Rapid and Reversible High-Affinity Binding of the Dinitroaniline Herbicide Oryzalin to Tubulin from Zea mays L

Oryzalin, a dinitroaniline herbicide, was previously reported to bind to plant tubulin with a moderate strengthe interaction (dissociation constant [Kd] = 8.4 [mu]M) that appeared inconsistent with the nanomolar concentrations of drug that cause the loss of microtubules, inhibit mitosis, and produce herbicidal effects in plants (L.C. Morejohn, T.E. Bureau, J. Mole-Bajer, A.S. Bajer, D.E. Fosket [1987] Planta 172: 252-264). To characterize further the mechanism of action of oryzalin, both kinetic and quasi-equilibrium ligand-binding methods were used to examine the interaction of [14C]-oryzalin with tubulin from cultured cells of maize (Zea mays L. cv Black Mexican Sweet). Oryzalin binds to maize tubulin dimer via a rapid and pH-dependent interaction to form a tubulin-oryzalin complex. Both the tubulin-oryzalin binding strength and stoichiometry are underestimated substantially when measured by kinetic binding methods, because the tubulin-oryzalin complex dissociates rapidly into unliganded tubulin and free oryzalin. Also, an uncharacterized factor(s) that is co-isolated with maize tubulin was found to noncompetitively inhibit oryzalin binding to the dimer. Quasi-equilibrium binding measurements of the tubulin-oryzalin complex using purified maize dimer afforded a Kd of 95 nM (pH 6.9; 23[deg]C) and an estimated maximum molar binding stoichiometry of 0.5. No binding of oryzalin to pure bovine brain tubulin was detected by equilibrium dialysis, and oryzalin has no discernible effect on microtubules in mouse 3T3 fibroblasts, indicating an absence of the oryzalin-binding site on mammalian tubulin. Oryzalin binds to pure taxol-stabilized maize microtubules in a polymer mass- and number-dependent manner, although polymerized tubulin has a much lower oryzalin-binding capacity than unpolymerized tubulin. Much more oryzalin is incorporated into polyment during taxol-induced assembly of pure maize tubulin, and half-maximal inhibition of the rapid phase of taxol-induced polymerization of 5 [mu]M tubulin is obtained with 700 [mu]M oryzalin. The data are consistent with a molecular mechanism whereby oryzalin binds rapidly, reversibly, and with high affinity to the plant tubulin dimer to form a tubulin-oryzalin complex that, at concentrations substoichiometric to tubulin, copolymerizes with unliganded tubulin and slows further assembly. Because half-maximal inhibition of maize callus growth is produced by 37 nM oryzalin, the herbicidal effects of oryzalin appear to result from a substoichiometric poisoning of microtubules.     

Resistance of Rosa microtubule polymerization to colchicine results from a low-affinity interaction of colchicine and tubulin

The inhibition of the polymerization of tubulin from cultured cells of rose (Rosa. sp. cv. Paul's scarlet) by colchicine and the binding of colchicine to tubulin were examined in vitro and compared with data obtained in parallel experiments with bovine brain tubulin. Turbidimetric measurements of taxol-induced polymerization of rose microtubules were found to be sensitive and semiquantitative at low tubulin concentrations, and to conform to some of the characteristics of a nucleation and condensation-polymerization mechanism for assembly of filamentous helical polymers. Colchicine inhibited the rapid phase of polymerization at 24°C with an apparent inhibition constant (K _i) of 1.4·10^-4 M for rose tubulin and an apparent K _i=8.8·10^-7 M for brain tubulin. The binding of [³H]colchicine to rose tubulin to form tubulin-colchicine complex was mildly temperature-dependent and slow, taking 2–3 h to reach equilibrium at 24°C, and was not affected by vinblastine sulfate. The binding of [³H]colchicine to rose tubulin was saturable and Scatchard analysis indicated a single class of low-affinity binding sites having an apparent affinity constant (K) of 9.7·10² M^-1 and an estimated molar binding stoichiometry (r) of 0.47 at 24°C. The values for brain tubulin were K=2.46·10⁶ M^-1 and r=0.45 at 37°C. The binding of [³H]colchicine to rose tubulin was inhibited by excess unlabeled colchicine, but not by podophyllotoxin or tropolone. The data demonstrate divergence of the colchicine-binding sites on plant and animal tubulins and indicate that the relative resistance of plant microtubule polymerization to colchicine results from a low-affinity interaction of colchicine and tubulin.Abbreviations MT microtubule - TC tubulin-colchicine complex     

