximately 2 sulfhydryl groups were lost. Increasing ECA preincubation times (0-60 min) with tubulin showed that the longer the preincubation time, the longer the lag time, and the slower the rate of assembly and that the percentage of inhibition was proportional to the ECA preincubation time. The number of blocked sulfhydryls also increased with preincubation time. Approximately two sulfhydryls were blocked at 50% inhibition of assembly. The critical concentration for assembly increased twofold when tubulin was preincubated with 0.1 mM ECA, suggesting a loss of active tubulin. Fifty percent inhibition of taxol-induced MTP and tubulin assembly occurred at 190 and 280 microM ECA, respectively, with 3.6 to 3.8 sulfhydryls blocked, respectively. Taxol protects microtubules from disassembly by ECA, suggesting that the ECA binding key sulfhydryls are blocked in the microtubule. These results suggest that ECA reacts slowly with tubulin and blocks sulfhydryl groups important for assembly. Microtubule-associated proteins and glycerol protect the sulfhydryls and so more ECA is necessary to inhibit assembly. Since the number of blocked sulfhydryls is greater at 50% inhibition for taxol-induced microtubules, sulfhydryl blocked tubulin incompetent to assemble under normal conditions may be induced to do so with taxol.     

Inhibition of microtubule assembly in vitro by TN-16, a synthetic antitumor drug

An antitumor drug, 3-(1-anilinoethylidene)-5-benzylpyrrolidine-2,4-dione (TN-16) inhibited the assembly of porcine brain microtubules in vitro. The assembly induced by taxol was also suppressed by the drug. However, the latter required much higher concentration of TN-16 than the former. Binding studies by means of the fluorometric method and the spun-column procedure indicate that the inhibition was caused by the reversible binding of the drug to the colchicine-sensitive site of tubulin. The affinity of TN-16 to tubulin was almost equal to that of nocodazole.     

Estimation of the diffusion-limited rate of microtubule assembly.

Microtubule assembly is a complex process with individual microtubules alternating stochastically between extended periods of assembly and disassembly, a phenomenon known as dynamic instability. Since the discovery of dynamic instability, molecular models of assembly have generally assumed that tubulin incorporation into the microtubule lattice is primarily reaction-limited. Recently this assumption has been challenged and the importance of diffusion in microtubule assembly dynamics asserted on the basis of scaling arguments, with tubulin gradients predicted to extend over length scales exceeding a cell diameter, approximately 50 microns. To assess whether individual microtubules in vivo assemble at diffusion-limited rates and to predict the theoretical upper limit on the assembly rate, a steady-state mean-field model for the concentration of tubulin about a growing microtubule tip was developed. Using published parameter values for microtubule assembly in vivo (growth rate = 7 microns/min, diffusivity = 6 x 10(-12) m2/s, tubulin concentration = 10 microM), the model predicted that the tubulin concentration at the microtubule tip was approximately 89% of the concentration far from the tip, indicating that microtubule self-assembly is not diffusion-limited. Furthermore, the gradients extended less than approximately 50 nm (the equivalent of about two microtubule diameters) from the microtubule tip, a distance much less than a cell diameter. In addition, a general relation was developed to predict the diffusion-limited assembly rate from the diffusivity and bulk tubulin concentration. Using this relation, it was estimated that the maximum theoretical assembly rate is approximately 65 microns/min, above which tubulin can no longer diffuse rapidly enough to support faster growth.     

The microtubule cytoskeleton and the rounding of isolated generative cells ofNicotiana tabacum

Summary Upon squashing of the pollen grain, the isolated generative cell ofNicotiana tabacum looses its spindle shape to become spherical; this phenomenon is independent of the sucrose concentration used. The time necessary for this change can vary from 1 min (0% sucrose) to 20 min (30% sucrose). The microtubular cytoskeleton was studied by means of immunofluorescence and electron microscopy. Just after isolation, 5 to 15 clearly visible bundles in microtubules organized in a basket-like structure are present. After 15 min in medium with 15% sucrose, the microtubular cytoskeleton disappears, and a diffusely spread tubulin can be observed. Neither the addition of 10–20 M taxol to the medium, nor the omission of Ca²⁺ to the medium has any effect on the changes in cell shape and loss of microtubular bundles after isolation.Abbreviations GC Generative cell - SC sperm cell - BK Brewbaker and Kwack - CLSM confocal laser scanning fluorescence micros copy     

