Assembly Of Microtubule Protein Research Articles

Effects of Allium hirtifolium (Iranian shallot) and its allicin on microtubule and cancer cell lines

Allium hirtifolim Boiss. (Iranian Shallot) belongs to Allium genus (Alliaceae family). Microtubule proteins (MTs) are crucial in maintenance of cell shape as well as cell division and mitosis. The present study aims at defining the anti-microtubule activities of A. hirtifolium and its allicin and examining its effects on nerve cell microtubules. MTs were prepared from sheep brain through two cycles of polymerization and depolymerization. The cell growth inhibition was measured by 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) after treatment with A. hirtifolium and its allicin on HeLa and MCF-7 and L-929 cell lines. A. hirtifolium displayed growth inhibitory activity against HeLa and MCF-7 cells with IC (50) value of 20 and 24 mg/L, respectively, for 72 h and obviously showed cell growth inhibition on these cell lines at non-toxic concentration (lower than 1 g/L). Inhibition of MTs polymerization induced by A. hirtifolium and its ability to bind to tubulin as a ligand was tested through turbidimetry assay then investigated by Transmission Electron Microscopy (TEM). The concentration of A. hirtifolium necessary to inhibit the assembly of MTs by 50% was 1.2 g/L, while an inhibition higher than 80% was observed in the presence of 4 g/L of A. hirtifolium. This plant decreased MTs polymerization; therefore we suggest A. hirtifolium can be an effective ligand for cancer therapy.

Alternative synthetic approach for (+)-phomopsidin via the highly stereoselective TADA reaction

This manuscript describes an alternative synthetic approach for (+)-phomopsidin, which shows strong inhibitory activity against the assembly of microtubule proteins. We observed that the TADA reaction of the macrolactone 5 with the reversed C11 configuration provided the desired cycloadduct 6 in 86% yield with excellent stereoselectivity (dr=16:1). Luche reduction of the ketone derived from the major product of the TADA reaction resulted in a 91% yield with excellent stereoselectivity (dr=21:1), and the major product was successfully converted to the known compound in the previously reported total synthesis of (+)-phomopsidin, thereby accomplishing the formal total synthesis of (+)-phomopsidin.

Curcuminoids Form Reactive Glucuronides In Vitro

Curcumin is of current interest because of its putative anti-inflammatory, anticarcinogenic, and anti-Alzheimer's activity, but its pharmacokinetic and metabolic fate is poorly understood. The present in vitro study has therefore been conducted on the glucuronidation of curcumin and its major phase I metabolite, hexahydro-curcumin, as well as of various natural and artificial analogs. The predominant glucuronide generated by rat and human liver microsomes from curcumin, hexahydro-curcumin, and other analogs with a phenolic hydroxyl group was a phenolic glucuronide according to LC-MS/MS analysis. However, a second glucuronide carrying the glucuronic acid moiety at the alcoholic hydroxyl group was formed from the same curcuminoids, but not hexahydro-curcuminoids, by human microsomes. Curcuminoids without a phenolic hydroxyl group gave rise to the aliphatic glucuronide only. The phenolic glucuronides of curcuminoids, but not of hexahydro-curcuminoids, were rather lipophilic and, in part, unstable in aqueous solution, their stability depending strongly on the type of aromatic substitution. The phenolic glucuronide of curcumin and of its natural congeners, but not the parent compounds, clearly inhibited the assembly of microtubule proteins under cell-free conditions, implying chemical reactivity of the glucuronides. These novel properties of the major phase II metabolites of curcuminoids deserve further investigation.