Competitive Inhibition of High-Affinity Oryzalin Binding to Plant Tubulin by the Phosphoric Amide Herbicide Amiprophos-Methyl

Amiprophos-methyl (APM), a phosphoric amide herbicide, was previously reported to inhibit the in vitro polymerization of isolated plant tubulin (L.C. Morejohn, D.E. Fosket [1984] Science 224: 874-876), yet little other biochemical information exists concerning this compound. To characterize further the mechanism of action of APM, its interactions with tubulin and microtubules purified from cultured cells of tobacco (Nicotiana tabacum cv Bright Yellow-2) were investigated. Low micromolar concentrations of APM depolymerized preformed, taxol-stabilized tobacco microtubules. Remarkably, at the lowest APM concentration examined, many short microtubules were redistributed into fewer but 2.7-fold longer microtubules without a substantial decrease in total polymer mass, a result consistent with an end-to-end annealing of microtubules with enhanced kinetic properties. Quasi-equilibrium binding measurements showed that tobacco tubulin binds [14C]oryzalin with high affinity to produce a tubulin-oryzalin complex having a dissociation constant (Kd) = 117 nM (pH 6.9; 23[deg]C). Also, an estimated maximum molar binding stoichiometry of 0.32 indicates pharamacological heterogeneity of tobacco dimers and may be related to structural heterogeneity of tobacco tubulin subunits. APM inhibits competitively the binding of [14C]oryzalin to tubulin with an inhibition constant (Ki) = 5 [mu]M, indicating the formation of a moderate affinity tubulin-APM complex that may interact with the ends of microtubules. APM concentrations inhibiting tobacco cell growth were within the threshold range of APM concentrations that depolymerized cellular microtubules, indicating that growth inhibition is caused by microtubules depolymerization. APM had no apparent effect on microtubules in mouse 3T3 fibroblasts. Because cellular microtubules were depolymerized at APM and oryzalin concentrations below their respective Ki and Kd values, both herbicides are proposed to depolymerize microtubules by a substoichiometric endwise mechanism.     

Effects of anti-microtubule drugs onin vitro polymerization of tubulin from mung bean

Effects of anti-microbule drugs on tubulin polymerizationin vitro were investigated using purified mung bean (Vigna radiata) tubuli. Colchicine induced the formation of macrotubules at the relatively low concentration of 10 μM. and the appearance of corkscrew-like filaments from the ends of the macrotubules at concentrations of more than 100 μM. Vinblastine substantially inhibited polymerization at 1 μM and caused the formation of paracrystals at concentrations greater than 10 μM. Oryzalin inhibited polymerization at 1 μM partially and at 10 μM completely. Paracrystal formation was also induced by cremart at 10 μM, but these paracrystals appeared to be more rigid than those induced by vinblastine. Amiprophos methyl (APM), with a chemical configuration similar to cremart, substantially inhibited polymerization at 1 μM, but the formation of paracrystals was weak. Griseofulvin at 10 μMalso inhibited the polymerization of tubulin while at higher concentrations aggregates of helices were formed. Inhibition of polymerization by phenylcarbamate herbicides was more effective than that caused by benzimidazoylcarbamate fungicides. The effects of drugs onin vitro preformed (MTs) were also investigated. Colchicine and vinblastine showed identical effects to those on the polymerization process. Griseofulvin, cremart and APM induced only macrotubule formation while the other drugs tested had no major effects     