Ammonia-Induced Extracellular Accumulation of Taurine in the Rat Striatum In Vivo: Role of Ionotropic Glutamate Receptors

Accumulation of taurine (Tau), glutamate (Glu) and glutamine (Gln) was measured in vivo in microdialysates of the rat striatum following a direct application to the microdialysis tube of 60 mM ammonium chloride which renders the final ammonia concentration in the extracellular space to 5 mM. The following compounds were coadministered with ammonia to distinguish between the different mechanisms that may underlie the accumulation of amino acids: ion transport inhibitors, diisothiocyanostilbene-2,28-disulfonate (DIDS) and furosemide, a Glu transport inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (PDC), an NMDA receptor antagonist dizocilpine (MK-801) and an 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate (KA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX). Ammonia stimulated Tau accumulation in the microdialysates to 250% of the basal value. Furosemide did not significantly affect the stimulation by ammonia and DIDS only moderately depressed the effect. The ammonia-dependent Tau accumulation was increased by 50% in the presence of PDC and reduced by 35% in the presence dizocilpine and DNQX. In the microdialysates ammonia stimulated Glu and Gln accumulation somewhat less than Tau accumulation. Except for stimulation of Gln accumulation by DNQX, the effects were not modified by any of the cotreatments. The results are consistent with the assumption that ammonia stimulates Tau efflux mainly via activation of ionotropic Glu receptors.     

Changes in the hydrodynamic properties of microtubules induced by taxol

Microtubule assembly was followed and monitored by (1) the turbidity at 350 nm, (2) the weight of the pelleted microtubules, (3) linear dichroism, LD tau, of the turbidity upon flow orientation, (4) the specific viscosity, eta spec, and (5) electron microscopy. These five methods showed the same features for normal microtubule assembly, but were different in the presence of taxol, a drug which binds to tubulin. The The apparent steady state of microtubule assembly in the presence of taxol as found by turbidity or the weight of pelleted polymer did not represent a stable state, as both LD tau and eta spec continued to change for a much longer time. Microtubules assembled in the presence of taxol from microtubule proteins as well as from purified tubulin were difficult to orient, as high flow gradients were needed and the maximal LD tau value represented only 20% of the LD tau for normal microtubules. In contrast to the slow relaxation of normal microtubules, rapid relaxation to random orientation was found in the presence of taxol. Low orientability was also indicated by electron micrographs, in which pelleted microtubules were seen to be randomly oriented in the presence of taxol. Taxol induced a very high eta spec, 4-times the steady-state value in the initial phase of assembly, which slowly declined again to a steady state, an effect which was also found for assembly of purified tubulin assembled in the absence of the microtubule-associated proteins. The presence of taxol did not change the relative amount and composition of the microtubule-associated proteins in the assembled microtubules. The results therefore suggest that taxol alters the hydrodynamic properties of the microtubules due to its interaction with tubulin and that this alteration is not an effect of the microtubule-associated proteins.     

Mechanical properties of brain tubulin and microtubules

We measured the elasticity and viscosity of brain tubulin solutions under various conditions with a cone and plate rheometer using both oscillatory and steady shearing modes. Microtubules composed of purified tubulin, purified tubulin with taxol and 3x cycled microtubule protein from pig, cow, and chicken behaved as mechanically indistinguishable viscoelastic materials. Microtubules composed of pure tubulin and heat stable microtubule-associated proteins were also similar but did not recover their mechanical properties after shearing like other samples, even after 60 min. All of the other microtubule samples were more rigid after flow orientation, suggesting that the mechanical properties of anisotropic arrays of microtubules may be substantially greater than those of randomly arranged microtubules. These experiments confirm that MAPs do not cross link microtubules. Surprisingly, under conditions where microtubule assembly is strongly inhibited (either 5 degrees or at 37 degrees C with colchicine or Ca++) tubulin was mechanically indistinguishable from microtubules at 10-20 microM concentration. By electron microscopy and ultracentrifugation these samples were devoid of microtubules or other obvious structures. However, these mechanical data are strong evidence that tubulin will spontaneously assemble into alternate structures (aggregates) in nonpolymerizing conditions. Because unpolymerized tubulin is found in significant quantities in the cytoplasm, it may contribute significantly to the viscoelastic properties of cytoplasm, especially at low deformation rates.     