Effects of Polybrominated Diphenol Ethers from a Marine Sponge Phyllospongia dendyi on IL-8 Production in a PMAstimulated Promyelocytic Leukemia Cell Line

The effects of five polybrominated diphenol ethers from a Marine Sponge, Phyllospongia dendyi, on the relative plating efficiencies in V79 cells and the cell proliferation and IL-8 production in PMA-stimulated HL-60 cells were examined. Three compounds, possessing two phenol units, were reported to inhibit the assembly of microtubule proteins and showed the inhibition of colony formation of V79 cells and increase in IL-8 production. Interestingly, a monomethoxy compound exhibited the stronger activity than the three active compounds in both bioassays. This compound did not inhibit the assembly of microtubule proteins. A dimethoxy compound was not active in either bioassays. In these experimental conditions, the biological activities were not high, but in future, these compounds will be expected to be made stronger by structural modifications.

Isolation and Characterization of Polybrominated Diphenyl Ethers as Inhibitors of Microtubule Assembly from the Marine Sponge Phyllospongia dendyi Collected at Palau

Two new polybrominated diphenyl ethers (1 and 2) were isolated by bioassay-guided separations together with nine known compounds (3-11) from the marine sponge Phyllospongia dendyi collected from Palau. The structures were assigned on the basis of their spectral data. Compounds 3, 7, and 10 showed inhibitory activities to the assembly of microtubule proteins (IC(50): 29.6, 33.5, and 20.9 microM, respectively) and to the meiotic maturation of starfish oocytes (IC(50): 3.6, 4.2, and 4.2 microM, respectively), while 1, 2, 4-6, 8, 9, and 11 were not active at 100 microM. Two phenolic hydroxyls are required for their bioactivities.

Antitumor activity of Z-ajoene, a natural compound purified from garlic: antimitotic and microtubule-interaction properties.

Ajoene, a garlic stable oil-soluble sulfur rich compound was generally isolated as a mixture of two isomers [(E, Z)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide]. It has been described essentially as a potent inhibitor of platelet aggregation in vitro and in vivo. The antiproliferative effects of ajoene and experiments using a single isomer had received little attention. The present study aims at defining the antitumor activities of cis-Z-ajoene in vitro and in vivo. Antiproliferative activity of Z-ajoene was demonstrated against a panel of human tumor cell lines with IC(50) values varying from 5.2 mM to 26.1 mM and at a lower extent in normal marsupial kidney cells (PtK2). Meanwhile, Z-ajoene arrested HL60 cells in G(2)/M phase of cell cycle in a dose and time-dependent way. In PtK2 cells, exposure to 20 microM Z-ajoene for 6 h induced a complete disassembly of the microtubule network, that was associated with an increased number of cells blocked in early mitotic stages. An IC(50) for microtubule disassembly of 1 microM was determined by a fully automated microplate-based multi-detection reader. In vitro, a reversible inhibition of the microtubule protein assembly was observed with an IC(50) of 25 microM Z-ajoene. In vivo, Z-ajoene inhibited tumor growth by 38% and 42% in mice grafted with sarcoma 180 and hepatocarcinoma 22, respectively. For the first time, Z-ajoene was shown to be a potent inhibitor of tumor cell growth both in vitro and in vivo. The microtubule cytoskeleton appeared to be one of the Z-ajoene targets, but the mechanisms by which Z-ajoene interacted with microtubule appeared different from those of other microtubule poisons such as those of the Vinca alkaloids family. The ability of Z-ajoene to preferentially suppress the growth of neoplastic cells could provide a new approach in tumor therapy.

GS-164, a small synthetic compound, stimulates tubulin polymerization by a similar mechanism to that of Taxol.