Pharmacokinetic studies of the herbicide and antitumor compound oryzalin in mice

Oryzalin [3,5-dinitro-N,N-di(n-propyl)benzensulfanilamide] is a widely used sulfonamide herbicide that selectively inhibits microtubule formation in algae and higher plants. Oryzalin has also been found to be an inhibitor of intracellular free Ca²⁺ signalling in mammalian cells and to have antitumor activity in animals. Despite its widespread use there have been no reports of the pharmacokinetics of oryzalin in animals or man. A reversed-phase high-performance liquid chromatographic (HPLC) method was developed to measure oryzalin in biological fluids. Following repeated daily administration of oryzalin to mice by the i.p. route to 200 mg/kg, or the p.o. route at 300 mg/kg, peak plasma concentrations of up to 25 μg/ml were achieved. The half life for oryzalin in plasma of mice given i.p. oryzalin was 14.3 h with a clearance of 0.07 1/h. A major metabolite of oryzalin, N-depropyloryzalin, was identified in plasma and its structure confirmed by mass spectral analysis (M+H⁺ = 305). This metabolite was cleared more rapidly than oryzalin with a half life of 1.15 h and a clearance of 0.17 1/h. N-Depropylorryzalin caused similar inhibition of colony formation by HT-29 colon cancer cells as oryzalin with IC₅₀ = 8 μg/ml. The results suggest that oryzalin and its N-depropyl metabolite can inhibit tumor colony formation at pharmacologically achievable levels.     

Comparison of [14C]oryzalin Uptake in Root Segments of a Sensitive and a Resistant Species

Uptake of the dinitroaniline herbicide oryzalin (3,5-dinitro-N⁴,N⁴-dipropylsulfamlamide) and its effect on root growth werestudied using 5 mm corn (Zea mays L.) and pea (Pisum sativum)root apices. Pea root growth was much less susceptible to oryzalinthan corn root growth. Uptake studies showed that pea root apicesalso accumulated much less [¹⁴C]oryzalin and had a lower bindingaffinity for this herbicide. [¹⁴C]oryzalin was not metabolizedin root apices from either species. Thus, the differential susceptibilityto oryzalin in the case of corn versus pea can be explained,at least in part, by differences in oryzalin uptake and accumulationby roots. Oryzalin, dinitroaniline herbicides, Zea mays, Pisum sativum     

Oryzalin bodies: in addition to its anti-microtubule properties, the dinitroaniline herbicide oryzalin causes nodulation of the endoplasmic reticulum

Oryzalin is a much-used pre-emergence herbicide which causes microtubules (Mt) to depolymerize. Here, we document that this dinitroaniline herbicide also leads to characteristic changes in the morphology of the endoplasmic reticulum (ER) and Golgi apparatus. These effects, which are reversible upon washing out the herbicide, are already elicited at low concentrations (2 μM) and become most pronounced at 20 μM. For our studies, we have employed roots of Arabidopsis thaliana, tobacco leaf epidermal cells, and BY-2 suspension cultures, all expressing the luminal ER marker GFP::HDEL. In all cell types, the typical cortical network of the ER assumed a pronounced nodulated morphology with increasing oryzalin concentrations. This effect was enhanced through subsequent application of brefeldin A (BFA). Thin sections of Arabidopsis roots observed in the electron microscope revealed the nodules to consist of a mass of anastomosing ER tubules. Oryzalin also caused the cisternae in Golgi stacks to increase in number but reduced their diameter. Oryzalin retarded ER mobility but did not prevent latrunculin B-induced clustering of Golgi stacks on islands of cisternal ER. While the mechanism underlying these changes in endomembranes remains unknown, it is specific for oryzalin since these effects were not elicited with other Mt-depolymerizing herbicides, e.g., trifluralin, amiprophosmethyl, or colchicine.     

Apigenin shows synergistic anticancer activity with curcumin by binding at different sites of tubulin

Apigenin, a natural flavone, present in many plants sources, induced apoptosis and cell death in lung epithelium cancer (A549) cells with an IC₅₀ value of 93.7 ± 3.7 μM for 48 h treatment. Target identification investigations using A549 cells and also in cell-free system demonstrated that apigenin depolymerized microtubules and inhibited reassembly of cold depolymerized microtubules of A549 cells. Again apigenin inhibited polymerization of purified tubulin with an IC₅₀ value of 79.8 ± 2.4 μM. It bounds to tubulin in cell-free system and quenched the intrinsic fluorescence of tubulin in a concentration- and time-dependent manner. The interaction was temperature-dependent and kinetics of binding was biphasic in nature with binding rate constants of 11.5 × 10⁻⁷ M⁻¹ s⁻¹ and 4.0 × 10⁻⁹ M⁻¹ s⁻¹ for fast and slow phases at 37 °C, respectively. The stoichiometry of tubulin–apigenin binding was 1:1 and binding the binding constant (K_d) was 6.08 ± 0.096 μM. Interestingly, apigenin showed synergistic anti-cancer effect with another natural anti-tubulin agent curcumin. Apigenin and curcumin synergistically induced cell death and apoptosis and also blocked cell cycle progression at G₂/M phase of A549 cells. The synergistic activity of apigenin and curcumin was also apparent from their strong depolymerizing effects on interphase microtubules and inhibitory effect of reassembly of cold depolymerized microtubules when used in combinations, indicating that these ligands bind to tubulin at different sites. In silico modeling suggested apigenin bounds at the interphase of α–β-subunit of tubulin. The binding site is 19 Å in distance from the previously predicted curcumin binding site. Binding studies with purified protein also showed both apigenin and curcumin can simultaneously bind to purified tubulin. Understanding the mechanism of synergistic effect of apigenin and curcumin could be helped to develop anti-cancer combination drugs from cheap and readily available nutraceuticals.     