Promotion of MAP/MAP interaction by taxol

The effects of taxol on microtubule-associated proteins of high molecular weight (MAPs) were studied in vitro. After negative staining, microtubules reconstituted in the presence of taxol from preparations of partially purified tubulin and MAPs, besides being bundled, displayed prominent elongated or globular extensions without apparent regularity. These extensions, but not the tubulin polymer, were heavily decorated after immuno-gold-labeling using antibodies to MAP-1 and MAP-2. Microtubules reconsituted in the absence of taxol showed a much more regular, and apparently helical, arrangement of MAPs along their surfaces. The formation of polymeric structures was also observed when preparation of MAPs free of tubulin were incubated with taxol. In this case in addition to large network-type aggregates with little apparent substructure, more regular structures seemingly consisting of approximately 5-nm-thick filaments arrayed in parallel were observed. Taxol-induced MAP aggregation occurred rapidly and was directly proportional to the concentration of protein, as revealed by optical density measurements. It is concluded that taxol, aside from promoting the assembly of tubulin and stabilizing microtubules, promotes MAP/MAP interaction.     

Taxol effect on tubulin polymerization and associated guanosine 5'-triphosphate hydrolysis

Taxol has been used as a tool to investigate the relationship between microtubule assembly and guanosine 5'-triphosphate (GTP) hydrolysis. The data support the model previously proposed [Carlier, M.-F., & Pantaloni, D. (1981) Biochemistry 20, 1918] that GTP hydrolysis is not tightly coupled to the polymerization process but takes place as a monomolecular process following polymerization. The results further indicate that the energy liberated by GTP hydrolysis is not responsible for the subsequent blockage of GDP on polymerized tubulin. When tubulin is polymerized in the presence of 10-100 microM taxol, the rapid formation of a large number of very short microtubules (l less than 1 micron) is accompanied by the development of turbidity to a lesser extent than what is observed when the same weight amount of longer microtubules (l = 5 microns) is formed. A slower subsequent turbidity increase corresponds to the length redistribution of these short microtubules into 3-5-fold longer ones without any change in the weight amount of polymer. The evolution of the rate of length redistribution with the concentration of taxol suggests a model within which taxol would bind to dimeric tubulin and to tubulin present at the ends of microtubules with a somewhat 10-fold lower affinity than to polymerized tubulin embedded in the bulk of microtubules. In agreement with this model, binding of taxol to the tubulin-colchicine complex in the dimeric form could be measured from the increase in the GTPase activity of the tubulin-colchicine complex accompanying taxol binding.     

Synthesis and biological evaluation of paclitaxel-thiocolchicine hybrids

The bifunctional taxoid-colchicinoid hybrids 6-8 were synthesized and evaluated in assays of cytotoxicity and tubulin assembly/disassembly. All compounds showed a high degree of cytotoxicity, but, while 6 and 7 behaved as bifunctional tubulin binders not unlike an equimolecular mixture of taxol and thiocolchicine, 8 was surprisingly devoid of tubulin activity, acting on a distinct and yet to identify molecular target.     

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.     

Magnesium requirements for guanosine 5'-O-(3-thiotriphosphate) induced assembly of microtubule protein and tubulin

Two conflicting interpretations on the role of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in microtubule protein and tubulin assembly have been previously reported. One study finds that GTP gamma S promotes assembly while another study reports that GTP gamma S is a potent inhibitor of microtubule assembly. We have examined the potential role of Mg2+ to learn if the conflicting interpretations are due to a metal effect. Turbidity, electron microscopy, and nucleotide binding and hydrolysis were used to analyze the effect of the Mg2+ concentration on GTP gamma S-induced assembly of microtubule protein (tubulin + microtubule-associated proteins) in the presence of buffer +/- 30% glycerol and in buffer with GTP added before or after GTP gamma S. GTP gamma S substantially lowers the Mg2+ concentration required to induce cross-linked or clustered rings of tubulin. These cross-linked rings do not assemble well into microtubules, and GTP only partially restores microtubule assembly. However, taxol will promote GTP gamma S-induced cross-linked rings of microtubule protein to assemble into microtubules. The effect of GTP gamma S on microtubule protein assembly in the presence of Zn2+ with and without added Mg2+ suggests that GTP gamma S also effects the formation of Zn2+-induced sheet aggregates. Purified tubulin was used in assembly experiments with Mg2+, Zn2+, and taxol to better understand GTP gamma S interactions with tubulin. The optimal Mg2+ concentration for assembly of tubulin is lower with GTP gamma S than with GTP.(ABSTRACT TRUNCATED AT 250 WORDS)