During our search for new microtubule effectors as anticancer agents, we have found that a small synthetic molecule designated GS-164 interferes with the assembly of porcine microtubule proteins and has cytotoxic activity against a wide range of human tumor cell lines. In this study, we investigated mode of action of the compound in comparison with Taxol and colcemid. To gain an insight into the mode of action of GS-164, we used an in vitro microtubule polymerization assay and a flow-cytometric measurement technique. Microtubule organization and the level of tubulin polymerization in HeLa cells were also examined by immunofluorescence microscopy and cytoskeletal protein analyses, respectively. GS-164 stimulated assembly of microtubule proteins in vitro in a concentration-dependent and a GTP-independent manner. Furthermore, as with Taxol, the microtubule polymerization induced by GS-164 was antagonized by podophyllotoxin, a tubulin polymerization inhibitor, and microtubules formed by GS-164 were resistant to disassembly by calcium or low temperatures. GS-164 in the micromolar range arrested the cell cycle of HeLa cells in the mitotic phase leading to cell death. GS-164 also increased the amounts of cellular microtubules in HeLa cells, resulting in the formation of microtubule bundles. These results indicate that GS-164 stimulates microtubule assembly by a similar mechanism to that of Taxol. A comparative conformational analysis of GS-164 and Taxol suggested that the structure of the former mimics the minimum essential sites of Taxol required to exert the Taxol-like activities described above. Although the cytotoxicity of GS-164 against human tumor cells was 1000-fold lower than that of Taxol and GS-164 was one-tenth as active as Taxol in vitro, these findings pave the way for synthesizing clinically useful anticancer agents using GS-164 as a lead compound.

Interaction of p-benzoquinone and p-biphenoquinone with microtubule proteins in vitro

p-benzoquinone (BQ) and p-biphenoquinone (BPQ) are metabolites of the human myelotoxin and leukemogen benzene, which has been reported to induce aneuploidy in mammalian cells. Because a possible mechanism for the aneuploidogenic effect of benzene may be the disruption of the mitotic spindle by covalent binding of BQ and BPQ to microtubule proteins (MTP), we have studied the reaction of these quinones with MTP and its consequences for microtubule (MT) formation under cell-free conditions. Both BQ and BPQ inhibit the assembly of MTP to MT in a concentration-dependent manner. This interaction is accompanied by a spectral change of the quinones and loss of free sulfhydryl groups of MTP. With 40 μM BQ or BPQ, 50% inhibition of MT assembly was observed and associated with the loss of 1.3 thiol groups per tubulin dimer. Further analysis showed that native MTP form monoadducts, but no diadducts nor disulfide bonds with both BQ and BPQ. The formation of covalent quinone/MTP monoadducts was unequivocally demonstrated by GC/MS analysis of the respective thioanisols liberated by alkaline permethylation. Denatured MTP or glutathione or 2-mercaptoethanol gave rise to the virtually exclusive formation of monoadducts with BQ but led to a high proportion of disulfide bonds with BPQ. Therefore, BQ and BPQ react differently with thiol compounds capable of disulfide bond formation. The fact that both quinones form only monoadducts with native MTP can be explained by the assumption that the thiol groups of native tubulin are not prone to oxidative disulfide bond formation. This proposition was supported by the lack of native MTP to form disulfide bridges upon treatment with hydrogen peroxide/horseradish peroxidase under conditions leading to a complete oxidation of glutathione. The covalent binding of the benzene metabolites BQ and BPQ to critical thiol groups of tubulin inhibits MT formation under cell-free conditions and may also interfere with the formation of a functional spindle apparatus in the mitotic cell, thus leading to the abnormal chromosome segregation and aneuploidy induction reported for benzene.

In vitro assembly of microtubule protein with GTP and 2'dGTP: kinetic evidence for a preassembly conformational change.

The assembly of chick brain microtubule protein in a NaCl-supplemented buffer has been examined with respect to nucleation and the subsequent elongation as a function of the nucleotide (GTP vs 2'dGTP), and the protein and nucleotide concentrations. The kinetics suggest that unassembled tubulin can exist in two conformational states (termed Tu1,GTP and Tu2,GIP when GTP is bound to the exchangeable site), with Tu1,GTP contributing to nucleation and Tu2,GTP participating in elongation. The extent of self-nucleation is proposed to be determined, in part, by the rate constant governing this conformational change. This analysis contrasts with that of earlier studies, which concluded that the number of subunits interacting to form an effective nucleus could be estimated from the dependency of self-nucleation on the initial concentration of unassembled tubulin.