微管骨架在轮藻节间细胞伸长生长中的作用

利用免疫荧光定位及激光共聚焦扫描显微镜,结合细胞生长曲线的定量测定,对不同生长阶段的轮藻节间细胞微管骨架进行了观察研究,结果如下：轮藻顶端生长活跃的新生细胞中,与细胞长轴垂直的周质微管(cortical microtubules)占绝对优势,随着生长速率的减慢,周质微管由垂直于细胞长轴逐渐转为平行排列;基部生长基本停止的节间细胞中,胞内微管则以平行细胞长轴为主;不同生长阶段节间细胞的微管骨架,对微管特异解聚剂黄草消(oryzalin)处理的敏感性表现不相同。顶端生长活跃的节间细胞经oryzalin处理40min后,绝大多数周质微管发生解聚;而基部生长基本停止的老细胞中,即使延长处理时间,仍残留一些尚未完全解聚的微管片段;10μmol／L微管解聚剂oryzalin处理轮藻顶端新生细胞,在高精度的细胞伸长生长测定装置监测下,发现oryzalin对细胞的伸长生长速率有明显的抑制作用,去掉药剂后,伸长生长又有一定的恢复。并且发现,经oryzalin处理后,微管的解聚(40min左右)与顶端节间细胞伸长生长的停止(100min左右)两者间存在着时间上的差异,即微管解聚在先,细胞伸长停止在后。以上结果均说明微管骨架在轮藻节间细胞生长中具有重要作用。     

Human Microtubule-Associated-Protein Tau Regulates the Number of Protofilaments in Microtubules: A Synchrotron X-Ray Scattering Study

Microtubules (MTs), a major component of the eukaryotic cytoskeleton, are 25 nm protein nanotubes with walls comprised of assembled protofilaments built from αβ heterodimeric tubulin. In neural cells, different isoforms of the microtubule-associated-protein (MAP) tau regulate tubulin assembly and MT stability. Using synchrotron small angle x-ray scattering (SAXS), we have examined the effects of all six naturally occurring central nervous system tau isoforms on the assembly structure of taxol-stabilized MTs. Most notably, we found that tau regulates the distribution of protofilament numbers in MTs as reflected in the observed increase in the average radius 〈R^MT〉 of MTs with increasing Φ, the tau/tubulin-dimer molar ratio. Within experimental scatter, the change in 〈R^MT〉 seems to be isoform independent. Significantly, 〈R^MT〉 was observed to rapidly increase for 0 < Φ < 0.2 and saturate for Φ between 0.2-0.5. Thus, a local shape distortion of the tubulin dimer on tau binding, at coverages much less than a monolayer, is spread collectively over many dimers on the scale of protofilaments. This implies that tau regulates the shape of protofilaments and thus the spontaneous curvature C_o^MT of MTs leading to changes in the curvature C^MT (=1/R^MT). An important biological implication of these findings is a possible allosteric role for tau where the tau-induced shape changes of the MT surface may effect the MT binding activity of other MAPs present in neurons. Furthermore, the results, which provide insight into the regulation of the elastic properties of MTs by tau, may also impact biomaterials applications requiring radial size-controlled nanotubes.     

Dynamics of microtubule reassembly and reorganization in the coenocytic green alga Ernodesmis verticillata (Kützing) Børgesen

The dynamics of microtubule (MT) disassembly and reassembly were studied in the green alga Ernodesmis verticillata, using indirect immunofluorescent localization of tubulin. This alga possesses two distinct MT arrays: highly-ordered, longitudinally-oriented cortical MTs, and shorter perinuclear MTs radiating from nuclear surfaces. Perinuclear MTs are very labile, completely disassembling in the cold (cells on ice) within 5–10 min or in 25 μM amiprophos-methyl (APM) within 15–30 min. Although cortical MTs are generally absent after 3 h in APM, it takes 45–60 min before any cold-induced depolymerization is apparent, and some cortical MTs persist after 6 h of cold treatment. The extent of immunofluorescence of cytoplasmic (depolymerized?) tubulin is inversely proportional to the abundance of cortical MTs. Recovery of MT arrays upon warming or upon removal of APM occurs within 30–60 min for the perinuclear MTs, but the cortical arrays take much longer to regain their normal patterns. The cortical MTs initially reappear in a random distribution with respect to the cell axis, but within 3–4 d of warming (or 24–36 h of removing APM) they are nearly parallel to each other and to the cell's longitudinal axis. Thus, although the timing differs, the actual patterns of depolymerization and recovery are similar, irrespective of whether physical or chemical agents are used. Longer-term treatments in 1 μM APM indicate that despite the rapid disappearance of perinuclear MTs, a loss of the uniform nuclear spacing occurs gradually over 1–6 d. Similar disorganization of nuclei is obtained with long-term treatment with 1 μM taxol, where a gradual loss of perinuclear MTs is accompanied by an increased abundance of mitotic spindles. This implies that perinuclear MTs can disassemble in vivo in the presence of taxol, and that they are not the sole components involved in maintaining nuclear spacing in these coenocytes. The results indicate that both nuclear and cortical sites of MT nucleation may exist in this organism, and that MT reassembly and re-organization are temporally distinct events in cells that have highly-ordered arrays of long MTs.     

Assembly of cortical microtubules during cold treatment of the coenocytic green alga,Chaetomorpha moniligera

The effects of cold treatment on the cortical microtubules (MTs) of Chaetomorpha moniligera Kjellman were investigated by immunofluorescence microscopy. Cortical MTs in Chaetomorpha thallus are arranged longitudinally. In this study, 70–75% of MTs disassembled within 4 h on ice while the others remained stable under these conditions. Reticulate background immunofluorescence, assumed to indicate the presence of a tubulin monomer, was distributed about the stable MTs. Immunofluorescence was prominent in only 50% of the cells. Tubulin polymerization was noted where the background and MT immunofluorescence was strong. New MTs grew transversely as single strings or clusters from the sides of MTs after cold treatment for 4 h and elongated with time to take on a reticulate form at 24 h. The significance of this tubulin polymerization under cold treatment is discussed.Abbreviations MT microtubule - MTOC microtubule-organizing center     

Effect of phenoxyalkanoic acid herbicides on the fermentative production of l-lysine

Summary The effect of the herbicides MCPA, MCPB, mecoprop, dichlorprop, 2,4-D, 2,4-DB, and 2,4,5-T on l-lysine fermentation was investigated using a lysine-producing mutant of Corynebacterium glutamicum. Stimulation of l-lysine production by 6% to 36% was observed in shaken flask experiments when the test herbicides were added at a concentration of 5 · 10^-4 M to growing cultures after 24 h of cultivation. The most effective stimulators were MCPA, mecoprop and dichlorprop.Detailed studies of the effect of MCPA (5 · 10^-6 M to 5 · 10^-3 M) showed that the degree of stimulation depended on medium composition and aeration. In the synthetic medium, maximum production of 50 g · l^-1 lys · HCl occurred at 5 · 10^-4 M MCPA and an oxygen transfer rate (OTR) of 1.97 g O₂ · l^-1 · h^-1, while 61.7 g · l^-1 of lys · HCL was formed at 5 · 10^-3 M MCPA and an OTR of 3.75 g O₂ · l^-1 · h^-1. In the amino-nitrogen rich medium, maximum production of 42 g · l^-1 lys · HCl was observed at 5 · 10^-6 M MCPA and an oxygen transfer rate of 1.5 g O₂ · l^-1 · h^-1. Results from batch l-lysine fermentation in a fermenter showed similar stimulatory effects, with an optimal concentration of MCPA for l-lysine production of 5 · 10^-5 M. Without herbicide addition, the test strain produced 16.25 g · l^-1 of product and with addition of 5 · 10^-5 M MCPA, the same strain produced 52.1 g · l^-1 lys · HCl after 72 h of fermentation.Abbreviations MCPA 2-methyl-4-chlorophenoxyacetic acid - MCPB 2-methyl-4-chlorophenoxybutyric acid - mecoprop 2-methyl-4-chlorophenoxypropionic acid - dichlorprop 2,4-dichlorophenoxypropionic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4-DB 2,4-dichlorophenoxybutyric acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