Interaction of tubulin and microtubule proteins with vanadate oligomers.

Microtubule assembly is known to be regulated by the phosphorylation of microtubule-associated proteins (MAPs), and is thus sensitive to phosphatase inhibitors. We have investigated the direct interaction between phosphatase inhibitors (vanadate, sodium fluoride, and okadaic acid) and microtubule proteins. Vanadate self-assembles into oligomers, primarily dimer, tetramer, and decamer in 0.1 M Pipes, pH 6.9. Oligomer concentrations and their direct binding to tubulin and MAPs were determined by 51V NMR. The assembly of microtubule protein (MTP) is strongly inhibited by decavanadate binding to MAPs and only weakly inhibited by tetravanadate binding to MAPs. Decavanadate will inhibit both MAP2 and tau-induced assembly. Decavanadate binds to MAP2 at 26 sites [Ka > or = (1.0-1.3) x 10(5) M-1]. The mechanism appears to involve competitive binding to MAPs, presumably at or near the microtubule binding domains, and reduced affinity for microtubules. The assembly of MAP-free, phosphocellulose-purified tubulin (PC-tubulin) is only weakly inhibited by decavanadate, although decavanadate binds to tubulin at four independent sites (Ka > or = 1.0 x 10(5) M-1). Monomeric vanadate, a strong phosphatase inhibitor, does not interact with tubulin or MAPs, and thus does not bind to the exchangeable nucleotide binding site on tubulin. Sodium fluoride stimulates both PC-tubulin and MTP assembly by a nonspecific effect, probably involving water structure formation. Wyman analysis suggests an absence of direct or specific binding to tubulin (d ln K/d ln [NaF] = 0.214). NaCl is nearly as effective in promoting assembly of PC-tubulin, but inhibits MTP assembly.(ABSTRACT TRUNCATED AT 250 WORDS)

Posttranslational modifications of nerve cytoskeletal proteins in experimental diabetes.

Axonal transport is known to be impaired in peripheral nerve of experimentally diabetic rats. As axonal transport is dependent on the integrity of the neuronal cytoskeleton, we have studied the way in which rat brain and nerve cytoskeletal proteins are altered in experimental diabetes. Rats were made diabetic by injection of streptozotocin (STZ). Up to six weeks later, sciatic nerves, spinal cords, and brains were removed and used to prepare neurofilaments, microtubules, and a crude preparation of cytoskeletal proteins. The extent of nonenzymatic glycation of brain microtubule proteins and peripheral nerve tubulin was assessed by incubation with 3H-sodium borohydride followed by separation on two-dimensional polyacrylamide gels and affinity chromatography of the separated proteins. There was no difference in the nonenzymatic glycation of brain microtubule proteins from two-week diabetic and nondiabetic rats. Nor was the assembly of microtubule proteins into microtubules affected by the diabetic state. On the other hand, there was a significant increase in nonenzymatic glycation of sciatic nerve tubulin after 2 weeks of diabetes. We also identified an altered electrophoretic mobility of brain actin from a cytoskeletal protein preparation from brain of 2 week and 6 week diabetic rats. An additional novel polypeptide was demonstrated with a slightly more acidic isoelectric point than actin that could be immunostained with anti-actin antibodies. The same polypeptide could be produced by incubation of purified actin with glucose in vitro, thus identifying it as a product of nonenzymatic glycation. These results are discussed in relation to data from a clinical study of diabetic patients in which we identified increased glycation of platelet actin. STZ-diabetes also led to an increase in the phosphorylation of spinal cord neurofilament proteins in vivo during 6 weeks of diabetes. This hyperphosphorylation along with a reduced activity of a neurofilament-associated protein kinase led to a reduced incorporation of 32P into purified neurofilament proteins when they were incubated with 32P-ATP in vitro. Our combined data show a number of posttranslation modifications of neuronal cytoskeletal proteins that may contribute to the altered axonal transport and subsequent nerve dysfunction in experimental diabetes.