Rapid assembly and collective behavior of microtubule bundles in the presence of polyamines

Microtubules (MTs) are cylindrical cytoskeleton polymers composed of α-β tubulin heterodimers whose dynamic properties are essential to fulfill their numerous cellular functions. In response to spatial confinement, dynamic MTs, even in the absence of protein partners, were shown to self-organize into higher order structures (spindle or striped structures) which lead to interesting dynamical properties (MT oscillations). In this study, we considered the assembly and sensitivity of dynamic MTs when in bundles. To perform this study, spermine, a natural tetravalent polyamine present at high concentrations in all eukaryote cells, was used to trigger MT bundling while preserving MT dynamics. Interestingly, we first show that, near physiological ionic strengths, spermine promotes the bundling of MTs whereas it does not lead to aggregation of free tubulin, which would have been detrimental to MT polymerization. Experimental and theoretical results also indicate that, to obtain a high rate of bundle assembly, bundling should take place at the beginning of assembly when rapid rotational movements of short and newly nucleated MTs are still possible. On the other hand, the bundling process is significantly slowed down for long MTs. Finally, we found that short MT bundles exhibit a higher sensitivity to cold exposure than do isolated MTs. To account for this phenomenon, we suggest that a collective behavior takes place within MT bundles because an MT entering into a phase of shortening could increase the probability of the other MTs in the same bundle to enter into shortening phase due to their close proximity. We then elaborate on some putative applications of our findings to in vivo conditions including neurons.     

Characterization of a 200 kDa microtubule-associated protein of tobacco BY-2 cells, a member of the XMAP215/MOR1 family

A microtubule-associated protein composed of a 200 kDa polypeptide (MAP200) was isolated from tobacco-cultured BY-2 cells. Analysis of the partial amino acid sequence showed that MAP200 was identical to TMBP200, the tobacco MOR1/XMAP215 homolog. Although several homolog proteins in animal and yeast cells have been reported to promote MT dynamics in vitro, no such function has been reported for plant homologs. Turbidity measurements of tubulin solution suggested that MAP200 promoted tubulin polymerization, and analysis by dark-field microscopy revealed that this MAP increased both the number and length of microtubules (MTs). Electron microscopy and experiments using a chemical crosslinker demonstrated that MAP200 forms a complex with tubulin. Throughout the cell cycle, some MAP200 colocalized with MT structures, including cortical MTs, the preprophase band, spindle and phragmoplast, while some MAP200 was localized in areas lacking MTs. Based on our biochemical and immunofluorescence findings, the function of MAP200 in MT polymerization is discussed.     

Mode of Dinitroaniline Herbicide Action: II. CHARACTERIZATION OF [C]ORYZALIN UPTAKE AND BINDING

The intracellular binding of dinitroaniline herbicides was studied in order to analyze the mechanism of their colchicine-like action. When corn root apices (5 millimeters) are incubated in [¹⁴C]oryzalin (a dinitroaniline herbicide), the ¹⁴C is taken up rapidly, reaching a plateau in about 4 hours, which corresponds to the minimum incubation time in oryzalin required to get maximum inhibition of elongation. At 4 hours, the [¹⁴C]oryzalin concentration inside the roots is 35 times higher than that in the incubation medium. Since this accumulation of [¹⁴C]oryzalin is not affected by 1 millimolar sodium azide and there is no metabolism of [¹⁴C]oryzalin under these conditions, the [¹⁴C]oryzalin must be accumulated (bound) in corn root apices by a process not requiring metabolic energy.     