Increase in cytoplasmic casein kinase II-type activity accompanies neurite outgrowth after DNA synthesis inhibition in NIA-103 neuroblastoma cells.

Whereas cells from most clonal lines derived from the murine neuroblastoma C1300 tumor can be induced to differentiate by serum withdrawal from culture medium, the NIA-103 clonal cell line has been considered unable to extend axon-like processes (neurites). Neurite growth depends on microtubule protein assembly, and although NIA-103 cells have essentially the same amounts of microtubule-associated protein (MAP)-1B and the neuronal-specific class beta 3-tubulin isoform as other neuroblastoma cell lines, these proteins are not phosphorylated in NIA-103 cells on serum withdrawal. The lack of microtubule protein phosphorylation may be due to the different sorting between the nucleus and the cytoplasm of the casein kinase II-related enzyme that is possibly involved in the modification of microtubule proteins. It is interesting that addition of DNA synthesis inhibitors to serum-starved NIA-103 cell cultures induces an increase in the level of cytosolic casein kinase II, an augmented in situ phosphorylation of MAP-1B, and the extension of neurites. Thus, the level of cytoplasmic casein kinase II appears to be controlled by the growth status of neuroblastoma cells. The shift to an increased cytoplasmic concentration of casein kinase II in nonproliferating, differentiating neuroblastoma cells is consistent with its putative role in the regulation of the cytoskeletal rearrangements underlying neuronal morphogenesis and plasticity.

Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization

Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of [14C]NAD+ and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the alpha and beta chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight mirotubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated [14C]ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD+ resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.

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)

Control of nucleation in microtubule self-assembly

The inhibition of the rate and amplitude of assembly of microtubule protein at low GTP concentration is shown by measurement of microtubule length distributions to be due to the suppression of microtubule nucleation. This inhibitory effect is enhanced by GDP added before assembly, but can be overcome by a number of molecules such as pyrophosphate or ADP. The selective inhibition of nucleation by GDP in vitro, which occurs in addition to inhibition of elongation, could provide a mechanism for the control of spontaneous microtubule nucleation in vivo.

Double and single exponential kinetics of microtubule assembly in vitro

1. 1. The kinetics of the microtubule protein assembly were studied in Mes buffer, pH 6.6, at 28°C. 2. 2. The assembly under above conditions follow a kinetic expression containing two exponential terms. The observed two rate constants depend on protein concentration, and are on the order of 10 −2 sec −1 and 10 −3sec −1. 3. 3. When CaCl 2 is added to the system in low concentration, the kinetic expression becomes single exponential. The observed rate constant is independent of protein concentration and its value is 5 × 10 −3 sec −1. 4. 4. It is concluded that the double exponential kinetics correspond to favorable assembly conditions, probably to a high extent of nucleation, whereas the single exponential kinetics is a slower process which occur under hindered assembly conditions.

Calcium-sensitivity of brain microtubule proteins in the presence of S-100 proteins

In the presence of the usual 0.1 M Mes buffer, pH 6.7, mM free Ca 2+ levels are required for half-maximal decrease in the rate and extent of brain microtubule protein (MTP) assembly in the absence of ox brain S-100, while μM free Ca 2+ levels are sufficient in the presence of S-100. At the same pH 6.7, but in the presence of 0.12 M KCl, as low as 1.5 μM free Ca 2+ is sufficient for S-100 to produce half-maximal reduction in the rate of assembly, while as high as 0.5 mM free Ca 2+ is required in the absence of S-100. Similar results are obtained with rat brain S-100 (S-100b), indicating that single S-100 isoforms are equipotent in affecting the MTP assembly. At pH 7.5, MTPs are remarkably resistant to Ca 2+ in the absence of S-100. In the presence of S-100, not only is the free Ca 2+ concentration required for complete inhibition of assembly at least one order of magnitude smaller than that required in the absence of S-100, but significant S-100-dependent inhibition of assembly occurs in the absence of Ca 2+. Under the two conditions where S-100 is particularly effective in inhibiting the assembly, i.e. at pH 6.7 in the presence of KCl and at pH 7.5, S-100 increases the disassembly rate even in the presence of μM Ca 2+ levels. Our results suggest that the free Ca 2+ concentration regulates the way S-100 disassembles microtubules (MTs): at μM Ca 2+ levels, S-100 sequesters tubulin with concomitant increase in the disassembly rate; at mM Ca 2+ levels, the S-100-Ca 2+ complex probably interacts with MTs producing endwise disassembly.