Thermoresponsive microtubule hydrogel with high hierarchical structure

A thermoresponsive 3D microtubule hydrogel (MT gel) was prepared by simultaneous polymerization and chemical cross-linking of tubulins. The main chain of this gel is composed of cross-linked MTs, which consists of a cylindrical assembly of tubulin covalently connected by polyethylene glycol. This gel, which contains 10 mg/mL of tubulin, exhibits a storage modulus G' as high as 1 × 10(3), which is 10 times higher than the loss modulus G' over a wide range of frequencies. The MT gel exhibits a reversible sol-gel transition by temperature changes at 4-37 °C via depolymerization and polymerization of the MT network. Notable effects of the presence of the cross-linkage on the process of polymerization and depolymerization of tubulin were experimentally observed, and the role of the cross-linkage was discussed.     

Interaction of anthelmintic benzimidazoles and benzimidazole derivatives with bovine brain tubulin

The binding and inhibitory properties of 11 benzimidazoles for bovine brain tubulin were investigated. The effects of the benzimidazoles on the initial rates of microtubule polymerization were determined by a turbidimetric assay. The median inhibitory concentrations (I₅₀) for nocodazole, oxibendazole, parbendazole, mebendazole and fenbendazole ranged from 1.97 · 10⁻⁶ to 6.32 · 10⁻⁶ M. Benomyl, cambendazole and carbendazim had I₅₀ values from 5.83 · 10⁻⁵ to 9.01 · 10⁻⁵ M. Thiabendazole had an I₅₀ value of 5.49 · 10⁻⁴ M. Inhibitor constants (K_i) were determined by the colchicine binding assay. Oxibendazole, fenbendazole, and cambendazole had K_i values of 3.20 · 10⁻⁵, 1.73 · 10⁻⁵ and 1.10 · 10⁻⁴ M, respectively. Oxibendazole and fenbendazole were competitive inhibitors of colchicine. In contrast, cambendazole was a noncompetitive inhibitor of colchicine. The ability of these benzimidazoles to inhibit microtubule polymerization and the mode of action for the anthelmintic benzimidazoles is discussed.     

Binding of 45Ca2+ to particulate fractions of coleoptile tissue

Summary Using recently developed techniques, we have investigated the binding of ⁴⁵Ca²⁺ to membrane preparations from corn (Zea mays L) and oat (Avena sativa L) coleoptile tissue. Scatchard plot analysis reveals at least two Ca²⁺-binding sites in each tissue, a high affinity binding site (K _m=7.7×10^-7 M, n=6.9×10^-10 mol·0.5 g f.w.^-1 in corn, K _m=4.93×10^-6 M, n=2.29×10^-9 mol·0.5 g f.w.^-1 in Avena) and a low affinity binding site (K _m=9.01×10^-5 M, n=5.4×10^-8 mol·0.5 g f.w.^-1 in corn; K _m=1.03×10^-4 M, n=3.40×10^-8 mol·0.5 g f.w.^-1 in Avena). There is also some evidence of a third, lower affinity binding site in each tissue, especially corn.More detailed studies with corn coleoptile homogenates show that they contain a potent dialyzable inhibitor of Ca²⁺ binding. Monovalent cations were observed to be ineffective as inhibitors of Ca²⁺ binding in corn. However, of six divalent cations tested, all were capable of strong inhibition of Ca²⁺-binding and there appeared to be a relationship between size of the atomic radius of the ion and potency as an inhibitor of calcium binding.Abbreviations CSM corn suspensiom medium - EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide - GEE glycine ethyl ester     

Oxygen consumption in Tardigrada from Spitsbergen

Summary Oxygen consumption was measured in seven species of Tardigrada (Doryphoribius smreczynskii, Diphascon spitzbergensis, Macrobiotus islandicus, M. echinogenitus, M. harmswothi, M. spectabilis and M. dispar) from the Spitzbergen tundar. The metabolic rate was measured at 2°, 6° and 10°C. At 6°C it ranged from 0.055 (D. smreczynskii) to 0.101 mm³·10^-3/g·10^-6h (M. harmswothi). In D. smreczynskii the phenomenon of relative thermal independence was observed at a temperature range of 2°–6°C. The dependence of oxygen consumption (R in mm³·10^-3·individ^-1·h^-1) on body weight (W in g·10^-6) for the latter species at 2°C was R=0.088·W^0.82.