Incorporation of GDP-tubulin during elongation of microtubules [formula omitted

Removal of GDP from tubulin E-site is not obligatory for the in vitro assembly of microtubule protein in 0.5 mM GMPPCP. This assembly, which is significantly enhanced by glycerol, produces microtubules of normal morphology and with normal composition of microtubule-associated proteins (MAPs). [ 3H]-GDP initially present at the E-site is shown to be incorporated directly into microtubules during assembly; this incorporation, maximally 60% of the assembled polymer, is dependent upon MAPs. These results are consistent with oligomeric species composed principally of GDP-tubulin plus MAPs, being incorporated directly into microtubules. The finding that stoichiometric GTP-tubulin formation is not an essential prerequisite for microtubule assembly may have important implications for the energetics of microtubule formation.

The assembly of microtubule protein in vitro. The kinetic role in microtubule elongation of oligomeric fragments containing microtubule-associated proteins

The kinetics of assembly were studied for bovine and pig microtubule protein in vitro over a range of conditions of pH, temperature, nucleotide and protein concentration. The kinetics are in general biphasic with two major processes of similar amplitude but separated in rate by one order of magnitude. Rates and amplitudes are complex functions of solution conditions. The rates of the fast phase and the slow phase attain limiting values as a function of increasing protein concentration, and are more stringently limited at pH 6.5 than pH 6.95. Such behaviour indicates that mechanisms other than the condensation polymerization of tubulin dimer become rate-limiting at higher protein concentration. The constancy of the wavelength-dependence of light-scattering and ultrastructural criteria indicate that microtubules of normal morphology are formed in both phases of the assembly process. Electrophoretic analysis of assembling microtubule protein shows that MAP- (microtubule-associated-protein-)rich microtubules are formed during the fast phase. The rate of dissociation of oligomeric species on dilution of microtubule protein closely parallels the fast-phase rate in magnitude and temperature-dependence. We propose that the rate of this process constitutes an upper limit to the rate of the fast phase of assembly. The kinetics of redistribution of MAPs from MAP-rich microtubules may be a factor limiting the slow-phase rate. A working model is derived for the self-assembly of microtubule protein incorporating the dissociation and redistribution mechanisms that impose upper limits to the rates of assembly attainable by bimolecular addition reactions. Key roles are assigned to MAP-containing fragments in both phases of microtubule elongation. Variations in kinetic behaviour with solution conditions are inferred to derive from the nature and properties of fragments formed from oligomeric species after the rapid temperature jump. The model accounts for the limiting rate behaviour and indicates experimental criteria to be applied in evaluating the relative contributions of alternative pathways.

Mechanism of action of S-100 protein(s) on brain microtubule protein assembly

The inhibitory effect of S-100 on microtubule protein assembly is inversely related to the microtubule protein concentration and/or the temperature of assembly. Moreover, the S-100-induced decrease in the rate and extent of assembly is positively correlated with the length of the lag of assembly. When microtubule fragments are added to the microtubule protein solution, the S-100 effect is reduced but not abolished. These data suggest that S-100 interferes with both the nucleation and the elongation of microtubules. Since S-100 also inhibits the assembly of purified tubulin, S-100 is suggested to affect the microtubule assembly by interacting with and sequestering tubulin.